ISSN isflD-3iss Pages 255-509 ■ Year 2019, Vol. 66, No. 2 Acta ChimicaSlc Acta Chimica Slc Slovenica ActaC 66/2019 http://acta.chem-soc.si EDITOR-IN-CHIEF KSENIJA KoGEJ Slovenian Chemical Society, Hajdrihova 19, SI-1000 Ljubljana, Slovenija, E-mail: ACSi@fkkt.uni-lj.si, Telephone: (+386)-1-479-8538 ASSOCIATE EDITORS Janez Cerkovnik, University of Ljubljana, Slovenia Krištof Kranjc, University of Ljubljana, Slovenia Ksenija Kogej, University of Ljubljana, Slovenia Franc Perdih, University of Ljubljana, Slovenia Aleš Podgornik, University of Ljubljana, Slovenia Helena Prosen, University of Ljubljana, Slovenia Damjana Rozman, University of Ljubljana, Slovenia Melita Tramšek, Jožef Stefan Institute, Slovenia Irena Vovk, National Institute of Chemistry, Slovenia ADMINISTRATIVE ASSISTANT Marjana Gantar Albreht, National Institute of Chemistry, Slovenia EDITORIAL BOARD Wolfgang Buchberger, Johannes Kepler University, Austria Alojz Demšar, University of Ljubljana, Slovenia Stanislav Gobec, University of Ljubljana, Slovenia Marko Goličnik, University of Ljubljana, Slovenia Günter Grampp, Graz University of Technology, Austria Wojciech Grochala, University of Warsaw, Poland Danijel Kikelj, Faculty of Pharmacy, Slovenia Janez Košmrlj, University of Ljubljana, Slovenia Blaž Likozar, National Institute of Chemistry, Slovenia Mahesh K. Lakshman, The City College and The City University of New York, USA Janez Mavri, National Institute of Chemistry, Slovenia Friedrich Srienc, University of Minnesota, USA Walter Steiner, Graz University of Technology, Austria Jurij Svete, University of Ljubljana, Slovenia Ivan Švancara, University of Pardubice, Czech Republic Jiri Pinkas, Masaryk University Brno, Czech Republic Gašper Tavčar, Jožef Stefan Institute, Slovenia Christine Wandrey, EPFL Lausanne, Switzerland Ennio Zangrando, University of Trieste, Italy ADVISORY EDITORIAL BOARD Chairman Branko Stanovnik, Slovenia Members Josef Barthel, Germany Udo A. Th. 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Graphical Contents «5- ■■ ' S 4 *. - ml ■«B r * Act aC/z im ica Sic ActaChimicaSi c fa SlovenicaActaC Year 2019, Vol. 66, No. 2 FEATURE ARTICLE 255—275 Inorganic chemistry Fluoride in Human Health and Nutrition Dona Stepec and Maja Ponikvar-Svet Fluoride in Human Health and Nutrition In small amounts it prevents dental 1 ) caries. m Excessive intakes can result in / development of i dental and/or '/¡r skeletal fluorosis. iu The adequate intake is set at 0.05 mg/day/kg body weight. w <4K Main sources: - water - _ -tea - food, especially fish - toothpaste SCIENTIFIC PAPER 276-283 Orga nic chemistry Green Synthesis of Bromo organic Molecules and Investigations on Their Antibacterial Properties: An Experimental and Computational Approach Naruti Longkumer, Kikoleho Richa, Rituparna Karmaker, Visekhonuo Kuotsu, Aola Supong, Latonglila Jamir, Pranjal Bharali and Upasana Bora Sinha 284-293 Analytical chemistry Electrochemical Degradation of Reactive Blue 21 and Synthetic Textile Effluent by Using Co475/C475-PVC5 Composite Electrode Norazzizi Nordin, Mohamad Anis Farith Pisal, Nur Izzatie Hannah Razman and Nur Farhana Jaafar Graphical Contents 294-307 Orga nic chemistry Design, Synthesis, Biological Evaluation and Molecular Docking Studies of Some New Sulfonamides Possessing 1,4-Benzodioxane Nucleus Misbah Irshad, Muhammad Athar Abbasi, Aziz-Ur-Rehman, Qamar Ali, Muhammad Aslam, Fozia Iram, Muhammad Shahid, Muhammad Ashraf, Muhammad Arif Lodhi and Syed Babar Jamal 308—314 Inorganic chemistry Three Chiral Cyanide-Bridged Cr-Cu Complexes: Synthesis, Crystal Structures and Magnetic Properties Xia Chen, Wen-Long Lan, Xiao-Yun Hao, Yu Liu, Zhen Zhou, Shu-Juan Zhuang, Lu Yang, Qing-Yun Liu, Wei-Jiang Si and Dao-Peng Zhang 315-325 Chemical, biochemical and environmental engineering Application of Inverse QSAR/QSPR Analysis for Pesticides Structures Generation Belgacem Souyei, Abdelkader Hadj Seyd, Faouzi Zaiz and Abdelkrim Rebiai 326—336 Chemical, biochemical and environmental engineering Adsorptive Performance of Soy Bran and Mustard Husk Towards Arsenic (V) Ions from Synthetic Aqueous Solutions Doina Humelnicu, Laurentiu Valentin Soroaga, Cecilia Arsene, Ionel Humelnicu and Romeo Iulian Olariu 337—343 Chemical, biochemical and environmental engineering Protein Release from Biodegradable Poly(s-Caprolactone)-Chitosan Scaffolds Prepared in scCQ, Gregor Kravanja, Maja Globočnik, Mateja, Primožič, Željko Knez and Maja Leitgeb 344—350 Organic chemistry Synthesis and Biological Evaluation of Some Novel S-^-D-Glucosides of 4-Amino-5-alkyl-1,2,4-triazole-3-thiones Derivatives Anila Rahimi Aghkand, Karim Akbari Dilmaghani, Zahra Dono Ghezelbash and Behvar Asghari 351—359 Applied chemistry Exploring Bikaverin as Metal Ion Biosensor: A Computational Approach Zakir Hussain, Haamid Rasool Bhat, Tahira Naqvi3 Malay K. Rana and Masood Ahmad Rizvi 360—366 Inorganic chemistry Ion-Associated Complex of the Anionic Chelate of Germanium(IV) with Nitro Derivative of the Catechol and the Cation of Monotetrazolium Salt Kirila Stojnova and Vanya Lekova 367-377 chemical, biochemical and environmental engineering Effect of Microwave-Assisted Extraction on Polyphenols Recovery from Tomato Peel Waste Marina Tranfic Bakic, Sandra Pedisic, Zoran Zoric, Verica Dragovic-Uzelac and Antonela Nincevic Grassino 378—387 Inorganic chemistry Cyclometalated Iridium(III) Complexes Containing 2-Phenylbenzo[d]oxazole Ligand: Synthesis, X-ray Crystal Structures, Properties and DFT Calculations Xiao-Han Yang, Qian Zhang, Hui Peng, Zi-Cen Zuo, Ding Yuan, Yan Chen, Qin Chen, Guang-Ying Chen, Zhi-Gang Niu and Gao-Nan Li 2a 2b 385—394 Analytical chemistry A Vortex-Assisted Deep Eutectic Solvent-Based Liquid-Liquid Microextraction for the Analysis of Alkyl Gallates in Vegetable Oils Hasan Çabuk, Yasemin Yilmaz and Elif Yildiz 395-401 physical chemistry Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-Competitive Inhibitors Stefano Rendine, Christian Orrenius, Federico Dapiaggi, Stefano Pieraccini, Ilaria Motto, Roberto D'Alessio, Paola Magnaghi, Antonella Isacchi, Eduard Felder and Maurizio Sironi 402-413 chemical, biochemical and environmental engineering Sandpaper Wastes as Adsorbent for the Removal of Brilliant Green and Malachite Green Dye Yasemin Içlek Coçkun, Nur Aksuner and Jale Yanik 414—420 Inorganic chemistry Preparation, Structure, Photoluminescent and Semiconductive Properties, and Theoretical Calculation of a Mononuclear Nickel Complex with 3-Hydroxy-2-Methylquinoline-4-Carboxylato Ligand Xiao-Niu Fang, Jia Li, Xiu-Guang Yi, Qi Luo, Jia-Yi Chen and Yong-Xiu Li 421—426 Organic chemistry Synthesis and in vivo Anti-inflammatory Evaluation of Piperazine Derivatives Containing 1,4-Benzodioxan Moiety Zhi-Ping Liu, Chang-Da Gong, Long-Yan Xie, Xiu-Li Du, Yang Li and Jie Qin 427—434 General chemistry Growth of Silver Nanoparticles Using Polythiocyanatohydroquinone in Aqueous Solution Valentina A. Litvin, Boris F. Minaev, Rostislav L. Galagan, Glib V. Baryshnikov and Hans Agren 435-442 physical chemistry On Topological Indices of OT [m, n] Octagonal Tillings and TiO2 Nanotubes Hafiz Usman Afzal and Tahzeeb Fatima 443-454 chemical, biochemical and environmental engineering Adsorption Mechanism of Congo Red on Mg-Al-layered Double Hydroxide Nanocompound Narges Safar Beyranvand, Babak Samiey and Abbas Dadkhah Tehrani 455-465 Materials science Preparation of Magnetite by Thermally Induced Decomposition of Ferrous Oxalate Dihydrate in the Combined Atmosphere Josef Kopp, Petr Novak, Josef Kaslik and Jiri Pechousek - Magnetite (T) I - Magnetite (O) - Maghemite Fit Signal -5 0 5 Velocity (mm/s) 466—472 Materials science Phase Equilibria in the MnGa2Te4-MnIn2Te4 System, Crystal Structure and Physical Properties of MnGaInTe4 Faig Mamedagha Mammadov, Imamaddin Rajabali Amiraslanov, Yegana Rasul Aliyeva, Sadiyar Sultan Ragimov, Leyla Farkhad Mashadiyeva and Mahammad Baba Babanly 473-483 chemical, biochemical and environmental engineering Subcritical Water for Recovery of Polyphenols from Comfrey Root and Biological Activities of Extracts Jelena Vladic, Natasa Nastic, Tatjana Stanojkovic, Zeljko Zizak, Jelena Cakarevic, Ljiljana Popovic and Senka Vidovic 484—489 Inorganic chemistry Synthesis, Crystal Structure and Antimicrobial Activity of a Linear Trinuclear Nickel(II) Complex with Schiff Base Ligand Cui-Lin Zhang, Xiao-Yang Qiu, Shu-Juan Liu 490-500 Organic chemistry Synthesis, X-Ray Structure Determination and Related Physical Properties of Thiazolidinone Derivative by DFT Quantum Chemical Method Youcef Megrouss, Fayssal Triki Baara, Nourdine Boukabcha, Abdelkader Chouaih, Antonis Hatzidimitriou, Ayada Djafri, Fodil Hamzaoui 501—509 Biochemistry and molecular biology Feedback Regulation of Cathepsin C by the Propeptide Dipeptides of Granzymes A and B Janja Božič and Iztok Dolenc DOI: 10.17344/acsi.2019.4932 Acta Chim. Slov. 2019, 66, 255-275 ^creative ^commons Feature article Fluoride in Human Health and Nutrition Dona Štepec1,2 and Maja Ponikvar-Svet1,2,* 1 Department of Inorganic Chemistry and Technology, Jožef Stefan Institute, Jamova cesta 39, 1000 Ljubljana, Slovenia 2 Jožef Stefan International Postgraduate School, Jamova cesta 39, 1000 Ljubljana, Slovenia * Corresponding author: E-mail: maja.ponikvar-svet@ijs.si Tel.: +386 1 477 32 03 Received: 03-13-2019 Abstract Fluorine is in the form of fluorides ubiquitous in nature and as such an inevitable part of our environment. In small amounts, it is known to have beneficial effects on dental health. On the other hand, excessive chronic intakes can result in adverse effects including the development of dental fluorosis in children and/or skeletal fluorosis in both children and adults. The adequate intake has been set, based on empirical observations, at 0.05 mg/day/kg body weight however, the threshold between beneficial and harmful effects is narrow. Despite numerous studies, knowledge on the fluoride toxicity is still relatively poor. In this review, the role and the effects of fluoride on human health are enlightened. Some of the fluoride controversies are discussed and future research directions suggested. Keywords: Adequate Intake; Adverse effects; Exposure; Fluoride; Fluorine; Human Health 1. Introduction Fluorine is chemically most reactive of all the elements and is never or rarely encountered in nature as elemental fluorine. Two extremely rare exceptions where fluorine as gas could be trapped within the crystal of antozonite1 and villiaumite2 were recently reported. In combination with other elements, it comprises 0.065% of the earth's crust, being the thirteenth element in abundance on the planet.3 A brief note on terminology is in order before proceeding. In this paper, the term fluorine (F) is used to denote the element in any of its forms and fluoride (F-) to denote the predominant chemical form of inorganic fluoride in which the element is found in nature and to which a fluoride ion selective electrode (ISE) responds. Fluoride is an inevitable part of the biosphere and human life. The average F contents in soil range from about 100-600 mg/kg4,5 from which about 0.05-0.5% represents water soluble fluorides.6,7 Natural waters contain fluoride in varying concentrations, from trace amounts to some mg/l and even toxic concentrations. Waters with high concentrations of fluoride are usually found at the foot of high mountains and in areas with geological deposits of marine origin. The highest fluoride levels have been recorded in the Kenyan lakes Elementaita (1640 mg/l) and Nakuru (2800 mg/l).8 Fluoride concentrations in ambient air are generally lower than 1 ^g/m3.9 The F is released into the air in a form of gaseous or particulate fluorides from natural sources, like dissolution of minerals, volcanoes, marine aerosols, and forest fires10,11 and from different industrial activities, like phosphate fertilizers and elemental phosphorus production, aluminium smelting, petroleum refining, glass, brick, and ceramic manufacturing.12 Combustion of the coal and fuel and controlled fluoridation of drinking-water supplies also contribute to the fluoride dispersion.13,14 Use of organofluorine compounds serving as pharmaceuticals, agro chemicals, refrigerants, pesticides, surfactants, fire extinguishing agents, fibers, membranes, ozone depletors, and insulating materials is in increase. Over 20% of approved pharmaceutical agents including several of the top drugs and 30-40% of commercially available agrochemicals are organofluorine com-pounds.15,16 All these processes and uses result in accumulation of fluoride compounds in soils, surface waters and groundwater reserves, air, and in the living organisms. The effect of fluoride on humans is a dual one. For over eight decades, it has been recognized that small amounts of fluoride have proven benefits for dental health. This resulted in widespread use of fluorides in public health practices.17 Continuous exposure to high levels, on the other hand, leads to severe adverse effects in humans, which include the development of dental fluoro- Štepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 256 Acta Chim. Slov. 2019, 66, 255-275 sis in children or skeletal fluorosis in both, children and adults.17-20 The "optimal" (or "adequate") daily intake of fluoride for children between 0.05 and 0.07 mg/kg body weight,21 that is still accepted worldwide, is based on empirical observations. In Europe, the European Food Safety Authority (EFSA) set, because of proven benefits of small amounts of fluoride on the prevention of caries, the adequate intake (AI) of fluoride from all sources (including non-dietary sources) at 0.05 mg/day/kg body weight for both children and adults.22 The primary sources of ingested fluorine for humans are water, beverages and food.23 In some countries, fluoride is deliberately added to salt24 or milk25 or public water supplies.26,27 Intake with fluoride containing dentifrices, especially in children, should be considered.28,29 In endemic fluorosis areas intake of fluoride with drinking wa-ter30 or brick tea31 and coal-burning32 were reported to result in development of skeletal fluorosis.33-36 The adequate intake of fluoride in relation to its beneficial/adverse effects and the main sources of fluoride for humans are graphically depicted in Figure 1. Figure 1: The adequate intake of fluoride in relation to its beneficial/ adverse effects and the main sources of fluoride for humans Fluoride remains a subject of debate, time and time again, especially in view of the current knowledge on its possible adverse effects. The aim of this paper was twofold: first to provide the adequate background information on fluoride, which would assist in understanding its role and effects on humans, and, second, to present some of the analytical problems associated with the determination of fluoride at low levels as present in human and environmental samples. In addition, some of the fluoride controversies related to the intake of fluoride by humans and its impact on health were discussed. gets. Beneficial effects of fluoride on dental health and on the increase of bone mineral content were suggested. 2. 1. Effects on Dental Health Until the 1980's the paradigm was that, to exert its maximum cariostatic effect, fluoride had to become incorporated into dental enamel during teeth development by forming hydroxyfluorapatite, which is more resistant to ingested acids or to acids generated by oral bacteria from ingested sugars.37 The new paradigm, which surprisingly slowly modified fluoride caries research, was introduced in 1981.38 It explains that fluoride controls caries lesion development predominantly, if not entirely, via its topical effect on de-and re-mineralization processes taking place at the interface between the tooth surface and the oral fluids.38,39 Thus, topical use of fluoride is recommended, rather than systemic. Fluoride also interferes with the metabolism of oral microbial cells by a direct inhibition of cellular enzymes (directly or in combination with metals) or enhancing proton permeability of cell membranes in the form of hydrogen fluoride (HF).40,41 2. 2. Osteoporosis Treatment Since the 1960s, fluoride at high dose levels (approximately 20-30 mg/day) has been used to treat age-dependent osteoporosis.12 Such treatment is currently not recommended. Although it may increase bone mass, the newly formed bone may lack normal structure and strength.42-44 The effect is more apparent in trabecular bone where volume and thickness is increased but without a concomitant increase in trabecular connectivity resulting in reduced bone quality.45 2. 3. Other Functions No average requirement (AR) of fluoride for the performance of essential physiological functions can be defined, i.e. fluoride is not essential for tooth development and has no known essential function in human growth and development.22 A statement that "no fluoride deficiency disease has ever been documented for humans"46 is controversial. An experimental diet completely free of fluoride, capable of provoking fluoride deficiency, is difficult to obtain.23 It is also difficult to prove that it is free of fluoride, because of methodological and analytical problems in determining fluorine at low levels (see also section Flu-orin(d)e analytical methods).23 2. Beneficial Effects of Fluoride on Human Health Fluoride in the body is mainly associated with calcified tissue, i.e. teeth and bone which are its primary tar- 3. Adverse Effects of Fluoride The terms adverse effects and side effects are often used interchangeably although their meaning is different. Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 257 Acta Chim. Slov. 2019, 66, 255-275 Adverse effects are unintended effects that occur when a medication is administered correctly while a side effect is a secondary unwanted effect that occurs due to drug therapy. Side effects are most often mild in nature and often self resolving but adverse effects can be fatal and need to be reversed or antidote immediately. Adverse effects reduce either by reducing the dose of the medicines or by stopping the administration of the drug altogether.47,48 There are many fluoride related adverse effects. Their symptoms are nonspecific and are very much similar to the adverse and/or side effects of many other drugs, vitamins, minerals or dietary supplements. We should be therefore not surprised if fluoride related adverse effects are ascribed to other causes than fluoride. 3. 1. Symptoms of Fluoride Toxicity The toxicity of fluorides is due to the toxicity of the fluoride ion, a direct cellular poison that binds calcium and interferes with the activity of proteolytic and glycolytic enzymes.49 As such, the toxicity of fluoride depends on the type of compound ingested. Generally, weakly soluble or insoluble salts of inorganic fluorides, such as calcium fluoride, are less toxic than those that are more soluble, such as sodium fluoride. 3. 1. 1. Acute Exposure Ingested fluoride forms hydrofluoric acid at a pH typical of gastric juice. Acute high oral exposure to fluoride may lead to (with increased seriousness of observed symptoms) nausea, vomiting, abdominal pain, diarrhea, drowsiness, headaches, polyuria and polydipsia, coma, convulsions cardiac arrest, muscle paralysis, carpopedal spasm, spasm of the extremities occurred and death.50 The most frequently cited range for the certainly lethal dose (CLD) of sodium fluoride is based on a review of case reports prepared by Hodge and Smith.51 It is set between 32 and 64 mg/kg body weight, which corresponds to 5-10 g of sodium fluoride for a 70 kg person).52 The probable toxic dose (PTD) for children, defined as the dose of ingested fluoride that should trigger immediate therapeutic intervention and hospitalization, because of the likelihood of serious toxic consequences, is set at 5.0 mg/kg body weight.51 Contact of liquid HF with the skin can produce severe burns; the gas is corrosive to the eyes and mucous membranes of the respiratory tract.53 The basis of the treatment of fluoride poisoning is intravenous or intramuscular calcium therapy.54-56 3. 1. 2. Chronic Exposure Development of dental fluorosis in children and/or skeletal fluorosis in both children and adults are the most obvious adverse effects associated with chronic excessive fluoride intakes. Both are an ancient problem because hu- mans settled since ever in areas having high concentrations of fluoride in drinking water.18 A threshold of 0.03 mg/day/kg body weight has been suggested for the appearance of dental fluorosis however even this intake will result in a certain, although low, level of fluorosis in a popula-tion.39,57 Enamel fluorosis and primary dentin fluorosis would begin with the lower incisors, which complete mineralization at approximately 2-3 years of age, and end after mineralization of the third molars.58 Development of dental fluorosis has been controversial at times - on the one hand it is considered as cosmetic and, on the other, as having an adverse effect.59 The early symptoms of skeletal fluorosis include stiffness and pain in the joints. In severe cases, the bone structure may change and ligaments may calcify, with resulting impairment of muscles and pain. Constriction of vertebral canal and intervertebral foramen exerts pressure on nerves, blood vessels leading to paralysis and pain.60 A review paper in which fluoride has been linked to causing neurodevelopmental harm,61 has been a subject of debates.62,63 Recent research however evidenced, that children living in fluorosis prevalent areas have five times higher chances of developing a low intelligence quotient (IQ) than those living in less fluorosis areas64 and that IQ level was negatively correlated with fluoride concentration level in drinking water.65 Higher prenatal fluoride exposure was associated with lower scores on tests of cognitive function.66 Earlier studies suggested a protective effect of fluoride against Alzheimer's disease. 67,68 This is in contrast to the later studies. The greatest impairments of structure and function may come about through the actions of charged and uncharged Al-F complexes. These complexes may cross the blood brain barrier and accumulate in the brain thus inducing brain neurotoxicity.69-71 Many other associations between increased fluoride concentrations in fluoridated drinking water and possible adverse effects were suggested, e.g., decreased total fertility rate in both females and males,72 diabetes73 and greater impairment of thyroid function.74 3. 2. Mechanisms of Fluoride Toxicity Fluoride exerts different effects on the cell machinery leading to cell death, apoptosis and/or necrosis both in vivo and in vitro. Necrosis has been observed as a primary mechanism of cell death after a short exposure (~ 1 h) to fluoride at relatively high concentrations (~ 100 ^m).75 At relatively lower concentrations (around few mM) different molecular mechanisms lead to fluoride-induced cytotox-icity and eventual apoptotic cell death of different cells from different organs and tissues, e.g. lungs, kidneys, liver, brain, pancreas, thymus, endometrium, bone marrow, hair follicles, erythrocytes, leukemic cells. The molecular mechanisms underlying fluoride-induced apoptosis are Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 258 Acta Chim. Slov. 2019, 66, 255-275 different by nature and include the stimulation of G protein-dependent signaling systems, oxidative stress, ATP depletion, activation of the cell surface death receptors, disruption of outer mitochondria membrane, activation of caspases, alterations in the ratio of anti-apoptotic-apoptot-ic Bcl-2 proteins, upregulation of p53 expression, expression of apoptosis-related genes, endoplasmic reticulum stress and disturbances in protein synthesis. Reviews on the intracellular molecular mechanisms proven to be responsible for cytotoxicity and development of cell death induced by inorganic fluoride were recently reported.19,20 4. Absorption, Metabolism, Distribution and Excretion of Fluoride In humans, the predominant route of fluoride absorption is via the gastrointestinal tract. Except for occupational exposure or exposure to fluoride by coal or fuel burning, exposure to fluoride by inhalation is negligible.14,76 Dermal absorption is insignificant except in cases of hydrofluoric acid burns.77 When ionic fluoride enters the acidic environment of the stomach lumen, it is largely converted to weak acid hydrogen fluoride with a pKa of 3.19.3 The higher acidity of the stomach speeds up the process of absorption by passive diffusion.78 The coefficient of permeability of lipid bi-layer membranes to HF is 1 million times higher than that of F-79 so there is no need for specialized enzymatic systems to be involved.80 Around 70-75% of fluoride not absorbed from the stomach will be rapidly absorbed from the small intestine in a pH-independent process.81,82 Factors like bioavailability, amount of ingested food, emptying the stomach, the presence of the bile salts, concentrations of pepsin and pancreatin all affect the absorption from the stomach. Bioavailability (absorption and utilization) is the most important among them. Bioavailability is a measure of the amount of an administered dose that reaches the blood stream. It is 100%, by definition, when a medication is administered intravenously. The bioavailability of fluoride from sodium fluoride (NaF) tablets, as used in many caries-prevention programmes, from a fasting stomach, is almost 100%.83 It is also high from other soluble fluoride compounds that occur naturally or are added to drinking water and yield fluoride ions on dissolution, e.g. KF, Na2SiF6, H2SiF6.84 The degree of fluoride absorption is highly affected by the presence of diet containing high contents of calcium and certain other divalent (Mg2+) or trivalent (Al3+, Fe3+) cations with which fluoride can form insoluble or poorly soluble compounds.85 In studies on adults, this bioavailability was decreased to 50-79% by co-administration of milk or calcium rich products.86,87 The poor fluoride bioavailability, in the range of 4-24%, observed from food such as bone meal, fish bone meal, canned sardines and chicken bone meal, was ascribed to the high content of calcium in these foods.88 Fluoride bioavailability from typical meals eaten in different regions of India was found to be low (2-32%).89 The halftime for fluoride absorption is approximately 30 minutes and peak plasma concentration usually occurs within 30-60 minutes.83,87-91 Fluoride is then rapidly distributed in plasma and deposited in bone and other calcified tissues containing approximately 99% of the body's fluoride.92 The remainder of fluoride is distributed between blood and soft tissues, where a steady-state distribution between extracellular and intracellular fluids is estab- lished.92 In adults, about 50% of daily fluoride intake is associated with the calcified tissues within 24 hours and the remaining 50% is excreted in urine.93 This 50:50 distribution is strongly shifted to greater retention in the very early and probably towards greater excretion in the later years of life.93 In adults, about 40-60%94 of the daily intake of fluoride is excreted in the urine and in children about 45%.95 Less than 10% of the daily intake of fluoride is excreted in faeces.96,97 It was estimated that 1% or less of an ingested dose is excreted in saliva, which returns back to systemic circulation.98 Sweat provides only a minor route of fluoride excretion.80 General features of fluoride metabolism (fluoride flow through the organism) are schematically presented in Figure 2. Figure 2: General features of fluoride metabolism 5. Biomarkers of Fluoride Exposure Identifying and monitoring the human exposure to fluoride is of especial importance in children at the age of risk for development of dental fluorosis and in populations exposed to fluoride, i.e. with tea, via occupational exposure, in areas of the world with endemic fluorosis. Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 259 Acta Chim. Slov. 2019, 66, 255-275 Only absorbed fluoride is involved in the development of fluoride related adverse effects. Identification and monitoring deficient or excessive intakes of biologically available fluoride can be achieved through the use of bio-markers of fluoride exposure.99 According to the US National Academy of Sciences (US NAS)100 definition, accepted for fluoride also by the World Health Organization (WHO),101 biomarkers are defined in a broad sense to include almost any measurement reflecting an interaction between a biological system and a potential hazard, which may be chemical, physical or biological. The measured response may be functional and physiological, biochemical at the cellular level, or a molecular interaction. For fluoride, several biological fluids and tissues can serve as biomarkers, which can be according to time-perspective classification classified as: (1) contemporary; (2) recent; and (3) historical markers.101 Extensive information on biomarkers of exposure to fluoride can be found in recent reviews.102-105 In this section, only a concise information will be presented. 5. 1. Contemporary Markers The concentrations of fluoride in body fluids (plasma, saliva, urine, milk, sweat) and body tissues (bone surface) were all suggested to assess present or very recent exposure to fluoride. Their fluoride content depends on the intake via water, diet, fluoride supplements and fluoride-containing dentifrices. Fluoride ion does not produce any metabolites, so it is itself the measured indicator. Both ionic and non-ionic fluoride forms exist in plasma. The ionic fluoride form is of far greater significance when evaluating the effect of fluoride on human's health.106,107 For less than optimal water fluoride concentrations, the resting ionic fluoride concentrations in plasma ranged from 9.3 to 24.0 ng/ml,102 however, the data across various age groups enabling to determine the "usual" concentrations are insufficient.108 Urine is regarded as the most suitable biomarker for predicting fluoride intake for groups of people, but not the individuals.109 Using available data it was estimated that the ratio between the excretion and the intake of fluoride is 0.35 and 0.54 in children and adults, respectively above a threshold of total daily intake of 0.5 and 2 mg/day, respec- tively.95 The whole saliva tends to be contaminated by fluoride from the oral environment. Saliva is therefore usually collected from parotid and submandibular/sublingual ducts. Fluoride concentrations in saliva closely follow the plasma concentration but at a lower level. In adults, the ratios of saliva to plasma fluoride concentrations, under resting conditions, varied from 0.32-0.55 for parotid saliva110 and from 0.61 to 0.88 for submandibular saliva.98 In the same series of experiments, the ratio for fluoride concentrations in whole saliva and plasma was 1.10.111 The data on a normal range of fluoride concentrations in duc- tal saliva as a basis for recommending saliva as a marker of fluoride exposure are however insufficient.102 Data on fluoride contents in surface bone, sweat and human milk are rather limited and seem unsuitable for estimating contemporary exposure of humans to fluoride.102 5. 2. Recent Markers In contrast to contemporary markers, whose fluoride concentrations provide a snapshot at a certain point of time and are subject to change due to recent fluoride intake and certain physiological variables, the concentration of fluoride in nails and hair is cumulative. It reflects the average level of intake over a time period taking into account their growth rate. Their major advantage over fluids and tissues as biomarkers for fluoride exposure is that they can easily be obtained in a non-invasive manner. The main issues related to fluoride in nails or hair, as biomarkers seem to be a preparation of the sample for the analysis and high possibility of external contamination. The content of fluoride in fingernails or toenails is about 0.5-5 ^g/g112 and in hair about 0.2-15 ^g/g.113 Extremely higher values in both nails and hair were also reported in populations occupationally exposed to fluoride or populations living in fluoride endemic areas.112-115 5. 3. Historical Markers The main historical biomarkers that could indicate total fluoride body burden are a non-exchangeable inner compartment of bone and dentin.102,103 Inner bone and dentin both increase with age due to continuous fluoride uptake throughout life.106,116 In contrast, bulk enamel fluoride concentrations mainly reflect the level of systemic exposure to fluoride during tooth formation.93 Bone sampling is next to ethical limitations, difficult and invasive. The X-ray screening for increased bone density could be performed, however only when the need for information justifies the radiation dose involved. In addition, bone density might not be related only to fluoride exposure or to bone fluorine content. Thus, teeth in particular third molars or premolars that are commonly extracted have emerged as potential historical biomarkers of exposure to fluoride. 6. Fluorin(d)e Analytical Methods Fluoride of high concentrations in water or soil and gaseous fluoride emissions, which are all a consequence of natural or anthropogenic activities, represent a threat to vegetation and subsequently entire food chain in many parts of the world. Reliable analytical methods are therefore a prerequisite for the determination of human exposure to fluoride. The goal is to monitor the intake of fluoride and maintain it at adequate levels so that optimal protection against dental caries is achieved, without excessive intake Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 260 Acta Chim. Slov. 2019, 66, 255-275 resulting in the appearance of adverse effects. Basic requirements for accurate and precise determination of fluorine in any type of the sample are: (1) the sample has to be appropriately pre-treated so that the required form can be determined; (2) interfering reactions or ions have to be effectively suppressed; (3) the final concentration of fluorine must be above the detection limit of the method; and (4) the result should be reported together with a measurement uncertainty (MU) evaluated in accordance Guide to the Expression of Uncertainty in Measurement (GUM).117.117 6. 1. Forms of Fluorine As noticed in the Introduction of this paper, the term fluorine (F) is used to denote the element in any of its forms and fluoride (F-) to denote predominant chemical form inorganic fluoride in which the element is found in nature and to which a fluoride ion selective electrode (ISE) responds. Difference between F and F- in inorganic compounds, which leads to the amount of bound fluoride was discussed.118 In biological samples such as human and animal serum fluorine can be present also in the form of organic fluorine. This represents covalent fluorine that is bound to carbon in all organic fluorine compounds and results from exposure to certain fluorine-containing compounds from natural and/or industrial sources.119 Classification of different forms of fluorine in biological materials was extensively discussed.120-122 6. 2. Sample pre-treatment Procedures The approach to sample preparation is dependent on the type of matrix and the form of fluorine to be determined. Fluorine in samples can be determined as ionic, total or covalent fluorine. 6. 2. 1. Ionic Fluoride The concentration of fluoride in water can usually be determined directly without pre-treatment. Dissolution in water prior to fluoride measurement is a valid approach also for determination of soluble fluorides in solid samples. During interpretation of the results it is necessary to consider equilibria between F- and ions with which fluoride forms soluble or poorly soluble compounds, in relation to the employed analytical technique, e.g. Al3+, Fe3+, Si4+, Sn4+, Zr4+.85 Methods for determining gaseous fluorides are generally based on the absorption of gaseous fluorides into a train of absorbing solutions, or collection on a filter with a treated pad and subsequent determination of fluoride. 6. 2. 2. Total Fluorine Samples containing non-ionic inorganic fluoride and/or organic fluorine have to be decomposed prior to the measurement. Extraction or digestion with acids or mixtures of acids or digestion with alkaline solutions might dissolve the F in the samples only to varying degrees. Results of such studies are difficult or even impossible to compare, especially because there is no certified reference material (CRM) available to support the results of such measurements. Therefore, prior to quantification, samples must undergo a total decomposition to assure release of the inorganic or organic bound F to F- and bring the solid sample in a water soluble form. In the classical Willard-Winter123 distillation procedure introduced in 1933, decomposition of compounds and separation of resulting fluoride from interfering substances take place concurrently. This technique and its modifica-tions124-126 have provided a wealth of information through the middle decades of the 20th century but, mostly because of its cumbersome procedure, have been replaced by simpler methods, like pyrohydrolysis127,128 or diffusion methods.129-131 Commonly used procedures nowadays for total sample decomposition involve oxygen bomb combus-tion,132,133 open ashing134,135 fusion with alkali metal hydroxides or carbonates136,137 and microwave acid digestion.138,139 Reactions proceeding during decomposition of metal fluorides by pyrohydrolysis, oxygen bomb combustion and alkaline carbonate fusion and their related thermochemistry were discussed in our recent papers.140,141 Calcium and magnesium fluoride, which can be in variable amounts present also in human, animal, vegetation and other environmental samples, were suggested as the most resistant ones to decompose among all the metal fluorides studied. 6. 2. 3. Covalent Fluorine The basis for determination of organic fluorine in biological materials such as blood serum and soft tissues rely on the determination of total F with respect to F- using total decomposition methods. As a faster alternative for sample preparation use of sodium biphenyl was de-scribed.142 The method relies on reductive cleavage of the C-F bond, which is the strongest bond in organic chemis-try143,144. 6. 3. Methods for Determination of Fluoride The methods for determination of F- range from classical volumetric123-125 to spectrophotometric125,126 and fluorometric145,146 methods. A breakthrough in the analytical chemistry of fluorine was in 1966 when the fluoride ion selective electrode (ISE) was introduced by Frant and Ross147. The electrode consists of a single crystal lanthanum fluoride (LaF3) doped with europium (II) fluoride to improve its conductivity. The electrode is highly selective and covers a wide range of concentrations (1 mol/l to 10-5 mol/l). The limit of detection of fluoride ISE is limited by Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 261 Acta Chim. Slov. 2019, 66, 255-275 the solubility of LaF3 to ~0.02 mg/l.148 In our laboratory we developed a multiple known standard addition method for determination of fluoride based on spiking the samples with known amount of fluoride prior to the measurement. In this way the limit of detection (LOD) of ~0.01 mg/l was achieved.149 Various chromatographic methods for fluoride determination include ion chromatography (IC),150 gas chromatography (GC)151,152 and high-performance liquid chromatography (HPLC).153 The main issues related to the use of IC are that fluoride peak tends to overlap with the water negative dip and that monocarboxylic acids interfere because of co-elution with fluoride or only partial resolution. This makes IC suitable for determination of F- in aqueous samples.154-157 Use of GC has been reported for determination of fluoride in various kinds of biological matrixes however it is time consuming and requires trained personnel.158 Use of HPLC with reverse-phase mode columns for determination of fluoride is rarely re- ported.153 Determination of fluorine by atomic absorption spectrometry (AAS), laser-induced breakdown spectroscopy (LIBS), inductively coupled plasma optical emission spectrometry (ICP-OES), or inductively coupled plasma mass spectrometry (ICP-MS) is almost impossible task because of high excitation and ionization potential (17.42 eV) of F and the location of its resonance line in the vacuum ultraviolet range (95 nm).159 Use of molecular absorption spectrometry (MAS) methods with high-resolution continuum source (HR-CS) to determine fluorine through the absorption of diatomic molecules, such as CaF or GaF was reviewed.160 Its major limitation is that online coupling with chromatographic separation methods is not possible. Use of mass-shift strategy using tandem inductively coupled plasma quadrupole mass spectrometry (ICP-MS-MS) was reported for the determination of flu- orine.161 Less frequently used methods for determination of fluorine include polarography,162 voltammetry,163 sensors,164 capillary zone electrophoresis (CZE),165 near infrared spectroscopy (NIR),166 19F-nuclear magnetic resonance (19F-NMR),167 neutron activation analysis (NAA)168 and others. 6. 4. Measurement Uncertainty Evaluation A truly informed decision, based on a measurement result, can only be made with the consideration of the MU.169 Classically in analytical chemistry, the MU has been expressed as one standard deviation, thus including only random effects. A superior, more comprehensive approach is outlined in the definite document in the field of metrology GUM.117 In this approach, in addition to random effects, also systematic effects are taken into account and all uncertainty components of each step of analytical procedure are identified, quantified and traceable169 to the SI units. All these contributors are then combined and the MU is reported as an expanded uncertainty, which corresponds to the 95% level of confidence.117 Unfortunately, the only paper reporting procedure for evaluation of the MU according to the GUM for F is on determination of fluoride in water.170 Recently we suggested a procedure for the MU evaluation for determination of F in solid samples, which must undergo a total decomposition before the measurement.171 This makes evaluation of the MU much more complex, because: (1) in addition to the uncertainty of the measurement step also uncertainty of the decomposition step must be included; and (2) transformation of the sample from one chemical form to another cannot be traceable to any reference and therefore the highest form of traceability can be achieved by the use of matrix matched CRM.172 7. Sources of Oral Exposure to Fluoride Knowing total fluoride contents contributing to the oral exposure to fluoride, e.g. water and beverages, different food items, dental products and alternatives to water fluoridation, like fluoridated salt or milk and fluoride supplements, can assist in estimating the total daily intake of fluoride and the measure of exposure. 7. 1. Drinking Water Natural waters aimed also for human consumption contain varying concentrations of fluoride, from trace amounts to some mg/l and even toxic concentrations. Waters with high concentrations of fluoride are usually found at the foot of high mountains and in areas with geological deposits of marine origin. Large variations in natural fluoride contents of drinking water between and within countries can be observed.173,174 The WHO, based on earlier documents (from 1984, 1993 and 20 04),77,175,176 set in 2010 the guideline value for fluoride in drinking water, by taking into account drinking water consumption, at 1.5 mg/l.26 This is in conflict with the WHO recommendation from 1994 according to which the absolute upper limit for fluoride in drinking water is 1.0 mg/l.99 In the European Union (EU) drinking, water for human consumption may not contain more than 1.5 mg/l.177 The earlier recommended optimal fluoride concentration for fluoridated water in the US was recently reduced from 0.7-1.2 mg/l to 0.7 mg/l.178 It has to be pointed out that few subjects in medicine have proved more controversial than fluoridation of public water supplies. The controversy is illustrated by the fact that, while the US Centers for Disease Control and Prevention (CDC) claimed that water fluoridation is one of the ten great public health achievements in the US during the 20th century,179- 69% of the population receives fluoridated drinking water27 - water fluoridation is banned in most of Europe.173 Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 262 Acta Chim. Slov. 2019, 66, 255-275 Use of bottled water is in increase. In the EU, bottled water exceeding fluoride concentration of1.5 mg/l shall bear on the label "contains more than 1.5 mg/l of fluoride: not suitable for regular consumption by infants and children under 7 years of age" and a maximum limit of 5 mg/l is set for naturally present fluoride.180 The concentration of fluoride in bottled water in the US depends on whether fluoride is naturally present or added and on the annual average of maximum daily air temperatures at the location where the bottled water is sold at retail. Thus, the concentrations of fluoride in bottled water packaged in the US and imported water should not exceed 2.4 and 1.4 mg/l, respectively.181 7. 2. Tea and Beverages The tea plant (Camellia sinensis L.; family Theacea) (Figure 3) is known to take up fluoride from the soil and accumulate it in the leaves. The total fluorine content of the leaves typically ranges between 50 and 900 mg/kg.182-186 About 25-100% of fluorine is released during the infusion, thus the infusions may contain about 0.3 to 8.8 mg/l of fluoride, depending also on the amount of dry tea used, the granulation of the tea, the concentration of fluoride in the added water, the presence of milk, duration of infusion, etc.182-186 Concentrations of fluoride in ready-to-drink tea were reported to range between 0.01 to 4.1 mg/l187-189 and in one study an extraordinarily high average of 25.7 mg/l of fluoride was reported.190 Figure 3: Tea harvesting in Sri Lanka (Photo: M. Ponikvar-Svet) Concentrations of fluoride in soft drinks, e.g. nectars, juices, juice drinks, ranged between 0.10-2.0 mg/l in Portugal,191 0.07-1.14 mg/l in the Canary Islands,192 0.022.80 mg/l in the US193 and 0.07-1.42 mg/l in Mexico.194 Concentrations of fluoride in juices and juices-flavoured drinks for infants and children ranged between 0.01-0.25 mg/l in Poland195 and 0.11-1.81 mg/l in the US.196 Carbonated soft drinks analysed in Europe contained 0.10-0.38 mg/l of fluoride.191,192 The observed concentrations were higher in the US and Mexico and ranged from 0.02 to 1.28 mg/l197 and from 0.07 to 1.62 mg/l, respectively.194 The range of fluoride concentration in beer available in Europe was 0.067-1.12 mg/l.198-200 High concentrations, 1.77 and 1.66 mg/l of fluoride, were determined in beer from Ireland and the US, respectively.194,200 Wide variations in fluoride concentrations found in different studies can be explained either by low natural presence of fluoride or by high presence of fluoride due to naturally occurring or artificially added fluoride in water used in manufacturing process. Fluoride concentration in bottled wines of the different types from the Canary Islands ranged between 0.03 and 0.70 mg/l201-203 and from Turkey between 0.02-0.38 mg/l.204 High concentrations of fluoride between 0.23 to 2.80 mg/l in nineteen California wines apparently resulted from the use of cryolite as a pesticide.205 7. 3. Milk and Baby Formulas The reported concentrations of fluoride in commercially available milk were generally low and did not exceed 0.1 mg/l.206,207 High concentration of fluoride in pasture or drinking water of livestock might however contribute to fluoride levels in milk of different cattle.208-210 Reported fluoride concentrations in soya milk varied between 0.01 and 0.964 mg/l.211-213 Fluoride concentrations in breast milk are generally low even at very high intakes of fluoride by mothers. The average concentrations range from 0.002 to 0.073 mg/l.214 Surprisingly high concentrations of fluoride in breast milk were determined in one study conducted in the high altitude (>2000 m). In that study the fluoride contents of breast milk of mothers with dental fluorosis and children with dental fluorosis, in the high altitude ranged between 0.13-0.99 mg/l (average 0.55 mg/l), while breast milk mothers in the control group without dental fluorosis contained 0.001-0.10 mg/l (average 0.006 mg/l).215 These results suggested that there is more to understand about the factors affecting the level of fluoride in breast milk, such as the effect of altitude.216 Concentrations of fluoride between 0.01 and 0.75 mg/l were reported in a review on fluoride concentration in infant formula products reconstituted with fluoride-free water according to the manufacturer's instructions.217 When formulae were prepared with water of differing fluoride concentrations, the fluoride concentration was found to be a simple linear function of water fluoride con- centration.217 7.4. Foods The fluorine contents in different foods and drinks in this paper are illustrated using the data from the USDA National Fluoride Database218 and the United Kingdom (UK) Fluoride Database.219,220 The USDA Database218 is based on the data extracted from reviews of existing scientific literature and unpublished results mainly from the Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 263 Acta Chim. Slov. 2019, 66, 255-275 Table 1: The averages and the ranges of total fluorine contents (wp) in different food groups on a fresh weight basis listed in the USDA National Fluoride Database218 and UK Fluoride Database219,220 analyzed as ready-to-eat items USDA Database UK Database Wf (^g/100g) wF (^g/100g) ^products Average (SD) Range ^products Average (SD) Range Fruits, nutsb 19 5 (3) 1-12 19 4 (4) 1-19 Fats, oils 6 9 (10) 1-25 8 4 (8) 0-17 Vegetables, spices, herbsc 37 17 (15) 1-49 23 7 (6) 1-19 Meat and products 17 24 (14) 4-48 25 7 (7) 2-24 Milk, egg and productsd 13 12 (13) 1-35 28 7 (13) 1-59 Cereal products 9 27 (18) 6-51 16 31 (21) 4-57 Breakfast cereals 12 44 (19) 17-72 12 26 (30) 4-75 Soups, sauces, gravies 22 39 (34) 1-132 11 14 (21) 1-49 Sweets, snacks 46 33 (26) 1-106 81 15 (19) 1-90 Beverages, water 132 48 (34) 2-204 22 13(11) 0-45 Tea 25 262 (136) 9-584 3 30 (27) 12-61 Fish, shellfish and products 7 111 (87) 18-210 13 149 (298) 8-1054 Dishes 28 37 (20) 5-84 30 15 (12) 1-51 Infant food and drinks 50 12 (14) 0-67 251 15 (21) 1-120 a Number of analyzed items b The highest content was not considered (234 ^g/100 g of F in raisins) c The highest content was not considered (115 |ig/100 g of F in french fries, McDonald's) d The highest content was not considered (112 ^g/100 g of F in cream substitute, powdered) period between 1977 and 2003. The more recent UK Database219,220 was generated in a period between 2003 and 2015 through a range of research projects. These databases arrange food and drink items in different groups, therefore the F contents are difficult to compare. Thus, we re-positioned all food items and drinks from both databases within the same groups. The averages and the ranges of total fluorine contents in these food groups are presented in Table 1. The presented data compiled from the original databases (Table 1) show a wide variation in fluorine contents within and between individual food groups (see SDs and ranges). The average content of F of all food groups is 2.7fold higher in the USDA Database than in the UK Database, this is 47 ^g/100 g as opposed to 17 ^g/100 g. The average content of F in the USDA Database remains 1.9fold higher than in the UK Database even if tea is taken aside. This can be explained by different levels of fluoride in water used for the preparation of the products requiring water; results obtained using tap water containing 0.71 mg F-/l are reported in the USDA Database and results reported using tap water containing 0.05-0.13 mg F-/l were selected from the UK Database. The fluorine contents in individual food groups were, for easier visualization presented also in Figure 4. The content of fluorine in food is generally below 50 ^g/100 g (Figure 4 and Table 1). Exceptions to this include: (1) processed food (breakfast cereals, sweets, snacks, sauces) and beverages which can contain considerable amounts of fluorine if fluoridated water is used during the production process or for their preparation; (2) tea, which can during infusion release high amounts of fluoride accumulated in the leaves; and (3) fish and shellfish which might Figure 4: The average contents of fluorine in different food groups on a fresh weight basis listed in the USDA National Fluoride Data-base218 and the UK Fluoride Database219,220 analyzed as ready-to-eat items contain high fluorine contents, if analyzed samples, due to mechanical deboning, contain bones and exoskeleton remains, where fluoride is accumulated from the sea. Finally, it is worth mentioning that in addition to these databases many studies reported the contents of total and/or free fluoride in different food items.221-227 The results of these studies are difficult to compare, because the determined level of fluorine in food is among other influenced by: (1) the locality in which the food is grown; (2) the differences in the amount of fertilizer and pesticides applied or differences in feeding routine; (3) the type of processing the food receives; (4) the amount of water and the fluoride content of water used in their preparation; (5) different sample pre-treatment procedures; and (6) the differences in the analytical procedures. In addition, the re- Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 264 Acta Chim. Slov. 2019, 66, 255-275 suits are not reported according to the Guide to the Expression of Uncertainty in Measurement (GUM).117117 7. 5. Fluoride Containing Dental Products Fluoride containing dental products (toothpaste, rinses and geis) aithough not considered as a dietary source, can increase the total oral intake of fluoride considerably, especially when used inappropriately. Because of poor control of the swallowing reflex, fluoride ingestion by young children needs to be during the first six years of life carefully controlled and the best balance between risk and efficacy might be achieved by using small amounts of high fluoride toothpaste under close supervision from parents.28,29 Fluoridated toothpaste (Figure 5) for children usually contains 250 to 500 ^g/g of fluoride228 and for adults from 1000-1500 ^g/g of fluoride.229 7. 6. Fluoridated Salt or Milk and Fluoride Supplements Salt fluoridation which begun in 1956 in Switzerland24 is practiced as an alternative to water fluoridation. It has been estimated that between 40 million and 280 million people worldwide use fluoridated salt, mainly in European, South American and Central American countries, some Asian countries, including Cambodia and Laos and in Africa, Madagascar. For salt fluoridation, sodium fluoride and potassium fluoride are mostly used at a concentration of 250-300 mg/kg of fluoride.231,232 Milk as a relatively cost-effective vehicle for fluoride delivery in the prevention of dental caries was first proposed in the 1950s.25 As suggested in an update to a Co- chrane Review233 first published in 20 0 5,234 there is low quality evidence to suggest fluoridated milk may be beneficial to schoolchildren, contributing to a substantial reduction in dental caries in primary teeth. Systematic reviews235,236 have shown that there is weak and inconsistent evidence to demonstrate that fluoride supplements administered in the form of tablets, drops, lozenges or chewing gums can effectively prevent caries in primary teeth. However, there is strong evidence that fluoride supplements can prevent caries in permanent teeth. The use of fluoride supplements was associated with a high risk of mild-to-moderate fluorosis. Current recommendations on their use are controversial - on one hand European Academy of Paediatric Dentistry (EAPD) recommends that fluoride tablets and fluoride drops could be considered on an individual basis for children at high risk of caries237 and, on the other hand, the American Academy of Pediatric Dentistry (AAPD) recommends that fluoride dietary supplements should be considered for children at caries risk who drink fluoride-deficient (less than 0.6 mg/l) water.238 8. Daily Intake of Fluoride Estimating exposure to fluoride, especially in children, is of crucial importance to avoid potential problems associated with too low or too high intakes. The adequate intake (AI) of fluoride from all sources (including non-dietary sources) is because of proven benefits of fluoride on dental health set at 0.05 mg/day/kg body weight for both children and adults.22 Major contributors to the oral intake of fluoride are drinking water, tea, beverages, foods and fluoride containing dental products.23 The contribution of inhaled airborne fluoride, or fluoride from the soil, to the total fluoride intake is, under normal conditions, small23 and is beyond the scope of this paper. There is a lack of data on the total fluoride intake from dietary and non-dietary sources based on analyses of individual actual diets.22 The estimates are often based on the estimates of quantities of foods consumed, such as standard food tables or questioners, rather than actual quantities of food consumed.22 This might result in under or over estimates of actual fluoride intakes because of wide variations in fluoride contents of individual food items. 8. 1. Fluoride Intake in Children As reported the prevalence of enamel fluorosis is over the last three decades on the rise in the United States (US).58,239,240 The cases reported include also more cases of moderate-to-severe fluorosis.58,239-241 One of the possible reasons might be that fluoride appears to be more readily available for consumption nowadays during the critical window when enamel is most susceptible to fluo- rosis.240 Figure 5: Toothpaste contributes significantly to the daily intake of fluoride especially in children (Photo: M. Ponikvar-Svet) Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 265 Acta Chim. Slov. 2019, 66, 255-275 Table 2: Estimated intakes of total fluoride from diet and toothpaste for children expressed as averages in mg/day/kg body weight by F concentration in water (y(F-)water) and age groups; fluoride intake by breastfed infants is not listed mg/day/kg body weight Age (yr.) y(F-)water (mg/l) Food (SD) Toothpaste (SD) Total (SD) Note Ref. Nonfluoridated water 2-6 0.08 0.017 (0.009) NA NA a 242 up to 4 0.04 0.006 0.055 0.061 b 243 up to 4 0.04 0.011 NA 0.011 b, c 243 4 0.04-0.07 0.033 (0.012) 0.016 (0.011) 0.050 (0.019) a 244 4 0.06-0.07 0.033 (0.012) 0.029 (0.016) 0.062 (0.022) a 244 4.75 0.08-0.015 0.02 (0.01) NA NA b, d 245 5.25 0.08-0.015 0.018 (0.018) NA NA b, d 245 6-7 0.08 0.008 (0.004) 0.023 (0.026) 0.031 (0.025) a 246 8 0.04-0.07 0.033 (0.012) 0.012 (0.007) 0.048 (0.038) a 244 8 0.06-0.07 0.030 (0.014) 0.013 (0.006) 0.043 (0.016) a 244 Average (SD) 0.021 (0.011) 0.023 (0.015) 0.044 (0.018) Fluoridated water up to 4 0.64 0.011 0.037 0.048 b 243 up to 4 0.64 0.015 NA 0.015 b,c 243 2-5 0.5-0.7 0.033 (0.013) 0.01 (0.01) 0.043 (0.016) b 247 2-6 0.6-0.8 0.028 0.036 (0.028) 0.064 (0.035) a 248 3-4 <0.7 0.02 (0.01) 0.00 0.02 a 249 3-4 0.7-1.2 0.04 (0.01) 0.01 0.05 a 249 4 0.64 0.025 (0.010) 0.046 0.071 (0.036) b 250 4 0.8-1.0 0.041 (0.026) 0.018 0.059 (0.029) a 244 4-6 1 0.027 (0.014) 0.030 0.058 (0.042) b, e 251 4-6 1 0.026 (0.014) 0.028 0.054 (0.034) b, e 251 6-7 0.47 0.016 (0.005) 0.022 0.038 (0.038) a 246 6-7 0.82 0.025 (0.014) 0.022 0.047 (0.008) a 246 8 0.8-1.0 0.041 (0.039) 0.015 0.057 (0.045) a 244 Average (SD) 0.027 (0.010) 0.023 (0.013) 0.048 (0.017) High fluoridated water 3-4 >1.2 0.05 (0.03) 0.01 (0.02) 0.07 a 249 4 2.0-3.0 0.362 (0.181) 0.022 (0.014) 0.385 (0.184) a 244 8 2.0-3.0 0.307 (0.120) 0.019 (0.016) 0.326 (0.128) a 244 11 5 0.29 (0.10) 0.017 0.307 b,f 30 12 5 0.35 (0.10) 0.017 0.367 b,f 30 12 5 0.36 (0.20) 0.017 0.377 b,f 30 Average (SD) 0.287 (0.120) 0.017 (0.004) 0.305 (0.119) a Questionnaire b Double plate c No tooth brushing as well as non-fluoride-toothpaste user d Measured at a baseline and after 6 months e Weekly variation f The intake from toothpaste was estimated as an average of the intakes reported in refs. 244,249 Estimates for the total fluoride intakes for children from food and toothpaste, by water fluoridation status and the age groups as reported in the last decade are presented in Table 2. The data for breastfed infants, who may receive lower amounts of fluoride than those listed in Table 2, are not included. Wide variations in the intakes of fluoride within and between studies are evident from Table 2. This can be ascribed to the factors like: (1) considerable differences in the contents of fluoride in different food items; (2) large variation in the quantities consumed; (3) differences between the age groups studied; and (4) differences in the analytical methods used for total fluoride determination. The average total daily intakes of fluoride in children were estimated using data listed in Table 2 and illustrated in Figure 6. The estimated average total daily intake of fluoride from all studies in nonfluoridated areas is slightly higher than in fluoridated areas (0.044 and 0.048 mg/kg body weight (Figure 6 and Table 2). Neither of them exceeds the adequate intake of 0.05 mg/day/kg body weight. Review of data (Table 1) reveals that the contribution of fluoride with the toothpaste in nonfluoridated and fluoridated areas is substantial and represents about half of the estimated average total intake and that the estimates for the total daily intake of fluoride in some studies exceed Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 266 Acta Chim. Slov. 2019, 66, 255-275 0.45 0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 T èé . ' rim,. Nonfluoridated water Fluoridated water High fluoridated water ■ Food «Toothpaste «Total — Adequate intake Figure 6: Estimated average total daily intake of fluoride in children from food and toothpaste in areas with nonfluoridated, fluoridated and high fluoridated water the AI in both nonfluoridated and fluoridated areas.30,243-251 The average estimated daily intake of fluoride in nonfluoridated areas is higher and in fluoridated areas lower, than that reported with diet and toothpaste a decade ago (0.036 and 0.071 mg/kg body weight in nonfluoridated and fluoridated areas, respectively).23 Possible explanations for these differences are that the recent research (Table 1) has been more focused on: (1) the actual intakes of fluoride, as determined by duplicate diet technique; (2) the total oral fluoride intake including actual intake of fluoride with the toothpaste; and (3) the use of more accurate and precise analytical methods for total fluorine determination. A comment has to be put on the average estimated total fluoride intakes of 0.260 mg/kg body weight in areas with fluoride contents in water above 1.2 mg/l.30,244,249 These intakes extremely exceed the AI and also the upper limit50 of fluoride daily intake set at 0.10 mg/kg body weight for children up to the age of eight years. 8. 2. Fluoride Intake in Adults The major source of fluoride intake in adults is diet. The data on the total fluoride intake in adults are of older date before 2007.23 The average daily total fluoride intake in nonfluoridated areas was then estimated to range from 0.56 to 1.50 mg (average 1.11 mg) (equivalent to 0.0080.021 (average 0.016) mg/kg body weight for a 70 kg man). The estimated daily intake of fluoride in fluoridated areas was almost 2-fold higher, being 0.91-3.78 (average 2.07) mg, equivalent to 0.013-0.054 (average 0.030) mg/kg body weight for a 70 kg man.23 The average total daily intake of fluoride estimated in our study conducted in Slovenia, where water is nonfluoridated and fluoride content is generally low was 0.73-2.50 mg (average 1.50 mg) (equivalent to 0.010-0.036 (average 0.021) mg/kg body weight for a 70 kg man). This study is of importance because it is one of the rare studies conducted in adults using duplicate diet technique.149 There are exceptions however showing higher fluoride intakes than those listed. Tea can significantly contribute to the total daily intake of fluoride. The content of F- in tea as determined in our recent study ranged between 0.32 and 3.55 mg/l (average 1.42 mg/l).163 Figure 7 illustrates average daily intakes of fluoride in adults from food and daily consumption of 1 l of tea. Water with F- concentration of 1 mg/l was considered for preparing tea infusions in fluoridated areas. Figure 7: Estimated average total daily intake of fluoride in adults from food and tea in Slovenia and areas with nonfluoridated and fluoridated water As illustrated in Figure 7, the consumption of tea can significantly contribute to the daily intake of fluoride; the AI for fluoride can be easily exceeded with consumption of higher quantities (~1 l) of low quality tea. The presented intakes can be however also extremely higher in fluoride endemic areas13,14 or in southern China, where brick tea-type fluorosis has even become an urgent public health problem.252,253 9. Adequate Intake of Fluoride The basis for setting the adequate intake of fluoride is research by Dean and others. Initial studies conducted in the 1930s and early 1940s were focused on fluoride in water in relation to the appearance of dental fluorosis254-259 and then turned to fluoride in water in relation to car-ies.260,261 A "dose response" relationship between fluoride concentration in water supplies and dental fluorosis was established in the 22 cities study261 and between fluoride concentration in water supplies and caries in the 21 cities study.261,262 Reduction in the average number of dental caries per child was nearly maximal in communities having water fluoride concentrations close to 1.0 mg/l. This is how 1.0 mg/l of fluoride became the "optimal" concentra- Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 267 Acta Chim. Slov. 2019, 66, 255-275 tion, i.e. it was associated with a high degree of protection against caries and a low prevalence of the milder forms of enamel fluorosis. The first conversion of the exposure to fluoride in water supplies to the exposure to fluoride by the intake from water and food was made by McClure.263 The daily fluoride intake in children at the age between 1 and 12 years ranged from 0.02-0.10 mg/kg body weight (average 0.05 mg/kg body weight).263 The way on how this information became interpreted as a recommendation was reviewed by Burt21, who concluded that "Despite its dubious genesis, however, empirical evidence suggests that 0.050.07 mg fluoride/kg body weight/day remains a useful upper limit for fluoride intake in children." The beneficial effects of fluoride on the prevention of dental caries were considered as an appropriate indicator for setting the adequate intake also by EFSA. Thus, the AI of fluoride from all sources (including non-dietary sources), was set at 0.05 mg/kg body weight per day for both children and adults, including pregnant and lactating women.22 At the same time, it was however noted that reliable and representative data on the European population's total fluoride intake are not available (see also section Daily intake of fluoride).22 The current guidelines on the AI of fluoride, which are widely used in authoritative advisory recommendations for many decades, have been recently questioned, mainly because: (1) they were established empirically; (2) sources of ingested fluoride have changed; and (3) the prevalence and severity of dental caries and dental fluorosis have changed.264 As a result, the appropriateness of current guidance was addressed, however, no firm conclusions were made.265-267 10. Conclusions - Enough or Too Much Fluoride? Many health authorities worldwide consider beneficial effects of fluoride on the prevention of dental caries as an appropriate indicator to set the adequate daily intake of fluoride from all sources between 0.05 and 0.07 mg/day/kg body weight. The AI was set based on empirical observations reported by McClure263 in 1943, when practically the only source of fluoride was fluoride in water. Today next to water, also tea and other beverages, diet, fluoridated food supplements and dental products can significantly contribute to the daily intake of fluoride. Among different food items processed food, fish and shellfish products might contain considerable contents of fluorine. Excessive chronic intakes of fluoride can result in the development of fluoride related adverse effects. The primary adverse effects associated with chronic, excess fluoride intake are dental and skeletal fluorosis. As reported, the prevalence of enamel fluorosis including more cases of moderate-to-severe fluorosis is in increase.58,239,240 A threshold as low as 0.03 mg/day/kg body weight has been suggested for the appearance of dental fluorosis, however even this intake will result in a certain, although low, level of fluorosis in a population.39,57 Other adverse effects related to the toxicity of fluoride for cells of different tissues include, among others, neurodevelopmental disorders, neurotoxicity, decreased total fertility rate and diabetes. A review of the recent studies showed that the average total daily intake of fluoride with diet and toothpaste in children living in nonfluoridated and fluoridated areas is (0.044 ± 0.026) and (0.048 ± 0.016) mg/kg body weight, respectively. These intakes are high enough to assure optimal protection against dental caries, but on the other hand, also high enough to pose a risk for the development of dental fluorosis. There is a lack of recent studies on the daily intake of fluoride in adults. Earlier reported estimates of the average total daily intake of fluoride are 0.016 and 0.030 mg/kg body weight in nonfluoridated and fluoridated areas, respectively. Attempts have been made to challenge the guidelines on the AI. However even the most notable ongoing recent study, which started between 1992 and 1995, on the association between fluoride intake, dental caries, and dental fluorosis (Iowa Fluoride Study)268-274 after years of extensive investigation, came to the following conclusion: "Given the overlap among caries/fluorosis groups in mean fluoride intake and extreme variability in individual fluoride intakes, firmly recommending "optimal" fluoride intake is problematic".272 It is therefore not surprising that the guidelines on the AI of fluoride have been recently questioned264 and addressed.265-267 Based on the available literature, current recommendations on fluoride intake and the fact that the majority of fluoride benefits can be ascribed to its topical, rather than systemic, effects, it is hard to say whether the current AI is appropriate, i.e. too low or too high. Knowing this is of crucial importance because the margin between the beneficial and deleterious effects of fluoride appears to be so narrow. 11. Our Research and Future Directions The majority of the fluoride debate is based on the results of the studies which can be regarded by scepticism. Only a few studies reported the use of certified reference materials (CRMs) as a part of the quality assurance system and only two studies with one being contributed by us reported the results together with the measurement uncertainty (MU) estimated according to the Guide to the Expression of Measurement Uncertainty (GUM).117,170,171 In addition, in more than eight decades, after McClure275 published his first study on fluorine contents in different food items, food growing and processing and our eating habits have changed dramatically. This is also evidenced by Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 268 Acta Chim. Slov. 2019, 66, 255-275 meaningful difference between the average contents of F in food consumed in the US and the UK (see Section 7.4.). Thus, more accurate and up to date information on fluoride content in food and possible adverse effects of fluoride in relation to other ions, especially those forming complexes with fluoride are a prerequisite to avoid possible problems related to high intakes. In our laboratory classical analytics used for determining the composition of bulk material of synthesised compounds containing fluorine118,276-283 was extended to thermochemical investigations,118,140,141,280-282,284-287 measurement uncertainty evaluation,171 determination of fluorine in food149,183 and environmental samples7,288,289 and nanomaterials.290-294 Principles of thermochemistry were used for the estimation of the entropies of formation of fluorine containing aqueous anions285 and to discuss ions containing solely fluorine atoms286 and oxidation potential of fluorine.287 Possible reactions proceeding during total decomposition of fluorine containing materials by pyrohydrolysis, oxygen bomb combustion and alkaline carbonate fusion were suggested and accompanying thermochemistry discussed. Based on this investigation some of the most resistant fluoride minerals towards the total decomposition were sug-gested.140,141 The importance of evaluating the MU according to the GUM in research laboratories was addressed and a procedure for the MU evaluation for determination of F in vegetation was suggested.171 Our study on fluorine contents in total diet samples is important, since it is one of the few studies on the intakes of fluoride from food in adults which is based on duplicate diet technique.149 There is a number of papers reporting the contents of fluoride in tea infusions (Camellia sinensis L.). In our study we extensively investigated contents of total fluorine in tea and its leaching in the form of F- into the infusion with respect to the type of tea and its manufacturing procedure.183 As concluded, more attention should be put on consumption of fluoride from food and tea containing beverages. Our laboratory is devoted also to the environmental issues. In a period of ten years two uncontrolled releases of gaseous fluorides from the industry, which caused damage to the environment and possibly humans via the food chain were identified.7,289 The first release also initiated a study on the stress syndrome response of nettle ( Urtica dioica L.) grown in fluoride contaminated substrate to fluoride.288 Based on these results nettle was suggested as a passive bioindicator for monitoring soil pollution with For for the phytoremediaton by the mechanism of phytoex-traction of F- polluted soils. We also participated in characterization of upcon-verting, lanthanide-doped, fluoride nanoparticles, which show a great potential in bioimaging.290-294 Fluorine research remains relatively sporadic also because of a lack of sound analytical techniques enabling fast and multielemental analysis of F. Thus the predominate method for F analysis remains fluoride ion selective electrode. We should be therefore not surprised if a lack of reliable data for F content in different food items and a lack of regulation regarding F content in human and animal diets can be noticed, i.e. the only EU directive related to fluorine in diet addresses the maximum content of F in animal feed.295-297 More research and actions regarding: (1) the content of F in different food items; (2) reporting the results in accordance to the GUM; (3) the intakes of fluoride and its possible adverse effects in relation to interaction with other food components should be encouraged to provide support for further development and implementation of legislation concerning fluorine. Despite all the knowledge we currently have we can conclude that one is currently left with the question "Enough or too much fluoride?". Whether the complex nature of the system precludes there ever being a definite answer, remains to be seen. Declaration of interest: none. Funding: This work was supported by the Slovenian Research Agency [ARRS Grant P1-0045, Inorganic Chemistry and Technology]. List of Acronyms AAPD, American Academy of Pediatric Dentistry; AAS, Atomic Absorption Spectrometry; AI, Adequate Intake; AR, Average Requirement; CDC, Centers for Disease Control and Prevention; CLD, Certainly Lethal Dose; CRM, Certified Reference Material; CZE, Capillary Zone Electrophoresis; EAPD, European Academy of Paediatric Dentistry; EFSA, European Food Safety Authority; EU, European Union; 19F-NMR, 19F-Nuclear Magnetic Resonance; F-, Fluoride Ion; F, Total Fluorine; GUM, Guide to the Expression of Uncertainty in Measurement; HF, Hydrogen Fluoride; HR-CS, High Resolution-Continuum Source; ICP-OES, Inductively Coupled Plasma Optical Emission Spectrometry; ICP-MS, Inductively Coupled Plasma Mass Spectrometry; ICP-MS-MS, Inductively Coupled Plasma Quadrupole Mass Spectrometry; IQ, Intelligence Quotient; ISE, Ion Selective Electrode; LIBS, Laser-Induced Breakdown Spectroscopy; MAS, Molecular Absorption Spectrometry; MU, Measurement Uncertainty; NAA, Neutron Activation Analysis; NIR, Near Infrared Spectroscopy; PTD, Probable Toxic Dose; SD, Standard Deviation; UK, United Kingdom; US, United States; US NAS, United States National Academy of Sciences; WHO, World Health Organization Author Biographies Dona Stepec finished her B.Sc. study (equivalent to M.Sc. after the Bologna reform) in 2013 at the Faculty of Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition 269 Acta Chim. Slov. 2019, 66, 255-275 Chemistry and Chemical Technology at the University of Ljubljana. After graduation, she was employed as a researcher at Faculty of Chemistry and Chemical Technology at the University of Ljubljana on the project in collaboration with BIA Separation d.o.o. for a year and a half. Since 2015 she is enrolled in postgraduate study of Eco-technology at the Jožef Stefan International Postgraduate School. She is a young researcher at the Department of Inorganic Chemistry and Technology at the Jožef Stefan Institute under mentorship of Prof. Dr. Maja Ponik-var-Svet. Her research activities are focused on determination of fluorine in food and environmental samples and evaluation of measurement uncertainty. Maja Ponikvar-Svet finished her B.Sc. study in 1994 at the Faculty of Chemistry and Chemical Technology at the University of Ljubljana. 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Lisjak, Acta Chim. Slov. 2015, 62, 789-795. D0I:10.17344/acsi.2015.1508 291. D. Lisjak, O. Plohl, M. Ponikvar-Svet, B. Majaron, RSC Adv. 2015, 5, 2046-2069. 292. D. Lisjak, O. Plohl, J. Vidmar, B. Majaron, M. Ponikvar-Svet, Langmuir 2016, 32, 8222-8229. D01:10.1021/acs.langmuir.6b02675 293. O. Plohl, S. Kralj, B. Majaron, E. Fröhlich, M. Ponikvar-Svet, D. Makovec, D. Lisjak, Dalton Trans. 2017, 46, 6975-6984. D0I:10.1039/C7DT00529F O. Plohl, M. Kraft, J. Kovač, B. Belec, M. Ponikvar-Svet, C. Würth, D. Lisjak, U. Resch-Genger, Langmuir 2017, 33, 553-560. D0I:10.1021/acs.langmuir.6b03907 294. European Commission, Off. J. Eur. Communities: Legis. 2002, 140, 10-21. 295. European Commission, Off. J. Eur. Communities: Legis. 2005, 318, 19-24. 296. European Union, Off. J. Eur. Communities: Legis. 2015, 31, 11-17. Povzetek Fluor v obliki fluoridov je v naravi zelo razširjen in zato predstavlja neizogiben del našega okolja. Znano je, da ima fluor v majhnih količinah koristne učinke na zdravje zob. Po drugi strani lahko čezmerni kronični vnos povzroči neželene učinke, vključno z razvojem zobne fluoroze pri otrocih in/ali kostne fluoroze pri otrocih in odraslih. Primeren dnevni vnos fluora, ki temelji na empiričnih raziskavah, je 0,05 mg/dan/kg telesne mase, vendar je prag med koristnimi in škodljivimi učinki ozek. Znanje o strupenosti fluorida je kljub številnim raziskavam še vedno pomanjkljivo. V tem preglednem članku so opisani vloga in učinki fluorida na zdravje ljudi. Podanih je nekaj s fluoridom povezanih kontroverznosti in predlog smernic za bodoče raziskave. Stepec and Ponikvar-Svet: Fluoride in Human Health and Nutrition DOI: 10.17344/acsi.2018.4580 Acta Chim. Slov. 2019, 66, 276-283 ^creative ^commons Scientific paper Green Synthesis of Bromo Organic Molecules and Investigations on Their Antibacterial Properties: An Experimental and Computational Approach Naruti Longkumer,1 Kikoleho Richa,1 Rituparna Karmaker,1 Visekhonuo Kuotsu,2 Aola Supong,1 Latonglila Jamir,2 Pranjal Bharali2 and Upasana Bora Sinha^* 1 Department of Chemistry, Nagaland University, Lumami-798627, Nagaland, India 2 Department of Environmental Science, Nagaland University, Lumami-798627, Nagaland, India * Corresponding author: E-mail: upasanabsinha@gmail.com; Tel: +91-9436006754 Received: 07-01-2018 Abstract A simple, environmentally benign methodology has been developed to synthesize some bromoorganic compounds which have potential as antimicrobial agents. The required compounds were obtained through microwave (MW) irradiation, on-water reactions and using cetyltrimethylammonium tribromide (CTMATB) as the bromine source. The high yield of the product could be achieved within short reaction times, thus representing the main attribute of the present synthetic approach. The compounds were evaluated for in vitro antibacterial activity against Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Bacillus subtilis. Further, in silico studies were carried out to elucidate the interactions of the compounds with the bacterial proteins. Keywords: Bromo compounds; aqueous conditions; green chemistry; antibacterial activity, in vitro studies; molecular docking. 1. Introduction Bromo derivatives, both naturally occurring as well as synthesised compounds, have been reported to have biological activities, such as feeding deterrent, antimicrobial, anti-diabetic, antioxidant, anti-inflammatory and enzyme inhibition.1-7 Considering the importance of bromo organic compounds, new methods of their synthesis are always sought and literature enumerates a few reports of the use of quaternary ammonium tribromides (QATBs) under MW irradiation for their synthesis.8,9 Among the tribromides reported so far, the efficacy and versatility of cetyl-trimethylammonium tribromide (CTMATB) has been reported in many important organic transformations.10-17 Its proven mildness as a brominating reagent and versatility towards various organic substrates added to its compatibility with the aqueous media is what prompted us to choose this reagent for the present study. In recent years there has been an increasing emphasis on avoiding the use of solvents in organic reactions. It often happens that while many reaction strategies are ef- ficient as well as benign, use of organic solvents in these reactions prevents them from being considered as perfectly green.18 There is an extensive current debate over the relative "greenness" of the use of various solvent media, but water can undeniably be considered the cleanest solvent available, and the use and release of clean water clearly will have the least impact on the environment. Numerous publications report the combination of water as an environmentally benign solvent for chemical transformations with the use of MW irradiation as an efficient heating method.19,20 In fact, MW heating has become a broadly accepted non-conventional energy source for performing organic synthesis21-29 as well as in various aspects of inorganic chemistry and polymer chemistry.30,31 Microwave heating is preferred in the context of environmentally benign synthesis because it is a more homogenous method and accelerates reaction processes as compared to the traditional heating methods (e.g. in an oil bath, heating mantle or hot air oven),9,25,28,32 hence our choice of MW irradiation and use of water as the solvent medium. Longkumer et al.: Green Synthesis of Bromo Organic Molecules Acta Chim. Slov. 2019, 66, 276-283 277 While the presently synthesized compounds are examples of small molecules which are very common, small molecules have found significance as new-age pharmaceutical compounds due to their less challenging manufacturing procedure as compared to larger biologicals. Further, even though bromoorganic compounds have been commonly used as antimicrobial agents,3-7 there seems to be no reports on the anti-microbial essay of the presently synthesized compounds in the literature. This led us to consider the prospects of such an investigation through experimental and computational approaches. To explain the promising activity of these compounds, this work includes the molecular docking study of the synthesised compounds within the binding pockets of DNA gyrase subunit B (PDB ID: 1KZN) and dihydrofolate reductase (PDB ID: 3SRW). DNA gyrase is a bacterial protein of the topoisomerase family involved in DNA replication and transcription by catalysing the negative supercoil-ing of the closed-circular DNA. As this function is essential for DNA replication and transcription, gyrase is really a suitable target for antibacterial agents.33 Dihydrofolate reductase (DHFR) is an important target in a number of therapeutic areas, including cancer and search for antiin-fective compounds where it is used to generate antibacterial, antifungal and antiparasitic agents.34 2. Experimental 2. 1. General Chemistry All the solvents and substrates were purchased from Merck, Spectrochem, Sigma-Aldrich, and S. D. Fine Chem. Hexane and ethyl acetate were distilled before the use in column chromatography, while the substrates were used without further purification. All reactions were monitored by TLC on silica gel HF254. The microwave reactions were carried out in a scientific microwave system CATA 2R (single mode reactor) from Catalyst System (Pune, India). Melting points were determined by digital melting point apparatus. IR spectra were recorded with KBr pellets on a Perkin-Elmer FT-IR (spectrum two). 1H NMR and 13C NMR spectra were recorded on a JEOL ECS-400 using CDCl3 as the solvent and TMS as the internal standard. 2. 1. 1. Procedure for Synthesis of CTMATB CTMATB was synthesised using a modified version of our method reported earlier.17 In this procedure, a mixture of 4.89 g (41.07 mmol) of potassium bromide (KBr) and 5.00 g (13.74 mmol) of cetyltrimethylammonium bromide (CTMAB), and 0.057 g (0.53 mmol) of sodium carbonate (Na2CO3) were taken in a mortar and 10 mL (88.24 mmol) of 50% H2O2 added to the whole. The resultant mixture was grinded thoroughly and then was dissolved in 50 mL of water taken in a 100 mL beaker. The reaction solution was stirred at room temperature for 5 minutes and then 30 mL of 1 M H2SO4 was added drop-wise. An exothermic reaction followed and the CTMATB precipitated out. CTMATB formed was filtered using suction pump, washed with water many times till the filtrate contained no trace of acid (tested using litmus paper), and then initially air-dried and finally dried in a vacuum des-siccator. (1) The compound was then dried in a vacuum desiccator using anhydrous calcium chloride (CaCl2) as desiccant. The product was obtained as bright yellow micro-crystals which was further recrystallized from methanol. Yield of the product was 5.52 g (96%), m.p. 87-88 °C, m.p. (lit.) 87 or 88 °C.35 2. 1. 2. General Procedure for the Synthesis of Compounds 1a-9a. A homogenous mixture of the reagent CTMATB (2 mmol) and substrate 1-9 (2 mmol) were taken in 1:1 ratio in a 50 mL round bottomed flask equipped with reflux condenser in the microwave reactor. 10 mL H2O was added to the mixture and stirred thoroughly. The reaction mixture was placed inside the microwave reactor. The reactor was switched on and kept at a controlled power of P-7 which corresponds to 595 W. Reaction temperature was recorded using the flexible temperature probe attached to the microwave reactor, immediately after the completion of the reaction, and was found to be 90 °C. The progress of the reaction was monitored by TLC on silica gel HF254 using ethyl acetate-hexane solvent system (volume ratio varied for different substrates). After completion of the reaction, the product was extracted with 10 mL (2x) ethyl acetate and washed with 5 mL (2x) sodium bicarbonate solution. The crude product thus obtained was subjected to column chromatography over a pad of silica gel using ethyl acetate-hexane solvent system (volume ratio varied for different substrates) to obtain the desired products 1a-9a. 2. 2. Antibacterial Studies All the synthesized compounds were evaluated for their in vitro antibacterial activities against Escherichia coli and Klebsiella pneumonia as Gram negative bacteria, as well as Staphylococcus aureus and Bacillus subtilis as Gram positive bacteria. The antimicrobial properties of the syn-thesised compounds were evaluated by the determination of the zone of inhibition, using agar well diffusion meth-od.36 Subsequently minimum inhibitory concentrations (MIC) of the compounds were determined by the twofold broth dilution method in nutrient broth. DMSO was used as the control and the tests were performed at 10 mg/mL Longkumer et al.: Green Synthesis of Bromo Organic Molecules 278 Acta Chim. Slov. 2019, 66, 276-283 278 concentration using DMSO as the solvent. Streptomycin was used as the standard reagent. Each experiment was performed in triplicate and the average reading was taken. using MVD cavities were predicted and the ligands were docked against the target proteins and 30 independent runs were performed for each ligand.37-40 2. 3. Docking Studies Molecular docking studies were conducted so as to validate the obtained data and to provide comprehensible evidence for the observed antibacterial activity of all synthesized compounds. In this study, molecular docking simulations were performed using Molegro Virtual Docker (MVD). The pdb file format of enzymes DNA Gyrase B (Pdb id: 1KZN) and dihydrofolate reductase (Pdb id: 3SRW) as receptors were obtained from the RCSB Protein Data Bank and were prepared for molecular docking. All the 3D structure of the ligands was drawn using ChemBi-oDraw as mol2 file. For molecular docking simulation, water molecules were removed and charges were assigned. By 3. Results and Discussion Cetyltrimethylammonium tribromide (CTMATB), having the molecular formula C19H42NBr3 is a bright orange crystalline solid with sharp melting point at 87-88 °C. However, from thermogravimetric (TG) analysis it was revealed that the compound is stable even up to ca. 200 °C. One of the major implications of this property is that the tribromide may be very useful for the appropriate organic transformations at relatively high temperatures as well. It is an obvious fear that tribromides, upon heating, release bromine, which is an environmentally hazardous chemical. However, while investigating their thermal stability by Table 1. Aqueous microwave bromination of organic substrates with CTMATBa Substrate Productb Reaction time Yieldc 4 min 5 min 4 min 4 min 5 min 2 min 3 min 5 min 4 min 70% 75% 80% 80% 62% 69% 60% 69% 86% a Reactions maintained at 90 °C using controlled power P-7 of the MW reactor; all reactions were monitored by TLC; b Confirmed by IR, 1H NMR and 13CNMR; c Isolated yields. Longkumer et al.: Green Synthesis of Bromo Organic Molecules Acta Chim. Slov. 2019, 66, 276-283 279 TG experiments, it was observed that CTMATB loses Br2 as the tail fragment at the temperature of 265-267 °C, which is much higher than the temperature at which bro-minations take place.17 In order to determine the efficiency of CTMATB in aqueous condition, different types of organic compounds were used and the reactions were performed under microwave conditions. These reactions, when performed in an oil bath under the same conditions, took longer time. As an example, 3a took 8 minutes for its formation, while it took 4 minutes using MW reactor, thereby justifying our choice of MW irradiation. The results of the bromination reactions under microwave conditions are presented in Table 1. The products were identified by comparing their melting points and IR absorption spectra with that of authentic samples.8,9,17,18,41 The antibacterial activity of the synthesised compounds was tested against Escherichia coli and Klebsiella Table 2. Zone of inhibition values (mm) of the synthesised compounds Compounds Zone of inhibition (mm) (10 mg/mL) E. coli K. pneumoniae S. aureus B. subtilis 1a 19 12 18 14 2a 24 15 21 18 3a 18 17 19 16 4a 12 10 15 >10 5a 10 >10 12 10 6a 19 15 16 12 7a 12 17 >10 14 8a 16 18 16 >10 9a 19 12 17 14 Streptomycin 32 30 34 30 Table 3. MIC (minimum inhibitory concentration in mg/mL) of the synthesised compounds Compounds MIC (mg/ml) mg/mL) E. coli K. pneumoniae S. aureus B. subtilis 1a 0.117 0.468 0.117 0.234 2a 0.058 0.144 0.234 0.117 3a 0.117 0.144 0.117 0.144 4a 0.937 0.937 0.144 0.937 5a 0.937 0.937 0.468 0.937 6a 0.117 0.144 0.144 0.468 7a 0.468 0.117 0.937 0.234 8a 0.144 0.937 0.144 0.937 9a 0.117 0.937 0.144 0.234 Table 4. Docking score of the compounds with 1KZN Ligand Moldock score Rerank scorea Interactionb Internalc HBondd LE1e LE3f 1a -64.19 -49.54 -61.17 -3.01 -2.50 -8.02 -6.19 2a -66.34 -57.59 -76.74 10.40 -2.29 -6.63 -5.76 3a -56.97 -48.30 -66.03 9.06 -2.58 -7.12 -6.04 4a -63.30 -52.11 -72.49 9.19 -2.02 -7.03 -5.79 5a -62.87 -52.41 -72.86 9.99 -1.94 -6.99 -5.82 6a -62.12 -52.74 -72.86 11.71 -2.08 -6.90 -5.86 7a -62.72 -55.09 -75.60 12.87 0.00 -5.70 -5.01 8a -62.50 -50.56 -73.12 10.61 -2.12 -6.25 -5.06 9a -63.07 -54.09 -72.88 9.81 -3.31 -7.01 -6.01 Streptomycin -101.15 -29.04 -150.03 48.87 -8.81 -2.53 -0.73 a The rerank score is a linear combination of E-inter (steric, Van der Waals, hydrogen bonding, electrostatic) between the ligand and the protein, and E-intra. (torsion, sp2-sp2, hydrogen bonding, Van der Waals, electrostatic) of the ligand weighted by pre-defined coefficients. b The total interaction energy between the pose and the protein (kJ/mol). c The internal energy of the pose. d Hydrogen bonding energy (kJ/mol). e Ligand efficiency 1: MolDock score divided by heavy atoms count. f Ligand efficiency 3: Rerank score divided by heavy atoms count. Longkumer et al.: Green Synthesis of Bromo Organic Molecules 280 Acta Chim. Slov. 2019, 66, 276-283 Table 5. Docking score of the compounds with 3SRW Ligand Moldock score Rerank scorea Interactionb Internalc HBondd LE1e LE3f 1a -64.94 -49.76 -61.92 -3.02 -4.18 -8.12 -6.22 2a -62.98 -54.19 -73.36 10.38 -3.19 -6.30 -5.42 3a -50.44 -41.89 -59.50 9.06 -1.49 -6.30 -5.24 4a -55.92 -46.30 -65.12 9.19 -1.43 -6.21 -5.14 5a -55.86 -46.43 -65.85 9.99 -1.78 -6.21 -5.16 6a -54.38 -46.44 -66.10 11.71 -1.91 -6.04 -5.16 7a -64.85 -56.88 -78.04 13.19 -8.59 -5.90 -5.17 8a -61.56 -51.24 -72.18 10.62 -2.38 -6.16 -5.12 9a -60.34 -49.08 -70.15 9.81 -4.37 -6.70 -5.45 Streptomycin -134.60 -39.20 -178.12 43.52 -9.39 -3.36 -0.98 a The rerank score is a linear combination of E-inter (steric, Van der Waals, hydrogen bonding, electrostatic) between the ligand and the protein, and E-intra. (torsion, sp2-sp2, hydrogen bonding, Van der Waals, electrostatic) of the ligand weighted by pre-defined coefficients. b The total interaction energy between the pose and the protein (kJ/mol). c The internal energy of the pose. d Hydrogen bonding energy (kJ/mol). e Ligand efficiency 1: MolDock score divided by heavy atoms count. f Ligand efficiency 3: Rerank score divided by heavy atoms count. a) b) < • l&f V" * A 7a - — Figure 1. Molecular interaction of the compounds 2a and 7a at the active pockets of the protein 1KZN (a and b) and 3SRW(c and d) (green dotted lines indicate the mode of interaction with the protein). pneumonia as Gram negative bacteria, as well as Staphylococcus aureus and Bacillus subtilis as Gram positive bacteria. The results of the primary screening are shown in Table 2. The investigation of the screening revealed that the compounds tested showed varying degree of activity against all the investigated microorganisms. Almost all the compounds showed moderate to potent activity against the strains. Subsequently, minimum inhibitory concentrations (MIC) of the compounds were determined to quantify the antibacterial potency of the compounds. The results of the MIC values of antibacterial activity are given in Table 3. Compound 2a showed a better activity against E. coli with a MIC of 0.058 mg/mL and compounds 4a and 5a have lesser effectiveness against the bacterial strains. In comparison, compound 2a showed the best activity indicating its promising broad spectrum of antibacterial property. Longkumer et al.: Green Synthesis of Bromo Organic Molecules ... Acta Chim. Slov. 2019, 66, 276-283 281 Docking studies were performed on known target proteins to understand the antibacterial mechanisms of bromo compounds using Molegro virtual docker (MVD). The proteins used as target were DNA gyrase B (Pdb id: 1KZN) from Escherichia coli and dihydrofolate reductase (Pdb id: 3SRW) from Staphylococcus aureus. The best pose of each compound were selected for ligand-protein interaction energy analysis as shown in Tables 4 and 5. The interaction energies of 2a and 7a were -76.74 kJ/mol and -75.60 kJ/mol as compared to streptomycin with -150.03 kJ/mol. This indicates that 2a and 7a also have a favourable ligand-protein interaction energy at the binding cavity of 1KZN. Similarly, the interaction energies of 2a and 7a were -73.36 kJ/mol and -78.04 kJ/mol as compared to streptomycin with -178.12 kJ/mol. This indicates that 2a and 7a also have a favourable ligand-protein interaction energy at the binding cavity of 3SRW. The snapshots of li-gand-protein interaction depicting the binding mode of the best poses are shown in Fig. 1a, b, c and d. In this study, the molecular interaction analysis as shown in Table 6 established a common molecular interaction with Val71 and Thr165. Similarly, in Table 7, a common interaction with Thr122 and Asp121 in the compounds and streptomycin was established. Table 6. Molecular interaction analysis of the compounds with the active site of 1KZN Compound Interaction (Protein—Ligand) Interaction Energy (kJ/mol) Interaction distance (A) Hybridisation (Protein) Hybridisation (Ligand) 1a Asp73(OD1)—N(4) -2.5 2.785 sp3(A) sp2(D) 2a Val71(O)-N(8) -2.409 2.589 sp2(A) sp2(D) Thr165(O)-N(8) -2.209 2.901 sp2(A) sp2(D) 3a Thr165(O)-N(6) -2.5 2.928 sp2(A) sp3(D) Val71(O)-N(6) -2.5 2.815 sp2(A) sp3(D) 4a Val71(O)-N(Ü) -2.5 2.676 sp2(A) sp3(D) 5a Val71(O)-N(Ü) -2.5 2.663 sp2(A) sp3(D) 6a Val71(O)-N(Ü) -2.5 2.683 sp2(A) sp3(D) 7a Thr165(OG1)-O(14) -2.423 3.115 sp3(B) sp2(A) 8a Val71(O)-N(0) -2.5 3.020 sp2(A) sp3(D) 9a Val167(N)-O(0) -0.922 3.217 sp2(D) sp3(B) Val71(O)-O(0) -2.5 2.672 sp2(A) sp3(B) Streptomycin Asn46(ND2)-N(36) -2.5 2.866 sp2(D) sp2(A) Asn46(ND2)-O(11) -1.937 2.532 sp2(D) sp3(B) Asp49(OD1)-N(38) -0.377 3.524 sp3(A) sp2(D) Asp49(OD1)-N(39) -1.968 2.880 sp3(A) sp2(D) Asn46(O)-N(39) -1.153 3.396 sp2(A) sp2(D) Asn46(O)-O(20) -2.44 3.111 sp3(A) sp3(B) Asp73(OD1)-O(20) -2.5 2.676 sp3(A) sp3(B) Thr165(OG1)-O(14) -0.464 3.507 sp3(B) sp3(A) Thr165(O)-O(35) -0.335 3.009 sp2(A) sp3(B) Val71(O)-O(35) -2.5 2.621 sp2(A) sp3(B) Val167(N)-O(35) -0.890 3.112 sp2(D) sp3(B) (A): Acceptor (D): Donor (B): Both donor and acceptor 4. Conclusion To conclude, microwave assisted aqueous reactions for the bromination of organic compounds as an attractive protocol due to its eco-friendly, efficient and economic nature are presented. The use of a CTMATB in the bromination reactions which is less toxic compared to using molecular bromine makes the process more environmentally benign. The antimicrobial study of these novel bromoor-ganic derivatives against Gram positive and Gram negative species showed that synthetic mimics of naturally occurring bromoorganic compounds can be of promise against drug-resistant bacteria. Docking studies revealed that both streptomycin and the synthesized compounds have a com- mon interaction at the active sites of the protein and further studies on these compounds might increase their potency thereby enhancing their anti bacterial activity. Thus, this study adopts significance in view of simple molecules that are potent and easy to synthesize. Further studies on derivatives involving synthetic mimics of naturally occurring moieties would provide a lead in the development of novel bromoorganic-based antimicrobial compounds. 5. Acknowledgement The authors N. Longkumer and K. Richa are thankful to the UGC Non-NET fellowship and UGC Project Longkumer et al.: Green Synthesis of Bromo Organic Molecules 282 Acta Chim. Slov. 2019, 66, 276-283 Table 7. Molecular interaction analysis of the compounds with the active site of 3SRW Compound Interaction Interaction Energy Interaction Hybridisation Hybridistion (Protein...Ligand) (kJ/mol) distance (Â) (Protein) (Ligand) 1a Gln96(N)-N(1) -0.042 3.480 sp2(D) sp2(A) Thr97(N)-N(1) -2.210 3.157 sp2(D) sp2(A) Thr97(OG1)-N(1) -2.225 3.148 sp3(B) sp2(A) 2a Gly95(N)-O(7) -0.175 2.944 sp2(D) 2 sp2 Thr95(N)-O(7) -0.510 3.223 sp2(D) 2 sp2 Leu98(N)-O(7) -2.5 3.027 sp2(D) 2 sp2 3a Thr122(OG1)-N(6) -2.5 2.867 sp3(B) sp3(D) Asp121(OD1)-N(6) -2.5 3.052 sp3(A) sp3(D) 4a Thr122(OG1)-N(0) -2.5 2.883 sp3(B) sp3(D) 5a Thr122(OG1)-N(0) -2.5 2.971 sp3(B) sp3(D) 6a Thr122(OG1)-N(0) -2.5 2.956 sp3(B) sp3(D) 7a Thr97(OG1)-N(0) -1.813 2.882 sp3(B) sp3(D) Gly95(N)-N(13) -2.208 2.932 sp2(D) sp3(A) Thr47(OG1)-N(13) -0.699 3.460 sp3(B) sp3(A) Thr47(OG1)-O(14) -2.5 2.813 sp3(B) sp2(A) Thr47(N)-O(14) -2.5 2.713 sp2(D) sp2(A) Lys46(N)-O(14) -0.280 3.402 sp2(D) sp2(A) 8a Thr122(OG1)-N(0) -2.5 2.815 sp3(B) sp3(D) 9a Thr97(N)-O(0) -0.673 3.110 sp2(D) sp3(B) Gly95(N)-O(0) -1.197 2.639 sp2(D) sp3(B) Leu98(N)-O(0) -2.5 2.746 sp2(D) sp3(B) Streptomycin Ala8(N)-O(35) -0.660 3.225 sp2(D) sp3(B) Ala8(O)-O(34) -1.812 2.517 sp2(A) sp3(B) Gln20(O)-O(10) -0.676 2.601 sp2(A) sp3(B) Ser50(OG)-O(12) -2.5 2.702 sp3(B) sp3(B) Ser50 (OG)-O(22) -2.5 2.607 sp3(B) sp2(A) Thr47(OG1)-N(39) -2.5 2.775 sp3(B) sp2(D) Thr122(OG1)-N(3) -2.5 3.085 sp3(B) sp2(A) Thr122(OG1)-O(11) -2.5 2.772 sp3(B) sp2(B) Asp121(OD1)-N(2) -2.5 3.053 sp3(A) sp2(D) Asp121(OD1)-N (0) -2.5 2.817 sp3(A) sp2(D) Asn19(OD1)-N(2) -2.093 3.181 sp2(A) sp2(D) (A): Acceptor (D): Donor (B): Both donor and acceptor (UGC MRP No.43-192/2014), respectively, for financial support. 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DOI:10.1021/jo050059u Povzetek Razvili smo enostavno in okolju prijazno metodologijo za sintezo nekaterih brhttps://doi.org/10.1021/jo050059ustavljale potencialne antimikrobne učinkovine. Zaželjene spojine smo pripravili s pomočjo mikrovalovnega obsevanja v reakcijah »na vodi« ter z uporabo cetiltrimetilamonijevega tribromida (CTMATB) kot vira broma. Visoki izkoristki reakcij, ki smo jih dobili že pri kratkih reakcijskih časih, so glavni atributi predstavljenega sinteznega pristopa. Spojinam smo določil in vitro antibakterijsko aktivnost proti Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus in Bacillus subtilis. Dodatno so in silico študije razložile interakcije med raziskovanimi spojinami in bakterijskimi proteini. Longkumer et al.: Green Synthesis of Bromo Organic Molecules ... 284 DOI: I0.i7344/acsi.20i8.4705 Acta Chim. Slov. 2019, 66, 284-293 ©commons Scientific paper Electrochemical Degradation of Reactive Blue 21 and Synthetic Textile Effluent by Using Co475/C475-PVC5 Composite Electrode Norazzizi Nordin,1^ Mohamad Anis Farith Pisal,1 Nur Izzatie Hannah Razman2 and Nur Farhana Jaafar1 1 School of Chemical Sciences, Universiti Sains Malaysia, 11800 Gelugor, Pulau Pinang, Malaysia. 2 Centre of Foundation Studies, Universiti Teknologi MARA, Selangor Branch, Dengkil Campus, 43800 Dengkil, Selangor, Malaysia. * Corresponding author: E-mail: azzizi@usm.my (+604 653 4030) Received: 11-09-2018 Abstract In this study, cobalt/graphite-polyvinyl chloride (Co475/C475-PVC5) composite electrode was fabricated by mechanical alloying technique. The fabricated electrode and graphite rod were used as anode and cathode, respectively, in the decolourisation of aqueous C. I. Reactive Blue 21 (RB21) and synthetic textile effluent, containing a mixture of azo and anthraquinone dyes. The fabricated electrode showed high efficiency in the decolorisation of aqueous RB21 (99.95%) and synthetic textile effluent. This is further confirmed by the high reduction percentage (>75%) of COD and BOD5 for both treated samples. The elemental composition study by using FESEM-EDX showed no significant changes in the composition of elements (C, Co and Cl) in the freshly fabricated electrode and after electrolysis of synthetic textile effluent. This showed that the fabricated electrode has a high mechanical strength and strong binding ability between C and Co due to the use of PVC as a binder. Keywords: Composite electrode; decolorisation; electrochemical technique; C. I. Reactive Blue 21; synthetic textile effluent 1. Introduction Synthetic dyes are among the most widely used pollutants in various industries, including textiles, paper, food and cosmetics. In line with the increase in usage by various industries, more than 100000 synthetic dyes with different structures are synthesised and more than 0.7 million tonnes of dyestuffs are produced throughout the year.1 Synthetic dyes are categorised into several groups, depending on their applications (dyeing method) such as reactive dyes, basic dyes, acid dyes, vat dyes, mordant dyes, and disperse dyes. It also depends on their chemical structure, such as azo dyes, anthraquionone dyes, triphenyl-methane dyes, and phthalocyanine dyes.2 In the textile industry, only 60%-90% of the dyes used are fixed to textile fibre while the other 10%-40% may remain unfixed to the fibre and will be released into the effluent.3 This has an adverse effect on aquatic ecosystems due to the presence of synthetic dyes even in small quantities (<1 mg L-1). They can be seen with the naked eye and have negatively affected the aesthetic quality and transparency of lakes, rivers, and others.4 This in turn resulted in the destruction of aquatic ecosystems and the formation of dead deoxygenated zones in seas and oceans.5 Therefore, the treatment of dye containing effluents is very important to reduce the negative effects on aquatic life. Various strategies were reported on the treatment of textile effluents. They are classified into biological methods (enzymes and microorganisms), physical methods (filtration, flocculation, and adsorption), and oxidation methods (advanced oxidation and chemical oxidation).6,7 However, today the use of electrochemical oxidation technique in textile wastewater treatment is getting more attention as this method does not involve the use of additional chemicals.89 Previous studies have shown that this technique provided high efficiency in the removal of organic and inorganic pollutants from the effluents.10,11,12 According to Najafpoor et al., this technique requires simple equipment, easy implementation, and on-site treatment in less space.13 Nordin et al.: Electrochemical Degradation of Reactive Blue 21 Acta Chim. Slov. 2019, 66, 284-293 285 In the electrochemical treatment of industrial effluents, anode plays an important role as it acts as a site for electrochemical reactions. The material for electrode fabrication plays a major role in determining the efficiency and reactivity of the prepared electrode in an electrochemical reaction. Various electrodes were used in the treatment of synthetic dyes and textile industrial effluents, such as Pt,14-16 boron-doped diamond (BDD),17-19 and dimen-sionally stable anodes (DSA).20-22 In the current study, metal/graphite composite electrodes were prepared by mixing two different material (metal and graphite) powders with polyvinyl chloride (PVC). It is expected that the electrode performance in the electrochemical reaction will increase as the existence of two different materials in an electrode provides better synergistic effects and active bi-functional catalyst.4 The main objective of this study is to investigate the efficiency of Co475/C475-PVC5 composite electrode in the decolourisation of aqueous RB21 and synthetic textile effluents. The effect of electrolysis conditions was also examined in the current study by investigating the effect of metal:-graphite ratio in the prepared electrode, supporting electrolyte concentration, applied voltage, and electrolysis time on RB21 decolourisation. The surface characterisation of the prepared composite electrode was also performed in this study by using FESEM, EDX, and gas sorption analyser. 2. Experimental 2. 1. Chemicals All the chemicals used were of analytical research (AR) grade. Tin (Sn), copper (Cu), cobalt (Co), and aluminium (Al) powder with 99.9% purity were purchased from Aldrich. Sodium chloride (NaCl) solution, which acts as a supporting electrolyte in the electrolysis process, was purchased from R & M Chemicals. In this study, C. I. Reactive Blue 21 (RB21), C. I. Reactive Blue 19 (RB19), C. 1. Reactive Violet 5 (RV5), and C. I. Reactive Red 198 (RR198) (Figure 1) were used as synthetic dyes and purchased from Dylon. Besides, polyvinyl chloride (PVC) and graphite powder (C) were obtained from BDH Ltd. whereas tetrahydrofuran (THF) was obtained from Systerm. 2. 2. Preparation of Metal/Graphite-PVC Composite Electrode The anode was prepared by mixing 0.475 g of metal powder (Sn, Cu, Al, or Co) with 0.475 g of graphite (C) powder (M:C is 50:50 (wt. %), in which M = metal) and 0.05 g of PVC as a binder by using a mechanical alloying technique (MAT).4 The ratio of metal and C powders to PVC was 95:5 PVC (wt. %). Then, 4 mL of THF was used to dissolve the PVC. The mixture was stirred until homogenous and oven dried at 100 °C for 2 h. The mixture was placed in a stainless-steel mould of 1 cm diameter and pressed at 104 kg cm-2. The pellet obtained (approximately 1.5 g) was then connected to a silver wire with silver conducting paint (Sig-ma-Aldrich) and sealed in a glass rod. Subsequently, epoxy resin (Devcon) was applied to cover the silver wire connecting surface. The metal/graphite-PVC composite electrode was used in the decolourisation of RB21 solution. 2. 3. Preparation of Aqueous RB21 and Synthetic Textile Effluent In this study, two types of samples were used, namely aqueous RB21 and synthetic textile effluent. The RB21 Na03S C. I. Reactive Violet 5 Figure 1. Structures of RB21, RB19, RV5, and RR198. Nordin et al.: Electrochemical Degradation of Reactive Blue 21 286 Acta Chim. Slov. 2019, 66, 284-293 solution was prepared by dissolving a certain amount of RB21 in deionised water. Synthetic textile effluent was prepared by dissolving equal amount of RB21, RB19, RV5, and RR198 in tap water along with the other chemical additives, as described by Verma et al.23 2. 4. Decolourisation of Aqueous RB21 and Synthetic Textile Effluent The electrolysis of aqueous RB21 was performed by using a two-electrode system, which consisted of an anode (metal/graphite-PVC composite electrode) and a cathode (graphite rod electrode), with NaCl solution as a supporting electrolyte. Prior to electrolysis, both anode and cathode were rinsed with distilled water and a small amount of acetone. A direct current (DC) power supply (TTi PSU Bench CPX 400) and stirrer hotplate with magnetic bar were used throughout the electrolysis process. The electrochemical cell used was a simple and undivided cell made of Pyrex glass with a capacity of 250 mL. The electrodes were placed vertically and parallel to each other in the electrochemical cell, with a constant gap between electrodes at approximately 20 mm. The electrolysis of synthetic textile effluent was carried out by using similar procedures, as described above. The same anode from the previous electrolysis of aqueous RB21 was used in the electrolysis of synthetic textile effluent. 2. 5. Optimisation of Electrolysis Parameters for the Decolourisation of Aqueous RB21 Optimisation of electrolysis parameters was carried out by optimising the ratio of metal and C in the prepared electrode, supporting electrolyte concentration, electrolysis time and applied voltage. For the optimisation of metal and C ratio in the prepared electrode, the ratio and mass of metal, C, and PVC are summarised in Table 1. The procedure for preparation of anodes is similar as mentioned in Section 2.2. using 10 mm quartz cuvettes. The surface properties of the electrode were determined by using gas sorption analyser (ASAP 2020 V4.010). Brunauer-Emmett-Teller (BET) method was used to determine the surface area of the electrode. BJH model was used to calculate the pore size. Surface morphology of the electrode was observed by using Field Emission Scanning Electron Microscope (FESEM) (Leo Supra 50 VP). The elemental composition of prepared electrode was determined by using Energy Dispersive X-ray (EDX) spectrometer. The size and shape of the particles were observed by using a transmission electron microscopy (TEM) (Philips CM12-12796) with an accelerating voltage of 100 kV. Samples were prepared on carbon-coated copper grids covered with a polyvinyl formal polymer by adding a drop onto the grid and evaporating the solvent (ethanol) in ambient condition. 2. 7. Analytical Measurement Decolourisation of aqueous RB21 was determined based on the original concentration of RB21 (c;) and RB21 concentration after electrolysis process (ct) by measuring absorbance at the wavelength of 624 nm in the Vis region of RB21. Measurement of RB21 absorbance was performed by using UV-Vis spectrophotometer. Decolourisation was calculated in percentage (%) as in the following equation: Decolourisation (%) = x 100% (1) The absorbance versus RB21 concentrations graph in the range of 10 to 110 mg L-1 is a linear graph referring to the following equation: Absorbance = 0.015c + 0.033 (R2 = 0.996) (2) Where c represents the concentration of RB21 solution in mg L-1 unit. 2. 6. Instrumentation 2. 8. Reduction of COD and BOD5 To observe the decolourisation level of aqueous RB21 and synthetic textile effluent, the sample solution was characterised by using an UV-Vis (Perkin Elmer Lambda 35) spectrometer in the range of 200-900 nm by Chemical Oxygen Demand (COD) and Biochemical Oxygen Demand (BOD5) were performed on an aqueous RB21 and synthetic textile effluent before and after electrolysis by using the prepared electrode. Standard methods Table 1. The composition ratio and mass of Co, C, and PVC for the preparation of Co/C-PVC electrodes. Electrode Co:C ratio (%) Mass of Co (g) Mass of C (g) Mass of PVC (g) C095.0/C0.0-PVC5 100:0 0.950 - 0.05 Co66.5/C28.5- PVC5 70:30 0.665 0.285 0.05 Co47.5/C47.5- PVC5 50:50 0.475 0.475 0.05 Co28 5/C665- PVC5 30:70 0.285 0.665 0.05 Co0.0/C95.0- PVC5 0:100 - 0.950 0.05 Nordin et al.: Electrochemical Degradation of Reactive Blue 21 ... Acta Chim. Slov. 2019, 66, 284-293 287 508B and 507 were used for the COD and BOD5 analyses, respectively, as described by the American Public Health Association (APHA 1981).24 2. 9. Statistical Analysis The data were expressed as mean ± standard error (SE) with at least three times replication. The statistical analysis was carried out by using Statistical Package for Social Sciences (SPSS) Version 17.0 with one-way ANOVA. The data obtained were considered as statistically significant when the value of p < 0.05. 3. Results and Discussion 3. 1. Preliminary Experiment Four types of metal/graphite-PVC composite electrodes were prepared and used in the preliminary experiment to investigate the efficiency of the prepared electrodes in the decolourisation of RB21 solution. This was to identify the best metal-graphite mixture that could give a maximum decolourisation of aqueous RB21. The results in Figure 2 show that maximum decolourisation percentage Figure 2. Decolourisation percentage of aqueous RB21 after electrolysis by using different types of metal/graphite-PVC composite electrodes. [RB21]o = 50 mg L-1; [NaCl] = 1.0 mol L-1; E = 5 V; I = 0.62 A; t = 15 min. (85.09%) of RB21 is obtained by using Co475/C475-PVC5 electrode compared to other metal/graphite-PVC composite electrodes. This showed that the mixture of Co and C in the Co475/C475-PVC5 electrode showed the best synergistic effect with a maximum decolourisation percentage as compared to other electrodes. Therefore, this electrode will be used in the optimisation of electrolysis parameters for the decolourisation of aqueous RB21. 3. 2. Optimisation of Electrolysis Parameters for the Decolourisation of Aqueous RB21 Figure 3a shows the percentage of decolourisation on RB21 by using Co/C-PVC electrodes with different Co:C ratios (100:0, 70:30, 50:50, 30:70, 0:100). The electrode with a composition ratio of 50:50 (known as Co475/ C47.5-PVC5 electrode) exhibited the highest percentage of decolourisation, which was 85.27% as compared to other electrodes. This showed that the ratio of Co and C in the electrode provided better efficiency for RB21 removal. The composition ratio of 100:0 and 70:30 (known as Co95.0/ C0.0-PVC5 and Co66.5/C25.5-PVC5 electrodes, respectively) gave almost equal percentage of decolourisation (84.8% and 82.5%, respectively) as the decolourisation percentage obtained for 50:50 ratio. However, the use of more Co powder in both electrodes can increase the operating costs. Therefore, the electrode with a 50:50 ratio (Co475 / C47.5-PVC5 electrode) was considered as an optimum electrode based on the highest RB21 decolourisation percentage achieved in this study with less Co used in the preparation of the electrode. Figure 3b shows the effect of the supporting electrolyte concentration on the decolourisation of RB21 solution. The supporting electrolyte (NaCl solution) was added in the electrochemical system to modify the electrochemical conductivity and facilitate the electrical current flow.4,25 The presence of chloride ions from the addition of NaCl can lead to active oxidant formations, such as chlorine, hypochlorous acid, and/or hypochlorite, depending on the pH (Equations 3-5). These active oxidants are beneficial in the oxidation of organic pollutants at the anode or/and in bulk solution (Equation 6).8 2 Cl- - Cl2 + 2 e- Cl2 + H2O - HOCl + H+ + Cl- HOCl - H+ + OCl- Organics + OCl- — intermediates - CO2 + Cl- + H2O (3) (4) (5) (6) As shown in Figure 3b, the percentage of decolouri-sation increased from 74.57% to 87.45% with the concentration of NaCl from 0.01 mol L-1 to 2.0 mol L-1, respectively, after 15 min of electrolysis. Plus, electrolysis by using 0.5 mol L-1 and 2.0 mol L-1 NaCl resulted in almost equal percentage of decolourisation (85.09% and 85.27%, respectively). In addition, application of higher concentration of electrolyte (in this case 1.0 mol L-1 and above) resulted in electrode damage and heat production. As shown in Equations 3-6, the increased NaCl concentration has increased the electrogenerated OCl- anions. This electrode corrosion was also due to the presence of OCl- ions in the electrolysis solution. This was reported Nordin et al.: Electrochemical Degradation of Reactive Blue 21 288 Acta Chim. Slov. 2019, 66, 284-293 a) 100 a> Q b) 100 100:0 30:70 50:50 70:30 0:100 Composition ratio (Co:C) 1 NaCI concentration (mol L-1) c) 100 d) 10 25 40 55 70 Electrolysis tirne (min) Applied voltage (V) Figure 3. Decolourisation percentage of aqueous RB21 by using different (a) Co:C ratio in the prepared electrode ([RB21]o = 50 mg L [NaCl] = 1.0 mol L-1; E = 5 V; I = 0.62 A; t = 15 min); (b) NaCl concentration ([RB21]o = 50 mg L-1; I = 0.02-1.12 A (for 0.01 - 2.0 mol L-1 NaCl); E = 5 V; t = 15 min; anode = Co475/C475-PVC5); (c) electrolysis time ([RB21]o = 50 mg L-1; [NaCl] = 0.5 mol L-1; E = 5 V; I = 0.36 A; anode = Co475/C475-PVC5); (d) applied voltage ([RB21]o = 50 mg L-1; [NaCl] = 0.5 mol L-1; t = 45 min; I = 0.06-0.93 A (for 1-20 V); anode = Co475/C475-PVC5). by several researchers in previous studies.26-28 According to Galvan-Martinez et al.,26 OCl- ions are corrosive to some elements. This resulted in the corrosion of Co475/ C47.5-PVC5 electrode surface after electrolysis of RB21 by using NaCl electrolytes. Therefore, 0.5 mol L-1 was considered as an optimum concentration of NaCl for the deco-lourisation of RB21 with 85.09% of RB21 decolourisation percentage. The effect of electrolysis time on the decolourisation percentage of RB21 was investigated in the range of 15 min to 90 min. The increase in electrolysis time from this study showed an increased decolourisation percentage of up to 93.67% (Figure 3c). Due to the increase in generation of OCl- ions in the bulk solution resulting from the longer electrolysis time, better RB21 decolourisation efficiency was obtained as compared to a shorter electrolysis time. Electrolysis by using 60 min to 90 min leads to the electrode damage and corrosion of anode materials into the bulk solution. Due to the anode corrosion, precipitate was formed at the end of the electrolysis process and mixed with the electrolysis product. At 45 min, the electrode showed its ability to decolourise the aqueous RB21 without the formation of precipitate at the end of the electrolysis. Therefore, 45 min was considered as an optimum electrolysis time, with 86.87% of RB21 decolourisation percentage. The decolourisation efficiency was significantly affected by the applied voltage which was studied in the range of 1 V to 20 V. In Figure 3d, increasing the applied voltage from 1 V to 20 V increased the percentage of RB21 decolourisation after 45 min of electrolysis from 56.20% to 99.95%, respectively. Again, this was due to the increased generation of OCl- species by the application of high value of applied voltage. Therefore, 20 V of applied voltage was considered as an optimum value of applied voltage based on the complete decolourisation of RB21 obtained in Figure 3d. 3. 3. Decolourisation of Aqueous RB21 Figure 4 shows the UV-Vis spectra obtained for 50 mg L-1 of RB21 in 0.5 mol L-1 NaCl by using the optimum electrolysis parameters as obtained in the previous Section Nordin et al.: Electrochemical Degradation of Reactive Blue 21 ... Acta Chim. Slov. 2019, 66, 284-293 289 Figure 4. UV-Vis spectra of aqueous RB21 before and after electrochemical treatment by using Co475C475-PVC composite electrode. ([RB21]o = 50 mg L-1; [NaCl] = 0.5 mol L-1; t = 45 min; E = 20 V; I = 0.93 A). (3.2). The initial spectra (0 min) showed the appearance of three peaks at the wavelength of 339 nm in the UV region, and 624 nm and 663 nm in the visible region. The peak at 663 nm exhibited maximum absorption and was selected for subsequent study.29,30 The spectra obtained after 45 min of electrolysis showed the disappearance of all peaks after the electrolysis process. This suggested that the complex structure of RB21 was completely destroyed by the electrochemical process, and a colourless solution was produced after the electrolysis. 3. 4. Decolourisation of Synthetic Textile Effluent Optimum electrolysis conditions obtained in Section 3.2 were also applied in the decolourisation of synthetic textile effluent. This was to determine the efficiency of Co475/C475-PVC5 electrode in the degradation of various types of structurally different dyes and other pollutants. Figure 5 shows the UV-Vis spectra obtained for synthetic textile effluent before and after electrolysis. For untreated effluents (0 min), three peaks were observed at the wavelengths of 337 nm in the UV region, and 626 nm and 667 nm in the visible region. After 45 min of electrol- ysis with an applied voltage of 20 V in the presence of 0.5 mol L-1 of NaCl as a supporting electrolyte, the previous peaks completely disappeared. The colour of dyes mixture solution changes from blackish blue to colourless solution at the end of the electrolysis. It proved that the electrochemical treatment by using Co475/C475-PVC5 electrode was able to degrade the pollutants contained in the effluent. Figure 5. UV-Vis spectra for synthetic textile effluent before and after electrochemical treatment by using Co47.5/C47.5-PVC5 electrode. ([NaCl] = 0.5 mol L-1; t = 45 min; E = 20 V; I = 0.93 A). 3. 5. Reduction of COD and BOD5 The results for COD and BOD5 analyses of treated and untreated aqueous RB21 and synthetic textile effluent are summarised in Table 2. In addition to the colour removal, the electrochemical treatment by using the Co47.5/ C475-PVC5 electrode could also significantly reduce the COD and BOD5 (Table 2). Table 2 also shows that the removal percentages of COD and BOD5 for the synthetic textile effluents were lower than for the RB21 solution. This was due to the presence of more organic and inorganic compounds in synthetic textile effluents that created more challenging conditions for the COD and BOD5 removal, resulting in lower COD and BOD5 removal percentage than for the aqueous RB21. Table 2. COD, BOD5, and pH of untreated and treated 50 mg L 1 RB21 solution and synthetic textile effluent by using Co475/ C47.5-PVC5 electrode. ([NaCl] = 0.5 mol L-1, E = 20 V, t = 45 min). Sample Analysis Untreated Treated Removal (%) RB21 solution COD (mg L-1) 182.4 ± 7.1 15.5 ± 8.5 91.5 ± 6.0 BOD5 (mg L-1) 56.8 ± 6.7 10.7 ± 8.3 81.2 ± 5.7 pH 6.6 6.3 - Synthetic textile effluent COD (mg L-1) 753.0 ± 5.5 89.8 ± 5.0 88.1 ± 6.2 BOD5 (mg L-1) 81.6 ± 4.2 19.1 ± 7.9 76.6 ± 4.8 pH 12.3 11.9 - Nordin et al.: Electrochemical Degradation of Reactive Blue 21 290 Acta Chim. Slov. 2019, 66, 284-293 According to Chatzisymeon et al.,31 two mechanisms are involved in oxidation of pollutants by an electrochemical technique, namely direct anodic oxidation and indirect anodic oxidation. For direct anodic oxidation, pollutants are absorbed on anodic surfaces and destroyed by anodic electron transfer reactions. Indirect anodic oxidation involves the use of electrogenerated oxidants, such as Cl2, OCl-, OH-, O3 and H2O2.31,32 The effect of NaCl presence on mineralisation of RB21 and synthetic textile effluent can be explained by referring to the following equations (Equations 7-10). cals on the anode surface (M) (Equation 7) which then oxidized the organic matter (R) (Equation 8). Furthermore, molecular O2 and free Cl2 can be formed by the reaction between H2O and ClOH radicals on the anode surface (Equation 9). Then, the resulting Cl- ions will react with water to form OCl- ions (Equation 10). The presence of large amount of electrogenerated OCl- ions in the bulk electrolysis provided better mineralisation efficiency (COD and BOD5 reduction) as these electrogenerated species are beneficial in the degradation of organic pollutants in both samples.32 H2O + M + Cl-- M[ClOH-] + H+ + 2 eR + M[ClOH-] - M + RO + H+ + Cl- H2O + M[ClOH-] + Cl-- M + O2 + + Cl2 + 3 H+ + 4 e- H2O + Cl-- HOCl + H+ + 2 e- (7) (8) (9) (10) In the presence of NaCl, anodic water discharge resulted in the formation of chlorohydroxyl (ClOH^) radi- 3. 6. Characterisation of Co47.5/C47.5-PVC5 Electrode Figure 6 shows the FESEM micrograph obtained from the morphological study of freshly prepared Co475/ C475-PVC5 composite electrode and after electrolysis of synthetic textile effluent by using 20 V applied voltage and 45 min electrolysis. Figure 6a shows the distribution of Co and C in the electrode can be seen clearly and are mixed quite well. However, the surface of the electrode slightly changes after electrolysis of RB21 solution in Figure 6b. Nordin et al.: Electrochemical Degradation of Reactive Blue 21 ... Acta Chim. Slov. 2019, 66, 284-293 291 This is due to the degradation of the pollutant that occurred on the surface of the electrode. FESEM micrograph in Figure 6d shows a significant change in the surface of the electrode as compared to the freshly prepared electrode in Figure 6c. The surface of the electrode was no longer smooth as compared to the freshly prepared electrode. In addition, the surface of the prepared electrode showed the existence of voids that were not filled by the element and PVC. The presence of voids in the surface was very important as it increased the porosity characteristic of the electrode for a better electrochemical reaction.33 Figure 6c and 6d show the presence of voids in the surface of each electrode before and after electrolysis, respectively. The presence of voids in the electrode surface after electrolysis showed that this electrode can be used for further electrolysis process without reducing its efficiency. TEM micrograph of freshly fabricated Co475/C475-PVC5 powder in Figure 7 indicated that the mixture formed agglomerate of different shapes. The Co particles can be identified due to their almost regular shape while the C was formed in an irregular shape as demonstrated in Figure 7. Figure 7. TEM micrograph of fresh Co47.5/C47.5-PVC5 powder by using 100,000x magnification. The elemental composition of Co475/C475-PVC5 electrode was determined by using the EDX analysis and the results are presented in Table 3. The results obtained proved that the prepared electrode contained C, Co, and Cl. The presence of Cl elements was due to the use of PVC as a binder in the fabricated electrode. The composition of C for both samples (fresh and after electrolysis) was higher as compared to the Co due to the presence of polymeric carbon chain in PVC. Additionally, no significant changes were indicated by the weight percentage of each element in the fresh electrode and after the electrolysis of synthetic textile effluent. The difference in weight percentage may occur if the precipitate resulted after the electrolysis of synthetic textile effluent. The resulting precipitation was due to the corrosion of anode materials. However, no precipitate was obtained after the electrolysis of synthetic textile effluent. It can be concluded that PVC acts as a good binder agent between C and Co elements inside the electrode. The surface area and pore size of fresh Co475/C475-PVC5 powder and after electrolysis of synthetic textile effluent were analysed by using Brunauer-Emmett-Teller (BET) method. The surface area of fresh Co475/C475-PVC5 and after electrolysis was 2.45 m2 g-1 and 6.89 m2 g-1, while the pore size was 12.53 to 9.19 nm, respectively. The increased surface area and reduced pore size of the Co47.5/ C47.5-PVC5 powder after electrolysis were due to the adsorption of compounds on a solid surface. However, no significant difference was shown by the results obtained from the surface area and pore size of Co47.5/C47.5-PVC5 powder (p > 0.05). The results obtained were consistent with the observations made based on the FESEM micrographs. The pore sizes obtained from this study for freshly prepared Co47.5/C47.5-PVC5 and after electrolysis of synthetic textile effluent were referred to A mesopore type, as mentioned by Chen et al.34 4. Conclusions The Co/C-PVC electrode with a composition ratio of 50:50 (known as the Co475/C475-PVC5) was considered to be the best metal/graphite-PVC composite electrode because it gives the highest decolourisation percentage of aqueous RB21. Optimum electrolysis conditions for the decolourisation of aqueous RB21 by using Co475/C475- Table 3. Elemental composition of Co475/C475-PVC5 electrode before and after electrolysis of RB21 solution. ([RB21]o = 50 mg L-1; [NaCl] = 0.5 mol L-1; t = 45 min; E = 20 V). Element Before electrolysis After electrolysis Weight (%) Atomic (%) Weight (%) Atomic (%) C 77.37 93.96 75.25 91.38 Co 19.96 4.94 21.23 6.25 Cl 2.67 1.10 3.52 2.37 Nordin et al.: Electrochemical Degradation of Reactive Blue 21 292 Acta Chim. Slov. 2019, 66, 284-293 PVC5 composite electrode was by using 20 V of applied voltage for 45 min of electrolysis time in the presence of 0.5 mol L-1 NaCl solution as a supporting electrolyte. Under the optimum electrolysis conditions, 99.95% of RB21 decolourisation percentage was achieved. The Co475/C475-PVC5 composite electrode also showed high efficiency in the decolourisation of synthetic textile effluent by using similar optimum electrolysis conditions as mentioned above. This was further confirmed by COD and BOD5 analyses, in which a high percentage of decolourisation was achieved for COD and BOD5 analyses of RB21 solution and synthetic dye effluent. The electrode surface characterisation by using FESEM showed no significant changes in the presence of void on the electrode surface before and after RB21 electrolysis. The presence of voids on the electrode surface after electrolysis showed that this electrode can be used for further electrolysis process without reducing its efficiency. The results obtained for elemental composition by using EDX showed no significant changes in the composition of C, Co, and Cl elements in the prepared electrode before and after electrolysis of RB21 solution. This showed a good bonding between C and Co by using PVC as a binding agent. 5. Aknowledgements The funding from Universiti Sains Malaysia through short-term grants (304-PKIMIA/6315104) and Universiti Teknologi MARA through LESTARI grant (600-IRMI/ MyRA 5/3/LESTARI (075/2017)) are gratefully acknowledged. 6. Conflict of Interest The authors declare no conflict of interest. 7. References 1. A. R. Tehrani-Bagha, K. Holmberg, Materials 2013, 6, 580608. DOI:10.3390/ma6020580 2. N. Ž. Šekuljica, N. Ž. Prlainovic, A. B. Stefanovic, M. G. Žuža, D. Z. Čičkaric, D. Ž. Mijin, Z. D. Kneževic-Jugovic, Sci. World J. 2015, 2015, 1-12. DOI: 10.1155/2015/371625 3. V. Lopez-Grimau, M. C. Gutierrez, Chemosphere 2006, 62, 106-112. DOI:10.1016/j.chemosphere.2005.03.076 4. N. Nordin, S. F. Mohd Amir, M. R. Yusop, M. R. Othman, Acta Chim. Slov. 2015, 62, 642-651. DOI: 10.17344/acsi.2014.1264 5. S. Wijetunga, X. F. Li, C. Jian, J. Hazard. Mater. 2010, 177, 792-798. DOI:10.1016/j.jhazmat.2009.12.103 6. C. R. Holkar, A. J. Jadhav, D. V. Pinjari, N. M. Mahamuni, A. B. Pandit, J. Environ. 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Li, Desalination 2010, 252, 149-156. D0I:10.1016/j.desal.2009.10.010 Povzetek V tej raziskavi smo s tehniko mehanske tvorbe zlitin izdelali kompozitno elektrodo iz kobalta/grafita in polivinilklori-da (Co475/C475-PVC5). Novo elektrodo smo uporabili kot anodo, grafitno paličko pa kot katodo za razbarvanje vodne raztopine C. I. Reactive Blue 21 (RB21) in sintetične tekstilne odpadne vode, ki je vsebovala zmes azo in antrakinonskih barvil. Novoizdelana elektroda je pokazala visoko učinkovitost za razbarvanje RB21 v vodni raztopini (99,95%) in sintetične tekstilne odpadne vode. To smo še nadalje potrdili z visokim deležem zmanjšanja (>75%) KPK in BPK5 pri obeh obdelanih vzorcih. Elementna sestava (C, Co in Cl), določena s FESEM-EDX, ni bila bistveno različna med sveže izdelano elektrodo in po elektrolizi sintetične tekstilne odpadne vode. To je potrdilo visoko mehansko trdnost novoizdelane elektrode in visoko stopnjo vezave med C in Co zaradi uporabe PVC kot veziva. Nordin et al.: Electrochemical Degradation of Reactive Blue 21 DOI: 10.17344/acsi.2018.4714 Acta Chim. Slov. 2019, 66, 294-307 ^creative ^commons Scientific paper Design, Synthesis, Biological Evaluation and Molecular Docking Studies of Some New Sulfonamides Possessing 1,4-Benzodioxane Nucleus Misbah Irshad,1^ Muhammad Athar Abbasi,2 Aziz-Ur-Rehman,2 Qamar Ali,1 Muhammad Aslam,1 Fozia Iram,3 Muhammad Shahid,4 Muhammad Ashraf,5 Muhammad Arif Lodhi6 and Syed Babar Jamal7 1 Department of Chemistry, Division of Science & Technology, University of Education, Lahore-54770, Pakistan. 2 Department of Chemistry, Government College University, Lahore-54000, Pakistan. 3 Department of Chemistry, Lahore College for Women University, Lahore-75500, Pakistan 4 Department of Biochemistry, University of Agriculture, Faisalabad-38040, Pakistan. 5 Department of Pharmacy; The Islamia University of Bahawalpur, Bahawalpur-63100, Pakistan. 6 Department of Biochemistry, Abdul Wali Khan University, Mardan-23200, Pakistan. 7 Department of Bioinformatics, Islamic International University, Islamabad, Pakistan. * Corresponding author: E-mail: misbahchatha@hotmail.com; misbahirshad@ue.edu.pk Tel: (+92)-42-99262232 Received: 09-10-2018 Abstract In the current research work we have reported a series of N-aryl-2,3-dihydrobenzo[1,4]dioxine-6-sulfonamides 3 and their N-substituted derivatives 6 and 7, obtained from 3 with benzyl chloride and ethyl iodide, respectively. The synthesis was accomplished as a multistep sequence. The structural confirmations were established by 'H NMR, IR and EIMS spectral techniques. Butyrylcholinesterase (BChE), acetylcholinesterase (AChE) and lipoxygenase (LOX) enzymes were used in this study. It was observed that most of the compounds prepared exhibit a moderate activity against BChE and AChE but promisingly good activity against lipoxygenase. Among the parent sulfonamides 3a, 3b, 3c and 3e showed the proficient antimicrobial activities, while from the derivatives 6a, 6c, 7a, 7b and 7c were found active against the selected panel of bacterial and fungal species. Hemolytic activity was also conducted to check their therapeutic utility. All the compounds were computationally docked against LOX, BChE and AChE enzymes. Keywords: 2,3-Dihydrobenzo[1,4]dioxine-6-sulfonyl chloride; lipoxygenase enzyme; antimicrobial and hemolytic activities; molecular docking. 1. Introduction The very first prepared antimicrobial agents were sulfonamides, which are bacteriostatic agents and active against Gram negative and Gram positive bacteria. Basically, sulfonamides contain a benzene ring with sulfonyl and amino group at para position and have general formula Ar SO2NHR. The nature of Arl and R varies from simple hydrogen to aliphatic carboaromatic, heterocyclic, OH, NH2 or sugar scaffolds. SAR studies revealed that the liberated NH2 group of sulfonamides is responsible for their activity. Low activity was observed in the case of attached substituents at ortho and meta positions.1-5 Sulfonamides restrain folic acid in bacteria which facilitates the production of bacterial DNA and RNA. Dihydropteroic acid is precursor of folic acid. Dihydropteroate synthase is responsible for the transformation of dihydropteroate di-phosphate and P-aminobenzoic acid into dihydropteroic Irshad et al.: Design, Synthesis, Biological Evaluation Acta Chim. Slov. 2019, 66, 294-307 295 acid; on the other hand, sulfonamides hinder this enzyme.6,7 These sulfonamide drugs can be classified as oral (absorbed and unabsorbed) and topical. Depending on the action absorbable sulfonamides can be short-, intermediate- and long-acting. Their absorption takes place through stomach and small intestine.8 Commonly these sulfa drugs are synthesized by treating substituted sulfonyl chloride with suitable amine compounds. Sulfonamides are used to cure membrane infections, enteritis, ulcerative colitis and for skin treatments. In agriculture sulfonamides have anti-herbicidal and antifungal uses. They have wide applications as antiobesity,9 anticancer, antiviral,3 protease inhibitors,10 diuretic,11,12 hypoglycemic,13 antithyroid,14 antitumor,15-17 anti-neuropathic pain,18 antibacterial,19,20 cyclooxygen-ase21 and carbonic anhydrase inhibitory agents.22 Compounds containing a 1,4-benzodioxane skeleton display wider range of biological activities,23 such as anticancer,24,25 immunosuppressive,26 antiinflammatory,27 antibacterial28 and some others.29,30 Additionally, interaction modes of the synthesized compounds were examined by molecular docking approach. The purpose of docking methodologies was to forecast the ligand and target complex and to align the molecular database (designed inhibitors) on the basis of binding affinity to that of target. The MOE-Dock was used for docking of all the synthesized inhibitors with the binding site of target enzymes. The eventual objective of molecular docking was to get ligands with better characteristics displaying good inhibition potential.31 This research work is a productive effort to bring in close proximity the pharmacologically significant moieties. Keeping continuity in connection with the research work on sulfonamide molecules,32 the different N-substituted sulfonamides derived from 1,4-benzodioxine-6-sulfonyl chloride were synthesized. It was aimed that the new drugs bearing sulfamoyl functionality would have striking activity and be able to cope with the challenges. 2. Experimental 2. 1. General 2,3-Dihydrobenzo[1,4]dioxine-6-sulfonyl chloride, different aryl amines and the other electrophilic reagents were purchased from local suppliers of chemicals. All the solvents were used after distillation and purification. Griffin and George melting point instrument was employed to record melting points of the target compounds. The open capillary tube was used and melting points are reported uncorrected. The TLC was utilized to check the purity and reaction progress. TLC plates were finished from pre-coat-ed silica gel G-25-UV254. The gradient solvent systems of ethyl acetate (C4H8O2) and M-hexane (C6H14) were applied. The detection was carried out at 254 nm and TLC plates were developed by ceric sulphate reagent. On a Jas-co-320-A spectrophotometer the IR spectra were record- ed. The pellet of a sample was made with KBr and functional group stretchings are reported in wave numbers (cm-1). NMR spectra were recorded on Bruker spectrometers. The deutrated methanol and chloroform were used as the solvents. The operating frequencies thus utilized were 300 and 400 MHz and chemical shifts are given in ppm. On a JMS-HX-110 spectrometer the mass spectra (EIMS) were measured with a data system. 2. 2. Synthesis 2. 2. 1. General Procedure for the Synthesis of N-Aryl-2,3-dihydrobenzo[1,4]dioxine-6-sulfonamides (3a-e) in Aqueous Medium 0.85 mmol of various substituted aryl amines 2a-e were dispensed in 50 mL of distilled water contained in a 250 mL round-bottom flask. The pH of the reacting medium was maintained at 9.0-10.0. The aqueous solution of Na2CO3 was added drop wise at 25 oC. Subsequently 0.85 mmol (0.20 g) of 2,3-dihydrobenzo[1,4]dioxine-6-sulfonyl chloride (1) was added slowly to the reaction mass over 10-15 min. The reaction mixture was kept on stirring at RT till the TLC confirmation indicated the completion of the reaction. The workup was done by slowly adding conc. aq. HCl (2 mL) and dropping pH to 2.0. The title compounds 3a-e were collected as solid precipitates on filtration and washed with immense volume of distilled water. N-(2,3-Dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3a) White solid; yield: 80% (217.7 mg); m.p. 102-104 oC; molecular formula: C16H17NO4S; molecular weight: 319 g/ mol; HR-MS: [M]+ 319.3772 (Calcd. for C16H17NO4S; 319.3854); IR (KBr, cm-1): vmax stretching: 3419 (N-H), 3026 (C-H Ar ring), 2914 (CH2), 1613 (C=C Ar ring), 1325 (SO2),1125 (C-O-C of ether); 1H NMR (400 MHz,C-DCl3): 5 (ppm) 1.99 (s, 3H, CH3-2"), 2.27 (s, 3H, CH3-1"), 4.24-4.27 (m, 4H, CH2-2, CH2-3), 6.73 (brd, J = 7.6 Hz, 1H, H-4'), 6.78 (t, J = 7.6 Hz, 1H, H-5'), 6.80 (d, J = 8.0 Hz, 1H, H-6'), 6.95 (d, J = 8.4 Hz, 1H, H-8), 7.18 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, H-7), 7.25 (d, J = 2.0 Hz, 1H, H-5); EIMS: m/z 319 [M]+, 255 [M-SO2]+, 214 [C6H3C2H4O-2SO2NH]+, 199 [C6H3C2H4O2SO2]+, 135 [C^C^O^, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C^C^^, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HCH^+. N-(2,4-Dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3b) White solid; yield: 89% (242.2 mg); m.p. 108-110 oC; molecular formula: C16H17NO4S; molecular weight: 319 g/ mol; HR-MS: [M]+ 319.3772 (Calcd. for C16H17NO4S; 319.3854); IR (KBr, cm-1): vmax stretching: 3435 (N-H), 3021 (C-H Ar ring), 2917 (-CH2-), 1615 (C=C Ar ring), 1324 (-SO2-), 1140 (C-O-C of ether); 1H NMR (400 MHz, CDCl3): 5 (ppm) 1.99 (s, 3H, CH3-2"), 2.24 (s, 3H, CH3-1"), 4.24-4.26 (m, 4H, CH2-2, CH2-3), 6.80 (s, 1H, H-3'), Irshad et al.: Design, Synthesis, Biological Evaluation 3Q6 Acta Chim. Slov. 2019, 66, 294-307 6.84 (d, J = 8.0 Hz, 1H, H-5'), 6.92 (d, J = 8.4 Hz, 1H, H-6'), 7.12 (d, J = 8.0 Hz, 1H, H-8), 7.17 (dd, J1= 8.4 Hz, J2 = 2.0 Hz, 1H, H-7), 7.26 (d, J = 2.0 Hz, 1H, H-5); EIMS: m/z 319 [M]+, 255 [M-SO2]+, 214 [C6H3C2H4O2SO2NH]+, 199 [C6H3C2H4O2SO2]+, 135 [C^C^O^, 107 [C^O^, 105 [C^CH^^, 90 [C^CH^, 79 ^(CH^^, 75 [0^]+, 64 [C4HCH3]+. N-(2,5-Dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3c) White solid; yield: 82% (223.1 mg); m.p. 124-126 oC; molecular formula: C16H17NO4S; molecular weight: 319 g/ mol; HR-MS: [M]+ 319.3772 (Calcd. for C16H17NO4S; 319.3854); IR (KBr, cm-1): vmax stretching: 3415 (N-H), 3024 (C-H Ar ring), 2912 (-CH2-), 1619 (C=C Ar ring), 1325 (-SO2-), 1122 (C-O-C of ether); 1H NMR (300 MHz, CD3OD): 5 (ppm) 1.96 (s, 3H, CH3-2''), 2.20 (s, 3H, CH3-1''), 4.22-4.30 (m, 4H, CH2-2, CH2-3), 6.85 (d, J = 7.6 Hz, 1H, H-4'), 6.89 (d, J = 7.5 Hz, 1H, H-3'), 6.91 (s, 1H, H-6'), 6.99 (d, J = 8.1 Hz, 1H, H-8), 7.09 (d, J = 2.1 Hz, 1H, H-5), 7.13 (dd, J1 = 7.8 Hz, J2 = 2.1 Hz, 1H, H-7); EIMS: m/z 319 [M]+, 255 [M-SO2]+, 214 [C6H3C2H4O2SO2NH]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C^CH^, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HCH3]+. N-(2,6-Dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3d) White solid; yield: 71% (193.2 mg); m.p. 151-153 oC; molecular formula: C16H17NO4S; molecular weight: 319 g/mol; HR-MS: [M]+ 319.3772 (Calcd. for C16H-17NO4S; 319.3854); IR (KBr, cm-1): vmax stretching: 3410 (N-H), 3045 (C-H Ar ring), 2909 (-CH2-), 1614 (C=C), 1326 (-SO2-), 1125 (C-O-C of ether); 1H NMR (300 MHz, CDCl3): 5 (ppm) 2.02 (s, 6H, CH3-1'', CH3-2''), 4.25-4.30 (m, 4H, CH2-2, CH2-3), 6.93 (brd, J = 7.8 Hz, 1H, H-4'), 7.01 (d, J = 8.4 Hz, 2H, H-3, H-5'), 7.03 (d, J = 8.4 Hz, 1H, H-8), 7.38 (d, J = 2.4 Hz, 1H, H-5), 7.48 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H, H-7); EIMS: m/z 319 [M]+, 255 [M-SO2]+, 214 [C6H3C2H4O2SO2NH]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C^CH^^, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [QH^, 64 [C4HCH3]+. N-(3,4-Dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3e) White solid; yield: 86% (234.0 mg); m.p. 124-126 oC; molecular formula: C16H17NO4S; molecular weight: 319 g/ mol; HR-MS: [M]+ 319.3772 (Calcd. for C16H17NO4S; 319.3854); IR (KBr, cm-1): vmax stretching: 3422 (N-H), 3018 (C-H Ar ring), 2919 (-CH2-), 1615 (C=C Ar ring), 1323 (-SO2), 1118 (C-O-C of ether); 1H NMR (400 MHz,CDCl3): 5 (ppm) 2.15 (s, 6H, CH3-1'', CH3-2''), 4.234.25 (m, 4H, CH2-2, CH2-3), 6.76 (d, J = 7.6 Hz, 1H, H-5'), 6.81 (s, 1H, H-2'), 6.84 (d, J = 8.0 Hz, 1H, H-6'), 6.96 (d, J = 8.0 Hz, 1H, H-8), 7.22 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, H-7), 7.28 (d, J = 2.0 Hz, 1H, H-5); EIMS: m/z 319 [M]+, 255 [M-SO2]+, 214 [C6H3C2H4O2SO2NH]+, 199 [C6H-3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C^CH^, 79 ^(CH^^, 75 [C6H3]+, 64 [C4HCH3]+. 2. 2. 2. General Procedure for the Synthesis of Compounds 6a-e and 7a-e 0.40 mmol (0.01 g) of lithum hydride was added to a solution containing of 0.1 g of compounds 3a-e in 25 mL of aprotic solvent (DMF) and kept in round-bottom flask at 25 °C. On complete addition, the reaction mixture was stirred for half an hour. The benzyl chloride (4) and ethyl iodide (5) were added into the reaction mixture to establish 6a-e and 7a-e series, respectively. The stirring lasted for 1-2 h. The monitoring of the reaction completion was done by TLC. The reaction contents were quenched with cold distilled water after ensuring the complete conversion of the reactants. The corresponding N-benzyl/ethyl derivatives of N-aryl-2,3-dihydrobenzo[1,4]dioxine-6-sulfon-amides 6a-e and 7a-e were obtained as solid precipitates on filtration. The subsequent washing with distilled water and drying yielded our target molecules. The greasy and sticky compounds were isolated through solvent extraction by using chloroform. N-Benzyl-.N-(2,3-dimethylphenyl)-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (6a) Yellow sticky solid; yield: 86% (110.3 mg); molecular formula: C23H23NO4S; molecular weight: 409 g/mol; HR-MS: [M]+ 409.4991 (Calcd. for C23H23NO4S; 409.5187); IR (KBr, cm-1): vmax stretching: 3416 (N-H), 3015 (C-H Ar ring), 2918 (-CH2-), 1617 (C=C Ar ring), 1329 (-SO2-), 1135 (C-O-C of ether); 1H NMR (400 MHz, CD3OD): 5 (ppm) 2.00 (s, 3H, CH3-2'''), 2.06 (s, 3H, CH3-1'''), 4.254.29 (m, 4H, CH2-2, CH2-3), 4.63 (s, 2H, CH2-7''), 6.81 (d, J = 7.6 Hz, 1H, H-4'), 6.87-6.95 (m, 5H, H-2'' to H-6''), 7.02 (d, J = 7.2 Hz, 1H, H-6'), 7.10 (t, J = 7.6 Hz, 1H, H-5'), 7.31 (d, J = 8.4 Hz, 1H, H-8), 7.34 (d, J = 2.0 Hz, 1H, H-5), 7.38 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, H-7); EIMS: m/z 409 [M]+, 345 [M-SO2]+, 304 [C6H3C2H4O2SO2NCH2C6H5]+, 199 [C6H3C2H4O2SO2]+, 135 [C^C^O^, 107 [C6H3O2]+,105 [C6H3(CH3)2]+, 91 [C7H7]+, 90 [QH3 CH3]+, 79 [C4H(CH3)2]+, 75 [C^, 65 ^5]+, 64 [C4HCH3]+. N-Benzyl-.N-(2,4-dimethylphenyl)-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (6b) Light grey sticky solid; yield: 78% (100.0 mg); molecular formula: C23H23NO4S; molecular weight: 409 g/mol; HR-MS: [M]+ 409.4991 (Calcd. for C23H23NO4S; 409.5187); IR (KBr, cm-1): vmax stretching: 3423 (N-H), 3067 (C-H Ar ring), 2932 (-CH2-), 1617 (C=C Ar ring), 1326 (-SO2), 1145 (C-O-C of ether); 1H NMR (300 MHz,CD3OD): 5 (ppm) 1.90 (s, 3H, CH3-2'''), 2.22 (s, 3H, Irshad et al.: Design, Synthesis, Biological Evaluation ... Acta Chim. Slov. 2019, 66, 294-307 297 CH3-1"'), 4.19 (s, 2H, CH2-7"), 4.32-4.34 (m, 4H, CH2-2, CH2-3), 6.55 (d, / = 8.1 Hz, 1H, H-5'), 6.86 (d, J = 7.8 Hz, 1H, H-6'), 6.90 (s, 1H, H-3'), 6.92-7.01 (m, 5H, H-2'' to H-6''), 7.10 (d, J = 8.1 Hz, 1H, H-8), 7.17 (d, J = 2.1 Hz, 1H, H-5), 7.19 (dd, J1 = 8.1 Hz, J2 = 2.1Hz, 1H, H-7); EIMS: m/z 409 [M]+, 345 [M-SO2]+, 304 [C6H3C2H4O2SO2N-CH2C6H5]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+,105 [C6H3(CH3)2]+, 91 [^7]+, 90 [C^CH^, 79 [C4H(CH3)2]+, 75 [C6H3]+, 65 ^5]+, 64 [C4HCH3]+. N-Benzyl-.N-(2,5-dimethylphenyl)-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (6c) Yellow sticky solid; yield: 76% (97.4 mg); molecular formula: C23H23NO4S; molecular weight: 409 g/mol; HR-MS: [M]+ 409.4991 (Calcd. for C23H23NO4S; 409.5187); IR (KBr, cm-1): vmax stretching: 3431 (N-H), 3041 (C-H Ar ring), 2922 (-CH2-), 1612 (C=C Ar ring), 1328 (-SO2-), 1131 (C-O-C of ether); 1H NMR (300 MHz, CD3OD): 5 (ppm) 1.89 (s, 3H, CH3-2'''), 2.14 (s, 3H, CH3-1'"), 4.304.36 (m, 4H, CH2-2, CH2-3), 4.56 (s, 2H, CH2-7''), 6.95 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H, H-4'), 7.00 (d, J = 8.4 Hz, 1H, H-3'), 7.03 (s, 1H, H-6'), 7.09-7.20 (m, 5H, H-2'' to H-6''), 7.56 (d, J = 8.4 Hz, 1H, H-8), 7.62 (d, J = 2.1 Hz, 1H, H-5), 7.71 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H, H-7); EIMS: m/z 409 [M]+, 345 [M-SO2]+, 304 [C6H3C2H4O2SO2N-CH2C6Hs]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+,105 [C6H3(CH3)2]+, 91 [C7H7]+, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [QH^, 65 ^5]+, 64 [C4HCH3]+. N-Benzyl-.N-(2,6-dimethylphenyl)-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (6d) Creamy white crystalline solid; yield: 72% (92.3 mg); m.p. 156-158 oC; molecular formula: C23H23NO4S; molecular weight: 409 g/mol; HR-MS: [M]+ 409.4991 (Calcd. for C23H23NO4S; 409.5187); IR (KBr, cm-1): vmax stretching: 3422 (N-H), 3013 (C-H Ar ring), 2927 (-CH2-), 1643 (C=C Ar ring), 1325 (-SO2-), 1126 (C-O-C of ether); 1H NMR (400 MHz, CD3OD): 5 (ppm) 2.68 (s, 6H, CH3-1''', CH3-2'''), 4.23-4.33 (m, 4H, CH2-2, CH2-3), 4.61 (s, 2H, CH2-7''), 6.14-6.94 (m, 5H, H-2'' to H-6''), 6.96 (brd, J = 8.8 Hz, 2H, H-3', H-5'), 7.01 (brd, J = 8.8 Hz, 1H, H-4'), 7.27 (d, J = 8.0 Hz, 1H, H-8), 7.32 (d, J = 2.0 Hz, 1H, H-5), 7.58 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, H-7), EIMS: m/z 409 [M]+, 345 [M-SO2]+, 304 [C6H3C2H4O2SO2NCH2C6H5]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+,105 [C6H3(CH3)2]+, 91 [C7H7]+, 90 [C6H3 CH3]+, 79 [C4H(CH3)2]+, 75 [C6H3]+, 65 [C5H5]+, 64 [C4HCH3]+. N-Benzyl-.N-(3,4-dimethylphenyl)-2,3-dihydroben-zo[1,4]dioxine-6-sulfonamide (6e) White solid; yield: 90% (115.4 mg); m.p. 118-120 oC; molecular formula: C23H23NO4S; molecular weight: 409 g/ mol; HR-MS: [M]+ 409.4991 (Calcd. for C23H23NO4S; 409.5187); IR (KBr, cm-1): vmax stretching: 3416 (N-H), 3055 (C-H Ar ring), 2943 (-CH2-), 1622 (C=C Ar ring), 1322 (-SO2), 1134 (C-O-C of ether); 1H NMR (400 MHz,CDCl3): 5 (ppm) 2.10 (s, 3H, CH3-2'''), 2.14 (s, 3H, CH3-1'''), 4.28-4.31 (m, 4H, CH2-2, CH2-3), 4.65 (s, 2H, CH2-7''), 6.66 (d, J = 7.2 Hz, 1H, H-5'), 6.78 (s, 1H, H-2'), 6.81 (d, J = 7.2 Hz, 1H, H-6'), 6.88-6.90 (m, 5H, H-2'' to H-6''), 6.92 (d, J = 8.4 Hz, 1H, H-8), 7.12 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H, H-7), 7.21 (d, J = 2.0 Hz, 1H, H-5); EIMS: m/z 409 [M]+, 345 [M-SO2]+, 304 [C6H3C2H4O2SO2N-CH2C6HS]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+,105 [C6H3(CH3)2]+, 91 [C7H7]+, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [CgH^, 65 [C5HJ+, 64 [C4HCH3]+. N-(2,3-Dimethylphenyl)-.N-ethyl-2,3-dihydroben-zo[1,4]dioxine-6-sulfonamide (7a) Yellow sticky solid; yield: 82 % (89.2 mg); molecular formula: C18H21NO4S; molecular weight: 347 g/mol; HR-MS: [M]+ 347.4302 (Calcd. for C18H21NO4S;347.4587); IR (KBr, cm-1): vmax stretching: 3435 (N-H), 3032 (C-H Ar ring), 2929 (-CH2-), 1625 (C=C Ar ring), 1320 (-SO2), 1123 (C-O-C of ether); 1H NMR (400 MHz, CD3OD): 5 (ppm) 0.96 (t, J = 7.2 Hz, 3H, CH3-2''), 2.00 (s, 3H, CH3-2'''), 2.06 (s, 3H, CH3-1'''), 3.63 (q, J = 7.2 Hz, 2H, CH2-1''), 4.20-4.29 (m, 4H, CH2-2, CH2-3), 6.81 (d, J = 7.6 Hz, 1H, H-4'), 6.93 (t, J = 7.6 Hz, 1H, H-5'), 7.02 (d, J = 7.2 Hz, 1H, H-6'), 7.21 (d, J = 8.0 Hz, 1H, H-8), 7.63 (d, J = 2.4 Hz, 1H, H-5), 7.75 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H, H-7); EIMS: m/z 347 [M]+, 283 [M-SO2]+, 268 [C6H3C2H4O2NCH-2C6H3(CH3)2]+, 242 [C6H3C2H4O2SO2NC2H5]+, 240 [C6H3O2NCH2C6H3(CH3)2]+, 227 [C6H3C2H4O2SO2 NCH2]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HCH3]+. N-(2,4-Dimethylphenyl)-.N-ethyl-2,3-dihydroben-zo[1,4]dioxine-6-sulfonamide (7b) Greenish brown sticky solid; yield: 73% (79.4 mg); molecular formula: C18H21NO4S; molecular weight: 347 g/ mol; HR-MS: [M]+ 347.4302 (Calcd. for C18H21NO4S; 347.4587); IR (KBr, cm-1): vmax stretching: 3430 (N-H), 3044 (C-H Ar ring), 2918 (-CH2-), 1629 (C=C Ar ring), 1328 (-SO2-), 1120 (C-O-C of ether); 1H NMR (400 MHz, CD3OD): 5 (ppm) 1.00 (t, J = 7.2 Hz, 2H, CH3-2''), 2.20 (s, 3H, CH3-2'''), 2.27 (s, 3H, CH3-1'''), 3.77 (q, J = 7.6 Hz, 2H, CH2-1''), 4.28-4.33 (m, 4H, CH2-2, CH2-3), 6.48 (s, 1H, H-3'), 6.50 (d, J = 7.6Hz, 1H, H-5'), 6.90 (d, J =7.6 Hz, 1H, H-6'), 6.97 (d, J = 8.0 Hz, 1H, H-8), 7.10 (d, J = 2.4 Hz, 1H, H-5), 7.12 (dd, J1 = 8.2 Hz, J2 = 2.0 Hz, 1H, H-7); EIMS: m/z 347 [M]+, 283 [M-SO2]+, 268 [C6H3C2H4O2NCH-2C6H3(CH3)2]+, 242 [C6H3C2H4O2SO2NC2H5]+, 240 [C6H3O2NCH2C6H3(CH3)2]+, 227 [C6H3C2H4O2SO2 NCH2]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HCH3]+. Irshad et al.: Design, Synthesis, Biological Evaluation 3Q6 Acta Chim. Slov. 2019, 66, 294-307 N-(2,5-Dimethylphenyl)-.N-ethyl-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (7c) Light yellow sticky solid; yield: 69% (75.1 mg); molecular formula: C18H21NO4S; molecular weight: 347 g/ mol; HR-MS: [M]+ 347.4302 (Calcd. for C18H21NO4S; 347.4587); IR (KBr, cm-1): vmax stretching: 3438 (N-H), 3021 (C-H Ar ring), 2927 (-CH2-), 1618 (C=C Ar ring), 1326 (-SO2-), 1129 (C-O-C of ether); 1H NMR (500 MHz, CD3OD): 5 (ppm) 0.99 (t, J =7.0 Hz, 3H, CH3-2''); 2.16 (s, 3H, CH3-2"'), 2.27 (s, 3H, CH3-1'''), 3.60 (q, J = 7.5 Hz, 2H, CH2-1''), 4.28-4.34 (m, 4H, CH2-2, CH2-3), 6.39 (s, 1H, H-6'), 6.99 (d, J = 8.5 Hz, 1H, H-3'), 7.04 (dd, J = 2.0, 8.0 Hz, 1H, H-4'), 7.09 (d, J = 2.5 Hz, 1H, H-5), 7.13 (dd, J1 = 8.5 Hz, J2 = 2.0 Hz, 1H, H-7), 7.16 (d, J = 7.5 Hz, 1H, H-8); EIMS: m/z 347 [M]+, 283 [M-SO2]+, 268 [C6H3C2H4O2N-CH2C6H3(CH3)2]+, 242 [C6H3C2H4O2SO2NC2H5]+, 240 [C6H3O2NCH2C6H3(CH3)2]+, 227 [C^C^O.SO, NCH2]+, 199 [C6H3C2H4O2SO2]+, 135 [C^C^O^, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HC^]+. N-(2,6-Dimethylphenyl)-.N-ethyl-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (7d) White solid; yield: 78% (84.9 mg); m.p; 109-111 oC; molecular formula: C18H21NO4S; molecular weight: 347 g/ mol; HR-MS: [M]+ 347.4302 (Calcd. for C18H21NO4S; 347.4587); IR (KBr, cm-1): vmax stretching: 3445 (N-H), 3017 (C-H Ar ring), 2921 (-CH2-), 1612 (C=C Ar ring), 1325 (-SO2), 1134 (C-O-C of ether); 1H NMR (300 MHz, CD3OD): 5 (ppm) 1.07 (t, J = 7.2 Hz, 3H, CH3-2''), 2.06 (s, 6H, CH3-1''', CH3-2"'), 3.56 (q, J = 7.2 Hz, 2H, CH2-1"), 4.28-4.32 (m, 4H, CH2-2, CH2-3), 6.99 (d, J = 8.4 Hz, 2H, H-3, H-5'), 7.02 (d, J = 8.4 Hz, 1H, H-4'), 7.06 (d, J = 8.4 Hz, 1H, H-8), 7.12 (d, J = 2.1 Hz, 1H, H-5), 7.24 (dd, J1 = 8.4 Hz, J2 = 2.1 Hz, 1H, H-7); EIMS: m/z 347 [M]+, 283 [M-SO2]+, 268 [C6H3C2H4O2NCH2C6H3(CH3)2]+, 242 [C6H3C2H4O2SO2NC2H5]+, 240 [C^O.NCH.QH (CH3)2]+, 227 [C6H3C2H4O2SO2NCH2]+, 199 [C6H-3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C6H3(CH3)2]+, 90 [C^CH^, 79 ^(CH^^, 75 [C6H3]+, 64 [C4HCH3]+. N-(3,4-Dimethylphenyl)-.N-ethyl-2,3-dihydrobenzo [1,4]dioxine-6-sulfonamide (7e) Yellowish brown sticky solid; yield: 86% (93.6 mg); molecular formula: C18H21NO4S; molecular weight: 347 g/ mol; HR-MS: [M]+ 347.4302 (Calcd. for C18H21NO4S; 347.4587); IR (KBr, cm-1): vmax stretching: 3432 (N-H), 3017 (C-H Ar ring), 2923 (-CH2-), 1612 (C=C Ar ring), 1325 (-SO2-), 1134 (C-O-C of ether); 1H NMR (400 MHz, CD3OD): 5 (ppm) 1.02 (t, J = 7.2 Hz, 3H, CH3-2''), 2.19 (s, 3H, CH3-2"'), 2.24 (s, 3H, CH3-1'''), 3.55 (q, J = 7.2 Hz, 2H, CH2-1''), 4.26-4.36 (m, 4H, CH2-2, CH2-3), 6.62 (d, J = 8.4 Hz, 1H, H-5'), 6.71 (dd, J1 = 8.0 Hz, J2 = 2.0 Hz, 1H, H-6'), 6.81 (d, J = 2.0 Hz, 1H, H-2'), 6.94 (d, J = 8.4 Hz, 1H, H-8), 7.01 (d, J = 2.0 Hz, 1H, H-5), 7.06 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 1H, H-7); EIMS: m/z 347 [M]+, 283 [M-SO2]+, 268 [C6H3C2H4O2NCH2C6H3(CH3)2]+, 242 [C^C^O-2SO2NC2HS]+, 240 [C6H3O2NCH2 Q^CH^^, 227 [C6H3C2H4O2SO2NCH2]+, 199 [C6H3C2H4O2SO2]+, 135 [C6H3C2H4O2]+, 107 [C6H3O2]+, 105 [C^CH^^, 90 [C6H3CH3]+, 79 [C4H(CH3)2]+, 75 [C6H3]+, 64 [C4HCH3]+. 2. 3. Enzyme Inhibition Studies 2. 3. 1. Cholinesterase Assay The BChE and AChE inhibition activities were assayed in concurrence with the reported method after making minor changes.33 Overall total volume of 100 ^L was pertained to reaction mixture; made by mixing 60 ^L of Na2HPO4 buffer (pH 7.7), 10 ^L of each test sample and the enzyme. The sample and buffer strength was kept at 0.5 mM per well. BChE was added 0.5 unit per well and a quantity of 0.005 units was made constant for AChE in each well. After mixing the contents, the reading was recorded at 405 nm. Before this, incubation of 10 min at 37 °C was ensured. On adding 10 ^L of both substrate (0.5 mM per well) and DTNB (0.5 mM per well) the reaction was allowed to start. The substrates for BChE and AChE were butyrylthio-choline chloride and acetylthiocholine iodide, respectively. Again the incubation was done for 15 min at 37 °C. Later on, the absorbance was measured at 405 nm. The instrument used was 96-well plate reader Synergy HT, Biotek, USA. 0.5 mM per well of Eserine was exploited as the positive control. The experiments were carried out in triplicate. The % inhibition was calculated as: (1) Here, total enzyme activity without inhibitor is described as control, whereas test corresponds to its activity in the presence of our synthesized molecules. EZ-Fit Enzyme Kinetics software was employed to determine IC50 values. The software was provided by Perrella Scientific Inc. Amherst, USA. 2. 3. 2. Lipoxygenase Assay For lipoxygenase assay the total volume of the mixture was maintained at 200 ^L. It contained 150 ^L of 100 mM Na3PO4 buffer having pH 8.0, 10 ^L of 0.5 mM per well of the test compound and 15 ^L of the enzyme. The lipoxygenase was added in 600 units in each well. After mixing and pre-incubation (10 min; 25 °C) the contents were pre-read at 234 nm. On adding 25 ^L of substrate solution the reaction was initiated. The absorbance was measured at 234 nm using 96-well plate reader Synergy HT, Biotek, USA. Baicalein was used as the positive control having 0.5 mM per well in the assay. The % inhibition and rest of calculation was executed by the same method as described above.34-36 Irshad et al.: Design, Synthesis, Biological Evaluation ... Acta Chim. Slov. 2019, 66, 294-307 299 2. 3. 3. Statistical Analysis All experimental measurements were recorded three folds. The statistical analysis was achieved by Microsoft Excel 2010. 2. 4. Antimicrobial Activity 2. 4. 1. Microbial Strains A set of microorganisms were used to conduct antimicrobial activity. All the synthesized compounds were tested against fungal, Gram negative and Gram positive bacterial strains. In this study Bacillus subtilis (B. subtilis) JS 2004 and Staphylococcus aureus (S. aureus) API Staph TAC 6736152 were used as Gram positive bacteria, whereas Pasteurella multocida (P. multocida) and Escherichia coli (E. coli) ATCC 25922 were among Gram negative bacteria. The four pathogenic fungi: Microsporum canis (M. canis), Candida albicans (C. albican), Aspergillus flavus (A. flavus) and Fusarium solani (F. solani) were also the constituents of the assay. All the pure strains were provided by CMS department of UAF, Faisalabad. The Department of microbiology checked their purity and confirmed their identification. In nutrient agar (NA, Oxoid) bacterial strains were cultured overnight at 37 °C. For fungal strains Potato Dextrose Agar (PDA, Oxoid) was chosen and subjected to culture at 28 °C overnight.37 2. 4. 2. Disc Diffusion Method Disc diffusion method was used to determine antimicrobial activity of synthesized compounds. Suspension of tested microorganisms (100 ^L) comprised 107 colony-forming units (CFU)/mL of bacterial cells. They were expanded on NA medium. The suspension with 106 spores/ mL of fungi were expended on PDA medium. The solution of compound was applied to saturate filter discs. Sample free discs were used for negative control. For the comparison of sensitivity of strain/isolate in the analyzed microbial species, positive reference used for fungi and bacteria were Flumequine (30 ^g/disk) (Oxoid, UK) and Amoxycillin (30 ^g/dish) (Oxoid, UK), respectively. After keeping it at 4 °C for 2 hours, plates were incubated for 24 hours at 28 °C for fungal strains and for 18 hours at 37 °C for bacteria. Antimicrobial activity was assessed by calculating the diameter (mm of growth) of inhibition zones using zone reader. Later on it was compared with the controls.37 2. 4. 3. Hemolytic Activity To study the hemolytic activity of the compounds 3 mL of fresh blood of heparinized human was collected. After consent the bovine from volunteers of the Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan was bleeded. Centrifugation of blood was done at 1000 x g for 5 min. Plasma was discarded. 5 mL of chilled (4 °C) sterile isotonic Phosphate Buff- ered Saline (PBS) with pH 7.4 was used to wash the cells. This task was performed for three times. For each assay erythrocytes were maintained 108 cells per mL. 100 ^L of each compound was taken and mixed with 108 cells/mL of human separately. The incubation of samples was done at 37 oC for 35 min. After incubation these samples were settled for 10 min and later on kept on agitation. The samples were placed on ice for 5 min immediately after incubation then centrifuged at 1000 x g for 5 min. From each tube 100 ^L of supernatant was taken and then diluted with chilled (4 °C) PBS 10 times. As the positive control, Triton X-100 (0.1% v/v) was used; while phosphate buffer saline (PBS) acted as the negative control. The ^Quant's life science instrument licensed by Biotek, USA was used to record the absorbance. The absorbance was taken at 576 nm. The % RBCs lysis for each sample was measured.38,39 2. 5. Molecular Docking The structures of all the synthesized inhibitors were constructed using MOE-Builder tool. The default parameters of MOE-Dock program were used for the molecular docking of the ligands. Ligands were allowed to be flexible in order to find the accurate conformations of the ligands and to obtain minimum energy structures. At the end of docking, the best conformations of the ligands were analyzed for their binding interactions.40 3. Results and Discussion 3. 1. Chemistry We report herein the synthesis of a series of hetero-cyclic compounds containing 1,4-benzodioxane nucleus. The precursor molecules N-aryl-2,3-dihydrobenzo[1,4] dioxine-6-sulfonamide 3a-e, were prepared by condensing 1,4-benzodioxane-6-sulfonyl chloride (1) and aryl amines 2a-e in basic aqueous medium. Two series of N-substituted derivatives (6a-e and 7a-e) were formed by the reactions of 3a-e with two different electrophiles. The substitution reactions yielded N-benzyl (6a-e) or N-ethyl (7a-e) derivatives of N-aryl-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamides as represented in Scheme 1. The derivatives 6a-e and 7a-e were obtained in DMF as the solvent and with LiH as the base and activator. The reaction products were obtained within 30 to 70 min of stirring at room temperature. By adding cold distilled water along with vigorous shaking of the reaction mixtures, the products were precipitated; isolation through filtration yielded pure solid targets. On the other hand, some sticky and greasy compounds were recovered through solvent extraction method using chloroform as the extracting solvent. Here the description of the parent compound 3a is given as an example for elaborating the spectral data. 3a was synthesized as a white powder with good yield (80%) and m.p. was recorded as 102-104 °C. High Resolution Mass Spectrome- Irshad et al.: Design, Synthesis, Biological Evaluation 3Q6 Acta Chim. Slov. 2019, 66, 294-307 try (HR-MS) showed M'+ (molecular ion peak) at m/z 319.3772. Therefore, molecular formula of the compound was established as C16H17NO4S. The total number of pro- tons was confirmed from JH NMR spectrum. The IR spectrum of 3a confirmed the presence of different functionalities. Like the absorption bands were observed at 3419 Reagents and conditions: (I) Na2CO3 (aq), pH 9-10, stir, 3-4h, rt, (II) C6H5CH2Cl (4), DMF, LiH, stir, 4-5 h, rt (III) CH3CH2I (5), DMF, LiH, stir, 4-5 h, rt Scheme 1. Synthetic scheme and sulfonamide compounds bearing benzodioxane nucleus. Irshad et al.: Design, Synthesis, Biological Evaluation ... Acta Chim. Slov. 2019, 66, 294-307 301 cm-1 for N-H stretching of sulfamoyl group and at 3026 cm-1 for aromatic C-H stretching. A peak at 1613 cm-1 depicted the presence of C=C stretching of Arl ring. A stretching band at 1325 cm-1 confirmed the SO2 stretching of sulfonyl group in the molecule. A characteristic band at 1125 cm-1 was assigned to C-O-C stretching of ether, respectively. The EI-MS gave characteristic peaks at m/z 199 and 90. These were attributed to the formation of Figure 1a. 1H NMR spectrum of N-(2)5-dimethylphenyl}-N-ethyl-2)3-dihydrobenzo[1)4]dioxine-6-sulfonamide 7c (aromatic region) Irshad et al.: Design, Synthesis, Biological Evaluation 3Q6 Acta Chim. Slov. 2019, 66, 294-307 Figure 1b. 1H NMR spectrum of N-(2,5-dimethylphenyl)-N-ethyl-2,3-dihydrobenzo[1,4]dioxine-6-sulfonamide 7c (aliphatic region) Irshad et al.: Design, Synthesis, Biological Evaluation ... Acta Chim. Slov. 2019, 66, 294-307 303 C6H3C2H4O2SO2+ and QH^CH^ cations, respectively. In the aromatic region of the :H NMR spectrum signals appeared at 6 7.25 as doublet (small coupling constant) confirming H-5, doublet of doublet at chemical shift of 7.18 showed presence of H-7 and another doublet at 6 6.95 (large coupling constant) indicated H-8 of phenyl ring attached to the sulfonyl group. Whereas three aromatic signals at 6 6.80 (brd), 6.78 (t) and 6.73 (brd) were assigned to the benzene ring of 2,3-dimethylphenyl group. In the shielded, upfield and aliphatic region of the spectrum three distinct signals appeared. The multiplet ranging from 4.244.27 (CH2-2, CH2-3), singlet at 2.27 (CH3-I'') and relatively higher upfield singlet at 1.99 (CH3-2") indicated the presence of 1,4-dioxane nucleus and two methyl groups attached to the second and third position of aniline in the molecule. The structure of 3a was established as (2,3-di- Figure 2. Mass fragmentation pattern of N-aryl-2)3-dihydrobenzo[1)4]dioxine-6-sulfonamides 3a-e Figure 3. Mass fragmentation pattern of N-ethyl-N-(dimethylphenyl}-2)3-dihydrobenzo[1)4]dioxine-6-sulfonamide 7a-e Irshad et al.: Design, Synthesis, Biological Evaluation 3Q6 Acta Chim. Slov. 2019, 66, 294-307 methylphenyl)-2,3-dihydrobenzo[1,4]dioxine-6-sul-fonamide on these collective confirmations. The structures of other derivatives were also ascertained on the similar pattern. The 1H NMR spectrum of molecule 7c is shown in Figure 1. The mass fragmentation pattern of parent sulfonamides 3a-e and their N-ethyl derivatives 7a-e are provided in Figures 2 and 3. 3. 2. Enzyme Inhibition The screening of all the synthesized compounds against butyrylcholinesterase enzyme revealed that only three compounds showed better activity; N-(2,3-dimethylphenyl)-2,3-dihydrobenzo[1,4]diox-ine-6-sulfonamide (3a), N-benzyl-(2,5-dimethyl-phenyl)-2,3-dihydro- benzo[1,4]dioxine-6-sulfon-amide (6c) and N-ethyl-(2,4-dimethylphenyl)-2,3 -dihydro- benzo[1,4]dioxine-6-sulfonamide (7b) having IC50 values of 374.11 ± 0.01, 387.51 ± 0.48 and 353.13 ± 0.86 ^mol/L respectively, relative to eserine, a reference standard with IC50 value of 0.85 ± 0.0001 ^mol/L (Table 1). The activity of these compounds was most probably due to the presence of methyl groups at different positions of phenyl ring in 3a, benzyl group in 6c along with alkyl groups and an additional ethyl group for 7b attached to the nitrogen of sulfonamide. The screening against acetylcholinester-ase enzyme of all the synthesized compounds showed that only four compounds were moderately active against it; i.e. N-(3,4dimethylphenyl)-2,3-dihydro-benzo[1,4]dioxine-6-sulfonamide (3e), N-ben-zyl-N-(2,4-dimethylphenyl)-2,3-dihydrobenzo[1,4] dioxine-6-sulfonamide (6b), N-ethyl-N-(2,4-dimeth-ylphenyl)-2,3-dihy-drobenzo[1,4]dioxine-6-sulfon-amide (7b) and N-ethyl-N-(2,6-dimethylphenyl) -2,3-dihydrobenzo[1,4]dioxine-6-sulfonamide (7d) having IC50 values ranging from 178.51 ± 0.14 to 364.27 ± 0.54 ^mol/L. The relative reference standard was eserine, with IC50 value of 0.04 ± 0.0001 ^mol/L. The % inhibition associated with these compounds arrayed from 59.91 ± 0.96 to 76.56 ± 0.19 ^mol/L respectively. The proficient activity was observed for 7b and 7d; this was most likely due to the occurrence of N-ethyl group in these compounds in comparison to the other series members. Against lipoxygenase enzyme, all the synthesized compounds showed beneficially good activity but the most active were N-(2,6-di-methylphenyl)-2,3-dihydrobenzo[1,4]dioxine-6-sulfonamide (3d), N-(2,4-dimethylphenyl)-2,3-di-hydrobenzo [1,4]dioxine-6-sulfonamide (3b), N-ben-zyl-N-(2,6-dimethylphenyl)-2,3-dihydrobenzo [1,4] dioxine-6-sulfonamide (6d) and N-(3,4-dimethyl-phenyl)-2,3-dihydrobenzo[1,4]dioxine-6-sulfon-amide (3e) having IC50 values of 34.21 ± 0.12, 64.21 ± 0.05, 84.61 ± 0.11 and 87.61 ± 0.14 ^mol/L respectively, relative to baicalein, a reference standard with IC50 T3 G 3 .g J3 c c w O 1 HH ^ Ü a o o ; s LH O y s " i u a o u ; s a o o T3 a s 0 01 E o O m o £ 2o(I)] 0.0535 0.0545 0.0769 wR2 (all data) 0.1475 0.1536 0.2233 Largest diff. peakl hole (elA3) 0.622/-0.296 0.513/-0.334 0.778/-0.347 and refined anisotropically. In ligand L4 in complex 2 chlorine atoms Cl1 and Cl2 were refined as disordered over two positions with 0.80:0.20 and 0.50:0.50 occupancy ratios, respectively. Hydrogen atoms were assigned isotropic displacement coefficients (7(H) = 1.2U(C) or 1.5U(C) and their coordinates were allowed to ride on their respective carbon atoms or nitrogen atoms using SHELXL-97 except of the solvent H atoms. For the latter, they were refined isotropically with fixed U values and the DFIX command was used to rationalize the bond parameter. CCDC 1861738-1861740 for these three complexes contain the supplementary crystallographic data for this paper, which can be obtained free of charge from the Cambridge Crys-tallographic Data Centre via www.ccdc.cam.ac.uk/data_ request/cif. Details of the crystal parameters, data collection, and refinement of complexes 1-2 are summarized in Table 1. complexes 2 indicate that the structure of the cyanide precursor has some effects on the structure of the cyanide-bridged complex formed. The three cyanide-bridged complexes have been characterized by IR spectroscopy. In the IR spectra of 1a and 1b, two sharp peaks due to the cyanide-stretching vibration were observed at about 2125-2130 and 2155-2160 cm-1, respectively, indicating the presence of bridging and non-bridging cyanide ligands in these complexes. The strong broad peak centered at about 1100 cm-1 for these two complexes is attributed to the free ClO4- anion. To confirm the optical activity and enantiomeric nature, the circular dichroism (CD) spectrum were measured in KBr pellets for complexes 1-2. The CD spectrum of 1a and 1b exhibit positive and negative Cotton effect at the same wavelengths (Figure. 1). 3. Results and Discussion 3. 1. Synthesis and General Characterization The recent works have proved that trans-dicyanome-tallates are good building blocks for synthesizing cyanide-bridged magnetic complexes.33-38 The relatively large planar pyridinecarboxamide ligand at the equatorial position can not only effectively lower the dimensionality of the resulted complex, but also weaken the supramolecular intermolecular magnetic interactions. With this in mind and also for the purpose of the preparation of chiral magnetic complexes, we investigated the reactions of trans-di-cyanidochromium(III) with chiral amine copper(II) compounds and obtained three new chiral cyanide-bridged Cr(III)-Cu(II) complexes. The different balance anions, i.e. ClO4- for complexes 1a, 1b and the cyanometallate for Wavelength /nm Figure 1. CD spectra of 1a (S isomer, black) and 1b (R isomer, blue) in KBr pellets. Chen et al.: Three Chiral Cyanide-Bridged Cr-Cu Complexes: Acta Chim. Slov. 2019, 66, 308-314 311 3. 2. Crystal structures of complexes 1a, 1b and 2 Some important structural parameters for complexes 1a, 1b and 2 are collected in Table 2. The perspective view of the enantiomeric structure of complexes 1a and 1b is demonstrated in Figure 2. The cell packing diagram of the complex 1a is given in Figure 3, which is similar to that for the complex 1b. For the complex 2, its cationic binucle-ar structure and the cell packing diagram are shown in Figures 4 and 5, respectively. Table 2. Selected bond lengths (A) and angles (°) for complexes 1a, 1b and 2. 1a 1b 2 Cu1- -N1 2.346(8) 2.344(6) 2.324(12) Cu1- -N3 2.049(8) 2.029(6) 2.028(9) Cu1- -N4 1.993(8) 2.012(6) 2.022(10) Cu1- -N5 2.062(8) 1.992(7) 1.983(11) Cu1- -N6 2.001(7) 2.000(6) 2.022(11) Cr1- C1 2.078(11) 2.121(9) 2.103(11) Cr1- -C2 2.048(12) 2.058(8) 2.068(11) Cr1- -N7 1.984(8) 1.967(6) 1.971(9) Cr1- -N8 1.976(7) 1.971(6) 2.123(10) Cr1- -N9 2.063(7) 2.083(6) 1.972(9) Cr1- -N10 2.070(8) 2.093(6) 2.098(9) Cu1- -N1-C1 139.0(9) 141.7(7) 159.2(10) Cr1- -C1-N1 165.7(10) 165.6(7) 172.7(11) Cr1- -C2-N2 176.7(11) 178.8(7) 175.2(12) The complexes 1a and 1b as a pair of enantiomer, containing Cr2Cu2 unit in the unit cell with a dimer structure, crystallize in monoclinic cell setting with the non-central space group P2^ while complex 2 crystallizes in triclinic cell setting with the non-central space group P1. All the three complexes are with the similar cationic cyanide-bridged binuclear structure and the different balance anion, i.e. the free ClO4- for complexes 1a, 1b and the free cyanide building block for complex 2. The distances between the O atom of the ClO4- ion and the Cu(II) ion is about 3.273 A, indicating the existed weak interaction. In Figure 3. The cell packing diagram along c axis of the complex 1a. All the H atoms and the solvent molecules have been omitted for clarity. Figure 4. Perspective view of the cationic structure of the complex 2. All the H atoms, the balanced anion and the solvent molecules have been omitted for clarity. Figure 2. The perspective view of the enantiomeric structure of complexes 1a and 1b. All the H atoms, the balanced anion and the solvent molecules have been omitted for clarity. Figure 5. The cell packing diagram along b axis of the complex 2. All the H atoms and the solvent molecules have been omitted for clarity. all the reported complexes, each cyanide-containing building block acting as a monodentate ligand through one of its two trans cyanide groups connects the Cu(II) ion with the other cyanide group as terminal. The Cr(III) ion Chen et al.: Three Chiral Cyanide-Bridged Cr-Cu Complexes: 312 Acta Chim. Slov. 2019, 66, 308-314 is six-coordinates with four equatorial nitrogen atoms from the pyridinecarboxamide ligand and two carbon atoms from the two cyanide groups with a trans position, so that forming a slightly distorted octahedral geometry, which can be proven by the bond parameters around the Cr(III) ion (Table 2). The Cr-C=N bond angles in these three complexes are in a comparatively narrow range of 165.6(7)°-178.8(7)°, showing almost the linear conformation for these three atoms. The Cu atom in complexes 1a, 1b and 2 is five-coordinated by a N5 unit, in which four N atoms come from the two chiral amine ligands and the additional one N atom from the bridging cyanide group. The Cu atom is only out of the plane formed by four N atoms 0.16(2), 0.17(4) and 0.058(2) A toward to the fifth coordinated Ncyanido atom in these three complexes, indicating that these five atoms are almost located in a plane. The average Cu-Namine bond lengths in complexes 1a, 1b and 2 are 2.009, 2.008 and 2.014 A, respectively, obviously shorter than the Cu-Ncy-anido bond length with the values of 2.346, 2.344 and 2.324 A, clearly showing the markedly distorted square pyramid surrounding of the Cu(II) ion. Additionally, it should be pointed out that there exists conspicuous difference for the C=N-Cu bond angles in these three complexes. The C=N-Cu bond angle in complexes 1a and 1b are only 139.0(9)°, 141.7(7)°, respectively, while the corresponding angle in the complexes 2 is obviously larger than those in complexes 1a and 1b with value 159.2(10)°. The intramolecular Cr(III)-Cu(II) separation through bridging cyanide group are 5.076, 5.098 and 5.412 A for these three complexes, which are obviously shorter than the shortest intermolecular metal-metal distance with the values of 6.519, 7.543, 8.108 and 7.511 A in the three complexes, respectively. 3. 3. The Magnetic Properties of Complexes Figure 6 shows the temperature dependences of magnetic susceptibility of complexes 1a and 2 measured in the temperature range of 2-300 K in the applied field of 2000 Oe. The room temperature cmT values are 2.13 and 4.09 emu K mol-1 for these two complexes, respectively, which are slightly lower than the spin only value of 2.25 emu K mol-1 for one uncoupled Cu(II) (S = 1/2) ion and one Cr(III) (S = 3/2) ion in complex 1a and 4.125 emu K mol-1 for one uncoupled Cu(II) (S = 1/2) ion and two Cr(III) (S = 3/2) ion in complex 2 based on g = 2.00. With the temperature decreasing, the cmT values increases gradually and attains the value of 2.37 and 6.76 emu K mol-1 about 15 K, then decreases sharply to 1.51 and 4.51 emu K mol-1 at 2 K, which indicated the characteristic of ferromagnetic coupling between the cyanide-bridged Cr(III)-Cu(II) center. The magnetic susceptibility for these two complexes conforms well to Curie-Weiss law in the range 2-300 K and give the positive Weiss constant q = 1.38 K and Curie constant C = 2.15 emu K mol-1 for complex 1a and q = 7.01 K and Curie constant C = 4.15 emu K mol-1 for complex 2, further proves the ferromagnetic coupled Cr(III)-Cu(II) through the cyanide bridge. On the basis of the binuclear model, the magnetic susceptibility of complex 1a can be fitted accordingly by the following expression (1) derived from the isotropic exchange spin Hamilton H = -2JSCuSCr. For complex 2, its magnetic susceptibility has been analyzed based-on also the binuclear model but by introducing the additional isolated Cr(III) ion with the expression (2). Ng2/?2 2 + 10cxp(4J/^r) kT ' 3 + 5exp(4J/kT) (1) By using the above model, the susceptibilities over the temperature range of 2-300 K for these two complexes were simulated, giving the best-fit parameters J = 0.74(2) cm-1, g = 2.01(2), R = Z(cobsdT - cCaldT)2/Z(cobsaT)2 = 2.30 x 10-5 for 1a and J = 2.37(2), g = 2.01(8), R = 3.21 x 10-5 for 2, respectively, which can further proven the weak ferro- Figure 6. The cmT and cm 1 vs T curves for complexes 1a (left) and 2 (right). Chen et al.: Three Chiral Cyanide-Bridged Cr-Cu Complexes: Acta Chim. Slov. 2019, 66, 308-314 313 magnetic coupling between the Cr(III) ion and Cu(II) ion through the bridging cyanide group. 4. Conclusion In summary, three new chiral cyanide-bridged het-erobimetallic complexes, in which two of them are a pair of enantiomers, have been designed and successfully synthesized based on the chiral amine copper(II) compounds and the fra«s-dicyanidochromium(III)-containing building blocks. 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Povzetek Tri heterokovinske Cr(III)-Cu(II) komplekse z mostovnim cianido ligandom s formulami {[Cu(L1)2Cr(L3)(CN)2] ClO4}2 • CH3OH • H2O (1a, L1 = (S,S)-1,2-diaminocikloheksan, H2L3 =1,2-bis(piridin-2-karboksamido)benzen), {[Cu(L2)2Cr(L2)(CN)2]ClO4}2 • CH3OH • H2O (1b, L2 = (£„R)-1,2-diaminocikloheksan) {[Cu(L3)2Cr(L4)(CN)2][Cr(L4) (CN)2]} • CH3OH-2H2O (2), (H2L4 = 1,2-bis(piridin-2-karboksamido)-4-klorobenzen) smo pripravili s kombiniranjem fra«s-dicianidokromovega(III) strukturnega motiva in kiralnega bakrovega(II) kompleksa. Vse tri komplekse smo ka-rakterizirali z elementno analizo, IR spektroskopijo in rentgensko strukturno analizo. Monokristalna rentgenska analiza razkrije, da oba enantiomerna kompleksa 1a in 1b ter kompleks 2 sodijo med s cianidnim mostom povezane kationske dvojedrne zvrsti s prisotnim ClO4- ali cianidnim anion. Magnetne lastnosti kompleksov 1a in 2 kažejo šibko feromag-netno sklopitev med sosednjima Cr(III) in Cu(II) ionoma preko mostovnega cianido liganda. Chen et al.: Three Chiral Cyanide-Bridged Cr-Cu Complexes: DOI: 10.17344/acsi.2018.4793 Acta Chim. Slov. 2019, 66, 315-325 ^creative ^commons Scientific paper Application of Inverse QSAR/QSPR Analysis for Pesticides Structures Generation Belgacem Souyei,1,2 Abdelkader Hadj Seyd,3^ Faouzi Zaiz4 and Abdelkrim Rebiai5 1 Faculty of Applied Sciences, University of Kasdi Merbah-Ouargla, 30000, Algeria 2 Chemistry Department, Echahid Hamma Lakhdar University of El Oued. 3 Faculty of Renewable Energy, Hydrocarbons, Earth and Universe Sciences, University of Kasdi Merbah- Ouargla 4 Informatics department, Echahid Hamma Lakhdar University of El Oued. 5 VTRS Laboratory, University of El Oued, P.O. Box 789, 39000, El-Oued, Algeria. * Corresponding author: E-mail: seydtg@gmail.com Tel: +213 662 726024 Received: 10-16-2018 Abstract The present work has focused on the application of the inverse-QSAR/QSPR problem for generating new structures of pesticides; this is in view of its extremely important and widespread use in several areas, particularly the agricultural field. For this reason, we implemented a methodology containing nine detailed successive steps that include a quantitative structure-activity/property relationship (QSAR/QSPR) study performed to develop a model that relates the structures of 190 pesticides compounds to their n-octanol-water partition coefficients (logk,w). We used the unique atomic signatures which represent the structures and acts as independent variables while the property (logkow) as the dependent variable. The model was constructed using 130 molecules as training set, and predictive ability tested using 60 compounds. Modeling of logk^ of these compounds as a function of the signatures descriptors was established by multiple linear regression (MLR) using (LOO) cross-validation. As a result, a QSAR/QSPR equation with 14 atomic signatures was hereby obtained with a R2 = 0.659273, Q2 = 0.65617 and RMSEtraining= 0.930192, s = 1.37297 for the training set and in leave-one-out (LOO) cross-validation experiment set value, q2 = 0.605676, RMSELOO = 1.0936 respectively. In addition to all of the above, new structures have been generated for a range of pesticides that can be included as future search topics. Keywords: Atomic Signatures; I-QSPR algorithm; multiple linear regression (MLR); n-Octanol-water partition coefficients; pesticides. 1. Introduction Pesticides are a large group of substances used to kill insects. These substances are mainly used to control pests that infest cultivated plants and crops or to eliminate disease-carrying insects in specific areas.1 The definition of pesticides according to Food Agriculture Organization of the United Nations (FAO 1989), a pesticide is any substance or mixture of substances intended for preventing, destroying, or controlling any pest including vectors of human or animal diseases, unwanted species of plants or animals causing harm during, or otherwise interfering with, the production, processing, storage, or marketing of food, agricultural commodities, wood and wood products, or animal feedstuff's, or which may be administered to animals for the control of insects, arachnids or other pests in or on their bodies.2 Pesticides and agrochemicals, in general, became an important component of worldwide agriculture systems during the last century, allowing for a noticeable increase in crop yields and food production.3 Poisoning from pesticides is a global public health problem and accounts for nearly 300,000 deaths worldwide every year.4 Pesticides have numerous beneficial effects. These include the protection of crop, preservation of food and ma- Souyei et al.: Application of Inverse QSAR/QSPR Analysis 316 Acta Chim. Slov. 2019, 66, 315-325 terials and prevention of vector-borne diseases. For example pesticides may be used in the prevention of malaria, which kills up to 1 million children per year,5 and for preventing other vector-borne diseases such as dengue, leish-maniosis and Japanese encephalitis. Sorption, volatilization, solubility in water, hydrolysis or oxidation, photo degradation and biodegradation are some of the important factors dealing with the fate of OPPs in the environment.6 Pesticides vary by source, structure and usage, for example, we find Botanical Ps and Neonicotinoid pesticides. Botanical Ps are naturally occurring chemical compounds extracted or derived from plants to manage field and storage crop pests.7 The BPs can easily degrade in the environment, and they are easily available, less toxic to human and non-targeted organisms and are compatible with different human cultures.8,9 Studies have shown that, plants are very good source of crop protectants against pests.10,11 Neonicotinoid pesticides were first introduced in the mid-1990s and since then their use has grown rapidly so that they have become the most widely used class of insecticides in the world, with the majority being used as seed coatings.12 As for the distribution of pesticides, it is concluded that lipo-philicity is the chief determinant of pesticide distribution in sediment/water systems.13 Accordingly lipophilicity kow (n-octanol-water partition coefficients) is a physico-chemical property that characterizes the ability of a chemical compound to dissolve in fats (lipids) and non-polar solvents.13 Lipophilicity plays an important role in the development of drugs and pesticides, since this parameter affects the phar-macokinetic and pharmacodynamic behavior of a biologically active substance.15,16 According to IUPAC, lipophilicity reflects the affinity of a molecule or a fragment thereof with a lipophilic medium.17 Due to the importance of lipophilicity parameter k„w in the distribution of these compounds (pesticides) between the water and organic phases in the organism, numerous studies report kow values for ionisable compounds.18-20 However, most of them determined a single kow value, reflecting the lipophilicity of the neutral species only. Lipophilicity is expressed by the octanol-water partition coefficient (kow), estimates the solubility in both aqueous and organic phases.21 The values of k„w generated using these various methods may vary by several orders of magnitude hence kow is usually expressed in the logarithmic form (log-k„w).22 Given all the above mentioned importance of the logkow, it is necessary to study the shape and characteristics of the relationship between this important property of (log-k„w) and the molecular structure of these compounds. The aim of this work is the application of the Inverse-quantitative structure-property relationship (I-QSPR) study. This method is based on a nine-step methodology. The first is a selection of database compound. The second is the generation of the 2D structures, while the third step is the QSPR analysis after translation of the database compounds into unique atomic signatures. In the fourth one, we construct constraint equations, specifically the graphicality and consistency equations, which facilitate the reconstruction of the solution compounds directly from the signatures. Fifth, we solve constraint equations while the sixth step is the database solutions checking. The seventh, eighth and ninth steps are respectively, keeping solutions with desired range, new structures generating, and finally, the database focused. Souyei et al.: Application of Inverse QSAR/QSPR Analysis ... Acta Chim. Slov. 2019, 66, 315-325 317 As for important uses of the QSPR analysis step is to develop a QSPR model that relates the structures of 190 pesticides compounds to their n-octanol-water partition coefficients using multiple linear regression technique and to generate new pesticides structures with novel physico-chemical and QSAR properties. 2. Methodology Inverse-QSAR/QSPR is known as the technical uses values for the independent variables of a particular compound in the QSAR /QSPR to solve for the activity /property of that compound (the dependent variable). In contrast, the goal of the inverse-QSAR/QSPR problem is to determine values for the independent variables given a desired activity /property.23 An inverse-QSPR(I-QSPR) problem is a signature-based CAMD (Computer Aided Molecular Design ) algorithm that identifies compounds possessing a certain performance (or property) of interest predicted using a developed QSPR model.24 The I-QSPR technique is interchangeable with the molecular signature descriptor CAMD algorithm. To achieve the I- QSPR algorithm in our study, we provided the steps detailed in Figure 1 below, which explains how this algorithm is performed in nine fundamental stages: (1) The selection of database compounds ; (2) Generation of the 2D structures; (3) Translation of the database compounds into signatures in addition to QSPR analysis; (4) Generation of constraint equations ; (5) Constraint equations solving then inverse solutions obtaining;(6) Check solutions for database; (7)Store solutions within desired range ; (8) New structure generation;(9) Focused database. 2. 1. Step 1: Selection of Database Compounds In this work the database contains 190 compounds (Pesticides) of different classes, which have an important role in human life. The corresponding experimental data («-octanol/water partition coefficients logkow) are obtained from the literature (www.chemspider.com chemical structures and www.pubchem.com). 2. 2. Step 2 : Generation of the 2D Structures We have developed a code that allows us to calculate the atomic signatures of molecules after generating 2D structures. 2. 3. Step 3 : Translation of the Database Compounds into Signatures in Addition to QSPR Analysis The structural representation of the studied compounds is of great importance for describing, circulating and explaining the significant structural information depending on their characteristics. Based on this representation, the extent to which this structure is related to the activity /physochemical properties of the studied molecule. The structural information of a molecule is evaluated by entities called molecular descriptors. The descriptor which is distinctive and in accordance with the applicable conditions to this technique (I-QSPR) is called signature.25,26 a) Signature The signature is a fragment based descriptor that encodes the local topology of an atom in a molecule.27 Degeneracy, when using signature, is controlled by the height of the signature, which represents the level of branching in a structure. Signature at height-1 or height-2 has lower degeneracy than height-0, and shows high correlation ability for atomic signatures of a molecule to its corresponding property of interest.28 b) Definition of the Atomic Signature Signature, which has its origins in structural elucidation studies of Faulon, 29 is based on the molecular graph of a molecule, G = (VG, EG), where the elements in VG denote the atoms in the molecule, and the edges of EG correspond to the bonds between those atoms. We define an atomic signature, hoG(x), as the canonical sub-graph of G consisting of all atoms a distance h from the root x.29 Once a signature height is specified, the molecular signature of each of the N compounds identified in step 1 is calculated using an in-house translator program. c) Definition of the Molecular Signature Descriptors Descriptors encoding significant structural information are used to present the physicochemical characteristics of compounds to build the relationship between structure and property in this study. The molecular descriptor used in this project was the molecular descriptor called signature because of its success to address the I-QSAR problem. The success of signature is threefold, First, signature performs the QSAR analysis as well as conventional molecular descriptors.30,31 Second, signature has a lower degeneracy than other molecular descriptors and can be controlled by the user by a variable termed height. The molecular signature for a compound is the sum of each atomic signature multiplied by the occurrence vector of that atomic signature in the given compound and it can be calculated using the following equation.32 Where the elements of VG (matrix of the vertices) are the atoms (X), i is the basis set of all atomic signatures of height h and haG is the vector of occurrence number of atomic h-signatures of the graph G. Example of molecular signatures for Ethephon is given in Figure2. Souyei et al.: Application of Inverse QSAR/QSPR Analysis 318 Acta Chim. Slov. 2019, 66, 315-325 Figure 2. Illustration of the atomic and molecular signatures of Ethephon (C2H6ClO3P) d) QSAR/QSPR analysis: The QSPR analysis was performed according to the organizational chart below: Quantitative structure-activity / property relationship (QSAR/QSPR) as an important area of chemometrics has been the subject of a series of investigations.32,33 The main aim of (QSAR/QSPR) studies is to establish an empirical rule or function relating the structural descriptors of compounds under investigation to properties. This rule or function is then utilized to predict the same properties of the compounds not involved in the training set from their structural descriptors. Figure 3: The Steps of QSPR proposed methodology 2. 4. Step 4: Generation of Constraint Equations In this step, we constructed the constraint equations, which serve in the construction of new compounds by reconnecting atomic signatures into molecular signatures with desired properties determined by the QSPR equa- tion.34 Constraint Equations are generated from the atomic signature database. Graphicality Equation is developed from the height 0 atomic signatures, and is a necessary condition for a connected graph.35 2. 5. Step 5: Solve Constraint Equations Since the space of solutions is infinite we limit the range that these solutions (independent variables) can take, based on their range in the original training set, minimum and maximum value (per atomic signatures) provides the additional constraints necessary to solve the system. Due to the large number of equations, we have used the min/max values in the Particle Swarm Optimization algorithm called PSO. This algorithm seems to satisfy the constraint equations in a step-wise manner such that the iterations involving those variables which occur in the equations go from least to most iterations. 2. 6. Step 6: Check Solutions for Database Since the constraint equations are derived from the number of used compounds, this number should represent solutions to the constraint equations, these are evaluated according to the bonding of the atoms within the molecule. 2. 7. Step 7 : Keep Solutions With Desired Range In this step the solutions must be scored for fitness relative to a desired property value. The solutions which have the desired fitness are kept, while the unsuitable atomic signature must be removed from the solution. It is at this stage where various heuristics can be applied to focus the solution space based on expert knowledge or other means.26 2. 8. Step 8: Generate New Structures The molecular Signatures (solutions) which emerge from Step 7 are the molecular signatures from which structure generation will occur. Structure generation is performed using an algorithm developed by Faulon and Coworkers,36 which is based on an earlier isomer enumeration algorithm developed by Faulon (Appendix 1 in supplementary files). In this step we have selected suitable structures after various filters to remove those undesirable candidate structures. 2. 9. Step 9: Focused Database The structures which have survived until this point become part of the focused database. These are the high-quality structures which are worthy of further investigation. It is here where experiments run on a select num- Souyei et al.: Application of Inverse QSAR/QSPR Analysis ... Acta Chim. Slov. 2019, 66, 315-325 319 ber of compounds to verify the predictions of the algorithm would be employed. Often, the results of the experimentation can be used to refine the QSPRs and the focused database itself.26 3. Results and Discussion 3. 1. Atomic Signatures Calculation As the first step in our I-QSPR analysis, is the translation of the 2D molecular structures into atomic signatures illustrated in Table 1 bellow. Table 1. The unique height-1 atomic signatures used in the QSPR analysis. Variable Height-1 Atomic Occurrence Signature [Min, max] X2 [H]( [N]) [0,5] X5 [O](=[C]) [0,3] X6 [C]([C] [H] [H] H]) [0,5] Xs [S]([P] [C]) [0,3] X9 [O]([P][C]) [0,4] X11 [C] ([O] [H] [H ] [H]) [0, 4] X12 [H] ([C]) [0,29] X14 p[C] ([N] p[C] p[C]) [0,4] X18 p[C] ( p[C] p[C] [C]) [0,5] X19 [Cl] ([C]) [0,4] X20 p[C] ( p[C] p[C] [H]) [0,10] X21 [H](p[C]) [0,11] X23 pC( p[N] p[N] [N]) [0,3] X24 p[N] ( p[C] p[C]) [0,3] X33 [O] ( p[C] [C]) [0,3] X35 [O] (=[S]) [0,4] X37 C( N] H] H] H]) [0,4] X39 [N] ([C] [C] [C]) [0,3] X43 [C](p[C][H][H][H]) [0,4] X44 [pN] ( pC] pN]) [0,4] X49 [C]([C] H] [H]S]) [0,3] X52 p[C](p[C]p[C]Cl]) [0,5] X53 [Cl](p[C]) [0,5] X57 p[C]([pC] p[C][O]) [0,3] X64 [C]([O] [C] [H] [H]) [0,4] X74 [C]([C][C][H][H]) [0,7] X75 [F] ([C]) [0,6] X89 [H] ([O]) [0,3] X104 [H](=[C]) [0,3] The QSPR analysis was calculated on the basis of a descriptor matrix. The descriptor matrix for the height -1 atomic signature contained 190 rows and 253 columns, one column for the logkow and 252 columns for the unique atomic signatures. The QSPR equation, however, was only calculated on the basis of 29 atomic signatures, hereby removing 223 atomic signatures, in order to perform the LOOCV.37 For performing the forward stepping MLR 223 unique atomic signatures removed and we leaved 29 with occurrence numbers greater than or equal to 3 in order to perform the LOOCV analysis. The most significant atomic signatures were then added one at a time, on the basis of the R2 and Q2 values were calculated for each step resulting in Figure 4, which depicts the R2 and Q2 values as function of the number of independent variables, i.e. atomic signatures. Figure 4. Impact of pesticides height-1 atomic Signatures on the QSPR statistics, which is plotted as function of the number of independent variables QSPR statistics consists in analyzing the improvement of the correlation with the increase of the number of variables of the model. The representation of the values of R2 and Q2 as a function of the number of descriptors (Fig. 4) brings out an asymptotic behavior, the model is considered optimal when the improvement of the correlation becomes maximal, that is to say representing the better compromise between correlation and parameterization. The calculations were terminated at 14 atomic signatures, thus the 15th atomic signature was insignificant. Statistically QSPR model using MLR was obtained, the QSPR equation was chosen on the basis of the best predicting model, i.e. highest Q2 value. A QSPR equation with 14 atomic signatures was hereby obtained with a R2 = 0.659273, Q2 =0.65617 and RMSEtraining = 0.930192, s = 1.37297 for the training set, and in leave-one-out (LOO) cross-validation experiment set value, q2 = 0.605676, RM-SEloo = 1.0936 respectively. Where R2 represents the determination coefficient, Q2: square validation coefficient, s: standard deviation, RMSE: the root square error. 3. 2. The model Equation The model equation can be written as fellow: logkow = - 0.167497 + 0.444669 * X52 + 0.417366 * X6 + 0.785521 * X20 + 0.461849 * X75 + 0.0716288 * X12 -0.419674 * X5 - 0.409265 * X21 + 0.528737 * X19 - 0.220096 r X44 + 0.25 1 8 59 * X74 - 0.365269 * X9 + 0.226272 * X14 0.445708 * X53 + 0.0357723 * X2 Souyei et al.: Application of Inverse QSAR/QSPR Analysis 320 Acta Chim. Slov. 2019, 66, 315-325 The 14 atomic signatures included in the QSPR equation are marked with bold in Table 1. Statistical results and significance of this final model, illustrate that the positive high value coefficient is for atomic signature X20. It was also suggested from this model that the atomic signature X24 was necessary contributor to logkow, the atomic signature X5 was assigned as an effective variable on log-k„w, but with a negative coefficient. Using the QSPR equation to predict the logk„w of the pesticides in the same training set and plotting these values against the experimental data, resulted in Figure 5. The plot shows the predicted logkow values based on the model equation which is validated to be statistically significant by the leave-one-out cross-validation versus ex- 7.- -2 O 2 * 6 S logkow(exp) Figure 5: The experimental- versus predicted values for the QSPR equation based on the logk„w with 29 height-1 atomic Signatures (logKow(Pred)=0.909412+0.704451.logKow(exp)) Table 2. Constraint equations N° Constraint equation perimental ones. Obviously, the predicted logkow values are in a good agreement with experimental ones. The 14-parameters of model provide a high statistical quality: R2 = 0,66 and Q2 = 0.65 , and this shows that the condition of predictability according to the consideration of R. Veerasamy,37 and A. Golbraikh,38 is satisfied. 3. 3. The Constraint Equations The following constraint equations are written in the order of the smallest parameters number to the greatest, thus in the order they were solved: (i) Consistency equations which ensure the alignment of atoms in the construction of molecular signatures. (ii) Graphicality equation which represents the valence of each atom. To solve these equations, a method developed by Weis and Visco.28 was adopted, and because of the wide database and the large number of constraint equations, we have used a program based on PSO algorithm. 3. 4. Generating of new Pesticides Structures Base on the inverse solutions obtained from solving the system of constraint equations, new structures can be constructed from molecular signatures. It is worth mentioning that for the same molecular signature there are multiple structures. Solving the constraint equations (Table 2) a total number of 5500 solutions (new molecular signatures) which will be a new structures. Since it would be difficult to examine over 5500 structures, the newly generated structures were refined according to the different chemical Eq.1 Mod(+X217,2) = 0 Eq.2 Mod(+X242,2) = 0 Eq.3 +X44+X169 = 2 Eq.4 -X45+X48 = 0 Eq.5 -X91+X92 = 0 Eq.6 -2X136+X137 = 0 Eq.7 -X138+X139 = 0 Eq.8 -X144+X145 = 0 Eq.9 -X191+X193 = 0 Eq.10 Mod(+X213+X215,2) = 0 Eq.11 -X243+X244 = 0 Eq.12 -X41+X42-X63 = 0 Eq.13 -X70+X72-X185 = 0 Eq.14 -X70+X73-X185 = 0 Eq.15 -X93+X95+X159 = 0 Eq.16 -X113+X114-X208 = 0 Eq.17 -X173+2X174-X175 = 0 Eq.18 -X206+X207+X209 = 0 Eq.19 -X52+X53-X119-X187 = 0 Eq.20 -X88+X89-X153-X195 = 0 Souyei et al.: Application of Inverse QSAR/QSPR Analysis ... Acta Chim. Slov. 2019, 66, 315-325 321 N° Constraint equation Eq.21 -X149+X150+X226+X250 = 0 Eq.22 -X168+X169-X186-X246 = 0 Eq.23 "X121+2X122"X123"X234"X235 = 0 Eq.24 -X151+X152+X170-X176-X252 = 0 Eq.25 -X1+X3+X160-X172+X188-X205 = 0 Eq.26 -X20+X21-X47-X58-X123-X175 = 0 Eq.27 -X50+X51-X116-X144-X170-X188 = 0 Eq.28 -X62+X63+X198-X199-X212-X247 = 0 Eq.29 -X101+X104-X150-2X155-X165-X226 = 0 Eq.30 -3X71+X75-X133-3X167-2X219-X220-2X239 = 0 Eq.31 -X109+X112-X142-X207-X215-X250+X251 = 0 Eq.32 -X3+X7-X59-X96-X134-X152-X160-X232 = 0 Eq.33 -X17+X19-2X80-3X103-X115-3X117-2X133-X245 = 0 Eq.34 -X32+X33-X57+2X85+X93+X118-X124+X195 = 0 Eq.35 -X3+X8-X50-X59-2X96+X146-X170-3X232+X241 = 0 Eq.36 -X27+X28-X67+X120+X146-X218+X230+2X236+2X240 = 0 Eq.37 Mod(+X60+X62+X140+X141+X184+X185+X201+X202+X238,2)= 0 Eq.38 -2X34+X35-2X38-2X95-2X120-2X159-2X223-X224-2X230-2X240-2X241 = 0 Eq.39 -X4+X5-X30-X56-X61-X65-X66-X82-X163-X171-X177-X222 = 0 Eq. 40 -X31+2X34-2X36+X38-X83-X84+X95+X99+2X107+X120-2X132-X221 =0 Eq.41 -X1+X2-X25-X31-2X46-X69-X79-X83-X162-X184-2X202-2X205-2X221-X238 =0 Eq.42 -X13+X14+X23-X25-X46+X55-X70-X76-X77-X83-X132-X140-2X162+X234-X238 = 0 Eq.43 -X3+X9-2X50-2X59-X96-3X116+X118-3X134-2X144-2X152+X153-2X160-X170-2X188+2X231+X251 = 0 Eq.44 -2X23+2X24-2X27-X32+X44+2X45-2X47-X55-X58-X94-X119-2X124-2X125-2X126+X169-2X187-X218-X234-X235 = 0 Eq.45 -X40+X41-X105+X149-X177+2X178-X181-X182-X183-X192+X193-X196-X197+X198-X210-X216+X217-X228-2X248-X249 = 0 Eq.46 -X76-X77+3X78-X79-X141+X142+X150+X165-2X166-X180+X194-X199+2X200-X201+X207-X208+X209-X211-X212-2X214+X215+X250 = 0 Eq.47 +2X14+2X18+2X20+X32+2X42+2X52+X55+2X57+X58+2X67+3X86+2X91+X94+X119+X121+X123+X126+2X138+2X145+X173+X-175+X218 = 2 Eq.48 -X18+X22+X43+X61+X71+X81+X82+2X87-X94-X121-X125+X127+2X130+X131+X147+X148+X157+3X161+X164-X173+X- 179+X197-X235= 0 Eq.49 -X8+X10-X28-X38+X49+X54+X66+X81+X97-X99+X103+X108-2X111+X133+2X156-X159+X179+X182+X196-X206+X210-2X223-2X224-X230+X237-X241-X242+X243= 0 Eq.50 -X100+2X101-X102-X108+2X109-X110-X135+X136+X142-X143-X154+X155-X164+X165-X181-X189+2X190-X192+2X194+X200- 2X204+X209+X213-X222-X225+X226-X227-X228-2X229-X245 = 0 Eq. 51 -X9+X11+X15-2X16-X33+X56+X64+X65+2X68+X82+X87-X88+X90+X106-X112 X114+X115+X135+X147+X148+2X158+X161+X- 167+X189-X191+X196+X219+X220+X228+X246+X252 = 0 Eq.52 -X1+X4-2X13+X15-X25+X26+X29+2X30-X31-X36+X37-3X39+X40+X54+X56-2X60+X61+X66-2X69-X76-X79-2X84+2X105-2X113+X-127+X129-X140-X166+X171-2X172+X176+X177-2X180-X184+X189-X201+2X203+X210-X211-X212+X222+X225+2X227+X233+X243-2X247+X249 = 0 Eq.53 -3X6-3X10-3Xn+X12-2X15-2X17-2X22-2X26-X29-3X37-X40-3X43-2X49-2X54-2X64-2X74-X80-2X81-X90-X97-X98-X100-2X102- 3X110-X115-2X127-X130-X131-X135-X148-2X151-2X156-X158-2X168-X171-2X176-X179-X181-2X183-X186-2X203-X204-X220-X225-X239- X246-2X249-X252 = 0 Eq.54 +X4+X6+X17+X22+X26+2X29+X49+X64+X65+2X68+2X74+X80+X87+2X90+2X97+3X98+X100+X102+3X106+X108+X115+X- 117+4X128+2X129+X130+2X131+3X143+2X147+X148+X151+2X154+3X157+X158+2X163+X164+X168+X179+X182+X183+2X186+X-192+X197+2X216+X219+X220+X229+3X233+3X237+X239+X245+X246+X248+X252 = 2 Eq.55 Mod( X1-X2+2X3+X4-X5+2X6-X7+X8+X9+2X10+2X11-X12+X13+X14+2X15+X16+2X17+X18-X19+X20-X21+2X22+X23+X- 24+X25+2X26+X27+X28+2X29+X30+X31+X32+X33+2X34-X35+X36+2X37+2X38+X39+X40+X41+X42+2X43+X44+X45+X- 46+X47-X48+2X49+2X50-X51+X52X53+2X54+X55+X56+X57+X58+2X59+X60+X61+X62+X63+2X64+X65+X66+X67+2X68+X69+X- 70+2X71-X72-X73+2X74-X75+X76+X77+X78+X79+2X80+2X81+X82+X83+X84+X85+X86+2X87+X88-1X89+2X90+X91-X92+X- 93+X94+2X95+2X96+2X97+2X98+X99+X100+X101+2X102+2X103-X104+X105+2X106+X107+X108+X109+2X110+X111+X112+X- 113+X114+2X115+2X116+2X117+X118+X119+2X120+X121+X122+X123+X124+X125+X126+2X127+2X128+X129+2X130+2X131+X- 132+2X133+2X134+X135+X136-X137+X138-X139+X140+X141+X142+2X143+2X144+X145+X146+2X147+2X148+X149+X150+2X- 151+2X152+X153+X154+X155+2X156+2X157+2X158+2X159+2X160+2X161+X162+X163+X164+X165+X166+2X167+2X168+X169+2X- 170+X171+X172+X173+X174+X175+2X176+X177+X178+2X179+X180+X181+X182+2X183+X184+X185+2X186+X187+2X188+X189+X- 190+X191+X192+X193+X194+X195+X196+X197+X198+X199+X200+X201+X202+2X203+X204+X205+X206+X207+X208+X209+X210+X- 211+X212+X213+X214+X215+X216+X217+X218+2X219+2X220+X221+X222+2X223+X224+X225+X226+X227+X228+X229+2X230+X- 0+X251+2X252 ,2) = 0 231+2X232+2X233+X234+X235+X236+2X237+X238+2X239+2X240+2X241+X242+X243-X244+X245+2X246+X247+X248+2X249+X- Souyei et al.: Application of Inverse QSAR/QSPR Analysis 322 Acta Chim. Slov. 2019, 66, 315-325 Table 3. Example of a solved molecular Signature, note that only atomic Signatures with occurrence numbers greater than 0 are depicted. Molecular Signature X6 X8 X9 X10 Xn X12 X49 X50 X51 X64 no. 1 1 1 2 1 1 15 1 1 1 1 Table 4. Values of logk^ of the 20 new structures calculated by I-QSPR theory and those predicted by Hyperchem software with other QSAR properties simulated by molecular mechanic MM+ and semi-empirical PM3 calculations. Formula 3D Structure (a) logkow(b) logkow(c) Surface area Volume Refractivity Mass MM+ PM3 (KJ/mol) (KJ/mol) c7h17o2ps4 C6H15O2PS3 C7H15 N2O4PS3 C8H17N2O4PS3 'T* fr-^. 2.93 2.62 536.71 760.29 76.60 292.42 22.67 -2641.14 2.17 2.61 531.90 730.74 64.50 246.34 24.518 -2302.97 6.05 2.19 580.95 882.26 77.57 318.36 146.488 -2905.23 6.74 2.20 577.82 882.76 81.80 332.39 131.31 -3174.98 6.30 2.19 564.48 874.47 77.39 353.22 26.47 -2922.12 C7H18NO2PS2 C5H12NO5PS C7H16NO4PS C6H14OS2 C6H16NO2PS2 C9H22N3O3PS3 //■-■y-^J 1.87 1.36 573.67 483.36 65.30 243.32 54.01 -2703.03 2.65 2.61 451.91 657.32 52.07 229.19 21.28 -2288.44 4.56 2.20 510.26 729.30 59.29 241.24 19.674 -2737 1.46 1.88 410.80 566.15 47.31 166.30 -0.46 -1997.52 6.00 2.20 651.1 992.59 95.46 378.47 32.8 -3488 3.34 2.61 600.25 1074.63 106.29 366.51 44.67 -4527.12 1.55 2.61 606.55 978.56 81.77 347.45 31.30 3549.116 5.02 2.62 651.7 982.59 95.42 346.48 32.8 -3458 4.71 2.19 499.29 791.32 73.09 307.40 14.102 2539.87 Souyei et al.: Application of Inverse QSAR/QSPR Analysis ... C9H17Cl2N2O3PS2 C9H19N2O4PS4 C13H23N4O5PS2 C6H14NO3PS4 Acta Chim. Slov. 2019, 66, 315-325 323 Formula C10H15N2O4PS2 C6H16NO5PS2 3D Structure (a) logkow(b) logk„w(c) Surface area Volume Refractivity Mass MM+ PM3 (KJ/mol) (KJ/mol) 5.2 2.20 488.23 853.74 76.81 322.33 27.922 -3329.07 4.27 2.61 493.77 731. 63.56 277.23 49.9 -2743.26 C16H26N3O7PS3 C8H18N3O2PS3 C8H20NO3PS2 C5H15N2O4PS 8.66 2.59 1.51 5 .52 1.47 2.61 1.46 2 .20 693 506.16 543.54 492.04 1249 858.53 803.91 72.69 115.85 74.30 70.45 57.54 499.5 46.36 -5356.36 315.40 43.37 -3075.79 273.35 26.001 -3074.00 242.23 40.15 -26.21 ["): J; Carbon (C); 0 : Chlorine (CI); ft : Nitrogen (N); ft:0xygen(0); 4 - Phosphorus; ^: Sulfur (S); (the H atoms are hidden) (b): logkow predicted by Hyperchem software; (e): logkow calculated by l-QSPR theory structures of all existing pesticides. All newly generated structures were passed through the ChemSpider, PUB Chem and LookChem (structure search) which are reliable database to identify commercially available compounds. After all these steps, a set of 20 samples was selected (Appendix 2 in supplementary files) according to their logkow, which is close to the average value of the database of the 190 pesticides studied (logkow = 2.94). The 20 identified compounds became part of the focused database. In order to assess the diversity level among the newly generated structures, they were compared to the training set structures.In addition to the work done, we compared the values of the logk„w predicted by our model to those calculated by Hyperchem software (ver. 8), it was concluded that the values for the two results (Table 4) are close in most cases. 4. Conclusion The high interest in pesticides and their uses in diverse fields, especially in agriculture, requires us to study these pesticides extensively and in depth . This is done by identification and focus on the characteristics including the physico-chemical properties, then the attempt to establish new chemical structures. The identification of new pesticides with desired properties was done by developing an inverse-quantitative structure-property relationship on the basis of octanol-water partition coefficient (logkow). We processed a database of 190 pesticide compounds, after developing molecular signatures calculated from atomic signatures. And in order to perform LOOCV. Only 29 atomic signatures from out of 252 are used as independent variables and logkow as a dependent variable in the QSPR realization, then the resolution of the constraint equations to the number of 55 by a computation code developed for this purpose, based on the successful PSO method to find 5500 solutions which represent new structures. Based on the goal of the inverse-QSPR method was to predict, if any, novel compounds structures possessing a logkow values are close to those in the training set. There were 20 new compounds classified as pesticides. We have presented and studied these new structures that do not yet exist in the databases of chemical compounds based on our search of reliable databases for this purpose. This work indicates that the inverse-QSPR method can be used as a reliable approach to generate new compound structures, since, on the one hand, the coefficient of determination R2 of the model is greater than 60%, and on the other hand, the predicted results are close to the values calculated by other software such as Hyprchem. This research is envisaged to serve as a base for further studies. Souyei et al.: Application of Inverse QSAR/QSPR Analysis 324 Acta Chim. Slov. 2019, 66, 315-325 5. Acknowledgments We thank Dr. Labbi Yacine, from Electrical Engineering Department, Echahid Hamma Lakhdar University of El Oued for assisting in the numerical analysis and calculating method. 6. References 1. A. Sharma, P. J. Gadi , V. P. Reddy, Science of The Total Environment, 2018, 643, 1522-1532. DOI:10.1016/j.scitotenv.2018.06.312 2. S. Knillmanna, P. Orlinskiyabc, O. Kaskea, K. Foita, M. Lies-sad, Science of The Total Environment, 2018, 630, 1619-1627. DOI:10.1016/j.scitotenv.2018.02.056 3. R. Münze, P. Orlinskiy, R. Gunold, A. Paschke, O. Kaske, M. A. Beketov, M. Hundt, C. Bauer, G. Schüürmann, M. Möder, M. Liess, Science of The Total Environment, 2015, 537, 69-80. DOI:10.1016/j.scitotenv.2015.07.012 4. A. Sabarwal, K. Kumar, R. P. Singh, Environmental Toxicology and Pharmacology, 2018, 63, 103-114. DOI:10.1016/j.etap.2018.08.018 5. V. Sujitha, K. Murugan, D. Dinesh, A. Pandiyan, R. Aruliah, J.Hwang, K. Kalimuthu, C. Panneerselvam, A. Higuchi, A. T. Aziz, S. Kumar, A. A. 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Inf. Comput. Sci. 2003, 43, 721-734. DOI:10.1021/ci020346o 37. R. Veerasamy, H. Rajak, A.Jain, S. Sivadasan, C. P. Varghese, R.K. Agrawal , International Journal of Drug Design and Discovery . 2011, 2, 511-519 38. A. Golbraikh, A. Tropsha, J. Mol. Graphics Mod. 2002, 20, 269-276. DOI:10.1016/S1093-3263(01)00123-1 Souyei et al.: Application of Inverse QSAR/QSPR Analysis ... Acta Chim. Slov. 2019, 66, 315- 325 325 Povzetek Predstavljena raziskava preučuje uporabo inverznega-QSAR/QSPR pristopa za generiranje novih struktur pesticidov, kar je izredno pomembno v luči njihove široke uporabe, še posebej na področju kmetijstva. S tem namenom smo uporabili metodologijo devetih zaporednih korakov, ki vključujejo kvantitativno študijo relacije med strukturo in aktivnostjo/ lastnostmi (QSAR/QSPR) s ciljem razviti model, ki povezuje strukturo 190 pesticidov z njihovim porazdelitvenim koeficientov za sistem n-oktanol-voda (logk^). Uporabili smo enolične atomske deskriptorje, ki predstavljajo strukture in nastopajo kot neodvisne spremenljivke, medtem kot je vrednost logk,w odvisna spremenljivka. Model smo razvili na učenju z nizom 130 molekul, njegovo sposobnost napovedovanja pa smo preverili na ostalih 60 komponentah. Modeliranje logk,w vrednosti teh komponent kot funkcije deskriptorjev smo izvedli z večkratno linearno regresijo (MLR) z uporabo pristopa izpusti-enega (LOO) navzkrižne validacije. Rezultat je QSAR/QSPR enačba s 14 atomskimi deskriptorji z R2 = 0.659273, Q2 = 0.65617 in RMSEjraining = 0.930192, s = 1.37297 za niz na katerem smo model učili ter q2 = 0.605676, RMSEloo = 1.0936 s pristopom izpusti-enega (LOO) navzkrižne validacije na testiranem nizu. Generirali smo tudi nove strukture pesticidov, ki bi bili lahko vključeni v nadaljnje študije. Souyei et al.: Application of Inverse QSAR/QSPR Analysis DOI: 10.17344/acsi.2018.4821 Acta Chim. Slov. 2019, 66, 326-336 ^creative ^commons Scientific paper Adsorptive Performance of Soy Bran and Mustard Husk Towards Arsenic (V) Ions from Synthetic Aqueous Solutions Doina Humelnicu,^* Laurentiu Valentin Soroaga,1 Cecilia Arsene,1 Ionel Humelnicu1 and Romeo Iulian Olariu1,2,* 1 Alexandru loan Cuza" University of Iasi, Faculty of Chemistry, Bld. Carol I, nr. 11, Iasi, 700506, Romania 2 Alexandru loan Cuza" University of Iasi, CERNESIM, Bd. Carol I, nr. 11, Iasi, 700506, Romania * Corresponding author: E-mail: doinah@uaic.ro, oromeo@uaic.ro, phone: +40232201136 Received: 10-29-2018 Abstract Recently, there is growing attention on the use of low-cost sorbents in the depollution of contaminated waters. As a consequence, the present work investigates the potential of soy bran and mustard husk as possible sorbent for the removal of arsenic(V) from residual water. Effects of various operating parameters such as: contact time, pH, initial arsenic concentration, pH, sorbent dose, temperature were investigated to determine the removal efficiency of arsenic(V). Thermodynamic parameters that characterize the process indicated that the adsorption is spontaneous and endothermic. The values for the separation factor, RL were less than one which confirms that the adsorption process was favorable. Equilibrium data fitted well to the Langmuir model with a higher adsorption capacity of soy bran (74.07 mg g-1) towards arsenic(V) ions than mustard husk (65.79 mg g-1). It was found that the pseudo-second order kinetic model was the best applicable model to describe the adsorption kinetic data. Keywords: Adsorption; arsenic; isotherm; soy bran; mustard husk 1. Introduction Arsenic is an element that reaches into the environment from a variety of natural sources (volcanic emission, minerals) and anthropogenic activities (mining activities, burning of fossil fuels and the use of arsenical pesticides which have a longer residence time and also an increased capacity of accumulation.1-4 Also, by erosion, decomposition and due to the action atmospheric factors, arsenic can be released into groundwaters and surface waters.5,6 Stable inorganic arsenic species in water include arsenic acid anions (H2AsO4-, H3AsO4,HAsO42- §i AsO43-). Arsenious acid is also stable in water as H3AsO3 and H2AsO3- in moderately reducing conditions (< 200 mV).7 Inorganic arsenic in oxidation states +V (arsenate) and +III (arsenite) is found in a variety of mineral in natural waters. Chemical arsenic behavior is related to the ease transformations between +III and +V oxidation states. The oxidation state affects the toxicity of arsenic compounds. The toxicity of different arsenic species decreases in the order arsenite > arsenate > monomethylarsonate > dimethy-larsinate).8,9 There is clear evidence that chronic exposure to inorganic arsenic increases the risk of cancer.10 Studies have shown that inhalation of arsenic leads to an increased risk of lung cancer whereas the ingestion of arsenic has been associated with an increased possibility of skin cancer and cancer of the bladder, liver and lungs.11,12 Because of that in 2006 the World Health Organization (WHO) has decided to change the maximum admissible concentration of arsenic from 0.05 mg/L to 0.01 mg/L in drinking water.13 In order to eliminate arsenic from water has been used various methods: i) precipitation/co-precipitation, (method that allows the removal of arsenic up to 0.05 mg/L and in some cases even less than 0.01 mg/L)14,15; ii) membrane filtration (that may remove a variety of contaminants from water but for arsenic compounds this method can reduce their concentration up to 0.05 mg/L.16 Howev- Humelnicu et al.: Adsorptive Performance of Soy Bran Acta Chim. Slov. 2019, 66, 326-336 327 er, this method presents some disadvantages, such as low efficiency, large amounts of waste and high cost, as well16; iii) ion exchange (method that are nowadays frequently used in the treatment of containing arsenic groundwater and drinking water because of its of high efficacy advantage.1,13,14,16-21 By using this method the level of the arsenic compounds in water is less than 0.01 mg/L. By adsorption, the contaminants are concentrated at the sorbent surface. Nature of the adsorption process could be explained based on two theories: one physical and one chemical. Physical theory, the most widespread theory is the so-called potential theory or concentrated layer theory according to which the reaction between atoms that are found on the surface of the solid (adsorbent) and adsorbed molecules is determined by the van der Waals forces of attraction. Chemical theory of the adsorption admits the existence of a single monomolecular layer on the surface of the solid (adsorbent); adsorption forces act only on a very short distance which not exceeding the diameter of a mol- ecule.22 Thus, the adsorbent material must fulfill certain conditions such as: a type of particle size, high adsorption capacity, high selectivity, and high degree of adsorption, water strong physical connection, and low price. In recent times, more attention is paid to cheap biomass such as powdered eggshell,23 pine leaves,24 rice husk.25 These biomasses appear to be a possible alternative for heavy metals removal due to their economic and environmental characteristics, the chemical composition, availability, low price, and high efficiency in removal of heavy metals from dilute solutions. Recently, the need for an economical method for the removal of pollutants from contaminated waters involves researches on low cost sorbents such as agricultural waste by-products. In this regard, various type of agricultural waste by-products such as palm oil fruit shell,26 coffee grains,27 fir tree sawdust,28 rose petals,29 rice husk,30 cellulose dust31 etc. have been investigated for the removal of the pollutants from the wastewaters. The agricultural by-products may be different parts of plant, such as bark, stem, leaves, root, flower, fruit biomass, husk, hull, shell and may contain compounds such as cellulose, lignin, hemi-cellulose. These compounds have potential functional groups such as hydroxyl, carboxyl, amino, amido and alkoxy with a great affinity for the metal 32 ions.32 The aim of the present study is to analyse the sorption capacity of mustard husk soy bran as low cost agricultural by-products towards arsenic(V) ions from the residual waters in different experimental conditions. During this study, effect of some parameters such as the dose of adsorbent, pH, temperature, initial metal concentration and contact time were studied. Moreover, various isotherm and kinetic models were used to explain the adsorption process. 2. Materials and Methods All chemicals were of analytical reagent grade and no further purification was carried out. The agricultural by-products used in these adsorption experiments were soy bran and mustard husk resulting from the milling and baking. Sorbents were collected from a local mill, ground, were prepared and characterized as shown by Humelnicu and colab.33 The stock solution containing the arsenic(V) was prepared from Na2HAsO4 7H2O (Sigma-Aldrich). The adsorption experiments were performed in a batch system by stirring at 350 rpm a suspension that contained arsenic(V) ions solution and the sorbent. The pH values were in range 2 and 10, the initial concentration of the solution varied from 50 to 350 mg L-1, at a temperature between of 25 °C - 45 °C, and the sorbent dose varied from 1.5 to 4 g L-1. The pH of the solution was adjusted with NaOH or HNO3 0.1 M solution and measured with a HANNA pH/tem-perature meter HI 991001. After the equilibrium has been reached the supernatant was used for arsenic quantification by using Hydride Generation-Atomic Absorption Spectrometry (HG-AAS). HG-AAS is powerful analytical techniques that provide information of the level of concentrations of As with low interferences and a lower LOQ because the analyte is separated from the sample matrix before the quantifica-tion.34,35 Experiments were conducted on a High Resolution Continuous Source Spectrometer ContrAA 700 (Analytik Jena). The amount of arsenic adsorbed per unit mass by the sorbent under equilibrium conditions was calculated by the equation (1). where: C0 is initial concentration of solution, (mg L-1), Ce is equilibrium As(V) concentration (mg L-1), V is volume of solution (L), and m is sorbent mass (g). The distribution coefficient, Kj, is defined as the ratio of the concentration of arsenic retained in the sorbent and the one in the solution at equilibrium being calculated with equation (2). where C0, Ce,V and m have the same meaning as in Eq (1). The adsorption capacities of the two adsorbent were analyzed through the use of Langmuir, Freundlich, Tem-kin and Flory-Huggins models. The kinetics of arsenic adsorption on the soy bran and mustard husk were analyzed by using pseudo first-order, pseudo second-order, and in-tra-particle diffusion kinetic models. Humelnicu et al.: Adsorptive Performance of Soy Bran 328 Acta Chim. Slov. 2019, 66, 326-336 Desorption experiments were carried out in batch system by using the sorbent loaded with arsenic immediately after the adsorption processes. Four common eluents have been tested, namely: NaOH, NaHCO3, HCl and HNO3 0.01 M. The sorbent loaded with As and eluent solution was kept in contact for 24 hours. The following abbreviations have been used: M-mus-tard husk, S-soy bran, As-M- mustard husk after As(V) adsorption and As-S-soy bran after As(V) adsorption, respectively. 3. Results and Discussion 3. 1. pH Effect on the Adsorption Process pH is one of the most important factor that influences the chemistry of arsenic in aqueous solution and surface of the adsorbents. The effect of pH on the adsorption process of As on the mustard husk and soy bran was investigated in the range of values between 2-10. Figure 1 illustrates the effect of pH on As(V) adsorption on the studied adsorbents. The amount of retained As(V) increased slightly with increasing pH and reached a maximum value at pH 6, after that decreased slightly. Consequently, in further experiments pH 6 value was selected as an optimum pH condition. 1 3 5 7 9 11 pH Figure 1. pH dependence of arsenic(V) adsorption on mustard husk and soy bran. The possible centers on the surface of the sorbents that could be responsible for the adsorption include -OH and -COOH functional groups.36 Mamindy-Pajany et al.37 denotes that in according to the arsenic speciation, H2AsO4- is predominant for pH values between 2 and 5, whereas HAsO42- is predominant for pH values between 7 and 10. On the other hand, at higher pH condition active centers were not protonated and were both neutral and anionic by releasing H+ ions (-COO-, -O) which leads to a less adsorption. 3. 2. Effect of Sorbent Dosage The adsorption process is efficient if it requires a small amount of sorbent. Effect of sorbent dosage on As(V) adsorption was investigated by changing the sorbent dose from 1.5 to 4 g L-1 with the initial metal concentration 250 mg L-1 at pH 6.0, temperature of 25 °C and contact time 60 min. Figure 2 shows that the adsorption capacity increases with the increase of adsorbent dose from 1.5 to 3.5 g L-1 followed by a slightly decrease. Increase of the adsorption capacity was due to the greater availability of the exchangeable sites or surface area at the higher concentrations of the adsorbent. 80 -i- 40 -I-I-1-I- 1 2 3 4 5 m:V, g/L Figure 2. Effect of sorbent dosage on the adsorption process of As(V) on the mustard husk and soy bran. From the experimental results it was found that the adsorption process has higher efficiency in the case of soy bran as adsorbent in comparison with mustard husk. 3. 3. Effect of Contact Time on the Adsorption Process of As(V) Influence of the contact time on the adsorption process of As(V) ions on the two sorbents has been studied for a period time between 15 to 180 minutes, all the other parameters being kept constant. In these studies the As ions concentration have been varied from 50 mg L-1 to 250 mg L-1. The obtained results are depicted in Figure 3. The results indicate that the amount of the retained ions increases with the increasing of the contact time and the equilibrium is reached after about 75 minutes. From Figures 3 on can conclude that the adsorption of As is more effective on soy bran as adsorbent. Humelnicu et al.: Adsorptive Performance of Soy Bran Acta Chim. Slov. 2019, 66, 326-336 329 a) 200 b) 80 -50 mg/L -150 mg/l -250 mg/L 150 200 Figure 3. Contact time dependence of adsorption process of As(V) on: a) mustard husk, b) soy bran. 3. 4. Effect of As(V) Initial Concentration on the Adsorption Process The effect of the initial concentration of the As(V) solution on the adsorption has been investigated, too. The initial concentration was varied from 25 to 350 mg L-1, all other parameters have been maintained constant. Figure 4 shows that the adsorption capacity increases with the increasing of the initial concentration of As(V). Thus, for mustard husk adsorption capacity increases form 31.25 to 59.47 mg g-1 and for soy bran from 36.98 to 70.39 mg g-1. In both cases after 250 mg L-1 as initial As(V) concentration the adsorption capacities decrease. These results are in good agreement with Asif and Chen25 that explained this variation due to a raise in the driving force of the concentration gradient and low concentration, the driving force of adsorbent is reduced due to low concentration gradient. In the diluted solutions the mobility of ions is high, and for Figure 4. Effect of initial concentration of As(V) on the adsorption process on mustard husk and soy bran. this reason, the interaction of As(V) ions with the adsorbents was amplified. 3. 5. Effect of the Temperature on the Adsorption of As(V) The effect of temperature on the adsorption process of the As(V) on the mustard husk and soy bran was investigated from the range of 25-45 °C. All the other parameters have been kept constant and the results are depicted in Figure 5. Figure 5 indicated that with the increasing of the temperature adsorption capacity of the adsorbents increase due to the increasing of the attractive forces between adsorbents surface and arsenic ions that is typical for the adsorption of most metal ions from their solutions Figure 5. Effect of temperature on the adsorption process of As(V) on the mustard husk and soy bran. Humelnicu et al.: Adsorptive Performance of Soy Bran 330 Acta Chim. Slov. 2019, 66, 326-336 onto natural materials.38,39 Rate of the adsorbate's molecules distribution along the external layer, as well as in the internal pores of the adsorbent increases with the increasing of temperature. 3. 6. Thermodynamic Parameters Determination of the thermodynamic parameters: enthalpy (AH°), entropy (AS°), and free Gibbs energy (AG°), was based on experiments performed in a batch system, at temperatures between 25-45 °C. For this reason equations (3) and (4) have been applied. (3) AG0 = AH0 - TAS° (4) where: K is distribution coefficient for adsorption that was calculated with the equation (2). AH° and AS° values have been estimated from the slope and intercept of the plot of lnKd versus 1/T (Figure 6). The obtained results are presented in Table 1. Figure 6. Plot of lnKd function of 1/T. The data from Table 1 reveal that AH° and AS° have positive values which indicates the sorbent's affinity for arsenic (V) ions and the adsorption is an endothermic process. The positive values of entropy suggest an increase in the disorder degree in the system. The spontaneity of the adsorption process is confirmed by the negative value of Gibbs energy. This parameter values decrease with the increasing of temperature which indicates the efficiency of adsorption at higher temperature. The activation energy of the adsorption process (Ea) was obtained from the slope of plotting ln(1-0) vs. 1/T, where sorbent surface coverage (0) was calculated using the equation (Eq. 5): (5) C, C0 are final and initial concentration of arsenic in aqueous solution (mg/L). According to the modified Arrhenius equation,40 the plot of ln(1-0) vs. 1/T gives a straight line with the slope Ea/R. Activation energy values were calculated from the slope of plot and have values of 59.38 kJ mol-1 and 31.97 kJ mol-1 for mustard husk and soy bran, respectively. The positive values of Ea were consistent with the positive values of AH° and confirm once again the endothermic nature of the adsorption process. 3. 7. Kinetic Models In order to obtain information on the mechanism of adsorption of arsenic on soy bran and mustard husk three different models were applied, that is: the pseudo-first order model (Eq. 6), pseudo-second order model (Eq. 7) and the intraparticle diffusion model (Eq. 8).41-43 A relatively high correlation coefficients value indicates that the model successfully describes the kinetics of arsenic adsorption. (6) (7) (8) where: qt and qe and are the amounts of arsenic adsorbed (mg g-1) at time t and at equilibrium, respectively, k1 is the rate constant of pseudo-first order kinetic (min-1), k2 is the rate constant of pseudo-second order kinetic (g mg-1 min-1), and kid is the intra-particle diffusion rate constant (mg Table 1. Thermodynamic parameters for the adsorption of arsenic (V) on mustard husk and soy bran. Sorbent AH°, AS°, AG°, kJ mol-1 kJ mol-1 J mol-1 K-1 25 °C 30 °C 35 °C 40 °C 45 °C Mustard husk 56.04 234.39 -13.84 -15.01 -16.18 -17.35 -18.52 Soy bran 70.36 286.27 -14.98 -16.41 -17.84 -19.27 -20.70 Humelnicu et al.: Adsorptive Performance of Soy Bran Acta Chim. Slov. 2019, 66, 326-336 331 g-1 min-05). The plot of log(qe - qt ) vs. time (Figure 7) give a linear relationship from which k1 and qe can be determined from the slope and intercept, respectively. The plot of (t/qt) vs. time (Figure 8) gives a linear relationship from which qe and k2 can be determined from the slope and intercept of the plot, respectively. a) -0.5 b) 2.5 2 1.5 -1 - IT | 0.5 -j ui o 0 -0.5 -i ^ -1.5 time, min R2 = 0.9471 ♦ 50 mg/L ■ 150 mg/L A 250 mg/L R2 = 0.9512 Time, min 100 a) 5 200 200 Figure 7. The pseudo-first order kinetics of arsenic (V) adsorption on mustard husk (a) and soy bran (b). Figure 8. The pseudo-second order kinetics of arsenic (V) adsorption on mustard husk (a) and soy bran (b). Table 2. Kinetic parameters for the adsorption of arsenic on mustard husk and soy bran. Initial concentration, mg/L Model 50 150 250 Mustard husk Soy bran Mustard husk Soy bran Mustard husk Soy bran Pseudo-first order qe, exp (mg g-1) 34.86 44.39 46.52 61.43 65.79 71.94 qe, calc (mg g-1) 35.70 46.08 44.66 62.23 73.63 122.71 k1 (min-1) 2.92 • 10-2 2.7 • 10-2 3.52 • 10-2 3.1 • 10-2 5.25 • 10-2 7.0 • 10-2 R2 0.957 0.947 0.979 0.951 0.969 0.898 Pseudo-second order qe, exp (mg g-1) 34.86 44.39 46.52 61.43 65.79 71.94 qe, calc (mg g-1) 38.76 50.0 49.75 67.56 69.44 75.75 k2(g mg-1 min-1) 1.35 • 10-3 8.88 • 10-4 1.71 • 10-3 8.6 • 10-4 1.78 • 10-3 1.77 • 10-3 R2 0.977 0.971 0.992 0.918 0.995 0.995 Humelnicu et al.: Adsorptive Performance of Soy Bran 332 Acta Chim. Slov. 2019, 66, 326-336 An intra-particle diffusion model was used to predict the rate controlling step but in this case a non-linear relationship has been obtained. The pseudo-first-order and pseudo-second-order rate constants determined are listed in Table 2 along with the corresponding correlation coefficients. From these results it can be seen that the values of correlation coefficient decreases from pseudo second-order to pseudo first-order. 3. 8. Adsorption Isotherms It is well known that the adsorption isotherms express the interaction between the adsorbent and adsorbate in the adsorption processes. In order to study the adsorption of arsenic ions on the two sorbents, Langmuir, Freundlich, Temkin and Flory-Huggins adsorption models have been used. Langmuir isotherm characterizes a monolayer adsorption on a surface with a finite number of identical centers which are homogeneously distributed on the surface of the sorbent. In our study a linearized Langmuir isotherm form (Eq. 9) has been used:44 Ce qc kL • qm qm (9) where: qe represents the amount of adsorbed arsenic per sorbent unit (mg g-1); Ce is arsenic ion concentration at equilibrium (mg L-1); qm is a parameter that express the maximum adsorption capacity (mg g-1) corresponding to monolayer coverage; KL is constantly referring to the adsorption energy (g L-1). KL and qm parameters values were calculated from the intercept and the slope of the plot Ce/qe vs. Ce (Figure 9). An important characteristic of Langmuir isotherms can be expressed by the dimensionless constant (Eq.10) called equilibrium parameter or separation factor. (10) where: KL is the Langmuir constant, C0 is the initial concentration of As(V) ions (mg L-1). For a favorable adsorption process RL value must be between 0 and 1. In our study Rl obtained values were less than one (Table 3) which indicates that the arsenic(V) adsorption process was favorable. The Freundlich isotherm is based on the multilayer adsorption that means a heterogeneous surface of the sor-bent and a non-uniform distribution of heat of adsorp-tion.45 A logarithmic form of this model (Eq. 11) was applied in our study: Figure 9. Langmuir isotherms for the arsenic(V) adsorption on mustard husk and soy bran. In the above equation, qe and Ce have the same meaning as in Eq (9); KF (mg(1-1/n) L1/n g-1/n) and n are Freundlich constants that indicate the relative adsorption capacity of the sorbent, and the adsorption intensity, respectively. The slope and intercept of Freundlich model (Figure 10) have been used to calculate KF and factor n. A value for 1/n less than 1 indicates a normal isotherm while 1/n > 1 suggests a cooperative adsorption. In the case of arsenic adsorption on both mustard husk and soy bran 1/n values are 0.233 and 0.179, respectively, indicating a normal isotherm adsorption. 1ogqc = log KF +—log C( (11) Figure 10. Freundlich isotherms for the arsenic (V) adosrption on mustard husk and soy bran. The third adsorption isotherm model used in the present work was the Temkin model. In this case, the main assumption is that the heat of adsorption decreases linearly with coverage due to sorbent-sorbate interactions.46 The Humelnicu et al.: Adsorptive Performance of Soy Bran Acta Chim. Slov. 2019, 66, 326-336 333 linear Temkin isotherm equation (Eq. 12) used in our study was: (12) where: A is the equilibrium constant (L g-1) corresponding to the maximum binding energy and constant B (J mol-1) is correlated to the heat of adsorption as follows: (13) where: b^ is the Temkin isotherm energy constant (J mol-1) and R is the universal gas constant (8.3146 J mol-1 K-1). The Temkin isotherm plots for both sorbents are presented in Figure 11 and the isotherm parameters extracted are listed in Table 3. Figure 11. Temkin isotherms for the adsorption of arsenic (V) on mustard husk and soy bran. For a most comprehensive characterization of the arsenic (V) adsorption process was used the fourth adsorption model, Flory-Huggins, in order to calculate the surface coverage of sorbent by sorbate.47 log — = log Km + n log(l - 9) (14) cess. The parameters of equation (14) were calculated from the slope and intercept of the plot log9/C0 vs. log(1-0) that is depicted in Figure 12 and are presented in Table 3. where 0 represents surface coverage and was calculated by Eq. (5), Kfh is equilibrium constant of the adsorption pro- Figure 12. Flory-Huggins isotherms for the arsenic adsorption on mustard husk and soy bran. Considering all extracted parameters for all four adsorption isotherm models (see Table 3) it can be concluded that for the arsenic (V) adsorption on the mustard husk and soy bran the best fit shows the Langmuir isotherm model. In addition, between the two analyzed materials, in terms of adsorption capacity the best candidate seems to be soy bran than of the mustard husk for arsenic (V). The efficiency of the two studied sorbents, soy bran and mustard husk, on the arsenic (V) was highlighted by a comparison with the results from the literature for other sorbents (Table 4). As can be observed the adsorption capacity of the investigated sorbents for the arsenic (V) is higher compared with some other sorbents and its low cost and abundance make it as possible materials for the use in residual waters decontamination. The fact that in the sorption stage there are a series of processes that can affect the morphology of the adsorbent materials is pointed out by the images obtained by using a Electronic Scanning Microscope, SEM Quanta 250. Images presents the morphology of sorbens samples (Figure 13) Table 3. Parameters for the adsorption models. Sorbent Langmuir Freundlich Flory-Huggins Temkin qm, Kl R2 Rl Kf n R2 Kfh n r2 A bf r2 (mg g-1) (L g-1) Mustard husk 65.79 0.032 0.982 0.111 16.15 4.29 0.899 1.99 • 103 1.565 0.886 1.158 242.21 0.866 Soy bran 74.07 0.058 0.993 0.065 26.09 5.56 0.949 1.29 • 103 2.647 0.911 0.648 265.57 0.915 Humelnicu et al.: Adsorptive Performance of Soy Bran 334 Acta Chim. Slov. 2019, 66, 326-336 Figure 13. SEM images of the mustard husk and soy bran before (a, c) and after (b, d) adsorption experiments. Table 4. Comparison of maximum adsorption capacity of different sorbents towards arsenic (V). Sorbent qm (mg g-1) Reference Rice polish 0.15 48 Rice husk 0.225 25 Coconut shell carbon 2.40 49 Pine leaves 3.27 24 Tea fungal biomass 4.95 48 Polymeric alginate beads 8.33 50 Coconut coir pith 13.75 51 Withan ia fru tescens 16.88 52 Calami rhizoma 22.04 52 Orange juice residue 67.43 53 Mustard husk 65.79 This work Soy bran 74.07 This work before and after adsorption processes with enlarge X2500, scale 40 ^m. 3. 9. Desorption Results Once the sorbent is used, it needs to be regenerated. Desorption processes are important from two points of view: first, to recover metal ion and its subsequent use in industrial and secondly, in the regeneration of sorbent for new use processes. The amount of As released from the sorbent was determined by HG-AAS and the percentage of arsenic de-sorbed was calculated with equation Eq. 15: , , „, amount.i,,,. desorbed ion % =-— ■ 100 amount, ads (15) Humelnicu et al.: Adsorptive Performance of Soy Bran Acta Chim. Slov. 2019, 66, 326-336 335 where: amountdes is the amount of desorbed arsenic and amountads is the amount of arsenic adsorbed by the sor-bent. The results of desorption experiments reveal that the best regeneration eluent may be aqueous solution of NaOH 0.01 M, 87.95% for mustard husk and 90.67% for soy bran, respectively. 4. Conclusions The adsorption of arsenic (V) ions on mustard husk and soy bran was studied as a function of contact time, initial arsenic ion concentration, pH, sorbent mass and temperature, the conclusion being that the sorption capacity of the soy bran was higher than that of mustard husk. The thermodynamic parameters indicate that adsorption of arsenic (V) ions on mustard husk and soy bran is a spontaneous (AG° < 0) and endothermic (AH° > 0) process. This study indicates that arsenic (V) adsorption is better described by Langmuir isotherm model and the kinetic of the process obeys the pseudo second-order model. 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Abou-Yousef, M. Yousef, M. El-Sakhawy, Carbohydrate Polym. 2012, 88, 250-256. DOI:10.1016/j.carbpol.2011.11.090 33. D. Humelnicu, M. Ignat, F. Doroftei, Environm. Monit. Assess. 2015, 187, 187-198. DOI:10.1007/s10661-015-4454-1 34. H. M. Anawar, Talanta 2012, 88, 30-42. DOI:10.1016/j.talanta.2011.11.068 35. A. N. Anthemidis, G.A. Zachariadis, J.A. Stratis, Anal. Chim. Acta 2005, 547, 237-242. DOI:10.1016/j.aca.2005.05.039 Humelnicu et al.: Adsorptive Performance of Soy Bran 336 Acta Chim. Slov. 2019, 66, 326-336 36. L. C. Ajjabi, L. Chouba, J. Environ. Manag. 2009, 90, 34853489. DOI:10.1016/j.jenvman.2009.06.001 37. Y. Mamindy-Pajany, C. Hurel, N. Marmier, M. Roméo, C.R. Chimie 2009, 12, 876-881. DOI:10.1016/j.crci.2008.10.012 38. K. H. Chong, B. Volesky, Biotech. Bioeng. 1995, 47, 451-460. DOI:10.1002/bit.260470406 39. U. Kumar, Sci. Res. Essay 2006, 1, 33-37. DOI:10.5958/j.0974-4487.11.1.001 40. C. S. Sundaram, N. Viswanathan, S. Meenakshi, J. Hazard. Mater. 2008, 155, 206-215. DOI:10.1016/j.jhazmat.2007.11.048 41. S. Lagergren, KSvenska Vetenskapsakad Handl 1898, 24, 1-39. 42. G. McKay, Y. S. Ho, Process Biochem. 1999, 34, 451-465. DOI:10.1016/S0032-9592(98)00112-5 43. W. J. Jr. Weber, J. C. Morris, J. Sanit. Eng. Div. ASCE, 1963, 89, 31-59. 44. I. Langmuir, J. Am. Chem. Soc. 1916, 38, 2221-2295. DOI:10.1021/ja02268a002 45. H. Freundlich, Phys. Chem. Soc. 1906, 40, 1361-1368. 46. M. Temkin, Zk Fiz. Khim. 1941, 15, 296-332. DOI: 10.2307/40085313 47. K. Vijayaraghavan, T. V. N. Padmesh, K. Palanivelu, M. Velan, J. Hazard. Mater. 2006, B133, 304-308. DOI:10.1016/j.jhazmat.2005.10.016 48. D. Ranjan, M. Talat, S. H. Hasan, J. Hazard. Mater. 2009, 166, 1050-1059. DOI:10.1016/j.jhazmat.2008.12.013 49. L. Lorenzen, J. S. J van Deventer, W. M. Landi, Miner. Eng. 1995, 8, 557-569. DOI:10.1016/0892-6875(95)00017-K 50. T. Dewangan, A. Tiwari, A. K. Bajpai, Toxicol. Environ. Chem. 2009, 91, 1055-1067. DOI: 10.1080/02772240802585012 51. T. S. Anirudhan, M. R. Unnithan, Chemosphere 2007, 66, 60-66. DOI: 10.1016/j.chemosphere.2006.05.031 52. M. Chiban, G. Carja, G. Lehutu, F. Sinan, Arabian J. Chem. 2016, 9, S988-S999. DOI:10.1016/j.arabjc.2011.10.002 53. K. N. Ghimire, K. Inoue, K. Makino, T. Miyajima, Sep. Sci. Technol. 2002, 37, 2785-2799. D0I:10.1081/SS-120005466 Povzetek V zadnjem času je velika pozornost namenjena uporabi cenovno ugodnih adsorbentov za čiščenje kontaminiranih voda. S namenom smo preverili potencial sojinih lupin in gorčičnih luščin za odstranjevanje arzena (V) iz odpadnih voda. Preučili smo vpliv procesnih parametrov kot so kontaktni čas, pH vrednost, začetna koncentracija arzena, količina adsorbenta in temperatura na odstranjevanje arzena (V). Termodinamski parametri so pokazali, da je proces pod preučevanimi pogoji spontan in endotermen. Separacijski faktor je bil manjši kot ena kar kaže, da je adsorpcija ugodna. Adsorpcijsko ravnotežje lahko dobro opišemo z Langmuirjevo izotermo z maksimalno kapaciteto vezave arzenovih (V) ionov na sojine lupine 74.07 mg g-1 in na gorčične luščine 65.79 mg g-1. Kinetiko lahko opišemo z reakcijo psevdo-prve-ga reda. Humelnicu et al.: Adsorptive Performance of Soy Bran DOI: 10.17344/acsi.2018.4822 Acta Chim. Slov. 2019, 66, 337-343 ©ccmmons Scientific paper Protein Release from Biodegradable Poly(£-Caprolactone)-Chitosan Scaffolds Prepared in scCO2 Gregor Kravanja,1 Maja Globočnik,1 Mateja, Primožič,1 Željko Knez1,2 and Maja Leitgeb1,2^ 1 University of Maribor; Faculty of Chemistry and Chemical Engineering; Laboratory of separation processes and product design; Smetanova ul. 17; 2000 Maribor; Slovenia 2 University of Maribor, Faculty of Medicine, Taborska ulica 8, 2000 Maribor * Corresponding author: E-mail: E-mail: maja.leitgeb@um.si, Telephone: +386 2 2294 462 Received: 10-29-2018 337 Abstract To study the release patterns of protein bovine serum albumin (BSA), porous poly(£-caprolactone)-chitosan scaffolds with entrapped BSA were fabricated by using supercritical CO2 for its potential use in tissue engineering applications. An emulsion, consisting of a polymer-solvent solution and buffer protein solution was saturated with scCO2 at 12 MPa and 37 °C and then rapidly depressurized through a release valve causing bubble nucleation and precipitation of the composite material. The controlled total protein release from biodegradable poly(s-caprolactone) with 5% chitosan (w/w) scaffolds was assessed by Bradford protein assay. After 16 to 20 days of protein release testing, 58.8% of the protein was released from composite with PCL (Mw = 10,000 g/mol) and 43.9% from composite with PCL (Mw = 60,000 g/mol). Preliminary studies for characterization of the prepared composite biomaterials using FTIR spectra, ESEM photo analysis and DSC analysis have been carried out. Keywords: Protein release; PCL polymer; chitosan; scaffold; supercritical CO2 1. Introduction Biodegradable porous polymer scaffolds are often required for reconstruction or regeneration of organ function in tissue engineering applications, alone or in combination with a bioactive agent. They mimic the extracellular matrix (ECM), so they must provide a sustainable template for cell attachment, proliferation, and differentiation. Bio-compatibility, absorbability, appropriate mechanical strength, and porous structure are some of the basic requirements of scaffolds. Biodegradability is a very convenient feature of polyesters, and that is why they are so promising and extensively utilized, since ceramic and metal implants require a second surgical operation, to remove them from the body.1-3 Furthermore, in bone tissue engineering, the material should form an osteoconductive structural support for the newly formed bone and consist of interconnected pores, which have an important role in supporting cell penetration, new tissue ingrowth, nutrient diffusion, and neovascularization.4-6 Poly(glycolic acid), poly(lactic acid), poly(hydroxyl butyrate), poly(s-caprolactone) (PCL) and their copolymers are the most frequently used synthetic biodegradable polymers in tissue engineering. PCL is highly appealing because of its physical-chemical and mechanical characteristics. It has been broadly investigated on account of its soft- and hard-tissue compatibility. Its low melting point (ca. 60 °C) allows easy processing. Its non-toxic degradation products are easily metabolized or secreted from the body. PCL is approved by the Food and Drug Administration (FDA) for use in biomedical applications. However, as with other biodegradable polyesters, its use could be limited by some drawbacks. The absence of cell recognition sites on the surface of scaffolds can lead to poor cell affinity and adhesion. Its hydrophobicity can prevent the cells from penetrating into the porous structure. Neutral charge distribution can cause a lack of interaction with the ECM. Acidic degradation products are formed during hydrolysis of PCL and could possibly cause side effects. It has a very Kravanja et al.: Protein Release from Biodegradable 338 Acta Chim. Slov. 2019, 66, 337-343 slow degradation rate, due to high crystallinity. Therefore, PCL is often mixed with natural polymers to enhance its bioactivity.1,5,7 Chitosan (CS) is a linear polysaccharide. Its numerous good features, such as biocompatibility, biode-gradability, non-antigenicity, non-toxicity, antibacterial properties, and bio-adherence have induced its use in biomedical areas. When combined with PCL, hydrophilic chi-tosan can modulate the hydrophobicity of PCL, which increases the biocompatibility, cellular affinity, wettability and permeability of matrices.6,7 Tissue engineering applications require scaffolds with high surface area to volume ratios. This is crucial for effective cell seeding onto the biomaterial support and for efficient nutrient acquisition, so that rapid proliferation and sufficient physiological activity can be achieved. Porous polyester scaffolds can be formed by many techniques, each of which results in different biomaterial characteristics, therefore, the most convenient method may depend on the application.8 In the last two decades, there has been significant progress in using scCO2 for polymer foaming. This is a non-toxic, non-flammable, chemically inert and environmentally safe gas.9 Furthermore, it is inexpensive and offers a useful alternative to organic solvents that can be potentially harmful to cells. CO2, dissolved in the polymer, decreases the viscosity, acts as a plasticizer and allows processing at lower temperatures. This foaming technique enables control of the size and distribution of pores by selecting suitable processing conditions, like temperature, depressurization rate and solubili-sation pressure (gas concentration in the polymer). Good solubility of CO2 in polymers can be additionally influenced by temperature, pressure and weak interactions with functional groups of polymers.5,10 With the ability to control the morphology of the composite, supercritical fluid technology has also overcome the problem of incorporating biologically active species into polymeric composites without a change in activity. The combination of gas-like viscosity and liquid-like density makes scCO2 an ideal medium for making polymer-based materials containing bioactive species. Interaction of scCO2 with amorphous polymers leads to depression of the glass transition temperature (Tg); under these conditions, the polymer is plasticized, which significantly lowers the viscosity and allows incorporation of insoluble bioactive particles into the polymer. Additionally, no solvent residues remain in the material after processing.11,12 To create new composite material scaffolds with specific physical-chemical properties PCL (Mw = 10,000 and 60,000 g/mol) was mixed with 5% chitosan (w/w) and further, bovine serum albumin (BSA) was incorporated. We have used BSA as a model protein for preliminary studies of controlled release from prepared porous scaffolds. Measurement of total protein release was performed by Bradford protein assay. As a prior characterization of the prepared material, FTIR, ESEM and DSC analysis on clean PCL and CS and their composites were performed. 2. Experimental 2. 1. Materials Bovine serum albumin (BSA) (CAS: 9048-46-8) was provided by Sigma-Aldrich. Dimethyl chloride (CAS: 7509-2), was purchased from Fluka Analytical. Poly(s-capro-lactone) (PCL) of molar weight 10,000 g/mol (CAS: 2498041-4) and of 60,000 g/mol (CAS: 24980-41-4) and chitosan (CAS: 9012-76-4) were purchased from Sigma-Aldrich. The polymers were delivered as powders and were used without further purification. Moisture content in the polymers, determined gravimetrically by means of an HB43-S Compact Halogen Moisture Analyzer, was lower than 0.10 wt. % for each polymer. CO2 (99.998%) was supplied by Messer (Slovenia). 2. 2. Scaffold Preparation in scCO2 Before processing with scCO2, an emulsion consisting of a water (aqueous) phase and an organic composite solution phase was prepared by sonicating the immiscible phases. The water phase consisted of 20 mg BSA dissolved in 200 ^L of phosphate buffer at pH 7.4. The organic phase consisted of 500 mg PCL + CS mixture dissolved in 1 mL of dimethyl chloride. Prior to supercritical processing, the emulsion was pipetted into the small cylindrical mold and then immediately placed into a high-pressure view equilibrium cell made of stainless steel (Sitec AG, Zurich, CH) (Figure 1).13 The high-pressure cell was pressurized to 12 MPa with CO2 by a high-pressure pump (NWA PM-101). The pressure inside the cell was measured by an electronic pressure gauge (WIKA Alexander Wiegand GmbH & Co. KG, Alexander-Wiegand-Straße, Klingenberg, Germany). The temperature of the cell was kept constant using a heating jacket and was observed using calibrated thermocouple immersed in the cell. The pressure deviation was ± 0.01 MPa and the total temperature deviation was 0.1 °C. When the CO2 reached the desired working pressure, a stable 20 mL/h flow-rate of CO2 was produced through the pressure cell by a syringe pump manufactured by ISCO (Lincoln, NE) to fully extract the dimethyl chloride. The emulsion consisting of BSA and a biodegradable polymer was saturated with scCO2 for 18 h to easily incorporate insoluble bioactive particles into the polymer by lowering the glass transition temperature (Tg) and eliminating all solvent residues from the material. In the last step, the high pressure cell was rapidly depressurized at a controlled depressuriza-tion rate of 0.1 MPa/s by opening the release valve in order to create a microporous composite scaffold. After the depressurization step, the composite scaffold was removed from the mold and sectioned into three parts for further protein release studies. Prior to material characterization, additional scaffolds were prepared without incorporating BSA protein. Kravanja et al.: Protein Release from Biodegradable ... Acta Chim. Slov. 2019, 66, 337-343 339 Figure 1. Diagram of the system used to generate biodegradable scaffolds with scCO2. 2. 3. Characterization of Prepared Scaffolds The porosity of the prepared scaffolds was measured with a graduated cylinder.1,14 It was calculated as given in the equation (1): (1) where w1 is the weight of graduated cylinder filled with eth-anol, w2 the weight of the graduated cylinder, ethanol and scaffold, w3 the weight of the graduated cylinder and ethanol after removing the scaffold, ws the weight of the scaffold and pe the density of absolute ethanol used in the analysis. 2. 3. 1. DSC Analysis Differential scanning calorimetry (HP DSC 1, Mettler Toledo) was performed on pure PCL (Mw = 10 000, 60 000 g/mol) and on the prepared porous scaffolds to confirm the composition of PCL and CS and to define the percentage of crystallinity. Samples were placed in sealed aluminium pans. Measurements were held at ambient pressure at a temperature range from 25 °C to 600 °C with a temperature rate of 10 °C/min. Based on the DSC curves, the melting temperature (Tm), heat of fusion (AHm) and crystallinity of the samples were determined. The crystallinity of the PCL fracture (Xc) in the samples was calculated with equation (2) 15: (2) where AHm is the specific melting enthalpy of the sample, AHm is the melting enthalpy of 100% crystalline poly (s-caprolactone) (76.9 J/g), and w is the weight fraction of PCL in the blend.16 2. 3. 2. FTIR Spectra and ESEM Analysis The FTIR spectra were recorded on a Fourier Transform infrared instrument (Bruker Platinum-ATR) equipped with OPUS Optik GmBH software in the range from 400 to 4400 cm-1 of wavelength. Pores size for the resulting composite scaffolds generated in scCO2 were determined using environmental scanning electron microscopy (ESEM) Quanta 200 3D (FEI Company, Hillsboro, OR), which allows observation of translation and non-sample without prior preparation (polymer materials, biological and medical samples). 2. 4. Protein Release Studies Each of the porous PCL-CS composites incorporated with BSA was divided into three smaller units of 50 mg, to verify the uniformity of protein release. Each section was placed into 2 mL microcentrifuge tubes, containing 1 mL of phosphate buffer (PBS) with pH 7.4. In order to simulate the conditions in the human body, the tubes were incubated at 37 °C. The amount of the released BSA was determined by the Bradford method.17 First measurement was performed after 3 hours and second after 24 hours from the start of the study. Then, Bradford assay was repeated every few days. After each measurement, fresh PBS was added to the microcentrifuges containing parts of the composites. 3. Results and Discussion Results for the characterization of prepared composite material using FTIR spectra, ESEM photo analysis, DSC analysis and finally protein release studies are presented in the following subsections. 3. 1. DSC Analysis DSC analysis was performed on two pure PCLs with different molar masses (Mw = 10,000 and 60,000 g/mol) and on porous scaffolds after processing with CO2 to confirm the composition formation of the mixture between PCL and CS. As seen in Table 1, the melting point of both composites was lower than the melting point of pure PCL. Kravanja et al.: Protein Release from Biodegradable ... 340 Acta Chim. Slov. 2019, 66, 337-343 Table 1. Melting temperatures and specific melting enthalpies of the samples. Material Tm (°C) AHm (J/g) PCL 10,000 67.58 71.31 PCL 10,000 + 5% CS 62.88 65.17 PCL 60,000 73.04 66.62 PCL 60,000 + 5% CS 66.23 55.04 That coincides with the previously published results by She et al.18, where the melting point of the composites was decreasing with the increase of the amount of CS in the mixture. PCL 10,000 based composite material has a higher crystallinity (89.2%) than the PCL 60,000 based composite (75.3%). Degradation of materials was observed at a temperature of approximately 420 °C, and a slight decrease in decomposition temperature of the composites can be obtained, compared to pure PCL. 3. 2. FTIR Analysis The FTIR analysis for raw materials, pure PCL, CS, and other obtained composites was made. Figure 2 shows the spectra of raw materials, PCL (Mw = 10,000 g/mol), CS and the composite material. Figure 3 shows the spectra of pure PCL (Mw = 60,000 g/mol), CS and their composite. The characteristic peak of pure PCL that corresponds C=O stretching vibration of the carbonyl group in ester is at 1732 cm-1 for PCL (Mw = 10,000 g/mol) and at 1734 cm-1 for PCL (Mw = 60,000 g/mol). The spectra of pure CS shows a broad peak for the O-H stretch at 3408 cm-1. The peak at 1656 cm-1 represents N-H stretching. Another peak for 1° amine in CS at 1076 cm-1 represents the C-N stretch. C=O stretching vibrations were detected in both composites, at 1735 cm-1 for the composite with PCL (Mw = 10,000 g/mol) and at 1734 cm-1 for the composite with PCL (Mw = 60,000 g/mol). In the blend of PCL and CS, interactions between the carbonyl groups of PCL and the hydroxyl and amine groups of CS can occur, which leads to the formation of ester and amide bonds. However, there are no major absorptions shifts of the characteristic functional groups to be seen when the spectra of pure PCL is compared to the spectra of the composites. This could indicate that no molecular interactions occurred between functional groups, a finding which corresponds to results from the previous studies.19 Peaks in the range from 2850 cm-1 to 3000 cm-1 represent alkyl (sp3 hybridization of C atoms) stretching vibrations in pure materials and in the composites. This could indicate that no molecular interactions occurred between functional groups, a finding that corresponds to results of Neves et al.7 who have constructed composite materials by blending CS and PCL to make 3D fiber-mesh scaffolds for articular cartilage tissue repair. On the contrary, spectra in Wu et al.19, where composites were made with layer-by-layer assembly technique, show Figure 2. The FTIR spectra of raw materials (PCL (Mw = 10,000 g/ mol) and CS) and the composite material. Figure 3. The FTIR spectra of raw materials (PCL (Mw = 60,000 g/ mol) and CS) and the composite material. some new strong peaks when spectrum of pure CS is compared to blend (PCL+CS). FTIR of PCL and CS composites, prepared by She et al.18, have confirmed that the intensity of characteristic peaks increases with increase of CS content. 3. 3. ESEM Analysis and Porosity Figure 4 (a and b) shows an ESEM micrograph of PCL-CS scaffolds foamed with scCO2 at 12 MPa and 37 °C. ESEM analysis showed that resulting porous scaffolds have a closed cell structure, which enables better isolating properties because of its greater stiffness and toughness and lower permeability. The diameters of the pores vary from 50 to 130 ^m when PCL 10,000 + 5% CS is used; and from 40 to 140 ^m when PCL 60,000 + 5% CS is used, so they are considered to be macropores (Table 2). For the pore growth period, a constant moderate de-pressurisation rate of 0.1 MPa/s was selected. At quicker depressurization rates more pores could be generated but with undesired smaller pore sizes.20 Similarly, the lowest possible operating pressure of 12 MPa was used in order to Kravanja et al.: Protein Release from Biodegradable ... Acta Chim. Slov. 2019, 66, 337-343 341 Figure 4. Environmental scanning electronic microscope image of a) PCL 10,000 + 5% CS and b) PCL 60,000 + 5% CS scaffolds. Table 2. Preparation of biodegradable composite material at an applied pressure of 15 MPa and a temperature of 37 °C. Composite Molar mass Depressurization rate Pore size interval Porosity (g/mol) (MPa/s) (^m) (%) PCL 10,000 + 5% CS 10,000 0.1 50-130 80.1 PCL 60,000 + 5% CS 60,000 0.1 40-140 75.1 achieve the melting behavior of a polymer PCL in contact with scCO2 at 37 °C. At selected supercritical conditions, PCL undergoes to partial or complete melting at 37 °C which is well below its melting point at ambient pressure.21 Increasing pressure above the selected one results in higher rate of dissolved gas in biomaterial matrix that creates more nuclei and decreases the number of suitable pore sizes for tissue engineering.22 Porosity was calculated from. and was in both composites relatively high, above 75%. Biomaterials with a porosity greater than 70% and a size of pores around 100 ^m are required to allow vascularization and tissue ingrowth.23 Additionally, used supercritical foaming method generated high porous biomaterial scaffolds without toxic organic residuals and a waste of the materials.24 Supercritical foaming method can be easily combined with other scaffold fabrication techniques like salt leaching, breath figure method and thermal induced phase method to obtain better pore interconnectivity and higher poros-ity.25 3. 4. Protein Release Monitoring Total protein release monitoring was assessed for the PCL 10,000 + 5% CS and PCL 60,000 + 5% CS scaffolds by dividing each of them into three equal parts. The influence of molecular mass of PCL and time of protein release from composite polymers on total concentration of released BSA was studied. As presented in Figure 5, BSA loaded scaffolds showed two-stage release profiles. Larger concentrations of protein were released in the first few days from both investigated scaffolds, and then release of protein was constant, since the total protein concentration linearly increased with increase in release time. The main share of the protein from PCL-based scaffolds was released within the first 16 days. After that time, concentrations of protein in PBS solutions were zero or negligibly small, regardless of the tested composite. Higher total protein release was observed for the scaffold PCL 10,000 + 5% CS compared to the scaffold PCL 60,000 + 5% CS. This can be related to scaffold porosity, which is approximately 5% higher in the Figure 5. Total concentration of released BSA from PCL 10,000 + 5% CS and PCL 60,000 + 5% CS scaffolds vs. time of release, compared to the literature data.21 Kravanja et al.: Protein Release from Biodegradable ... 342 Acta Chim. Slov. 2019, 66, 337-343 can be attributed to better porosity of the material. 58.8% of the protein was released from the composite with PCL (Mw = 10,000 g/mol) and 43.9% from composite with PCL (Mw = 60,000 g/mol) after 16 or 20 days of protein release testing. 4. Conclusion Porous PCL-CS composite scaffolds with entrapped BSA were generated in scCO2 as constructs for tissue engineering. Before the protein release studies, scaffolds were prepared for characterization of the composite material without incorporated BSA. DSC analysis confirmed the miscibility of the blend with a decrease in the melting points of the composites, compared to pure PCL. ESEM analysis showed that the resulting porous scaffolds have a closed cell structure that yields better isolating properties because of higher toughness and lower permeability. When used as a model protein loaded into scaffolds, BSA showed two-stage release profiles. Larger concentrations of protein were released in the first few days in both investigated scaffolds. After 16 to 20 days of protein release testing, 58.8% of the protein was released from composite with PCL (Mw = 10,000 g/mol) and 43.9% from composite with PCL (Mw = 60,000 g/mol). The ability to simply incorporate and release thermolabile proteins at a controlled rate demonstrates the potential for using scCO2 as a blowing agent for creating scaffolds and opens up the frontier for investigating encapsulation of healing agents and therapeutic enzymes for tissue engineering applications. Table 3. Concentrations of protein, released from three equal parts of scaffolds (PCL 10,000 + 5% CS and PCL 60,000 + 5% CS). Time Protein Protein Protein Average Total (days) concentration concentration concentration concentration roteinp in scaffold part 1 in scaffold part 2 in scaffold part 3 (mg/mL) release (mg/mL) (mg/mL) (mg/mL (mg/mL) PCL 10,000 + 5% CS 0 0 0 0 0 0 0.125 0.249 0.204 0.205 0.219 0.219 1 0.169 0.162 0.320 0.217 0.436 4 0.113 0.680 0.550 0.448 0.883 8 0.106 0.291 0.121 0.173 1.056 12 0.122 0.144 0.096 0.121 1.177 16 0 0 0 0 1.177 20 0 0 0 0 1.177 PCL 60,000 + 5% CS 0 0 0 0 0 0 0.125 0.385 0.118 0.302 0.268 0.268 1 0.090 0.103 0.281 0.158 0.426 4 0.123 0.398 0.152 0.225 0.651 8 0.108 0.115 0.121 0.115 0.765 12 0.018 0.079 0.080 0.113 0.878 16 0.060 0.068 0.083 0.070 0.949 20 0 0 0 0 0.949 Kravanja et al.: Protein Release from Biodegradable ... case of PCL 10,000 + 5% CS. With high porosity, the lightly trapped BSA in the micropores can be easily released.26 Similar controlled release protein patterns were observed when BSA and basic fibroblast growth factor (bFGF) was encapsulated in poly(lactic-co-glycolic) acid (PLGA 65:35) generated in scCO2.27 The overall release rates from the scaffolds foamed with CO2 were up to twice as high compared to release patterns of PLGA 65:35 by the salt leaching method. This can be related to protein loss due to the distilled water required for salt leaching.28 The BSA release was normalized to the initial protein entrapped in the scaffold, as shown in Figure 6. At the beginning, the release of proteins follows the same curve for both tested scaffolds and after 4 days of protein release, the PCL 10,000 + 5% CS shows better release profile, which ¿J 70 .2 = 60 o . O £ Q.-D 3 € ® m U> 5 re £ 50 40 20 S 10 -□-n _____-o-o -n-PCL 10,000 + 5% CS —i—i—■—i— -o-PCL 60,000 + 5% CS 10 i(days) 15 20 Figure 6. Total protein release normalized to the initial protein entrapped in the scaffold (for PCL 10,000 + 5% CS and PCL 60,000 + 5% CS) vs. time of release. Acta Chim. Slov. 2019, 66, 337- 343 343 5. Acknowledgement The authors would like to acknowledge the Slovenian Research Agency (ARRS) for financing this research within the frame of program P2-0046 (Separation Processes and Production Design). 6. References 1. Y. Wan, H. Wu, X.Cao, S. Dalai, Polym. Degrad. Stab. 2008, 93, 1736-1741. DOI:10.1016/j.polymdegradstab.2008.08.001 2. H.-Y. Cheung, K-T. Lau, T.-P. Lu, D. Hui, Composites, Part B. 2007, 38, 291-300. DOI:10.1016/j.compositesb.2006.06.014 3. M. Leitgeb, K. Heržič, G. Hojnik Podrepšek, A. Hojski, A. Crnjac, Ž. Knez, Acta Chim. Slov. 2014, 61, 145-152. 4. S. Kovačič, N. B. Matsko, G. Ferk, C. Slugovc, Acta Chim. Slov. 2014, 61, 208-214. 5. B. Dariš, P. Ferk, E. Markočič, Ž. Knez, Acta Medi. Bio. 2016, 9, 42-48. 6. R. M. Jin, N. Sultana, S. Baba, S. Hamdan, A. F. Ismail, J. Na-nomater. 2015, 2015. DOI:10.1155/2015/357372 7. S. C. Neves, L. S. Teixeira Moreira, L. Moroni, R. L. Reis, C. A. Van Blitterswijk, N. M. Alves, M. Karperien, J. F. Mano, Biomaterials. 2011, 32, 1068-1079. DOI:10.1016/j.biomaterials.2010.09.073 8. M. Whitaker, R. Quirk, S. Howdle, K. Shakesheff, J. Pharm. Pharmacol. 2001, 53, 1427-1437. DOI: 10.1211/0022357011777963 9. M. Škerget, Ž. Knez, Acta Chim. Slov. 2007, 54, 688-692. 10. E. Markočič, M. Škerget, Ž. Knez, Ind. Eng. Chem. Res. 2013, 52, 15594-15601. DOI:10.1021/ie402256a 11. S. M. Howdle, M. S. Watson, M. J. Whitaker, V. K. Popov, M. C. Davies, F. S. Mandel, J. D. Wang, K. M. Shakesheff, Chem. Commun. 2001, 109-110. DOI:10.1039/b008188o 12. A. Salerno, J. Saurina, C. Domingo, Int. J. Pharm. 2015, 496, 654-663. DOI:10.1016/j.ijpharm.2015.11.012 13. G. Kravanja, M. K. Hrnčič, M. Škerget, Ž. Knez, J. Supercrit. Fluids. 2016, 108, 45-55. DOI:10.1016/j.supflu.2015.10.013 14. J. Yang, G. Shi, J. Bei, S. Wang, Y. Cao, Q. Shang, G. Yang, W. Wang, J. Biomed. Mater. Res. 2002, 62, 438-446. DOI:10.1002/jbm.10318 15. A. R. C. Duarte, J. F. Mano, R. L. Reis, Polym. Degrad. Stab. 2010, 95, 2110-2117. DOI:10.1016/j.polymdegradstab.2010.06.020 16. E. de Paz, A. n. Martin, S. Rodríguez-Rojo, J.Herreras, M. a. J.Cocero, J. Chem. Eng. Data. 2010, 55, 2781-2785. DOI:10.1021/je900997t 17. Kruger, N. J. in The protein protocols handbook, Springer, 2009, pp. 17-24. DOI:10.1007/978-1-59745-198-7_4 18. H. She, X. Xiao, R.Liu, J. Mater. Sci. 2007, 42, 8113-8119. DOI:10.1007/s10853-007-1706-7 19. H. Wu, Y. Wan, X. Cao, S. Dalai, S. Wang, S. Zhang, Matter. Lett. 2008, 62, 2733-2736. DOI:10.1016/j.matlet.2008.01.029 20. V. N. Malheiro, S. G. Caridade, N. M. Alves, J. F. Mano, Acta Biomater. 2010, 6, 418-428. DOI:10.1016/j.actbio.2009.07.012 21. L. J. M. Jacobs, M. F. Kemmere, J. T. F. Keurentjes, Green Chem. 2008, 10.7, 731-738. DOI:10.1039/b801895b 22. L. J. White, V Hutter, H. Tai, S. M. Howdle, K. M. Shakesheff, Acta Biomater. 2012, 8.1, 61-71. DOI:10.1016/j.actbio.2011.07.032 23. A. Salerno, S. Zeppetelli, E. Di Maio, S. Iannace, P. A. Netti, Macromol. Rapid Commun. 2011, 32(15), 1150-1156. DOI:10.1002/marc.201100119 24. M. A. Fanovich, J. Ivanovic, D. Misie M. V. Alvarez, P. Jaeger, I. Zizovic, R. Eggers, The J. Supercrit. Fluids. 2013, 78, 42-53. DOI:10.1016/j.supflu.2013.03.017 25. L. D. Gomez, A. Concheiro. C. A. Lorenzo, C. A. G. González, Carbohydr. Polym. 2016, 142, 282-292. DOI:10.1016/j.ijpharm.2017.05.038 26. K. Zhang, Y. Wang, J. Jiang, X. Wang, J. Hou, S. Sun, Q. Li. J. Mater. Sci. 2019, 54, pp 5112-5126. DOI: 1https://doi.org/10.1007/s10853-018-3166-7 27. H. Nie, S. T. Khew, L. Y. Lee, K. L. Poh, Y. W. Tong, C.-H. Wang, J. Controlled Release. 2009, 138, 64-70. DOI:10.1016/j.jconrel.2009.04.027 28. D. D. Hile, M. L. Amirpour, A. Akgerman, M. V. Pishko, J. Controlled Release. 2000, 66, 177-185. DOI:10.1016/S0168-3659(99)00268-0 29. S. B. Lee, Y. H. Kim, M. S. Chong, S. H. Hong, Y. M.Lee, Biomaterials. 2005, 26, 1961-1968. DOI:10.1016/j.biomaterials.2004.06.032 Povzetek Z namenom študija sproščanja proteina BSA iz kompozitnega materiala, smo pripravili s superkritičnim CO2 porozne pene iz polikaprolaktona in hitozana, ki bi bile potencialno uporabne v tkivnem inženirstvu. Emulzijo, ki sestoji iz polimera, topila in puferne raztopine s proteinom smo nasitili s superkritičnim CO2 pri 12 MPa in 37 °C in nato z odprtjem izhodnega ventila hitro zmanjšali tlak, ki je povzročil nukleacijo in nastanek poroznega kompozitnega materiala. Kontrolirano sproščanje proteina iz biorazgradljivih pen polikaprolaktona in 5 % hitozana (w/w) smo merili z Bradfordovo metodo za določevanje proteinov. Po 16 do 20 dneh sproščanja se je skupno izločilo iz kompozita s PCL (Mw=10,000 g/ mol) 58.8 % proteina in iz kompozita s PCL (Mw = 60,000 g/mol) 43 %. Preliminarno smo izvedli študije za karakterizaci-jo pripravljenih kompozitnih biomaterialov z uporabo FTIR spektrov, ESEM foto analize in DSC analize. Kravanja et al.: Protein Release from Biodegradable ... DOI: 10.17344/acsi.2018.4841 Acta Chim. Slov. 2019, 66, 344-350 ^creative ^commons Scientific paper Synthesis and Biological Evaluation of Some Novel S-ß-D-Glucosides of 4-Amino-5-alkyl-1,2,4-triazole-3-thiones Derivatives Anila Rahimi Aghkand,1 Karim Akbari Dilmaghani,1'* Zahra Dono Ghezelbash1 and Behvar Asghari2 1 Department of Organic Chemistry, Faculty of Chemistry, Urmia University, Urmia, 57159, Iran 2 Department of Horticultural Sciences Engineering, Faculty of Agriculture and Natural Resources, Imam Khomeini International University, Qazvin, Iran * Corresponding author: E-mail: k.adilmaghani@urmia.ac.ir Tel: (+98)914-443-1392. Fax: (+98)44-357153-165 Received: 11-10-2018 Abstract A novel series of 3-S-ß-D-glucosides-4-arylideneamino-5-alkyl-1,2,4-triazoles were designed and synthesized by reaction of 4-amino-5-alkyl-4H-1,2,4triazole-3-thiol Schiff bases and 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide. The structures of the target compounds have been characterized by 'H NMR, 13C NMR, FT-IR, and Microanalyses. All the newly synthesized compounds have been screened for their in vitro antibacterial and antifungal activities against two Gram-positive bacteria [Bacillus cereus (PTCC 1015) and Staphylococcus aureus (ATCC 25923)], two Gram-negative bacteria [Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (PTCC 1399) and two fungi [Aspergillus niger (PTCC 5012) and Candida albicans (PTCC 5027)]. Keywords: 4-amino-5-alkyl-4H-1,2,4-triazole-3-thiol; acetobromoglucose; thiocarbohydrazide; antimicrobial activity; antifungal activity; schiff base 1. Introduction Antibiotics are drugs used for treating infection caused by microorganism such as bacteria or fungi and antibiotic resistance is the ability of microorganism to stand the effect of antibiotic. The resistance of infective bacteria to present antibiotics remains a clinical obstacle in the chemotrapy of many cancers to overcome the rapid development of drug resistance, new agents should preferably have chemical characteristics that clearly differ from those of existing agents. Thus led to the design and synthesize the new antimicrobial agents. 1,2,4-Triazole and its derivatives are an important class of compounds which possess diverse biological activities including an-ti-microbial,1,2 antibacterial,3,4 antifungal,5,6 anti-inflam-matory,7 insecticidal,8 anticonvulsant,9,10 antitumor activity,11 anti HIV activity,12,13 hypoglycemic14 and anti-convulsant.15,16 Triadimefon and fluconazole which exhibits excellent fungicidal activities possessing 1,2,4-tri- azole nucleus.17,18 Compounds containing an azomethine group known as Schiff bases and are formed by the condensation of a primary amine with a carbonyl compound. Schiff bases attract much interest due to their synthetic availability along with antibacterial19-22 and antitumor23 properties. The synthesis and investigation of biological activity of 1,2,4-triazole glycosides have been stimulated by the finding that Ribavirin, ^-D-ribofuranosyl-1,2,4-triazole-3-car-boxamide, as potent drug against DNA and RNA viruses.24 Moreover, sulfur-containing heterocycles represent an important group of sulfur compounds that are promising for use in practical applications. Therefore, it is interesting to report the synthesis of a new series of compounds in which the glycosyl moieties have been used as carriers for the heterocycles having the oxadiazole or triazole ring. It has been reported that the attachment of carbohydrate moieties to the 1,2,4-triazole nucleus through a thioglyco-sidic linkage enhances its antimicrobial activity.25 Some novel S-^-D-glucosides of 5-aryl-1,2,4-tri-azole-3-thiones derivatives exhibited antibacterial and an- Aghkand et al.: Synthesis and Biological Evaluation Acta Chim. Slov. 2019, 66, 344-350 345 tifungal activities.26 In our previous work,27,28 we reported the synthesis and antibacterial properties of new series of thioglycoside derivatives of 1,2,4-triazole moieties. The above facts and our interest on design of potent antibacterial agents with 1,3,4-oxadiazoles and 1,2,4-tri-azole moieties, promoted us to synthesis novel substituted thioglycosides by reaction of a-D acetobromoglucose (6) with 4-amino-5-alkyl-4H-1,2,4-triazole-3-thiol Schiff bases 5(a-g). The newly synthesized multicompounds 7(a-g) are useful in probing biological activity such as antibacterial and antifungal effects. 1. 1. Antimicrobial Activity 1. 1. 1. Bacterial and Fungal Strains Two Gram-positive bacteria Bacillus cereus (PTCC 1015) and Staphylococcus aureus (ATCC 25923)], two Gram-negative bacteria [Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (PTCC 1399) and two fungi Aspergillus niger (PTCC 5012) and Candida albicans (PTCC 5027)] were used in antimicrobial assays. Micro-bial strains were obtained from the Pasteur Institute of Iran. 1. 2. Determination of Inhibition Zone The agar disc diffusion method was used for antimicrobial activity determination of the compounds using a previously described standard method.29 1 00 ^l of the tested microorganisms suspensions, adjusted to 106-108 CFU/mL were spread on the solid media plates. The compounds were dissolved in dimethyl sulphoxide and filtered by 0.45 ^m Millipore filters for sterilization. The paper discs (6 mm in diameter) werm saturated with 10 ^l of sample solution and placed on the inoculated agar. DMSO was used as the untreated control. These plates were incubated for 24 h at 37 °C for bacterial strains and 48 h at 30 °C for the yeasts. The diameter of inhibition zone (IZ) was measured in mm (including 6 mm diameter of paper disc). Gentamicin (10 ^g/disc) and nystatin (50 IU) were used as positive control in antibacterial and antifungal assays, respectively. Analyses were performed in triplicate and expressed as average values ± SEM. 1. 3. Determination of Minimum Inhibitory Concentrations (MICs) The MIC values of the compounds were determined using the method of micro-well dilution assay.30-31 Briefly, the inoculants of the microbial strains were prepared from freshly cultured microorganisms that were adjusted to 0.5 McFarland standard turbidity. Serial dilutions of the compounds and standard samples were made in a concentration range from 5 to 1000 ^g/mL in 96-well plates, containing Mueller-Hinton broth for bacterial strains and Sabouraud dextrose broth for yeast. Gentamicin and ny-statin were used as standard drugs in identical conditions to test materials. The plates were covered with sterile plate sealers and then incubated at 37 °C under normal atmospheric condition for 24 h for bacterial strains and at 30 °C for 48 h for yeasts. The MIC values were considered as the minimum concentration of the sample which could inhibit the growth of microorganisms. 2. Result and Discussion 4-amino-5-alkyl-4H-1,2,4-triazole-3-thioles 3(a-c) were prepared by the condensation of aliphatic carboxylic acids 2(a-c) with thiocarbohydrazide. The reaction is improved by using carboxylic acids at thiocarbohydrazide melting point. This reaction is the selective method for the preparation of 4-amino-5-aryl/alkyl-4H-1,2,4-triazole-3-thioles32 (Scheme 1). h2n-xta nh-z n n h h (1) O 2 + r-\>h" 2(a-c) Reflux n-nh rV> nh2 3(a-c) N^NH + R'cho reflux. R^n^S N {' 4(d-f) R. 5(a-g) OAc K2CO3 / Dry Aceton r- OAC Br-^T^OAc OAc (6) n-n n f R' 7(a-g) O OAc OAc OAc 5a,7a: R=Me(a), R'= 5b,7b: R=Me(a), R'= 5c,7c: R=Me(a), R'= 5d,7d: R=Et(b), R'= 5e,7e: R=Et(b), R'= 5f,7f: R=Et(b), R'= 5g,7g: R=Pr(c), R'= Pyridin-4-yl (d) Pyridin-3-yl (e) Pyridin-2-yl (f) Pyridin-4-yl (d) Pyridin-3-yl (e) Pyridin-2-yl (f) Pyridin-3-yl (e) Schemel. Synthesis of 3-S-ß-D-glucosides-4-arylideneamino-5-alkyl-1,2,4-triazoles. Aghkand et al.: Synthesis and Biological Evaluation 346 Acta Chim. Slov. 2019, 66, 344-350 Tablel. Diameter of inhibition zone (IZ) values of the compounds against 2 gram (+) bacteria, 2 gram (-) bacteria and 2 fungi (^g/mL) 5a 7a 5b 7b 5c 7c 5d 7d 5e 7e 5f 7f 5g 7g Gentamicin Nystatin Escherichia coli gram(-) Pseudomonas aeruginosa gram(-) Staphylococcus aureus gram(+) Bacillus cereus gram(+) Aspergillus niger fungi Candida albicans fungi 6.7 6.6 6.3 6.7 6.5 6.1 6.5 6.7 12.4 6.3 14.3 6.4 11.4 - 13.1 6.5 9.1 8.4 7.2 6.7 9.3 6.4 14.7 7.1 15.6 6.5 9.8 6.2 13.9 6.6 8.2 - 10.7 - 9.5 7.1 15.7 8.7 13.4 7.5 11.2 6.4 10.2 8.2 12.1 7.3 10.1 6.8 9.7 6.3 17.5 7.9 14.1 7.3 10.1 6.3 14.2 8.7 12.4 6.6 12.7 6.4 11.3 23.4 27.3 28.5 31.6 22.6 26.8 Schiff bases 5(a-g) have been synthesized by a reaction of 3-alkyl-4-amino-1,2,4-triazole-5-thione 3(a-c) with pyridine aldehydes 4(d-f) in absolute ethanol as solvent in presence of glacial acetic acid as a catalyst. The IR spectra of the compounds 3(a-c) showed the absorptions at 3100, 1330 cm-1 which were attributed to N-H and C=S stretching vibration and a strong absorption at 1580 cm-1 which was assigned to the C=N stretching vibration. The 1H NMR spectra showed a singlet signal at about 10 ppm due to (CH=N) and the absence of the chemical shift of 4-NH2 in the spectra of 3(a-c) proving that the title Schiff bases 5(a-g) were formed. In our attempt to obtain a-acetobromoglucose (6) at the first step D-glucose were treated with acetic anhydride in pyridine at room temperature gave 1,2,3,4,6-penta-O-acetyl-8-glucose and the anomeric bromination of this compound with hydrogen bromide in acetic acid gave 2,3,4,6-tetra-O-acetyl-a-glucopyranosyl bromide (6). The existence of thiol-thione tautomerism is known for the compounds 3(a-c) and generally one forms is pre-dominant.34-37 3-S-^-D-glucosides-4-arylideneamino-5-alkyl-1,2,4 -triazoles 7(a-g) were synthesized by the reaction of 3-al-kyl-4-amino-1,2,4-triazole-5-thione Schiff bases 5(a-g) and the peracetylated ^-pyranosyl bromide (6) in the presence of potassium carbonate as a weak base in dry acetone. (Scheme 1). Anomeric ^-configurations of the S-linked glycosides 7(a-g) were supported by their 1H NMR data. The chemical shifts of the anomeric proton signals of thiogly-cosides revealed around 5 (6.20) with a large coupling constant J12 values of (9.3) Hz which consistent with the reported data for S-^-D glycosides. The result of the antibacterial and antifungal activity shows that all the compounds have lesser activity than corresponding standard compounds and the target com- pounds exhibited better antifungal activity than anti-bacterial activity. However the anti-bacterial results showed that against Staphylococcus aureus compounds (7b) and (7d); against Bacillus cereus compounds (7b), (7a) and (7d); have comparable activity with Gentamicin as Standard. The antifungal study results revealed that against Aspergillus niger compounds (7a) (7b) and (7e); against Candida albicans (7a), (7b) and (7d) have comparable activity with Nystatin as standard. Though compound (7b) was found to have the highest activity against Bacillus cereus and compound (7a) was found to have the highest activity against Candida albicans among all the tested compounds. 3. Experimental 3. 1. General The melting points of all compounds were recorded on a Philip Harris C4954718 apparatus without calibra-tion.IR and 1H- and 13C-NMR spectra were recorded on Thermo Nicolet Nexus 670 FT-IR and Bruker Avance 300 MHz spectrometers, respectively. Thin layer chromatogra-phy (TLC) analyses were carried out on silica gel plates. All chemicals were purchased from Merck (Tehran, Iran) and used as received by standard procedures. All of the instruments, chemicals and solvents were dried according to standard methods. Freshly distilled solvents were used throughout, and anhydrous solvents were dried according to the method reported by Perrin and Armarego.38 Microanalyses were performed on a Leco Analyzer 932. 3. 2. General Procedure of Synthesis of 4-amino-5-alkyl-4H-1,2,4-triazole-3-thiol 3(a-c): Aghkand et al.: Synthesis and Biological Evaluation Acta Chim. Slov. 2019, 66, 344-350 347 A mixture of thiocarbohydrazide (0.01 mol) and car-boxylic acid (0.01 mol) was heated until it melted. The mixture was consistently refluxed for 40 min. The product obtained on cooling was treated with a sodium bicarbonate solution to neutralize the unreacted acid if any. The product was then washed with water and collected by filtration. The solid product was recrystallized from a distilled water.32 General Procedure for Synthesis of Schiff bases of (E) -5-alkyl-4-((pyridin-3-ylmethylene) amino)-2,4-dihy-dro-3H-1,2,4-triazole-3-thione 5(a-g): To a suspension of substituted pyridine carboxalde-hyde 4(d-f) (0.2 mol) in ethanol (1 mL), an equimolar amount of corresponding 4-amino-5-alkyl-4H-1,2,4-tri-azole-3-thiol (0.1mol) 3(a-c) was added. The suspension was heated until a clear solution was obtained. Then few drops of concentrated sulfuric acid were added, and the solution was heated for 6 hrs on a water bath, the precipitate solid was filtered off and recrystallized from in etha- nol.33 General Procedure for Synthesis of 2,3,4,6-tetra-O-ace-tyl-a-D-glucopyranosyl bromide (6): It was also prepared according to the literature procedure.39 General Procedure for Synthesis of 3-S-ß-D-gluco-sides-4-arylideneamino-5-alkyl-1,2,4-triazoles (7a-g): A mixture of compound 5(a-g) (1 mmol) and potassium carbonate (1 mmol, 0.138 g) in dry acetone (25 mL) and 2 drops of Dimethylformamide were stirred for 1 hrs, then glycosyl bromide (6) (1.2 mmol, 0.410 g) was added. Stirring was continued overnight, and then the reaction mixture was heated under reflux for 2-4 hrs. After cooling, the mixture was filtered, then the precipitate of the compound was submitted to column chromatography (SiO2, EtOAc/hexane, 3:8).27 (E)-5-methyl-4-((pyridin-4-ylmethylene) amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5a): (Yellow crystals), (Yield% = 74) (0.32 g) m.p: 267269 °C, FT-IR (KBr, u cm-1): 3042 (C-H, Ar), 2839 (CH3), 1592 (C=N), 1278 (C=S) cm-1, 1H NMR (300 MHz, DMSO): 6 (ppm), 2.36 (s, 3H, CH3), 7.82 (d, /=5.1 Hz, 2H Pyridin) 8.75 (d, /=5.1Hz, 2H Pyridin) 10.32 (s, 1H, HC=N) 13.75 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 6 (ppm), 11.15, 122.32, 139.97, 149.27, 151.38, 161.78, 162.33 (E)-5-methyl-4-((pyridine-3-ylmethylene) amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5b): (Light Yellow crystals) (Yield% = 76) (0.83 gr), m.p: 239-241°C, FT-IR (KBr, u cm-1: 3061 (C-H Ar), 2852 (CH3), 1590 (C=N), 1274 (C=S) cm-1, 1H NMR (300 MHz, DMSO): 6 (ppm), 2.35 (s, 3H, CH3), 7.53-7.57 (m, 1H, pyridine), 8.29 (d, /=7.8 Hz, 1H pyridine) 8.73 (d, /=4.5 Hz, 1H pyridine) 9 (s, 1H, pyridine) 10.19 (s, 1H, HC=N) 13.79 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 5 (ppm), 11.17, 124.71, 128.75, 135.34, 135.40, 149.05, 150.38, 153.36, 161.63. (E)-5-methyl-4-((pyridin-2-ylmethylene) amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione) (5c): (Pale green crystals), m.p :194-196 °C (Yield% = 82) (0.18 g) FT-IR (KBr, u cm-1): 3104 (NH), 3066 (C-H Ar) 2934 (CH3), 1586 (C=N), 1287 (C=S) cm-1, 1H NMR (300 MHz, DMSO): 5 (ppm), 2.37 (s, 3H,CH3) 7.56 (t, /=3.9, 1H, pyridine) 7.83 (t, /=7.5, 1H, pyridine) 8.14 (d, /=5.4 1H, Pyridine) 8.25 (d, /=4.8, 1H, Pyridin) 10.35 (s, 1H, HC=N) 13.79 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 5 (ppm), 10.87, 120.66, 127.64, 136.54, 138.66, 149.20, 151.44, 160.23, 161.74. (E)-5-ethyl-4-((pyridin-4-ylmethylene) amino)-2,4-di-hydro-3H-1,2,4-triazole-3-thione) (5d): (Yellow crystals), (Yield% = 73) (0.17 g) m.p: 246248 °C, FT-IR (KBr, u cm-1): 3041 (C-H Ar), 2973 (Et), 1591 (C=N), 1268 (C=S) cm-1. 1H NMR (300 MHz, DMSO): 5 (ppm), 1.22(t, /=7.2, 3H, CH3), 2.73-2.81(m, 2H, CH2) 7.82 (d, /= 4.5 Hz, 2H pyridine) 8.76 (d, /=4.5 Hz, 2H pyridine) 10.34 (s, 1H, HC=N) 13.85 (s, 1H, NH).13C-NMR (75 MHz, CDCl3): 5 (ppm), 11.34, 18.65, 121.23, 140.03, 150.21, 153.14, 160.71, 161.93. (5e): ( (E)-5-ethyl-4-((pyridin-3-ylmethylene) amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (Yellow crystals), m.p:188-190 °C, (Yield% = 72) (0.08 g), FT-IR (KBr, u cm-1): 3097 (NH), 3048 (CH-Ar), 2975 (Et), 1583 (C=N), 1278 (C=S) cm-1. 1H NMR (300 MHz, DMSO): 5 (ppm), 1.22(t, /=7.2, 3H, CH3), 2.72-2.80 (m, 2H, CH2) 7.56-7.60 (m, 1H, pyridine), 8.31 (d, /=6.6 Hz, 1H Pyridine) 8.76 (d, /=4.5 Hz, 1H pyridine) 9.02 (s, 1H, pyridine) 10.19 (s, 1H, HC=N) 14.81(s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 5 (ppm), 9.73, 18.66, 125.79, 128.78, 134.38, 136.47, 151.58, 152.01, 159.53, 161.82. (E)-5-ethyl-4-((pyridin-2-ylmethylene) amino)-2,4-di-hydro-3H-1,2,4-triazole-3-thione (5f): (Brown crystals), m.p: 168-170 °C, (Yield% = 86) (0.2 g), FT-IR (KBr, u cm-1): 3119 (NH), 3068 (C-H Ar), 2918 (Et), 1579 (C=N), 1285 (C=S) cm-1. 1H NMR (300 MHz, DMSO): 5 (ppm), 1.23 (t, /=7.5, 3H, CH3) 2.74-2.81 (m, 2H, CH2) 7.56 (t, /=6.6, 1H, pyridine) 7.97 (t, /=6.9, 1H, pyridine) 8.13 (d, /=8.1, 1H, Pyridin) 8.73 (d, /=4.2, 1H, Pyridin) 10.35 (s, 1H, HC=N) 13.83 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 5 (ppm), 11.33, 18.71, 120.71, 122.88, 127.77, 136.74, 138.88, 149.57, 159.87, 162.02. (E)-5-propyl-4-((pyridin-3-ylmethylene) amino)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5g): (Yellow crystals), (Yield% = 77) (0.38 gr) m.p:166-168°C, FT-IR (KBr, u cm-1): 3057 (CH-Ar), 1586 (C=N), Aghkand et al.: Synthesis and Biological Evaluation 348 Acta Chim. Slov. 2019, 66, 344-350 1278 (C=S) cm-1. XH NMR (300 MHz, DMSO): 5 (ppm), 0.94 (t, /=7.5, 3H, CH3), 1.63-1.72 (m, 2H, CH2) 2.73 (t, /=7.5,2H, CH2) 7.56-7.60 (m, 1H, pyridine), 8.31 (d, /=6.3 Hz, 1H pyridine) 8.76 (d, /=4.8 Hz, 1H pyridine) 9.02 (s, 1H, pyridine) 10.19 (s, 1H, HC=N) 13.81 (s, 1H, NH). 13C-NMR (75 MHz, CDCl3): 5 (ppm), 13.87, 19.34, 26.75, 124.79, 128.78, 135.40, 151.72, 153.44, 159.66, 161.85, 162.33. Synthesis of 1,2,3,4,6-penta-O-acetyl-a-D-glucopyra-nose: It was also prepared according to the literature procedure.36 (Yield% = 68) (7.5 gr)(White precipitate), FT-IR (KBr, u cm-1):1748, 1374, 1227, 1H NMR (300 MHz, CDCl3), 5: 2.01 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.08 (s, 3H, OAc), 2.11 (s, 3H, OAc), 2.18 (s, 3H, OAc), 3.82-3.85 (m, 1H, H-5), 4.08-4.12 (m, 1H, H-6a), 4.26-4.32 (m, 1H, H-6b), 5.09-5.28 (m, 3H, H-2, H-4, H-3), 5.71 (d, 1H, /1,2 = 8.4, H-1). 13C NMR (75 MHz, CDCl3), 5: 20.55 (3C), 20.69, 20.80 (5 x OCOCH3), 61.41 (C-6), 67.70 (C-4), 70.18 (C-2), 72.69 (C-3), 72.75 (C-5), 91.66 (C-1), 168.94, 169.23, 169.37, 170.08, 170.59 (5 x OCOCH3). Synthesis of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide (6): It was also prepared according to the literature procedure.39 White precipitate; 57% (1.2 g); FT-IR (KBr, u cm-1): 1745, 1377, 1236, 607. 1H NMR (300 MHz, CDCl3), 5: 2.03 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.10 (s, 6H, OAc), 4.12 (d, 1H, H-6a), 4.28-4.36 (m, 2H, H-6b, H-5), 4.81-4.86 (dd, 1H, /1,2 = 3.9, /2,3 = 9.9, H-2), 5.16 (t, 1H, H-4), 5.56 (t, 1H, H-3), 6.61 (d, 1H, /1,2 = 3.9, H-1). 13C NMR (75 MHz, CDCl3), 5: 20.53, 20.60 (2C), 20.63 (4 x OCOCH3), 60.93 (C-6), 67.15 (C-4), 70.14 (C-2), 70.58 (C-3), 72.12 (C-5), 86.54 (C-1), 169.44, 169.77, 169.82, 170.48 (4 x OCOCH3). Synthesis of 4-(pyridine-4-yl methylene-amino) -5-methyl-2-yl-3- (2,3,4,6-tetra-O- acetyl-0-D-glucopy-ranosyl Sulfonyl) -1,2,4-triazole (7a): yellow crystals 76% (1.24 g), m.p :74-76 °C; FT-IR (KBr, o cm-1): 2947 (C-H), 1753 (C=O), 1601 (HC=N), 1371 (CH3), 1047, 1230 (C-O), 1H NMR (300 MHz, CDCl3): 5 1.94 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.49 (s, 3H, CH3), 3.97-4 (m, 1H, H-6a), 4.15 (d, /=12.3 Hz, 1H, H-6b), 4.28-4.34 (m, 1H, H-5), 5.25 (t, /=9.9Hz, 1H, H-4), 5.42 (t, /=9.3 Hz, 1H, H-2), 5.74 (t, /=9.3 Hz, 1H, H-3), 6.21 (d, /=9.3 Hz, 1H, H-1), 7.70 (d, /= 4.8 Hz, 2H pyridine) 8.78 (d, /=4.5 Hz, 2H pyridine) 10.83 (s, 1H, HC=N). 13C-NMR (75 MHz, CDCl3): 5(PPM), 11.22, 20.57, 61.67, 67.73, 69.14, 73.61, 74.59, 81.28, 121.85, 139.95, 149.30, 150.65, 162.33, 164.04, 168.89, 169.35, 170.15, 170.61. Calcd: C, 50.27; H, 4.95; N, 12.74; S, 5.83%; Found: C, 50.37; H, 4.85; N, 12.94; S, 5.92%. Synthesis of 4-(pyridine-3-yl methylene-amino) -5-methyl-2-yl-3- (2,3,4,6-tetra-O-acetyl-^-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7b): White crystals 73% (0.8 g), m.p: 68-70 °C; FT-IR (KBr, u cm-1): 2958 (C-H), 1752 (C=O), 1599 (HC=N), 1370 (CH3), 1046, 1231 (C-O).1H NMR (300 MHz, CDCl3): 5 1.93 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.05 (s, 3H, OAc), 2.07 (s, 3H, OAc), 2.46 (s, 3H, CH3), 3.96-4 (m, 1H, H-6a), 4.06-4.16 (m, 1H, H-6b), 4.27-4.32 (m, 1H, H-5), 5.24 (t, /=9.9 Hz, 1H, H-4), 5.40 (t, /=9.6 Hz, 1H, H-2), 5.72 (t, /=9.3 Hz, 1H, H-3), 6.20 (d, /=9.3 Hz, 1H, H-1), 7.40-7.44 (m, 1H, pyridine), 8.19 (d, /=7.8 Hz, 1H pyridine) 8.74 (d, /=3.9 Hz, 1H pyridine) 8.99 (s, 1H, pyridine) 10.68 (s, 1H, HC=N). 13C-NMR (75 MHz, CDCl3): 5 (PPM), 11.27, 20.60, 61.64, 67.66, 69.10, 73.58, 74.50, 81.29, 123.92, 128.63, 134.80, 149.22, 152.94, 157.59, 163.90, 168.94, 169.38, 170.17, 170.65. Calcd: C, 50.27; H, 4.95; N, 12.74; S, 5.83 %; Found: C, 50.17; H, 4.82; N, 12.86; S, 5.98%. (Synthesis of 4-(pyridine-2-yl methylene-amino) -5-meth-yl-2-yl-3- (2,3,4,6-tetra-O-acetyl-ß-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7c): White crystals 76% (1.24 g), m.p: 105-107 °C; FT-IR (KBr, u cm-1): 2948 (C-HAr), 1752 (C=O), 1590 (HC=N), 1373 (CH3), 1045, 1234 (C-O), (C-1H NMR (300 MHz, CDCl3): 5 1.94 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.09 (s, 3H, OAc), 2.48 (s, 3H, CH3) 3.96-4.01 (m, 1H, H-6a), 4.12 (d, /=7.2 Hz, 1H, H-6b), 4.28-4.34 (m, 1H, H-5), 5.25 (t, /=9.9 Hz, 1H, H-4), 5.41 (t, /=9.3 Hz, 1H, H-2), 5.73 (t, /=9.3 Hz, 1H, H-3), 6.23 (d, /=9.3 Hz, 1H, H-1), 7.40-7.44 (m,1H, Pyridin) 7.83 (t, /=7.5 Hz, 1H, Pyridin) 8.09 (d, /=7.8 Hz, 1H, Pyridin) 8.75 (d, /=4.8 Hz, 1H, Pyridin), 10.63 (s, 1H, CH=N). 13C-NMR (75 MHz, CDCl3): 5(PPM), 11.26, 20.58, 61.70, 67.75, 69.20, 73.59, 74.52, 81.37, 121.96, 125.91, 136.74, 149.13, 150.15, 151.92, 162.33, 164.12, 168.97, 169.39, 170.13, 170.65. Calcd: C, 50.27; H, 4.95; N, 12.74; S, 5.83%; Found: C, 50.17; H, 4.82; N, 12.86; S, 5.98%. Synthesis of 4-(pyridine-4-yl methylene-amino) -5-eth-yl-2-yl-3- (2,3,4,6-tetra-O-acetyl-ß-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7d): Orange crystals 77% (0.085 g ), m.p: 59-61 °C; FT-IR (KBr, u cm-1): 2934 (C-H), 1754 (C=O), 1594 (HC=N), 1372 (CH3), 1042, 1228 (C-O), 1H NMR (300 MHz, CDCl3): 5 1.92 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.35 (t, /=7.2Hz, 3H, CH3), 2.83-2.87 (m, 2H, CH2) 3.97-3.99 (m, 1H, H-6a), 4.16 (d, /=12 Hz, 1H, H-6b), 4.28-4.33 (m, 1H, H-5), 5.26 (t, /=9.6 Hz, 1H, H-4), 5.41 (t, /=9.6 Hz, 1H, H-2), 5.79 (t, /=9 Hz, 1H, H-3), 6.17 (d, /=9.3 Hz, 1H, H-1),7.69 (d, /=4.5 Hz, 2H pyridine), 8.77 (d, /= 3.9 Hz, 2H pyridine) 10.81 (s, 1H, HC=N), 13C-NMR (75 MHz, CDCl3): 5(PPM), 10.11, 18.92, 20.60, 61.64, 67.71, 68.98, 73.65, 74.50, 81.37, 121.86, 140.03, 150.64, 153.15, 155.90, 164.11, 168.76, 169.40, 170.20, 170.66. Calcd: C, 51.15; H, 5.19; N, 12.43; S, 5.69%; Found: C, 51.01; H, 5.21; N, 12.53; S, 5.73%. Aghkand et al.: Synthesis and Biological Evaluation Acta Chim. Slov. 2019, 66, 344-350 349 Synthesis of 4-(pyridine-3-yl methylene-amino)-5-eth-yl-2-yl-3-(2,3,4,6-tetra-O-acetyl-ß-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7e): White crystals, 78% (0.43 g), m.p: 105-107 °C, FT-IR (KBr, u cm-1): 2947 (C-H), 1753 (C=O), 1591 (HC=N), 1371 (CH3), 1042, 1229 (C-O) ,1H NMR (300 MHz, CDCl3): 5 1.92 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.34 (t, /=7.2Hz, 3H, CH3), 2.792.88 (m, 2H, CH2), 3.97-4.01 (m, 1H, H-6a), 4.17(d, /=12.6, 1H, H-6b), 4.28-4.33 (m, 1H, H-5), 5.26 (t, /=9.9 Hz, 1H, H-4), 5.41 (t, /=9.3 Hz, 1H, H-2), 5.79 (t, /=9.3 Hz, 1H, H-3), 6.18 (d, /=9.3 Hz, 1H, H-1),7.41-7.45 (m, 1H, pyridine), 8.18 (d, /=7.8 Hz, 1H pyridine) 8.75 (d, /=4.5 Hz, 1H pyridine) 9.01 (s, 1H, pyridine) 10.69 (s, 1H, HC=N). 13C-NMR (75 MHz, CDCl3): 5 (PPM), 10.11, 18.93, 20.60, 61.65, 67.71, 68.99, 73.66, 74.46, 81.44, 123.92, 128.70, 134.79, 149.81, 153.05, 158.26, 164.02, 168.76, 169.40, 170.18, 170.66. Calcd: C, 51.15; H, 5.19; N, 12.43; S, 5.69%; Found: C, 51.07; H, 5.21; N, 12.60; S, 5.61%. Synthesis of 4-(pyridine-2-yl methylene-amino) -5-eth-yl-2-yl-3- (2,3,4,6-tetraO-acetyl-ß-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7f): yellow crystals 79% (0.87 g), m.p :55-57 °C; FT-IR (KBr, u cm-1): 2974 (C-H), 1753 (C=O), 1586 (HC=N), 1372 (CH3), 1044, 1229 (C-O). 1H NMR (300 MHz, CDCl3): 5 1.92 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.33 (t, /=7.2 Hz, 3H, CH3), 2.772.89 (m, 2H, CH2), 3.96-4 (m, 1H, H-6a), 4.16(d, /=11.4 Hz, 1H, H-6b), 4.27-4.33 (m, 1H, H-5), 5.25 (t, /=9.6 Hz, 1H, H-4), 5.40 (t, /=9.3 Hz, 1H, H-2), 5.77 (t, /=9.3 Hz, 1H, H-3), 6.19 (d, /=9.3 Hz, 1H, H-1), 7.39-7.43 (m,1H, pyridine) 7.81 (t, /=7.5 Hz, 1H, pyridine) 8.07 (d, /=7.8 Hz, 1H, pyridine) 8.73 (d, /=3.9 Hz, 1H, Pyridin), 10.61 (s, 1H, CH=N). 13C-NMR (75 MHz, CDCl3): 5(PPM), 10.10, 18.92, 20.56, 61.70, 67.82, 69.09, 73.68, 74.48, 81.52, 121.79, 125.84, 136.70, 150.18, 152.05, 152.95, 162.33, 164.27, 168.76, 169.38, 170.11, 170.63. Calcd: C, 51.15; H, 5.19; N, 12.43; S, 5.69%; Found: C, 51.25; H, 5.28; N, 12.33; S, 5.72%. Synthesis of 4-(pyridine-3-yl methylene-amino) -5-pro-pyl-2-yl-3- (2,3,4,6-tetra-O-acetyl-ß-D-gluco pyranosyl Sulfonyl) -1,2,4-triazole (7g): White crystals; 81% (0.93 g), m.p: 63-65 °C; FT-IR (KBr, u cm-1): 2962 (C-H), 1754 (C=O), 1590 (HC=N), 1369 (CH3), 1048, 1229 (C-O). 1H NMR (300 MHz, CDCl3): 5 1.92 (s, 3H, OAc), 2.03 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.08 (s, 3H, OAc), 1.01 (t, /=7.5Hz, 3H, CH3), 1.741.86 (m, 2H, CH2) 2.73-2.86 (m, 2H, CH2) 3.97-3.99 (m, 1H, H-6a), 4.13-4.19 (m, 1H, H-6b), 4.27-4.32 (m, 1H, H-5), 5.25 (t, /=9.6 Hz, 1H, H-4), 5.41 (t, /=9.3 Hz, 1H, H-2), 5.79 (t, /=9.3 Hz, 1H, H-3), 6.18 (d, /=9.6 Hz, 1H, H-1), 7.49-7.53 (m, 1H, pyridine), 8.25 (d, /=7.8 Hz, 1H pyridine) 8.77 (d, /=3.6 Hz, 1H pyridine) 9.04 (s, 1H, pyr- idine) 10.78 (s, 1H, HC=N): 13C-NMR (75 MHz, CDCl3): S(PPM), 13.49, 19.22, 20.73, 26.93, 61.66, 67.78, 69, 73.69, 74.51, 81.45, 124.35, 129.37, 135.99, 151.91, 155.24, 157.16, 162.33, 163.99, 168.69, 169.35, 170.14, 170.60. Calcd: C, 51.99; H, 5.41; N, 12.12; S, 5.55%; Found: C, 51.89; H, 5.50; N, 12.23; S, 5.42%. 4. Conclusions In summary, a series of 3-S-^-D-glucosides-4-arylideneamino-5-alkyl-1,2,4-triazoles 7(a-e) was prepared by reaction of 4-amino-5-alkyl-4H-1,2,4-triazole-3-thiol Schiff bases 5(a-f) and 2,3, 4,6-tetra-O-ace-tyl-a-D-glucopyranosyl bromide (6) in presence of potassium carbonate at room temperature for 24h and then 3h reflux. The structures of the target compounds have been characterized by 1H NMR, 13C NMR, and FTIR. All the newly synthesized compounds have been evaluated for their antimicrobial activities in vitro against two gram (+) bacteria, two gram (-) bacteria and two fungi. These compounds exhibited better antifungal activity than antibacterial activity. Though compound (7b) was found to have the highest activity against Bacillus cereus and compound (7a) was found to have the highest activity against Candida albicans among all the tested compounds. 5. 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Bahadori, LWT - Food Science and Technology. 2017, 75, 42-45. DOI:10.1016/j.lwt.2016.08.048 32. H. Beyer, C.F. Kroger, Liebigs Ann. Chem. 1960, 637, 135. DOI: 10.1002/jlac.19606370111 33. K. S. Ashok, R.N. Singh, R. N. Handa, S. N. Dubey, P. J. Squat-trito, J. Mol. Struct. 1998, 470, 61-69. DOI:10.1016/S0022-2860(98)00470-0 34. C. Guimon, G. Pfister-Guillouzo, Tetrahedron. 1974, 30, 3831-3838. DOI:10.1016/S0040-4020(01)97072-3 35. F. Malbec, R. Milcent, G. Barbier, J. Heterocycl. Chem. 1984, 21, 1689-1698. DOI:10.1002/jhet.5570210624 36. V. I. Kelarev, G. A. Shvekhgeimer, A. F. Lunin, Khim. Geterot-sikl. Soedin. 1984, 1271-1276. 37. S. Gopinathan, S. A. Pardhy, A. P. Budhkar, C. Gopinathan Synth. React. Inorg. Metal. Org. Chem. 1988, 18, 823-836. DOI: 10.1080/00945718808060824 38. D. D. Perrin, W. L. F. Armarego, Purification of Laboratory Chemicals, Pergamon Press, Oxford. 1988. 39. Vogel, I. Arthur, Vogel's Textbook of Practical Organic Chemistry. 1989, 5th ed. Povzetek Z reakcijami med 4-amino-5-alkil-4ff-1,2,4-triazol-3-tiolnimi Schiffovimi bazami in 2,3,4,6-tetra-O-acetil-a-D-glu-kopiranozil bromidom smo sintetizirali novo serijo 3-S-ß-D-glukozid-4-arilidenamino-5-alkil-1,2,4-triazolov. Strukture pripravljenih spojin smo določili z 'H NMR, 13C NMR, FT-IR in elementno mikroanalizo. Za vse novopripravljene spojine smo in vitro določili antibakterijsko in protiglivično delovanje na dve Gram pozitivni bakteriji (Bacillus cereus (PTCC 1015) in Staphylococcus aureus (ATCC 25923)), dve Gram negativni bakteriji (Pseudomonas aeruginosa (ATCC 27853) in Escherichia coli (PTCC 1399)) ter na dve glivi (Aspergillus niger (PTCC 5012) in Candida albicans (PTCC 5027)). Aghkand et al.: Synthesis and Biological Evaluation DOI: 10.17344/acsi.2018.4843 Acta Chim. Slov. 2019, 66, 351-359 ^creative ^commons Scientific paper Exploring Bikaverin as Metal Ion Biosensor: A Computational Approach Zakir Hussain,1 Haamid Rasool Bhat,2 Tahira Naqvi,3 Malay K. Rana2 and Masood Ahmad Rizvi1,* 1 Department of Chemistry, University of Kashmir, Hazratbal, Srinagar J&K, India. 2 Department of Chemical Sciences, IISER, Berhampur, Odisha, India. 3 Department of Chemistry, Degree College for Women M.A. Road Srinagar J&K, India * Corresponding author: E-mail: masoodku2@gmail.com Received: 11-12-2018 Abstract A computational exploration of fungi produced pigment bikaverin as a biosensor towards bioavailable metal ions is presented. Systematic studies of the optimized ground and excited state geometries were attempted for exploring metal ion binding pocket, comparative binding propensity and optical properties of the bikaverin and its adducts with studied metal ions. The screening of thirteen (13) bioavailable metal ions, revealed a range of binding strength towards bikaverin receptor with Ca2+, Mg2+ and Al3+as the strongest binders. Besides, upon binding to bikaverin receptor an enhancement in its fluorescence intensity was observed in the order Ca2+> Al3+> Mg2+. The computationally predicted selectivity of bikaverin receptor towards Ca2+was experimentally corroborated through the preliminary fluorescence studies. The bikaverin probe showed an enhancement of fluorescence emission in presence of Ca2+ ions in buffered aqueous medium. Keywords: Computational chemistry; Biosensors; Fluorescence Spectroscopy; Quantum chemical calculations; Electronic structure 1. Introduction The well designed computational studies can be good to predict the starting point for targeted experiments in a time and cost effective manner.1 Experimental chemists use theoretical calculations to supplement, support and guide their experiments particularly in the areas of conformational analysis,2 reaction mechanism,3 transition states,4 charge distributions,5 modeling larger molecules like DNA and proteins,6 structure-activity relationships,7 orbital interactions,8 and excited state studies.9 Metal ions are required for a plethora of functions in biosystems. However, the metal ions can be like double-edged swords; at their proper concentrations, these remain coordinated to their natural binding sites and perform the desired functions; but a change in their normal concentration can lead to their de-compartmentalization and consequently bring onset of deleterious functions.10 Therefore, selective sensors for detection and quantification of metal ions for the real time analytical monitoring and medical diagnosis are of considerable importance.11 Thus, it becomes imperative to investigate the natural bio-active compounds for their in vivo metal ion sensing applications in order to potentiate the diagnosis for better therapeutic action. In continuation of our computational chemistry interests12-15 and towards investigating the bio-activities of natural products,16-18 we attempted to survey the fungi derived fluorescent bioactive natural product bikaverin for metal ion sensing ability. Bikaverin is a reddish pigment produced by different fungal species of the genus Fusarium with the diverse biological activities.19-21 With the increasing reports on its pharmacological role, bikaverin is becoming a metabolite of biotechnological interest.22 Commercially bikaverin as pure compound is costly and synthesizing bikaverin through a total chemical process is less viable and environmentally unfavorable. However, obtaining bikaverin from microbial sources present an environment friendly, continuousand cheap source. Thus for obtaining bikaverin in a cost effective and an environment friendly manner, we extracted bikaverin from fungal extracts using the standardized procedure from literature reports.23 In this study, we present the Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: 352 Acta Chim. Slov. 2019, 66, 351-359 computational exploration of bikaverin as biosensor towards thirteen (13) bioavailable metal ions. The computational prediction was experimentally confirmed through preliminary florescence studies of studied metal ions as bikaverin adducts. 2. Experimental 2. 1. Computational Procedures and Materials All density functional theory (DFT) and time-dependent density functional theory (TD-DFT) calculations were performed using the Gaussian 09 program package.24 The geometries of all the studied molecules (bikaverin and its metals adducts) were fully optimized at the singlet ground (S0) and first excited (S1) states in the acetonitrile solvent using CAM-B3LYP, Coulomb-attenuating method based on Becke's three parameter hybrid exchange and nonlocal correlation functional of Lee, Yang and Parr (65% exchange and 35% correlation weighting at long-range).25,26 The standard 6-311G, split-valence atomic basis set function of DFT has been employed for all the atoms except for the transition metal ions; Nickel (Ni2+), Mercury (Hg2+), Cadmium (Cd2+), Manganese(Mn2+), Iron (Fe2+), Zinc(Zn2+), Copper(Cu2+) and Lead (Pb2+) atoms for which effective core potential (ECP) of Wadt and Hay pseudo-potential with a double-Z valence basis set LANL2DZ was used.27-29 It is because the LANL2DZ reduces the computational cost as it uses an effective core potential for the core electrons, thus the core electrons are not explicitly considered in the computation. For the structural calculations of receptor-transition metal complexes, the relativistic LANL2DZ pseudopotential is the typical basis set asit has been identified to give close agreement between calculated and experimentally observed geometries. Frequency calculations were carried out to verify that the optimized molecular structures correspond to the energy minima, thus only positive frequencies were expected. The TD-DFT studies of all the molecules were performed by utilizing the same functional and the basis sets as CAM-B3LYP has been shown to be good for the excited state property calculations.30 The Conductor-like Polariz-able Continuum Model (CPCM) was utilized for taking care of the effect of solvation in acetonitrile (dielectric constant, £ = 37.5) on all the calculations as implemented in Gaussian 09. Natural Bond Orbital (NBO) method was employed for the natural population analysis, using the NBO 3.1 version as implemented in Gaussian 09 program.31 Free energy changes (AG) and binding energies (AE) were calculated for the receptor-analyte complexes to comprehend the thermodynamic binding propensity of the selected bioavailable cations with receptor (1).The Gibb's free energy change (AG) of the fragments (1), Na+, K+, Ni2+, Al3+, Hg2+, Cd2+, Mn2+, Fe2+, Zn2+, Cu2+, Mg2+, Pb2+ and Ca2+and their complexes were calculated from equation 1: AG = Gcomplex - (Greceptor+ Gcation) (1) Where Gcomplexis the free energy of receptor-cation adduct, Greceptor and Gcation are free energies of isolated receptor and cation respectively.The binding energy changes were also calculated to check the binding selectivity of the studied cations towards bikaverin receptor using the equation 2: AE = (Ecomplex) - (Ereceptor+ Ecation) (2) Where Ecomplex is the total energy of the receptor-cation adduct, Ereceptor and Ecation are total energies of isolated receptor and cation respectively.In order to reduce basis set superposition error (BSSE) in these energy calculations, the Boys-Bernardi scheme was applied to yield the counterpoise corrected energies.32 2. 2. Extraction of Bikaverin from Fungal Extracts Fungal mycelium showed a growth up to 5-6 cm on a PDA (potato dextrose agar) plate after 7 days. The morphological features supported the genus Fusarium resulting in 99% homology with the Fusarium proliferatum. Di-chloromethane solvent was used to extract the crude bikaverin using National Cancer Institute's protocol. The extract was concentrated under vacuum and subjected to purification over column chromatography on silica gel using hexane-ethyl acetate (7:3 V:V) mobile phase leading to the isolation of pure bikaverin. 2. 3. Absorption and Emission Studies All reagents were purchased from Aldrich and used without further purification. UV-Vis and fluorescence spectra were recorded on a Shimadzu UV-2450 and Shi-madzu 5301 PC spectrophotometer respectively, with a quartz cuvette (path length 1 cm). A 10-3 M stock solution of bikaverin (probe 1) was prepared by dissolving the required amount of bikaverin in 10 mL of DMSO; 30^L of this stock solution was further diluted with CH3CN and HEPES buffer (0.05 M, pH = 7.4) to prepare 3 mL solution of probe (1) and this solution was used for each UV-Visi-ble and fluorescence experiment. The aliquots of freshly prepared standard solutions (10-2M) of metal chloride/ metal perchlorates {M = Hg2+, Fe2+, Pb2+, Cd2+, Cu2+, Zn2+, Ni2+, Al3+, Co2+, Mg2+, Ca2+, Na+, and K+}, in distilled water were added to 3 mL solution of probe (1) taken in quartz cuvette and spectra's were recorded. 3. Results and Discussions Quantum-chemical calculations are impressive for their step to step analysis and prediction whereas experi- Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: Acta Chim. Slov. 2019, 66, 351-359 353 Figure 1. Optimized ground (S0) and the first excited state (S1) geometries of bikaverin receptor(i), 1-Al3+, 1-Mg2+ and 1-Ca2+ and their corresponding energies [in electron volts (eV)]. (a) (b) Figure 2. (a) Optimized geometry and (b) Electrostatic potential map of ground state (S0) of bikaverin. bikaverin(1) and its receptor-analyte complexes with thirteen (13) studied bioavailable metal ions are shown in Figures 1, and S1 and S2 respectively (see Supplementary in-formation).The natural charge distribution (Table 1) and electrostatic potential map of bikaverin (1) were calculated to identify its appropriate cation binding site. The charge distribution data in Table 1 and electrostatic potential map of bikaverin (1) indicate that the pocket for the electrophilic addition of cations is the carbonyl and the hydroxyl oxygen atoms Figure2 (see the ground state structure of bikaverin). The calculated free energy change (AG) and the binding energy (AE) values of receptor analyte complexes are presented in Table 2. The data in Table 2 reveals that the binding of all the studied bioavailablecations to bika-verin is thermodynamically feasible and these ions bind the bikaverin receptor (1) with a range of strength. The trend in the binding propensity of studied metal ions can be correlated to their charge densities and hard soft acid base strength. It is evident from the binding and free energy data of Table 2 that the electrophillic binding of Ca2+, mental results are a net outcome of all the influencing factors in a blend. The optimized geometries and the corresponding energies of the ground and first excited state of Table 1. The NPA Charge distribution on some crucial atoms of bikaverin 1 and 1-Ca2+in S0 calculated at CAM-B3LYP/6-311G level of theory. Atom involved NPA charge distribution Bikaverin (1) 1-Ca2+ O22 -0.685 -0.776 O19 -0.534 -0.678 O23 -0.645 -0.635 O21 -0.698 -0.685 020 -0.588 -0.559 O24 -0.495 -0.495 O25 -0.532 -0.527 O26 -0.516 -0.513 H36 -0.523 -0.551 Ca43 1.964 Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: ... 354 Acta Chim. Slov. 2019, 66, 351-359 Table 2. Calculated free energy change (AG) and binding energy for receptor-analyte complexes using CAM-B3LYP/6-311Glevel of theory with basis set superposition error (BSSE) corrections. S. No Adduct AG Binding energy S. No Adduct AG Binding energy (Kcal/mol) (Kcal/mol) (Kcal/mol) (Kcal/mol) 01 1-Na+ -22.44 -41.94 08 1-Mn2+ -43.76 -31.81 02 1-K+ -19.92 -39.31 09 1-Fe2+ -55.39 -50.11 03 1-Al3+ -53.09 -52.96 10 1-Ni2+ -27.11 -29.72 04 1-Pb2+ -51.31 -22.72 11 1-Cu2+ -34.64 -39.94 05 1-Hg2+ -15.29 -34.20 12 1-Mg2+ -51.97 -58.63 06 1-Cd2+ -22.64 -45.29 13 1-Ca2+ -73.19 -69.21 07 1-Zn2+ -31.66 -23.77 Mg2+ andAl3+ with receptor (1) is stronger in comparison to the remaining cations. Among all the studied metal ions the binding of Ca2+ to bikaverin was found to be strongest which suggests a possible interaction between bikaverin and Ca2+ at lower concentrations. Thus a sensing application of bikaverin towards Ca2+ ions at its physiological concentrations can be possible Motivated by the binding of metal ions to bikaverin receptor(l), we attempted to explore the fluorescence behavior of the studied metal ions for predicted sensing applications. To get an insight into the origin of absorption and emission bands, the transitions of bikaverin(l) and its receptor-analyte complexes under consideration were simulated using preferred DFT/TD-DFT level of theory (Experimental section). The computationallypredicted spectra of studied metal ion bikaverin (1) adducts in the acetonitrile solvent depicted various peaks. However only the peaks of good intensity fluorescence were chosen for analysis. The major absorption and fluorescence intensity band of bikaverin (1) and its receptor-analyte adducts with studied metal ions are summarized in Table 3. The calculated absorption spectrumfor bikaverin (1) shows a major peak (Aabs) at 491 nm (2.52 eV). The metal ion bikaverin adducts showed varied emission responses in which the fluorescence intensities in some cases were seen to be attenuated and in some cases enhanced. The calculated fluorescence intensities of 1-Ca2+, 1-Mg2+ and 1-Al3+adducts were found to be significantly higher than that of bikaverin (1) while as the absorption intensities of other recep-tor-analyte complexes don't show much deviation in comparison to pure bikaverin (1). To have a further insight into the emission behavior of the bikaverin (1) metal ion adducts, the frontier molecular orbitals (FMOs) analysis was attempted. The FMOs of the bikaverin (1) and some representative receptor-analyte complexes are depicted in Fig. 3. The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbitals (LUMO) energies of bikaverin (1) and all its metal ion adducts have been summarized in (Table S1, see supporting information).The interactions between the metal ions and the bikaverin (1) can be clearly observed from the frontier molecular orbital contour distribution.33 Table 3. Summary of absorption and fluorescence spectral data of bikaverin(1) and its analyte complexes Calculated Oscillator Calculated Oscillator Molecule \abs(nm) strength ( f ) \flu(nm) strength ( f ) 1 491 0.2524 576 0.0102 1-Na+ 412 0.1960 426 0.0081 1-K+ 315 0.1910 370 0.0013 1-Ni2+ 441 0.2103 494 0.0014 1-Al3+ 342 0.1543 388 0.1976 1-Hg2+ 377 0.1130 435 0.0045 1-Cd2+ 389 0.1866 437 0.0043 1-Mn2+ 422 0.2010 497 0.0036 1-Fe2+ 360 0.2349 454 0.0045 1-Zn2+ 390 0.1873 437 0.0033 1-Cu2+ 391 0.1033 463 0.0002 1-Mg2+ 372 0.1537 422 0.1278 1-Pb2+ 418 0.1004 471 0.0037 1-Ca2+ 525 0.1629 588 0.3722 In case of bikaverin (1), the HOMO and LUMO are spread evenly over the benzoquinone and hydroquinone rings of the molecule. The observed changes in the HOMO and LUMO contours of bikaverin (1) indicate a pi to pi* transition in absorption. However, HOMOs and LUMOs in the receptor-analyte adducts are budged on different regions of the adducts indicating intramoleculor charge transfer due to the binding of cationic species. It can also be seen from the Fig. 3 that the orbital contour distribution of HOMO in case of bikaverin calcium ion adduct (1-Ca2+) gets shifted towards the opposite end compared to free bikaverin (1) or 1-Al3+ and 1-Mg2+ adducts. The calculated fluorescence wavelength for bika-verin (1) was found to be 576 nm (2.15 eV) with oscillator strength of 0.0102, depicting low intensity fluorescence peak in bikaverin (1). While the calculated fluorescence spectrum of 1-Ca2+ shows an intense peak at 588 nm (2.19 eV) with oscillator strength of 0.3722. Thus the observations of strong Ca2+ ion binding coupled with the enhancement in the fluorescence intensity of bikaverin (1) on Ca2+ ion binding predict the bikaverin Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: Acta Chim. Slov. 2019, 66, 351-359 355 Fig. 3. Frontier molecular orbitals of bikaverin (1), 1-Al3+, 1-Mg2+and 1-Ca2+. (1) as the new possible fluorescent biosensor for Ca2+ ion. The fluorescence imaging of Ca2+ ions in the form of bikaverin (1) calcium adduct can serve as a method for analyzing signaling pathways involving calcium ions. Besides, the changes in the intracellular Ca2+ ion concentrations have been related to different physiological, pathological and immune responses, therefore bikaverin based calcium ion sensing can also be an important tool in diagnostics. The calculated oscillator strengths of fluorescence intensities of studied bioactive metal ions with bikaverin Fig. 4. Predicted electronic excitations and de excitations of bikaverin receptor (1) and its calcium adduct. Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: ... 356 Acta Chim. Slov. 2019, 66, 351-359 (1) also predict a mild to medium increase in the intensity in case of 1-Mg2+ and 1-Al3+ adducts. But the intensities of their fluorescence peaks are predicted to be smaller in comparison to Ca2+ as depicted by their oscillator strengths of 0.1976 and 0.1278, respectively. For the other cationic species, there is no significant effect on the fluorescence intensities once they bind with the bikaverin receptor (1) as can be seen from their oscillator strengths in Table 3. It is therefore predicted from the DFT/TD-DFT calculations that receptor (1) can strongly sense Ca2+ ions fluorometri-cally with the milder sensing ability towards Mg2+ and Al3+ among the studied bioactive metal ions. The probable reason for the fluorescence intensity enhancement in case of 1-Ca2+, 1-Al3+, and1-Mg2+ adducts is that their absorption transitions involve electron shifting to higher singlet excited states like fourth singlet excited state (S4),third singlet excited state(S3) and second singlet excited state(S2) in case of 1-Ca2+, 1-Al3+, and 1-Mg2+ respectively. As per Kasha's rule,34 the fluorescence phenomenon occurs from the firstsinglet excited state (S1). In order for fluorescence to occur, the higher excited states have to relax to the first singlet excited state. For this relaxation process, internal conversions and the vibrational relaxations occur which lead to the loss of energy during the de-excitation. Due to this energy loss there is significant Stoke's shift (change between absorption and emission energies) While as in the other cations and in receptor (1), the transitions involve the lowest singlet excited state S1. So there is no internal conversion and hence no energy loss and eventually no Stoke's shift. Hence, in such cases, there is either no fluorescence or the intensity of the fluorescence is very weak. TD-DFT calculated results show that the main transitions for bikaverin(1), 1-Al3+, 1-Mg2+and 1-Ca2+ are from HOMO LUMO, HOMO LUMO+2, HOMO LUMO+3 and HOMO LUMO+1 respectively see Fig 4. In all other receptor-analyte complexes, the main transitions involve HOMO^LUMO. The experimental and theoretical fluorescence study of bikaverin (1) reveala major fluorescence peak corresponding to HOMO to LUMO transition with Àem 576 nm in the theoretical spectra and Àem 425 nm in experimental spectra. However on binding of Ca2+ the theoretical fluorescence peak gets shifted to a value Àem 588 nm which closely corroborateswith the observed experimentalpeak of Bikaverin Ca2+adduct around Àem of 620 nm.Thus, the theoretically predicted red shift in fluorescence of bikaver-in(1) on calcium ion binding was also experimentally observed and therefore can be rationalized with theoretical prediction of HOMO^LUMO+1 absorption in case bika-verin calcium ion adduct. 3. 1. Characterization of Bikaverin from Fungal Extract The developments in synthetic biology has allowed tuning of microbial systems for generating industrially viable strains as the green and sustainable sources of value-added compounds.35The composition and amounts of metabolites(bikaverin) are specific to the species of genus Fusarium. The morphological features of our crude fungal extract matched to the species Fusarium proliferatum. The chemical structure of purified bikaverin was confirmed through comparative analysis of NMR, mass, Àmax and TLC of isolated compound with bikaverin standard and further to the literature reported values.36 1H-NMR (CDCl3, 400MHZ):S 2.87 (3H, s, 1-Me), 3.93 xio 2 -ESI Scan:2 (0 2 min) Frag=80.0V CNE9V1.d 181 -00 22" MO 381 .00 112 90 I I il.il 1 10 4 +ESI Scan:1 (0,2 min) Frag=85.0V CNE9V1 .d IbJ UU 32.00 382.90 100 200 300 400 500 S00 700 800 900 1000 11 00 1200 1300 1400 1500 1600 1700 1800 Counts vs. Msss-to-Charge (nVz) Fig. 5. Mass spectra of isolated product Bikaverin Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: Acta Chim. Slov. 2019, 66, 351-359 357 (3H, s, 8-OMe), 3.96 (3H, s, 3-OMe), 6.35 (1H, s, H-9), 6.81 (1H, s, H-2), 6.93 (1H, s, H-4).HRMS (ESI+) calculated for C20H14O8 [M+H]+ : 381.0761, found: 382.90 Fig.5. 3. 2. Metal Ion Sensing Applications The preliminary metal ion sensing behavior of bika-verin (1) was studied towards selected bioactive metal ions as their chloride/perchlorate salts in CH3CN/H2O (6:4, v/v; buffered with PBS, pH 7.4) using fluorescence spec- troscopy Fig 6. The fluorescence spectrum of receptor (1) (10.0 ^M) exhibits a weak emission band at 620 nm corresponding to bikaverin moiety when excited at 596 nm in CH3CN/H2O (6:4, v/v; buffered with PBS, pH = 7.4). The weak fluorescence emission behavior of bikaverin is due to intramolecular charge transfer process as seen from HOMO- LUMO orbital contours. It is evident from Figure 7 that on binding of calcium ions to bikaverin (1) its fluorescence profile gets modified. The free bikaverin shows very little to no fluorescence when excited at 596 nm while as the calcium bound bika- 80 1 2 3 4 5 6 7 8 9 10 11 12 13 Metal ions (80 equivalent each) Fig. 6. Fluorescence response of bikaverin (1) towards various metal ions: Bars represent fluorescence selectivity (I/Io) of bikaverin (1) (10.0 |iM) towards various metal ions (80 equiv each) in CH3CN:H2O (6:4, v/v) buffered with 0.05 M HEPES, pH = 7.4; Xex = 596 nm. The experimental fluorescence spectra of bikaverin(l) and its calcium adduct under different excitation wavelengths are shown in Fig.7 (A,B) respectively. Fig. 7. Fluorescence response under different excitation wavelengths for A: bikaverin(l) B: Bikaverin Ca2+ adduct. Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: ... 358 Acta Chim. Slov. 2019, 66, 351-359 verin shows a major fluorescence when excited at the 596 nm wavelength. Among the fluorescence peaks obtained under the excitation wavelengths of 425 and 345 nm, the former gets an enhancement in the emission intensity while as the latter peak remains largely unchanged on calcium ion binding to bikaverin. 4. Conclusions In summary, the present work describes a thorough computational investigation of metal ion sensing ability of natural pigment bikaverin as a fluorescent probe. An exploration of comparative metal ion binding affinities and optical properties of the bikaverin adducts through systematic studies using the appropriate level of theory are presented. The Ca2+, Mg2+and Al3+ metal ions are shown to bind the bikaverin receptor more strongly and with an enhancement in its florescence intensity. The strongest binding affinity with the most intense fluorescence emission in case of Ca2+ predicts bikaverin pigment as a possible Ca2+ biosensor. The concept of isolation of bikaverin receptor (1) from the natural source as an economical and sustainable method is also presented. The computationally predicted metal ion fluorescence behavior of bikaverin receptor (1) towards the studied metal ions was experimentally corroborated through the preliminary fluorescence studies. Among the studied metal ions, the bikaverin receptor (1) was seen to strongly detect Ca2+ions with an enhancement of fluorescence emission in its ^M concentration range in the buffered aqueous medium due to charge transfer phenomena. The inclusive experimental studies of the bikaverin receptor (1) towards the selective calcium ion sensing under physiological conditions, the complete analytical profile of bikaverin as biosensor, the real time imaging of Ca2+ ions in cells through confocal microscopy and other related studies for the buildup of bikaverin as Ca2+ ion biosensor are underway in our laboratory. 5. Acknowledgement MAR thankfully acknowledges Dr Hemant J Puro-hit, Chief Scientist & Head Environmental Genomics Division, National Environmental Engineering Research Institute (NEERI), CSIR, Nagpur India for providing the Bikaverin crude extract for bikaverin extraction and University of Kashmir for assistance under UGC 12th innovative research activity. HRB and MKR are highly thankful to Indian Institute of Technology Kanpur, India for providing some computational facilities. Conflict of Interest All the authors declare no conflict of interest what so ever. 6. References 1. H. Nikoofard, M. Sargolzaei, F. Faridbod Acta Chim. Slov. 2017, 64, 842-848. DOI:10.17344/acsi.2017.3357 2. S. Krishnamurty, M. Štefanov, T. Mineva, S. Begu, J. M. De-voisselle, A. Goursot, R. Zhu, D. R. Salahub, J. Phys. Chem. B. 2008, 112, 13433-13442. DOI:10.1021/jp804934d 3. G. J. Cheng, X. Zhang, L. W. Chung, L. Xu, Y. D. Wu, J. Am. Chem. Soc. 2015, 137, 1706-1725. DOI:10.1021/ja5112749 4. F. 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Vanka, J Organomet Chem. 2016, 801, 30-41. DOI:10.1016/j.jorganchem.2015.10.015 14. M.V. Mane, M. A. Rizvi, K. Vanka, J. Org. Chem. 2015, 80, 2081-2091. DOI:10.1021/jo5023052 15. M. Kumar, A. Kumar, M. Rizvi, M. Mane, K. Vanka, S. C. Taneja, B. A. Shah, Eur. J. Org. Chem. 2014, 2014, 5247-5255. DOI:10.1002/ejoc.201402551 16. A. Pandey, M. Rizvi, B. A. Shah, S, Bani, Cytokine, 2016, 79, 103-113. DOI:10.1016/j.cyto.2016.01.004 17. A. Goswami, B. A. Shah, A. Kumar, M. A. Rizvi, S. Kumar, S. Bhushan, F. A. Malik, N. Batra, A. Joshi, J. Singh,Chem.-Biol. Interact., 2014, 222, 60-67. DOI:10.1016/j.cbi.2014.08.011 18. R Chib, M. Kumar, M. Rizvi, S. Sharma, A. Pandey, S. Bani, S. S Andotra, S. C. Taneja, B. A Shah, RSC Advances, 2014, 4, 8632-8637. DOI:10.1039/c3ra46412a 19. J. Balan, J. Fuska, I. Kuhr, V. Kuhrova, Folia Microbiol (Praha). 1970, 15, 479-484. DOI:10.1007/BF02880192 20. J. F. Henderson, M. L. Battell, G. Zombor, J. Fuska, P. Nemec, Biochem Pharmacol. 1977 26, 1973-1977. DOI: 10.1016/0006-2952(77)90004-1 21. D. Kjaer, A. Kjaer, C. Pedersen, J. D. Bulock, J. R. Smith, J Chem Soc Perkin 1. 1971, 16, 2792-2797. DOI:10.1039/J39710002792 22. M. C. Limon, R. Rodriguez, J. Avalos, Appl Microbiol Biotech-nol. 2010, 87, 21-29. DOI:10.1007/s00253-010-2551-1 Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: Acta Chim. Slov. 2019, 66, 351- 359 359 23. D. Arora, N. Sharma, V. Singamaneni, V. Sharma, M. Kush-waha, V. Abrol, S. Guru, S. Sharma, A. P. Gupta, S. Bhushan, S. Jaglan, P. Gupta, Phytomedicine. 2016, 23, 1312-1320. D01:10.1016/j.phymed.2016.07.004 24. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Men-nucci, B.; Petersson, G. A.; et al. Gaussian 09; Gaussian Inc: Wallingford, CT, 2009. 25. T. Yanai, D. P. Tew, N. C. Handy, Chemical Physics Letters, 2004, 393, 51-57. D0I:10.1016/j.cplett.2004.06.011 26. Jacquemin, D.; Planchat, A.; Adamo, C.; Mennucci, B. J. Chem. Theory Comput. 2012, 8, 2359-2372. D0I:10.1021/ct300326f 27. H. R. Bhat, P. C. Jha, Chem. Phys. Lett. 2017, 669, 9-16. D0I:10.1016/j.cplett.2016.12.025 28. H. R. Bhat, P. C. Jha, Phys. Chem. Chem. Phys. 2017, 19, 14811-14820. D0I:10.1039/C7CP02287E 29. H. R. Bhat, P. C. Jha, J. Phys. Chem. A. 2017, 121, 3757-3767. D0I:10.1021/acs.jpca.7b00502 Povzetek Članek predstavlja računalniško raziskovanje gliv, ki proizvajajo pigment bikaverin kot biosensor za biološko razpoložljive kovinske ione. Narejene so bile sistematične študije geometrije optimiziranega osnovnega in vzbujenega stanja za raziskovanje vezavnega žepa za kovinske ione, primerjalna vezavna nagnjenost in optične lastnosti bikaverina in njegovih aduktov s preiskovanimi kovinskimi ioni. Pregled trinajstih (13) biodostopnih kovinskih ionov je pokazal, da jakost vezanja na bikaverinski receptor ustreza zaporedju Ca2+, Mg2+ in Al3+. Po vezavi na bikaverinski receptor je bila opažena ojačitev fluorescence v vrstnem redu Ca2+ > Al3+ > Mg2+. Računsko napovedana selektivnost bikaverinskega receptorja za Ca2+ je bila eksperimentalno podprta s predhodnimi fluorescenčnimi študijami. Bikaverinska sonda je pokazala povečanje fluorescenčne emisije v prisotnosti Ca2+ ionov v zapufranem vodnem mediju. Hussain et al.: Exploring Bikaverin as Metal ion Biosensor: ... 30. S. F. Boys, F. Bernardi, Mol. Phys. 1970, 19, 553-566. D01:10.1080/00268977000101561 31. Reed, A. E.; Curtiss, L.; Weinhold, F. Chem. Rev. 1988, 88, 899-926. D0I:10.1021/cr00088a005 32. J. W. Cornforth, G. Ryback, P. M. Robinson, D. Park, J Chem Soc Perkin 1971, 1.16, 2786-2788. D0I:10.1039/j39710002786 33. R. M. Jagtap, M. A. Rizvi, Y. B. Dangat, Satish K. Pardeshi, J. Sulfur Chem. 2016, 37, 401-425. D01:10.1080/17415993.2016.1156116 34. M. Kasha, Discuss. Faraday Soc. 1950, 9, 14-19. D0I:10.1039/df9500900014 35. J. Du, Z. Shao, H. Zhao, J Ind Microbiol Biotechnol. 2011, 38, 873-890. D0I:10.1007/s10295-011-0970-3 36. D. Kjaer, A. Kjaer, C. Pedersen, J. D. BuLock, J. R. Smith, J. Chem. Soc. C 1971, 0, 2792-ww2797. D0I:10.1039/J39710002792 DOI: 10.17344/acsi.2018.4862 Acta Chim. Slov. 2019, 66, 360-366 ^creative ^commons Scientific paper Ion-Associated Complex of the Anionic Chelate of Germanium(IV) with Nitro Derivative of the Catechol and the Cation of Monotetrazolium Salt Kirila Stojnova and Vanya Lekova* Department of General and Inorganic Chemistry with Methodology of Chemistry Education, Faculty of Chemistry, Plovdiv University "Paisii Hilendarski", 24 Tsar Assen Street, Plovdiv 4000, Bulgaria * Corresponding author: E-mail: vanlek@uni-plovdiv.bg Tel.: +35932261420 Received: 11-21-2018 Abstract The formation of an ion-associated complex between of the anionic chelate of germanium(IV) with 4-nitrocatechol (4-NC) and the cation of 3-(2-naphtyl)-2,5-diphenyl-2H-tetrazolium chloride (TV) was investigated by spectrophotometry. The optimum conditions for the chelate formation and the extraction of the complex in the liquid-liquid extraction system Ge(IV)-4-NC-TV-H2O-CHCl3 were established. The validity of Beer's law was checked and some analytical characteristics were calculated. The association process in the aqueous phase and the extraction equilibria were studied and quantitatively characterized by the following key constants: association constant, distribution constant, extraction constant and recovery factor. The molar ratios of the components in the ion-associated complex were determined by independent methods. Based on this, a reaction scheme, a general formula and a structure of the ion-associated complex were suggested. Keywords: Ge(IV); ion-associated complex; chelate formation; extraction equilibriums 1. Introduction The germanium is a forth-period post-transition metal which forms complexes with various natural organic ligands, containing O, N and S donor atoms, such as polyphenols and their functional derivatives, polyhydroxycar-boxylic acids, aminopolyhydroxycarboxylic acids, thio-polycarboxylic acids, 8-hydroxyquinoline and its derivatives, aromatic derivates of hydroxyaldehydes and hy-droxyketones, hydroxyazodyes.1-9 The colored anionic chelates of Ge(IV) form ion-associated complexes with bulky organic cations, like tetradecyl(trihexyl)phosphoni-um, methyltrioctylammonium, cetylpyridinium, cetyl-trimethylammonium, tetraphenylammonium, tetraphe-nylarsonium.10-16 The structure and the properties of the tetrazolium salts determine their ability to form ion-associated com-plexes.17,18 The bulky hydrophobic organic substituents in the molecules of the tetrazolium salts increase the extract-ability of the ion-associated complexes. The presence of a quaternary nitrogen atom in the molecules of the tetrazoli-um salts determines the ability to form ionic associates with chelates of metals in aqueous phase without protonation, as opposed to the amines. The preparation and the application of ion-associated complexes of anionic che-lates of metals with various natural organic and inorganic ligands containing N- or O-donor atoms and with the participation of mono- and ditetrazolium cations is a special scientific research field of chemistry of the coordination compounds. Tetrazolium salts are used as reagents for the preparation of various ion-associated complexes of metals, e.g. Mo(VI), Ga(III), Co(II).19-26 The liquid-liquid extraction, as a part of the chemistry of the solutions and the coordination compounds, is applied to study the processes of complex formation and the extraction equilibria. The extraction spectrophotometry is a relatively simple, convenient, sensitive, selective, rapid to perform and inexpensive method for preparation and characterization of new complex compounds as well as for their application in the chemical analysis.27-32 The aim of this research was to study spectrophoto-metrically the formation of the ion-associated complex between the anionic chelate of germanium(IV) with 4-ni-trocatechol (4-NC) and the cation of of 3-(2-naph- Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate Acta Chim. Slov. 2019, 66, 360-366 361 tyl)-2,5-diphenyl-2H-tetrazolium chloride (TV) in the liquid-liquid system Ge(IV)-4-NC-TV-H2O-CHCl3 as well as to evaluate the possible applications of the system for determination of traces of germanium(IV) in alloys, biological, medical and pharmaceutical samples. 2. Experimental 2. 1. Reagents and Apparatus GeO2 (Sigma-Aldrich, Munich, Germany, p.a.): A 2.0 x 10-3 mol L-1 aqueous solution of Ge(IV) was prepared by dissolving GeO2 in water upon moderate heating. 4-Nitrocatechol (4-NC) (Sigma-Aldrich, p.a.): 4-NC was dissolved in distilled water to give a 1.0 x 10-2 mol L-1 solution. 3-(2-Naphthyl)-2,5-diphenyl-2H-tetr azolium chloride (TV) (Fluka, p.a.): A chloroform 3.0 x 10-3 mol L-1 solution was prepared. The acidity of the aqueous medium was set using a buffer solution prepared by mixing 2.0 mol L-1 aqueous solutions of CH3COOH and NH4OH. The organic solvent CHQ3 was additionally distilled. The pH was checked by HI 83140 pH meter (Romania). A Camspec M508 spectrophotometer (United Kingdom), equipped with 10 mm path length cells, was employed for measurement of the absorbance. 2. 2. Procedure for Establishment of the Optimum Extraction-Spectrophotometric Conditions Aliquots of Ge(IV), 4-NC, TV and buffer (pH = 3.54.5) solutions were filled into separatory funnels. The resulting solutions were diluted with distilled water to a total volume of 10 mL. Then 10 mL of chloroform was added and the funnels were shaken. A portion of the organic extract was filtered through a filter paper into a cell and the absorbance was measured against a blank sample.20,23 2. 3. Procedure for Determination of the Distribution Constant In order to determine the distribution constant KD, it is necessary to measure the light absorbances A1 and A3, which are the light absorbance after a single extraction in chloroform under the optimum conditions for complex formation (Table 1, column 1) and after a triple extraction performed under the same conditions, respectively. The distribution constant KD can be calculated according to the ratio Kd = A1/A - A1). Single extraction: the single extraction is conducted with 10 mL of chloroform. After the separation of the two phases, the organic extract is transferred into a 25 mL calibrated flask which is brought to volume with chloroform. The measurement of the light absorbance A1 is performed against a blank sample, pre- pared under the same conditions. Triple extraction: the first stage of the triple extraction is performed with 10 mL of chloroform and the extract is transferred into a 25 mL calibrated flask. During the second stage of the extraction, 8 mL of chloroform are added to the aqueous phase remaining after the first stage. The organic layer is added to that from the first stage. For the third stage of extraction, 7 mL of chloroform are added to the aqueous phase remaining after the second stage and an extraction is performed for the third time. The organic layer is transferred to the previous two. The calibrated flask is brought to volume with chloroform. The measurement of A3 is performed against a blank sample prepared in the same way.22 3. Results and Discussion 3. 1. Optimum Extraction- Spectrophotometric Conditions 3. 1. 1. Absorption Spectra The colored anionic chelate of germanium(IV)-4-NC can be efficiently extracted in chloroform in the presence of the bulky hydrophobic organic cations of monotetrazolium salt (TV). The absorption spectrum of the extract of the ion-associated complex, formed between the anionic che-late of Ge(IV) with 4-NC and the cation of monotetrazoli-un salt in CHCl3, is characterized by an absorption maximum in the visible range (Amax = 420 nm) (Figure 1). 0,700 - 0,600 - 0,500 - " 0,400 - £ 0,300 -n 1 0,200 - A < 0,100 -0,000 - 350 400 450 500 550 Wavelength nm Figure 1. Absorption spectra of the complex Ge(IV)-4-NC-TV and of the blank sample 4-NC-TV in CHC^; Cg^iv) = 2.0 x 10-5 mol L-1; C4-nc = 5.0 x 10-4 mol L-1; Ctv = 1.5 x 10-4 mol L-1; pH = 4.0; A = 420 nm; t = 2 min 3. 1. 2. Effect the Acidity of the Aqueous Phase and the Shaking Time The acidity of the aqueous phase has a substantial effect on the extraction of the anionic chelate Ge(IV)-4-NC into the organic phase. The maximum and constant extraction of the ion-associated complex is achieved in Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate 362 Acta Chim. Slov. 2019, 66, 360-366 the pH range from 3.5 to 4.5 (Figure 2). Acetate buffer solution with pH = 4.0 was used in all further experiments. The carried-out experiments, showed that the extraction equilibrium is achieved for shaking time of not less than 60 s. The longer shaking time did not affect the absorbance. The further experiments were performed with shaking time 2 min. 1.5 2.5 3.5 4.5 55 6.5 pH Figure 2. Absorbance of Ge(IV)-4-NC-TV extract against 4-NC-TV extract and of 4-NC-TV against CHCl3 vs. pH of the aqueous phase; CGe(IV) = 2.0 x 10-5 mol L-1; C4-NC= 5.0 x 10-4 mol L-1; CTV = 1.5 x 10-4 mol L-1; X = 420 nm; T = 2 min 3. 1. 3. Effect of Reagents' Concentrations The concentrations of the reagents are the most important factor, influencing the extraction equilibrium. The chelate formation of Ge(IV)-4-NC requires a 15.0-fold excess of 4-NC (>3.0 x 10-4 mol L-1). For a maximum association and extraction the amount of TV should not be lower than a 4.5-fold excess (>9.0 x 10-5 mol L-1). 3. 2. Beer's Law, Apparent Molar Absorptivity and other Analytical Characteristics The range of obedience to the Beer's law, i.e. the linear relationship between the germanium(IV) concentra- tion in the aqueous phase (CGe(IV), ^g mL-1) and the absorbance of the ion-association complex in the organic phase after extraction was studied using regression analysis under the optimum conditions for complex formation. The equation of a straight line was found to be Y = 0.3933 X + 0.0020 with a correlation coefficient squared 0.9995. Under the optimum conditions for complex formation, the linearity is observed for concentrations up to 4.07 ^g cm-3 Ge(IV). Further analytical characteristics, such as apparent molar absorptivity e', adherence to Beer's law, Sandell's sensitivity, limit of detection and limit of quantification, are shown in Table 1, column 2. 3. 3. Molar Ratios of the Ion-Associated Complex The molar ratios of the ion-associated complex were determined by three independent methods: the mobile equilibrium method, the straight-line method of Asmus and the method of continuous variations.33 The mobile equilibrium method and the straight-line method of Asmus were applied to prove the molar ratios Ge(IV):4-NC and Ge(IV):TV. The values of the correlation coefficient squared R2, determined by the straight-line method of Asmus are presented in Table 2. Table 2. Values of the correlation coefficient squared (R2), corresponding to various molar ratios of Ge(IV):4-NC and Ge(IV):TV (n and m, respectively) Values of correlation coefficient squared (R2), corresponding to molar ratios 1, 2 and 3, respectively Ge(IV):4-NC Ge(IV):TV (n = 1) R2 = 0.9303 (m = 1) R2 = 0.9841 (n = 2) R2 = 0.9814 (m = 2) R2 = 0.9959 (n = 3) R2 = 0.9988 (m = 3) R2 = 0.9653 The results from the application of the straight-line method of Asmus and the mobile equilibrium method are shown in Figure 3 and Figure 4, respectively. Table 1. Optimum extraction-spectrophotometric conditions and analytical characteristics of the system Ge(IV)-4-NC-TV-H2O-CHCl3 Optimum Conditions Analytical Characteristic Absorption maximum (Amax) 420 nm Volume of the aqueous phase 10 cm3 Volume of the organic phase 10 cm3 pH of the aqueous phase 3.5^4.5 Shaking time (t) 2 min Concentration of 4-NC > 3.0 x 10-4 mol L-1 Concentration of TV > 9.0 x 10-5 mol L-1 Apparent molar absorptivity (e') (2.87 ± 0.05) x 104 L mol-1 cm-1 True molar absorptivity (e) (2.95 ± 0.01) x 104 L mol-1 cm-1 Sandell's sensitivity (SS) 2.53 ng cm2 Adherence to Beer's law up to 4.07 |ig cm-3 Relative standard deviation (RSD) 1.98% Limit of detection (LOD) 0.10 |g cm-3 Limit of quantification (LOQ) 0.33 |g cm-3 Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate Acta Chim. Slov. 2019, 66, 360-366 363 Figure 3. Determination of the molar ratios by the straight-line method of Asmus; CGe(IV) = 2.0 x 10-5 mol L-1; pH = 4.0; X = 420 nm; T = 2 min; • Ge(IV):4-NC, CTV = 3.0 x 10-4 mol L-1, ■ Ge(IV):TV, C4 -nc = 5.0 x 10-4 mol L-1 log CR, mol L1 Figure 4. Straight lines by the mobile equilibrium method for determination of the molar ratios Ge(IV):4-NC and Ge(IV):TV; CGe(iv) = 2.0 x 10-5 mol L-1; pH = 4.0; X = 420 nm; T = 2 min; ♦ Ge(IV):4-NC, CTV = 3.0 x 10-4 mol L-1; • Ge(IV):TV, C4-NC = 5.0 x 10-4 mol L-1 Figure 5. Determination of the molar ratio (n) Ge(IV):TV by the method of continuous variations; CGe(Iv) + Ctv = 6.0 x 10-5 mol L-1; C4-NC = 5.0 x 10-4 mol L-1; pH = 4.0; X = 420 nm; T = 2 min On the basis of the results it can be concluded that Ge(IV), 4-NC and TV interact in molar ratio 1:3:2. The application of the method of continuous variations confirmed the molar ratio Ge(IV):TV = 1:2 (Figure 5).33 3. 4. Reaction Scheme and Suggested General Formula Ions containing germanate [Ge(OH)6]2- are already described in the literature although in dilute aqueous solutions the major determined ions appear to be [GeO(OH)3]-, [GeO2(OH)2]2- and {[Ge(OH)4]8(OH)3}3-.1 The performed experiments showed that the complex formation and the extraction of the ion-associated complex have occurred in the dilute solutions and the molar ratio established by the independent methods mentioned above was Ge(IV):4-NC:TV = 1:3:2. Therefore, the complex formation of anionic chelate Ge(IV)-4-NC can be given by equation (1): [GeO2(OH)2] ■ {Ge[O2C6H3(NO2)]3}2-aq + 4 H2O aq + 3 (HO)2C6H3(NO2)aq ■ (1) Having in mind the molar ratio indicated above and the reaction of chelate formation of Ge(IV)-4-NC, it can be suggested that the formation of the ion-associated complex in the aqueous phase, its distribution between the aqueous and the organic phases and its extraction in chloroform can be given by the following equations (2-4). 2 (TV)+(aq) + {Ge[O2C6H3(NO2)]3}2-(aq) ^ (TV)2{Ge[O2C6H3(NO2)U(aq) (TV)2{Ge[O2C6H3(NO2)]3}(aq) ^ ^ (TV)2{Ge[O2C6H3(NO2)]3}(org) (2) (3) 2 (TV)+(aq) + {Ge[O2C6H3(NO2)U2-(aq) ^ (4) ^ (TV)2{Ge[O2C6H3(NO2)]3}(org) () Therefore, the ion-associated anionic chelate of Ge(IV)-4-NC with the cation of monotetrazolium salt can be represented by the general formula (TV)2{Ge[O2C6H3 (NO2)]3}. 3. 5. Equilibrium Constants, True Molar Absorptivity, Recovery Factor and Structure of the Ion-Associated Complex The association process in aqueous phase and the extraction equilibria were investigated and quantitatively characterized with respect to the following key constants: distribution constant KD, association constant ft, extraction constant Kex and recovery factor R%. The distribution constant KD was determined by equation (5), where Aj and A3 are the absorbances (measured against blanks) obtained after a single and triple extraction, respectively. Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate 364 Acta Chim. Slov. 2019, 66, 360-366 KD = {(TV)2{Ge[O2C6H3(NO2)]3}}(org) / / {(TV)2{Ge[O2C6H3(NO2)]3}}(aq) = =A / (A3 - A1) (5) The recovery factor was determined from the equation (6): R% = 100 Kd / (Kd + 1) The extraction constant Kex independent methods: (6) was calculated by two (i) from the equation log Kex = log KD + log ft (ii) by the method of Likussar-Boltz. 34 (7) (i) The association constant ft was determined by two independent methods: Komar-Tolmachev method and Holme-Langmyhr method and their values are given in Table 3, column 2.33,34 The association constant ft was calculated by the method of Komar-Tolmachev from equation (8).33 ß = (l /n)n / [e (tg a)n+1] (8) where l is the cuvette thickness (l = 1 cm); n -the molar ratio between the components independently determined (e.g. by the mobile equilibrium method, the straight-line method of Asmus or the method of continuous variations) (n = 2), e - the true molar absorptivity. The true molar absorptivity e was determined by the method of Komar-Tolmachev (Figure 6) from the equation of a straight line Y = 0.4182 X + 3.3895 (e = 1 / (b x 10-5) and its value is given in Table 1, column 2.33 (ii) The method of Likussar-Boltz uses the data from the method of continuous variations (Figure 5). The extraction constant Kex was calculated by the equation of Li-kussar-Boltz for molar ratio 1:2 (equation 9):35 log Kex = 0,3522 - 2 log K + + log Ymax - 3 log (1 Ymax) (9) Figure 6. Dependency of (C.l/A) on A n/(n+1) (method of Komar- Tolmachev) ; C = Cge(iv) mol L 1; Ctv = 2 Cge(iv) mol L 1; C4-nc = 5.0 x 10-4 mol L-1; pH = 4.0; X = 420 nm; T = 2 min; A - absorbance; l - cell thickness, l = 1 cm; n = 2 CGe(iv) + CTV = 6.0 X 10 5 mol L-1); Ymax and (1-Ymax) are determined from the additionally plotted normalized absorption curve (Ymax = 0.900; (1 - Ymax) = 0.100). The values of the equilibrium constants and the recovery factor, describing quantitatively the equilibrium in the aqueous phase and the extraction of the ion- associated complex in the organic phase are presented in Table 3. The results obtained by the independent methods are statistically similar and confirm the proposed scheme of the process of formation of the ion-associated complex in the aqueous phase, its distribution between the aqueous and the organic phases and its extraction in chloroform. Based on this, the proposed structure of the ion-associated complex is represented in Figure 7. where K is the total concentration of the reagents (K = Figure 7. Structure of the ion-associated complex Ge(IV)-4-NC-TV Table 3. Values of the Equilibrium Constants and the Recovery Factor Equilibrium Constant and Recovery Factor Value Equilibrium (equation 2) - Association constant ß ß = (TV)2{Ge[O2C6H3(NO2)U(aq) / {[(TV)+]2(aq) X {{Ge^Cft^)^}2-}^)} Equilibrium (equation 3) - Distribution constant KD Kd = {(TV)2{Ge[O2C6H3(NO2)U} (org) / {(TV)2{Ge[O2C6H3(NO2)U} (aq) Equilibrium (equation 4) - Extraction constant Kex Kex = {(TV)2{Ge[O2C6H3(NO2)U} (org) /{{[TV]+}2(aq) X {{Ge[O2C6H3(NO2)U2-}(aq)} Recovery factor R% log ß = (11.06 ± 1.34)a log ß = (10.25 ± 1.01)b log Kd = (1.20 ± 0.01)c log Kex = (12.26 ± 1.35)d log Kex = (11.76 ± 0.01)e R = (94.02 ± 0.41)%f a Calculated by Komar-Tolmachev method (equation.(8)); b Calculated by Holme-Langmyhr method;34 c Calculated by equation (5); d Calculated by equation (7), where ft is determined by the Komar-Tolmachev method; e Calculated by Likussar-Boltz method (equation (9)); f Calculated by equation (6). Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate Acta Chim. Slov. 2019, 66, 360-366 365 and 4-nitrocatechol (4-NC) with the cation of of 3-(2-naph-tyl)-2,5-diphenyl-2H-tetrazolium chloride (TV) was studied by spectrophotometry. The yellow-colored anionic chelate of Ge(IV)-4-NC interacted with the organic cation of the monotetazolium salt (TV) leading to the formation of an ion-associated complex well soluble in chloroform. The bulky organic molecule of TV determined the extract-ability of the ion-associated complex in organic phase. The optimum conditions for the association in the aqueous phase and for the extraction of the ion-associated complex Ge(IV)-4-NC-TV were established. The equilibrium constants and analytical characteristics needed for the quantitative assessment of the extraction equilibrium were calculated, i.e. the association constant (8), the distribution constant (KD), the extraction constant (Kex), the recovery factor (R), the apparent molar absorptivity (e'), the true molar absorptivity (e), the limit of detection (LOD), the limit of quantification (LOQ) and the Sandell's sensitivity (SS). From the analytical characteristics of the extraction system Ge(IV)-4-NC-TV-H2O-CHCl3, it can be concluded that the ion-associated complex formed between the anionic chelate of Ge(IV)-4-NC and the mono-tetrazolium cation could allow determinations of Ge(IV) in various samples with a high sensitivity. The molar ratio of the components, determined by independent methods, showed that the ion-associated complex could be represented with the general formula (TV)2{Ge[O2C6H3(-NO2)]3}. A corresponding reaction scheme of the complex formation and a structure of the ion-associated complex were also suggested. 5. Acknowledgements The authors would like to thank the Research Fund of the University of Plovdiv for the financial support of the current research. 6. References 1. F. A. Cotton, G. Wilkinson, C. A. Murillo, M. Bochmann: Advanced Inorganic Chemistry, Wiley Publishers, New Jersey, 1999, pp. 392-394. 2. V. V. Skopenko, A. Y. Tsivadze, L. I. Sabranskiy, A. D. Garno-vskiy: Coordination Chemistry, Akademkniga, Moscow, Russia, 2007, pp. 78-81. 3. V. S. Sergienko, L. K. Minacheva, A. V. Churakov, Rus. J. Inorg. Chem. 2010, 55, 2001-2030. DOI:10.1134/S0036023610130012 4. W. Levason, G. Reid, W. Zhang, Coord. Chem. Rev. 2011, 255, 1319-1341. DOI:10.1016/j.ccr.2010.11.019 5. M. F. Davis, W. Levason, G. Reid, M. Webster, Dalton Trans. 2008, 17, 2261-2269. DOI:10.1039/b716765b 6. I. I. Seifullina, N. V. Shmatkova, E. E. Martsinko, Russ. J. Coord. Chem. 2010, 30, 214-220. 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Slov. 2016, 63, 654-660. DOI:10.17344/acsi.2016.2513 24. K. Stojnova, P. Racheva, V. Divarova, K. Bozhinova, V. Lekova, Russ. J. Inorg. Chem. 2017, 62, 249-256. DOI:10.1134/S0036023617020188 25. P. Racheva, K. Stojnova, V. Divarova, V. Lekova, Acta Chim. Slov. 2017, 64, 365-372. DOI:10.17344/acsi.2017.3214 26. K. Stojnova, V. Divarova, P. Racheva, K. Bozhinova, V. Lekova, Acta Chim. Slov. 2018, 65, 213-220. DOI:10.17344/acsi.2017.3860 27. A. K. Babko, A. T. Pilipenko: Photometric Analysis, Khimiya, Moscow, Russia, 1968, pp. 159-164. 28. V. A. Mikhaylov: Extraction Chemistry, Nauka, Novosibirsk, Russia, 1984, pp. 194-249. 29. G. Kristian: Analytical Chemistry, BINOM, Moscow, Russia, 2009, pp. 414-426. Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate 366 Acta Chim. Slov. 2019, 66, 360-366 30. Yu. A. Zolotov, V. A. Bodnya, A. N. Zagrusina, H. Freiser, Anal. Chem. 1982, 14, 93-174. DOI: 10.1080/10408348208085525 31. V. V. Divarova, K. T. Stojnova P. V. Racheva, V. D. Lekova, J. Appl. Spectrosc. 2017, 84, 231-236. D0I:10.1007/s10812-017-0456-9 32. V. Divarova, K. Stojnova, P. Racheva, V. Lekova, Russ. J. Inorg. Chem. 2018, 63, 974-977. D0I:10.1134/S0036023618070057 33. M. I. Bulatov, I. P. Kalinkin: Practical Handbook on Photometric Methods of Analysis, Khimiya, Leningrad, Russia, 1986, pp. 174-264. 34. A. Holme, F. J. Langmyhr, Anal. Chim. Acta 1966, 36, 383-391. DOI: 10.1016/0003-2670(66)80066-1 35. W. Likussar, D. F. Boltz, Anal. Chem 1971, 43, 1265-1272. DOI: 10.1016/0003-2670(66)80066-1 Povzetek S spektrofotometričnimi metodami smo raziskali tvorbo ionsko asociiranega kompleksa med anionskim kelatom ger-manija(IV) s 4-nitrokateholom (4-NC) in kationom iz 3-(2-naftil)-2,5-difenil-2ff-tetrazolijevega klorida (TV). Določili smo optimalne pogoje za tvorbo kelata in za ekstrakcijo kompleksa v tekočina-tekočina ekstrakcijskem sistemu Ge(IV)-4-NC-TV-H2O-CHCl3. Preverili smo veljavnost Beerovega zakona ter izračunali nekatere analizne karakteristike. Asociacijski proces v vodni fazi in ekstrakcijsko ravnotežje smo proučili in kvantitativno karakterizirali sledeče ključne konstante: konstanto asociacije, porazdelitveno in ekstrakcijsko konstanto ter izkoristek ekstrakcije. Molsko razmerje komponent v ionsko asociiranem kompleksu smo določili na podlagi neodvisnih metod. Na podlagi dobljenih podatkov je predlagana reakcijska shema, splošna formula in struktura kompleksa. Stojnova and Lekova: Ion-Associated Complex of the Anionic Chelate DOI: 10.17344/acsi.2018.4866 Acta Chim. Slov. 2019, 66, 367-377 ^creative ^commons Scientific paper Effect of Microwave-Assisted Extraction on Polyphenols Recovery from Tomato Peel Waste Marina Tranfic Bakic, Sandra Pedisic, Zoran Zoric, Verica Dragovic-Uzelac and Antonela Nincevic Grassino* Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000 Zagreb, Croatia * Corresponding author: E-mail: aninc@pbf.hr Phone: +385 1 4605 062 Fax: +385 1 4836 083 Received: 11-22-2018 Abstract This study describes the application of microwave-assisted extraction (MAE) as an innovative technique for isolation of polyphenols from tomato peel waste. Effects of solvents, temperatures (25, 55 and 90 °C) and times (5 and 10 min) were evaluated with regard to total phenols (TP), total flavonoids (TF) and phenolic compound contents. Tomato peel extracts contain high amounts of kaemferol-3-O-rutinoside (8.5 to 142.5 mg kg-1), p-coumaric acid (3 to 111.5 mg kg-1) and chlorogenic acid derivative (10.5 to 109 mg kg-1). Results revealed that extraction time has no significant (p > 0.05) influence on TP, TF and phenolic compounds recovery (exception is cis-p-coumaric acid hexoside). On the other hand, the influence of temperature and chosen solvent on polyphenols yield is significant. Considering interest of consumers to intake natural compounds exhibiting antioxidant properties, this work suggests that tomato peel waste can be used as one of sustainable resources for polyphenols production by MAE. Keywords: Tomato peel waste; microwaves-assisted extraction; polyphenols; HPLC-DAD 1. Introduction Tomato edible fruit of Solanum lycopersicum, which belongs to the Solanaceae family, is the second most produced and consumed vegetable in the world, either fresh or processed in the form of canned tomatoes, sauce, juice, ketchup and soup. Industrial processing of tomato generates significant amounts of waste, consisting mainly of tomato peels and seeds, but also leaves and stems. The management of tomato by-products is considered an important problem faced by tomato processing companies, due to its disposal to the environment. Usually, they are partially reused by composting or drying for animal feeding. However, modern eco-compatible technologies offer more efficient strategies to recycle these by-products and reuse them as a sustainable source of different nutrients and highly biologically active compounds, such as amino acids, fatty acids, minerals and carotenoids.1-3 Apart from carotenoids, i.e. lycopene and ^-carotene found in major amounts, tomato by-products are also rich in phenolic compounds.2,4-6 Variations in chemistry of phenolic compounds in fruits, vegetables and agri-industrial wastes are related to different proportions of simple and complex phenols, such as benzoic and cinnamic acids, coumarins, tannins, lignins, lignans and flavonoids.7 These diverse forms of phenolic compounds show variable responses to different extraction conditions. Furthermore, the optimum recovery of pheno-lics differs from one substrate to the other and depends on the type of fruit, vegetable and their by-products. In order to maximize the recovery of polyphenols from tomato waste, different conventional8-15 and non-conventional (sonication or ultrasonic bath)16,17 extraction techniques, as well as operation conditions (time, temperature, polarity of solvent and addition of hydrochloric acid or sodium hydroxide to organic solvents) were employed. Several researches4,5,18,19 have reported that conventional methods, such as Soxhlet extraction and re-fluxing, had significant disadvantages like long extraction times, evaporation of the huge amounts of solvents, and thermal decomposition of thermo-labile compounds. To overcome these limitations, new and promising extraction methods, such as ultrasound-assisted extraction, enzyme-assisted extraction, and pressurized liquid extraction, were recently introduced for polyphenols isola-tion.4,5,18,19 Another one of the innovative and advanced techniques is microwave-assisted extraction (MAE), which provides greater extraction efficiency in shorter time, at lower temperature and with smaller amount of Bakic et al.: Effect of Microwave-Assisted Extraction 368 Acta Chim. Slov. 2019, 66, 367-377 solvent.20,21 In this method, the separation of solutes from sample matrix is based on the conversion of microwave energy to heat, by ionic conduction and dipole rotation.22 The structure and polarity of target compound, environmental safety, human toxicity, and financial feasibility are main factors in the choice of solvent. Water, methanol, ethanol, acetone, hexane and their combinations, are commonly used for polyphenols extractions from tomato waste.8-10,12-17,23 The addition of hydrochloric acid or sodium hydroxide to water or water/organic solvent mixtures was also reported to enhance the extraction of phenolic compounds.10,17 Considering health benefits of phenolic compounds and growing interest of consumers to intake natural compounds exhibiting antioxidant properties, this work presents re-utilization of tomato peel waste from canning factory as an additional, low-cost, sustainable resources for the production of polyphenols. Apart from direct addition of dried tomato waste to various food products, such as meat, bread and cookies,24-26 the carotenoids, mostly lycopene, isolated from tomato by-products could be also utilized as a food colorants and functional ingredients.6 In that context and from the agro-industrial point of view, the phenolic compounds extracted in this work could be further used for enrichment of different foodstuffs. The fact that only a few papers16,17,27 reported extraction of polyphenols from tomato wastes using non-conventional extraction method inspired us to focus our attention on utilization of MAE as a novel, not yet explored method for polyphenols isolation from tomato peel. Hence, this paper describes the effect of solvent type, acidity, temperature, and time on extraction of polyphenols using MAE as simple, quick and environmentally friendly technique, and defines the optimal parameters for MAE that yield the highest possible amounts of polyphenols with minimum expenditure of extraction resources. 2. Materials and Methods 2. 1. Materials All the reagents, standards and solvents were of analytical grade. Folin-Ciocolateu reagent and all standards (gallic acid, rutin, caffeic acid, chlorogenic acid, p-couma-ric acid, quercetin and kaempferol) were purchased from Sigma Aldrich (Steinheim, Germany). Ethanol, methanol, hydrochloric acid, sodium carbonate, sodium nitrite and aluminium chloride hexahydrate were obtained from Kefo (Zagreb, Croatia). Formic acid and acetonitrile (HPLC grade) was purchased from Prolabo (Lutterworth, UK). Deionised water used for the preparation of reagents, standards and solvents was obtained with Millipore-MilliQ apparatus. Tomato peel waste was purchased from the manufacturing unit (after processing of fresh tomato) of Benin- Table 1. Microwave-assisted extraction (MAE) of polyphenols from tomato peel waste using water, 1% HCl, 50 and 70% methanol (MeOH) with and without addition of 1% HCl, and 50 and 70% ethanol (EtOH), at temperature of 25, 55 and 90 °C, and time of 5 and 10 min Run Sample Temperature/°C Solvent HCl Time/min 1 1A 25 2 1A 55 5 3 4 1A 1B 90 H2O 25 2 / 5 1B 55 10 6 1B 90 7 2A 25 8 2A 55 9 2A 90 10 2B 25 11 2B 55 12 2B 90 13 3A 25 14 3A 55 5 15 16 3A 3B 90 25 50% (v/v) MeOH 1 %(v/v) 17 3B 55 10 18 3B 90 19 4A 25 5 20 4A 55 21 22 4A 4B 90 25 70% (v/v) MeOH 1% (v/v) 23 4B 55 10 24 4B 90 25 5A 25 26 5A 55 5 27 28 5A 5B 90 25 50% (v/v) MeOH / 29 5B 55 10 30 5B 90 31 6A 25 32 6A 55 5 33 34 6A 6B 90 25 70% (v/v) MeOH / 35 6B 55 10 36 6B 90 37 7A 25 38 7A 55 5 39 40 7A 7B 90 25 50% (v/v) EtOH / 41 7B 55 10 42 7B 90 43 8A 25 44 8A 55 5 45 46 8A 8B 90 25 70% (v/v) EtOH / 47 8B 55 10 48 8B 90 5 H2O 1% (v/v) 10 Bakic et al.: Effect of Microwave-Assisted Extraction Acta Chim. Slov. 2019, 66, 367-377 369 casa S.r.l., canning industry located in the Agro Nocerina area of Campania (Angri, Italy). The tomato peel was dried, grounded and packed in polyethylene bags until analysis. 2. 2. Microwave-Assisted Extraction Procedure Microwave-assisted extractions (MAEs) were carried out in a Milestone START S Microwave Labstation for Synthesis (Bergamo, Italy), using water and water solution of HCl (1%, v/v), methanol (50 and 70%, v/v), with or without addition of 1% HCl and ethanol (50 and 70% v/v) (Table 1). The MAE parameters were times of 5 and 10 min, and temperatures of 25, 55 and 90 °C (Table 1). Tomato peel sample (1.00 g) was mixed with 50 mL of solvent in a round flask, placed in the MAE apparatus equipped with a condenser and subjected to microwave irradiation, at atmospheric pressure. The power of irradiation was varied from 0 to 500 W to maintain the set extraction temperature. After extraction, the mixture was left to cool down at room temperature and then filtered. The extracts were collected and stored at -8 °C until further analyses. 2. 3. Total Phenols Content Total phenols (TP) content was determined using the Folin-Ciocalteu method, according to the procedure obtained by Spanos and Worlstad.28 The detailed procedure is given in the Supporting Information (SI). TP content is expressed as g of gallic acid equivalent (GAE) per kg of tomato peel sample. All the experiments were performed in triplicate and the presented results are the mean values. 2. 4. Total Flavonoids Content Total flavonoids (TF) content was determined using the aluminum chloride colorimetric assay developed by Zhishen, Mengcheng and Jianming.29 The detailed procedure is given in the SI. TF content is expressed as g of rutin equivalent (RE) per kg of tomato peel sample. All the experiments were performed in triplicate and the results are reported as mean values. 2. 5. Phenolic Compounds Content HPLC analysis of individual phenolic compounds in the tomato peel extracts was performed by a liquid chromatograph (Agilent Technologies 1260 series) with a quaternary pump, equipped with a diode array detector (DAD). The HPLC system was controlled by OpenLAB ChemStation Software. Phenolic compounds were separated on a 250 x 4.6 mm, 5 ^m film thickness Nucleosil 100-5C18 column (Sigma Aldrich). The column tempera- ture was 35 °C. The gradient elution conditions were previously described by Barros et al.30 Identification of phenolic compounds was carried out by comparing the retention time and spectral data with those of standards, 1.e. caffeic acid, chlorogenic acid, p-coumaric acid, querce-tin and kaempferol (Sigma Aldrich). The results are expressed in mg of phenolic compounds per kg of tomato peel. 2. 6. Experimental Procedure and Statistical Analysis The evaluation of under-utilized tomato peel waste as a potential source of polyphenols and MAE as an advanced technique for their extraction was performed in three steps. Firstly, TP and TF were quantified spectropho-tometrically, screening the extraction parameters, i.e. solvent, time, temperature and addition of hydrochloric acid (Table 1), to determine which combination assures the highest amounts. Afterwards, selected phenolic compounds were identified and quantified by HPLC-DAD, and influence of extraction parameters on their yields was studied. Finally, multivariate analyses of variance (MANO-VA) were performed using Statistica 12 package (StatSoft) to compare the mean mass fraction values (N = 3) of TP, TF and individual phenolic compounds, depending on the used solvents, addition of hydrochloric acid, extraction time and temperature. Post-hoc Tukey HSD test provided additional insight by identifying the means that are significantly different from the other (p < 0.05). 3. Results and Discussion 3. 1. Influence of Experimental Conditions on Total Phenols and Flavonoids Recovery Content of total phenols (TP) and total flavonoids (TF) in tomato peel samples, acquired by MAE are presented in Table 2. Generally, all samples contained high amounts of TP with variations due to different extraction time, temperature, and solvent. The average TP content was 53.12 g kg-1, and the lowest value was obtained for samples extracted at 25 °C, while higher values were found in samples extracted at 55 and 90 °C. Evidently, the temperature (Figure 1 and Figure S1, SI) had a significant influence on the extractions of phenols (p = 0.000125 for 25 °C vs 90 °C and p = 0.000770 for 55 °C vs 90 °C). Regarding the TF content, the average amount was 50.36 g kg-1. Again, higher extraction temperatures yielded higher average contents of TF. Like for phenols, the increase of temperature also improved the solubility of flavonoids (Figure 1 and Figure S1, SI). A rise from 25 to 55 and 90 °C significantly (p = 0.000126 and p = 0.000223) en- Bakic et al.: Effect of Microwave-Assisted Extraction 370 Acta Chim. Slov. 2019, 66, 367-377 Table 2. Content of total phenols (TP) and total flavonoids (TF) obtained from tomato peel waste, extracted by MAE, at temperature of 25, 55 and 90 °C for 5 min (1-8A) and 10 min (1-8B), using water (1A-B), 1% HCl (2A-B), 50 and 70% methanol with (3A-B and 4A-B) and without (5A-B and 6A-B) addition of 1% HCl, and 50 and 70% ethanol (7A-B and 8A-B) Sample 25 w(TP)/(g kg-1) ± SD* 55 T (extraction)/°C 90 25 w(TF)/(g kg-1) ± SD* 55 90 t = 5 min 1A 54.01 ± 0.06 55.03 ± 0.04 54.50 ± 0.12 39.84 ± 0.19 57.05 ± 0.17 36.57 ± 0.07 2A 57.18 ± 0.11 48.63 ± 0.01 59.86 ± 0.09 39.30 ± 0.11 36.08 ± 0.08 46.30 ± 0.09 3A 57.98 ± 0.06 50.91 ± 0.13 57.81 ± 0.03 39.28 ± 0.19 34.46 ± 0.15 43.95 ± 0.08 4A 35.08 ± 0.17 55.29 ± 0.70 63.57 ± 0.19 45.56 ± 0.24 36.92 ± 0.13 55.98 ± 0.09 5A 40.82 ± 0.12 52.25 ± 0.03 61.65 ± 0.06 50.14 ± 0.29 46.77 ± 0.16 61.76 ± 0.15 6A 32.55 ± 0.16 44.81 ± 0.19 62.69 ± 0.09 29.92 ± 0.18 49.60 ± 0.08 62.88 ± 0.22 7A 44.32 ± 0.05 54.81 ± 0.13 63.36 ± 0.02 38.73 ± 0.24 51.45 ± 0.33 71.37 ± 0.47 8A 37.15 ± 0.15 46.66 ± 0.29 59.45 ± 0.05 46.87 ± 0.25 57.77 ± 0.07 107.47 ± 0.05 t = 10 min 1B 57.67 ± 0.03 51.07 ± 0.12 57.21 ± 0.07 41.20 ± 0.15 33.05 ± 0.04 47.30 ± 0.04 2B 58.40 ± 0.21 55.32 ± 0.56 60.06 ± 0.22 39.97 ± 0.30 35.47 ± 0.09 43.28 ± 0.09 3B 57.38 ± 0.17 53.39 ± 0.73 59.29 ± 0.07 42.30 ± 0.10 37.08 ± 0.15 54.18 ± 0.04 4B 39.03 ± 0.33 59.36 ± 0.17 67.33 ± 0.05 51.80 ± 0.32 38.86 ± 0.18 60.32 ± 0.32 5B 36.49 ± 0.06 55.44 ± 0.07 78.06 ± 0.05 35.58 ± 0.12 49.63 ± 0.13 65.85 ± 0.18 6B 29.44 ± 0.07 44.79 ± 0.36 70.12 ± 0.10 32.62 ± 0.07 54.56 ± 0.10 72.14 ± 0.45 7B 43.37 ± 0.03 60.12 ± 0.16 71.85 ± 0.08 42.83 ± 0.05 56.87 ± 0.17 76.16 ± 0.11 8B 36.16 ± 0.20 45.13 ± 0.11 52.90 ± 0.02 49.73 ± 0.77 66.41 ± 0.50 104.36 ± 0.09 mean 44.81 ± 10.50 52.06 ± 4.94 62.48 ± 6.59 41.60 ± 6.15 46.38 ± 10.48 63.11 ± 20.22 grand mean 53.12 ± 10.52 50.36 ± 16.26 *SD = standard deviation Figure 1. 3D categorized plots of temperature (25, 55 and 90 °C), time (5 and 10 min), and solvent interactions on contents of total phenols (TP) and total flavonoids (TF), extracted from tomato peel waste by MAE. Bakic et al.: Effect of Microwave-Assisted Extraction 371 Acta Chim. Slov. 2019, 66, 367-377 hanced TF content, due to acceleration of solvent diffusion into the sample matrix. However, some values obtained for TF were greater than the corresponding (same temperature, time and solvent) TP values, particularly that obtained after extraction at 90 °C, using water/organic solvent mixtures. This can be accounted by consideration that some of the phenolic compounds (isoflavone, antocyanin, cinnamic acid, flavanone, flavones, chalcone, flavonol and aurone),31 as well as other organic compounds presented in tomato cuticle32 were involved in complexation with AlCl3. Due to the fact that AlCl3 is unspecific reagent in its chelating power, we could suppose that in total sum of fla-vonoids were additionally included reactions with all of other mentioned compounds. In contrast to effects of temperature, increase of extraction time from 5 to 10 min had no significant influence on TP (p = 0.333826) and TF (p = 0.519694) contents (Figure 1 and Figure S2, SI). Therefore, extraction time of 5 min is sufficient for a successful microwave-assisted extraction of phenols from tomato peel waste, and could be considered as optimal for further MAE extraction. Considering solvents used for extractions, the data (Table 2 and Figure 1) revealed that higher proportion of water added to organic solvents provided better diffusion of phenols through tomato peel cuticle and consequently yielded higher amounts of TP. In that context, 50% methanol (5A-B) and ethanol (7A-B) provided better extraction of TP, compared to 70% methanol (6A-B) and ethanol (8A-B), with some exception (90 °C, 5 min, 5A and 6A). Although, slightly higher values were obtained using 50% ethanol than 50% methanol with somewhat lower values obtained at 90 °C (5B-7B), both solvents can be considered an adequate choice for further MAE, depending on applied temperatures. Several researches33,34 already suggested that a binary solvent system, with lower volume fractions of organic solvents were efficient for extractions of phenols. Moreover, pure water, particularly at lower temperatures of extraction (25 and 55 °C) acted as excellent solvent compared to organic solvent/water mixtures (Table 1 and Figure 1), depending on extraction time (5 min). Due to the fact that water has higher dielectric constant and can thus absorb more microwave energy (by ionic conduction and dipole rotation mechanisms) compared to less polar solvents, the result is higher extraction of phenols. Pure water was used by El-Mahan et al.27 for extraction of polyphenols from Egyptian tomato waste by MAE. They reported values of 355.5, 375.0 and 377.5 mg/100 g (TP) and 43.33, 46.66 and 49.89 mg/100 g (TF), for 30, 60 and 90 seconds of extraction, respectively. Except water, 1% aqueous solution of HCl as polar solvent at lower extraction temperature (25 °C) also gave the high amounts of TP, regardless of the extraction time. However, addition of HCl to other solvents, i.e. 50 and 70% methanol, did not significantly (p > 0.05) influence on TP and TF amounts (Figure 1 and Figure S3, SI). Com- pared to 1% aqueous HCl, organic solvents/water mixture, particularly at temperatures of 55 and 90 °C assured better extractions of flavonoids (Table 2 and Figure 1). Considering common un-polar structure of flavonoids, it was expected that organic solvents/water mixtures will be more appropriate solvents than 50 and 70% methanol with addition of 1% hydrochloric acids. In summary, tomato peel waste obtained from the canning industry possessed remarkable amounts of TP and TF, compared to values reported by other authors.8,12-14,23,35 In reference to other extraction parameters, temperature had the greatest effect on extraction yields of phenols and flavonoids. However, their recovery was also associated with the appropriate choice of solvent and its combination with temperature of extraction. In that context, the temperature of 90 °C and 50% methanol with addition of 1% HCl, as well as temperature of 90 °C and 70% ethanol could be considered as optimal extraction conditions for phenols and flavonoids recovery from tomato peel waste, at time of 5 min. 3. 2. Influence of Experimental Conditions on Individual Phenolic Compounds Recovery The quantification of individual phenolic compounds, namely phenolic acids and their derivatives: p-coumaric acid (p-CA), cis-p-coumaric acid derivative (cis-p-CA-der), p-coumaric acids hexoside (cis-p-CA hexoside), chlorogenic acid (ChA), chlorogenic acid derivative (ChA-der), caffeic acid hexoside I (CA hexoside I), and caffeic acid hexoside II (CA hexoside II), and fla-vonols quercetin-pentosylrutinoside (Q pentosylrutino-side), quercetin-3-O-rutinoside (Q-3-O-rutinoside), kae-mpferol-pentosylrutinoside (K pentosylrutinoside), and kaempferol-3-O-rutinoside (K-3-O-rutinoside), in tomato peel extracts obtained after MAE was performed by HPLC-DAD analysis and are presented in Figure 2. The studied compounds were identified in the majority of the extracts. Among them, CA hexoside I, CA hexoside II and ChA were not found in samples extracted by water, 1% HCl, and 50 and 70% ethanol, respectively. Furthermore, flavonols were not identified in samples extracted by water and 1% HCl. In addition, Q and K pentosylrutinoside, and Q-3-O-rutinoside was not found in 70% ethanol, and 50% methanol extracts, respectively. Among phenolic acids and its derivatives (Figure 2), p-CA (3 to 111.5 mg kg-1) and ChA der (10.5 to 109 mg kg-1) was predominant, compared to ChA (6 to 62 mg kg-1) and cis-p-CA-der (1 to 47.5 mg kg-1). Regarding other derivatives, cis-p-CA hexoside, CA hexoside I and CA hexoside II were found in lower amounts, i.e. 0.5 to 29 mg kg-1, 1.5 to 31 mg kg-1 and 9 to 25 mg kg-1, respectively. Several papers 8,10,15-17 reported the presence of phenolic acids (caffeic, chlorogenic, p-coumaric, ferulic, phloretic, sinapic, vannilic, syringic, gallic, and rosmarin- Bakic et al.: Effect of Microwave-Assisted Extraction ... 372 Acta Chim. Slov. 2019, 66, 367-377 Bakic et al.: Effect of Microwave-Assisted Extraction ... Acta Chim. Slov. 2019, 66, 367-377 373 Figure 2. Content of individual phenolic compounds extracted from tomato peel waste by MAE, at temperature of 25, 55 and 90 °C, time of 5 min (1A-8A) and 10 min (1B-8B), using water, 1% HCl, 50 and 70% methanol with and without addition of 1% HCl, and 50 and 70% ethanol. ic) and their derivatives (chlorogenic, p-coumaric, feru-lic-O-hexoside, and caffeic-O-hexoside), flavonoids (quer-cetin, rutin, chrysin, epicatechin, catechin, kaempferol, luteonin, and naringenin) and their derivatives, extracted from different tomato wastes. For instance, Kalogeropou-los et al.12 reported quantities of p-CA (10.7 mg kg-1, dw) and ChA (51.7 mg kg-1, dw) lower than ours. Navarro-Gonzalez et al.10 found p-CA and p-CA der in the range from 7.38 to 26.58 mg kg-1 (dw) and 33.00 to 141.10 mg kg-1, respectively, depending on used extraction method (enzymatic treatment, maceration and sonication). ChA was also found in the works published by Lavelli and Tor-resani9 in quantities of 97 and 121 mg kg-1 (dw) for raw and heat-treated tomato by-products, and Aires et al.17 in the range from 17.3 to 43.7 mg kg-1 (dw), depending on the used extraction method and solvents. Among flavonols (Figure 2) K-3-O-rutinoside was found in the highest amounts, 8.5 to 142.5 mg kg-1, depending on used solvent, temperature, and time. Q-3-O-ru-tinoside was also found in considerable amounts (3 to 78 mg kg-1), compared to Q pentosylrutinoside (4.5 to 21 mg kg-1) and K pentosylrutinoside (2.5 to 13 mg kg-1). For comparison, the work of Kalogeropoulos et al.12 showed quantities of K-3-O-rutinoside of 5.5 mg kg-1 (dw) in tomato processing by-products. Aires et al.17 found 57.8 to 259.3 mg kg-1 (dw) of K-3-O-rutinoside and 15.0 to 45.1 mg kg-1 (dw) of Q-3-O-rutinoside, depending on the used extraction method and solvents. With the purpose to estimate the significance of MAE experimental parameters and interactions between them that provide the highest contents of individual phenolic compounds, the results of statistical analyses are described below. The increase of extraction times from 5 to 10 min (Figure S4, SI) significantly influenced (p = 0.044719) the content of cis-p-CA hexoside. However, raise of time did not significantly affect the recovery of other isolated phenolic compounds. Therefore, the extraction time of 5 min is appropriate for their extraction, according to main advantage of MAE, i.e. applying shorter extraction time. Regarding applied temperatures of extraction, the increases from 25 vs 55 to 90 °C did not significantly influ- ence the p-CA, ChA, ChA der, K pentosylrutinoside, and CA hexoside II contents (Figure 3). However, the raise of temperature significantly influenced the recovery of cis-p-CA der (55 vs 90 °C, p = 0.005986) and Q pentosylrutinoside (25 vs 90 °C, p = 0.034721 and 55 vs 90 °C, p = 0.000742). In the case of these two compounds, the temperature of 90 °C should be avoided due to degradation effects. On the other hand, the temperature of 90 °C had a significant influence on the highest recovery of cis-p-CA hexoside (p = 0.000955 for 25 vs 90 °C and p = 0.000143 for 55 vs 90 °C), CA hexoside I (p = 0.029699 for 25 vs 90 °C and p = 0.028620 for 55 vs 90 °C), and K-3-O-rutinoside (p = 0.001415 for 25 vs 90 °C and p = 0.000466 for 55 vs 90 °C) from tomato peel extracts. Regarding solvents used for p-CA extraction from tomato peel (Figure 3), 50 and 70% methanol with and without addition of1% HCl, as well as 50% ethanol showed significant (p < 0.05) influence on p-CA recovery, compared to water or 1% HCl solution. Furthermore, significantly higher (p < 0.05) values were also obtained using 50 and 70% methanol compared to 50 and 70% ethanol. In the case of cis-p-CA der, several solvents, such as 1% HCl, 50 and 70% methanol with and without addition of HCl can be considered as a good choice for cis-p-CA-der extraction by MAE. Due to the fact that temperature of 90 °C had a significant effect on degradation of cis-p-CA der, the temperature of 25 °C in combinations with 1% HCl and 50 and 70% methanol with addition of 1% HCl could be recommended for its extraction, particularly for 10 min. In addition, high contents of cis-p-CA-der, i.e. 35 and 34 mg kg-1 for 5 and 10 min of extraction, using 50% methanol, at 55 °C revealed that this combination could be also applied for further successful extraction of this compound by MAE. High contents of cis-p-CA hexoside (Figures 2 and 3) were obtained at 90 °C, using 50 and 70% methanol with addition of 1% HCl, which confirmed these solvents efficiency not only for p-CA and cis-p-CA der extractions, but also for cis-p-CA hexoside. Mentioned solvents can be also recommended for extractions of ChA and its derivatives, due to significant (p < 0.05) influence on their quantities. Bakic et al.: Effect of Microwave-Assisted Extraction 374 Acta Chim. Slov. 2019, 66, 367-377 Bakic et al.: Effect of Microwave-Assisted Extraction ... Acta Chim. Slov. 2019, 66, 367-377 375 Figure 3. Influence of temperature (25, 55 and 90 °C) and solvent on contents of individual phenolic compounds extracted from tomato peel waste by MAE Bakic et al.: Effect of Microwave-Assisted Extraction 376 Acta Chim. Slov. 2019, 66, 367-377 For instance, using 50% methanol with addition of 1% HCl (3A-B) significant differences of ChA were observed vs water (p = 0.000486), 1% HCl (p = 0.020184), 50% methanol (p = 0.005655), 70% methanol (p = 0.001195), 50% ethanol (p = 0.000364) and 70% ethanol (p = 0.020184). By 70% ethanol with addition of 1% HCl (4A-B), the obtained p-values were also significant vs: water (p = 0.006096), 70% methanol (p = 0.015760), 50% ethanol (p = 0.004196) and 70% ethanol (p = 0.001284). Furthermore, 50 and 70% methanol or ethanol without addition of 1% HCl can also be used for ChA extraction, depending on temperature and extraction time. In the case of CA hexoside I and II, 50 and 70% methanol with and without addition of 1% HCl showed significant influence on their contents, compared to water or 1% HCl. Regarding flavonols, the extraction of Q-3-O-ruti-noside was significantly influenced by 50% (p = 0.004893) and 70% methanol (p = 0.000641). The highest amounts were obtained by 70% methanol, i.e. 75.5 and 78 mg kg-1, for 5 and 10 min of extraction. The recovery of K-3-O-rutinoside was significantly influenced by 70% methanol (p = 0.000283), 50 and 70% methanol with addition of HCl (p = 0.007673 and p = 0.002455), compared to other used solvents. High extraction yield of this compound was obtained at temperature of 90 °C for 5 min, using 70% methanol (142.5 mg kg-1), followed by 70% and 50% methanol with addition of 1% HCl, which gave values of 125 and 112.5 mg kg-1, respectively. Although found in minor amounts (Figures 2 and 3), extractions of Q pentosylrutinoside and K pentosylrutino-side were affected by application of organic solvents, regardless of addition of 1% HCl. To summarize, the HPLC-DAD analyses revealed that tomato peel waste presents an important source for exploitation of phenolic compounds. However, their contents depend on extraction conditions, with emphasis on interaction between the chosen temperature and solvent. The addition of 1% HCl to 50 or 70% methanol assured good recovery of majority of phenolic acids and their derivatives. However, 50% and particularly 70% methanol provided better recovery of some flavonols, such as K-3-O-rutinoside. In addition, particular care should be taken regarding the temperature applied during MAE, since an increase in temperature can have a profound influence on degradation of cis-p-CA-der and Q pentosylru-tinoside, as well as enhancement of amounts of cis-p-CA hexoside, CA hexoside I and K-3-O-rutinoside. Therefore, taking into account that extraction time had no significant influence on extraction of majority of phenolic compounds, we propose the extraction time of 5 min in combination with the follow solvent and temperature as optimal, i.e. that which exhibited the highest recovery of phenolic compound: i) 50% methanol with addition of 1% HCl and 25 °C for cis-p-CA der, ii) 70% methanol with addition of 1% HCl and 55 °C for ChA, and 90 °C for cis-p-CA hexoside and CA hexoside II, respectively, iii) 50% methanol and 90 °C for p-CA, and 55 °C for ChA der, Q pentosylrutinoside and K pentosylrutinoside, respectively and iv) 70% methanol and 90 °C for CA hexoside I, Q-3-O-rutinoside and K-3-O-rutinoside. 4. Conclusion The present study showed that tomato peel waste from the canning industry could be utilized as a sustainable, low cost source for polyphenols production. Results revealed that polyphenols were isolated in remarkable amounts with minimal expenditure of time (5 min), applying MAE as an innovative and eco-friendly technique. Among evaluated extraction parameter, temperatures and solvents have a considerable influence on total phenols, total flavonoids, and individual phenolic compounds yields. Various combinations of these parameters result with isolation of different amounts of individual phenolic compounds. Therefore, additional MAE extractions of tomato peel waste should be performed combining temperature and solvent type adequate for isolation of one or more target compounds with similar structures and properties. 5. Acknowledgements This work was supported by the Croatian Ministry of Science, Education, and Sports, No. 058-0580000-3071. The authors are grateful to Giovanni Paolo Buoninconti from Benincasa S.r.l., Angri (SA) canning industry for providing the tomato peel waste. 6. Reference 1. A. Zuorro, R. Lavecchia, F. Medici and L. 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Ferreira and C. Santos-Buelga, Food Chem. Toxicol. 2012, 50, 1576-1582. D0I:10.1016/j.fct.2012.02.004 31. J. B. Harborne, in J. B. Pridhan (Ed.): Methods in Polyphenol Chemistry, Elsevier, Oxford, England, 1964, pp. 13-36. D0I:10.1016/B978-0-08-010887-2.50006-2 32. K. H. Caffall and D. Mohnen, Carbohydr. Res. 2009, 344, 1879-1900. D0I:10.1016/j.carres.2009.05.021 33. M. Dent, V. Dragovic-Uzelac, M. Penic, M. Brncic, T. Bosil-jkov and B. Levaj, Food Technol. Biotechnol. 2013, 51, 84-91. 34. P. Putnik, D. B. Kovacevic, M. Penic, M. Feges and V. Drago-vic-Uzelac, Food Anal. Methods. 2016, 9, 2385-2394. D0I:10.1007/s12161-016-0428-3 35. S. Gharbi, G. Renda, L. La Barbera, M. Amri, C. M. Messina and A. Santulli, Nat. Prod. Res. 2017, 31, 626-631. DOI: 10.1080/14786419.2016.1209671 Povzetek V delu je preučena uporaba ekstrakcije z mikrovalovi (ang. MAE) kot inovativna tehnika za izolacijo polifenolov iz odpadnih olupkov paradižnika. Ovrednotili smo vpliv topila, temperature (25, 55 in 90 °C) in časa (5 in 10 min) glede vsebnosti celotnih fenolov (TP), celotnih flavonoidov (TF) in fenolnih spojin. Odpadni olupki paradižnikov vsebujejo visoke količine kaemferol-3-O-rutinozida (8.5 do 142.5 mg kg-1), p-kumarinske kisline (3 do 111.5 mg kg-1) in derivate klorogene kisline (10.5 do 109 mg kg-1). Izkazalo se je, da čas ekstrakcije nima znatnega vpliva (p > 0.05) na izkoristek TP, TF in fenolnih spojin (z izjemo heksazida cis-p-kumarinske kisline). Po drugi strani pa se je izkazalo, da je izkoristek polifenolov odvisen od temperature in izbranega topila. Upoštevaje znatno zanimanje uporabnikov za uživanje naravnih snovi z antioksidativnimi lastnostmi, predstavljena raziskava kaže, da lahko ostanke paradižnikovih lupin uporabimo kot trajnostni vir polifenolov z uporabe MAE. Bakic et al.: Effect of Microwave-Assisted Extraction DOI:10.17344/acsi.2018.4876 Acta Chim. Slov. 2019, 66, 378-384 /^creative ^commons Scientific paper Cyclometalated Iridium(III) Complexes Containing 2-Phenylbenzo[d]oxazole Ligand: Synthesis, X-ray Crystal Structures, Properties and DFT Calculations Xiao-Han Yang,1 Qian Zhang,1 Hui Peng,1 Zi-Cen Zuo,1 Ding Yuan,1 Yan Chen,1 Qin Chen,1 Guang-Ying Chen,2 Zhi-Gang Niu1,2 and Gao-Nan Li1,* 1 Key Laboratory of Electrochemical Energy Storage and Energy Conversion of Hainan Province, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China 2 Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, Hainan Normal University, Haikou 571158, PR China * Corresponding author: E-mail: ligaonan2008@163.com Received: 11-25-2018 Abstract Two new iridium(III) complexes were synthesized and fully characterized, [(bo)2Ir(pzpy)] (2a) and [(bo)2Ir(pzpyz)] (2b) (where bo = 2-phenylbenzo[d]oxazole, pzpy = 2-(1H-pyrazol-3-yl)pyridine, pzpyz = 2-(1H-pyrazol-3-yl)pyrazine). The single crystal structures of 2a-2b have been determined. Considering the relationship between their structures and photophysical properties, DFT calculations have been used to further support this inference. These Ir(III) complexes emit from the excited state of 3MLCT/3LLCT in the green and yellow region, and the quantum yields in the degassed CH2Cl2 solution at room temperature are 35.2% and 46.1%. Theoretical and experimental results show that iridium(III) complexes 2a-2b are promising phosphorescent material. Keywords: Iridium(III) complex; Crystal structure; 2-phenylbenzo[d]oxazole; Photoluminescence; DFT calculation 1. Introduction Neutral mononuclear cyclometalated iridium complexes have been found to be suitable for use in organic light emitting diodes (OLEDs).1-3 The privileged use is due to their interesting luminescence properties,4-5 such as high quantum yields, long excited-state lifetimes, and tunable emission color over the entire visible spectrum.6-7 Most often, the variation of their emission color was largely governed by the cyclometalated and/or ancillary ligands structures. 2-phenylbenzo[d]oxazole (bo) is one typical ligand framework for constructing Ir(III) complexes, and can be used to fine-tune the emission color of complexes by judicious modification.8-10 For example, in 2015, we reported Ir(bo)2(acac) derivatives with substituents on the benzox-azole ring and their emissions covered a narrow range from 560 to 566 nm.11 The color adjusting by the change of cyclometalated ligands structures were not very satisfactory, although the quantum yield was up to 53.5%. Afterward, we designed a series of bo-based iridium(III) com- plexes with different NAO ancillary ligands. They exhibited a wide range of emission wavelengths (Xmax = 531-598 nm) with high quantum yields (19%-94%).12 The research findings showed that the structures of ancillary ligands have obvious effect on tuning the emission color of bo-based iridium(III) complexes. Therefore, we wanted to further investigate other types of ancillary ligands. In this paper, we design two NAN ancillary ligands (a and b) and synthesize two bo-based iridium(III) complexes (2a and 2b) (Scheme 1). The photophysical and electrochemical properties of these complexes were investigated, and the lowest energy electronic transitions were analyzed based on density functional theory (DFT) and time-dependent DFT (TDDFT). 2. Experimental 2. 1. Materials and Instrumentations 2-aminophenol and benzaldehyde were obtained from Hebei Guanlang Biotechnology Co., Ltd.. 2-(prop-1- Yang et al.: Cyclometalated Iridium(III) Complexes Containing Acta Chim. Slov. 2019, 66, 378-384 379 en-2-yl)pyridine, 2-(prop-1-en-2-yl)pyrazine and 1,1-di-methoxy-N,N-dimethylmethanamine were obtained from Dayang Chemicals Co., Ltd., SAGECHEM LIMITED and Sigma-Aldrich, respectively. IrCl3 • 3H2O was industrial products. The target ligands 2-phenylbenzo[d]oxazole (1), 2-(1H-pyrazol-3-yl)pyridine (a) and 2-(1H-pyrazol-3-yl) pyrazine (b) were prepared according to the literature method.13-15 All commercial chemicals were used without further purification unless otherwise stated. Solvents were dried and degassed following standard procedures. 1H NMR spectra were recorded on a Bruker AM 400 MHz instrument. Chemical shifts were reported in ppm relative to Me4Si as internal standard. UV-Vis spectra were recorded on a Hitachi U3900/3900H spectrophotometer. Fluorescence spectra were carried out on a Hitachi F-7000 spectrophotometer. The FTIR spectra were taken on a Nicolet 6700 FTIR spectrometer (400-4000 cm-1) with KBr pellets. 2. 2. Synthesis of (bo)2Ir(pzpy) (2a) A mixture of IrCl3 ■ 3H2O (306 mg) and 2-phenyl-benzo[d]oxazole (500 mg) in 15 mL of 2-ethoxyethanol and H2O (v: v = 2:1) was heated at 120 °C for 12 hours under N2. After cooling to room temperature, the yellow precipitate was collected by filtration and washed with cooled ether and MeOH. After drying, the crude product of chlorine bridged dimer complex [(bo)2Ir(p-Cl)]2 can be used in the next step without further purification. This was followed by crude chlorine bridged dimer (207 mg), 2-(1H-pyrazol-3-yl)pyridine (54 mg, 2.2 eq ) and Na2CO3 (88 mg, 5.0 eq). In 2-ethoxyethanol (10 ml), the mixture was heated at 120 °C under N2 for 12 hours. After removing the solvent, the mixture was poured into water, extract- ed three times with CH2Cl2, and then evaporated. The residue was purified by flash column chromatography (DCM: MeOH = 50: 1) to obtain iridium complex 2a as a yellow solid (76 mg, yield: 64.6%). 1H NMR (400 MHz, CDCl3) 5 7.93 (s, 1H), 7.80-7.67 (m, 4H), 7.56 (dd, J = 17.5, 9.0 Hz, 2H), 7.31 (d, J = 6.9 Hz, 2H), 7.14 (s, 2H), 6.96 (dd, J = 44.3, 7.6 Hz, 6H), 6.71 (s, 2H), 6.65 (s, 1H), 5.97 (dd, J = 16.8, 7.8 Hz, 2H). MS (ESI): m/z = 726.0 [M+]. IR (KBr, cm-1): 2856(w), 1649(w), 1592(vs), 1520(m), 1448(vs), 1387(s), 1252(w), 1188(w), 1130(s), 1084(s) 1045(w), 816(w), 742(m), 474(m). Calcd for C33H21IrN5O2 (%): C 56.34, H 3.06, N 9.66; Found: C 55.67, H 3.22, N 9.37. 2. 3. Synthesis of (bo)2Ir(pzpyz) (2b) The complex 2b (70 mg, yield: 59.4%) was obtained using 2-(1H-pyrazol-3-yl)pyrazine instead of 2-(1H-pyra-zol-3-yl)pyridine by a method similar to that of preparing 2a. 1H NMR (400 MHz, CDCl3) 5 9.04 (s, 1H), 8.22 (d, J = 2.3 Hz, 1H), 7.92 (s, 1H), 7.82 (dd, J = 16.8, 9.6 Hz, 3H), 7.69-7.56 (m, 2H), 7.42-7.33 (m, 2H), 7.19 (t, J = 7.8 Hz, 1H), 7.14-6.91 (m, 5H), 6.88 (s, 1H), 6.74 (d, J = 7.5 Hz, 1H), 6.65 (d, J = 7.6 Hz, 1H), 6.01 (dd, J = 26.9, 8.0 Hz, 2H). MS (ESI): m/z = 727.0 [M+]. IR (KBr, cm-1): 2962(m), 2920(m), 2854(w), 1595(m), 1520(m), 1452(m), 1387(m), 1335(w), 1259(s), 1092(vs), 1030(vs), 804(vs), 742(m), 476(w). Calcd for C33H21IrN6O2 (%): C 54.61, H 2.92, N 11.58; Found: C 54.95, H 3.08, N 11.04. 2. 4. Crystallographic Studies X-ray diffraction data were collected with an Agilent Technologies Gemini A Ultra diffractometer equipped Yang et al.: Cyclometalated Iridium(III) Complexes Containing 380 Acta Chim. Slov. 2019, 66, 378-384 with graphite-monochromated Mo Ka radiation (X = 0.71073 A) at room temperature. Data collection and reduction were processed with CrysAlisPro software.16 The structure was solved and refined using Full-matrix least-squares based on F2 with program SHELXS-97 and SHELXL-9717 within Olex2.18 All non-hydrogen atoms were found in alternating difference Fourier syntheses and least-squares refinement cycles and, during the final cycles, refined anisotropically. Hydrogen atoms were placed in calculated positions and refined as riding atoms with a uniform value of (7iso. 2. 5. Computational Method The geometry of complexes 2a-2b was optimized starting from the X-ray data by the DFT (density functional theory) method with B3LYP (Becke three-parameter Lee-Yang-Parr) hybrid density functional theory and the 6-31G* basis set. All calculations were carried out with Gaussian 09 software package.19 3. Results and Discussion 3. 1. Description of Crystal Structure The single crystal structures of 2a-2b were obtained by X-ray diffraction studies, and ORTEP diagrams are shown in Fig. 1. The crystallographic data and structural details are given in Table 1. The selected bond lengths and bond angles are collected in Table S1. For the structures of these complexes, the Ir(III) centre adopts a twisted octahedral geometry, and the CAN li-gands are in cis-C,C and trans-N,N' configurations. The Ir-N average bond lengths of 2a (2.077Â) and 2b (2.100Â) are longer than the Ir-C average bond lengths of 2a (2.019Â) and 2b (2.041Â), which are reported in other iridium complexes.20 Furthermore, the Ir-N bonds between the iridium and NAN ligands (2.081-2.152 Â) are longer than those between the iridium and CAN ligands (2.037-2.073Â), consistent with strong trans influence of the CAN ligands. The octahedral para-orbital angles range from 171.0(5)° to 174.6(5)° for 2a and from 171.9(4)° to 174.9(4)° for 2b, which is close to a straight line. The metric parameters of the two iridium complexes are similar owing to the same cyclometalated ligands and analogous ancillary ligands. Table 1. Crystallographic data for complexes 2a-2b. 2a 2b Empirical formula C34H22IrN5O2 C33H21IrN6O2-H2 O Mr 724.76 743.77 Crystal system Monoclinic Monoclinic Space group P21/c P21/c Wavelength /  0.7107 0.7107 X-radiation Mo-Ka Mo-Ka (graphitemonochromator) T / K 293(2) 293(2) a (Â) 11.9035(5) 11.951(3) b (Â) 17.9960(9) 18.130(6) c (Â) 13.7103(7) 13.742(4) « (°) 90 90 s (°) 96.654(4) 98.00(2) y (°) 90 90 V (Â3) 2917.2(2) 2948.4(15) Z 4 4 Dcalcd (Mg/m3) 1.650 1.676 F(000) 1416 1456 Absorption 4.616 4.573 coefficient (mm-1) Index ranges -14 < h < 11 -14 < h <14 -21 < k < 22 -22 < k < 21 -15 < l < 17 -17 < l < 17 Rint 0.0484 0.1096 GOF (F2) 1.037 1.028 Ri", wR2b (I>2o(I)) 0.0634, 0.1601 0.0694, 0.1674 Rl", wR2b (all data) 0.1146, 0.2001 0.1262, 0.2336 Fig. 1. ORTEP view of 2a (up) and 2b (down) with the thermal ellipsoids drawn at the 50% probability level. Hydrogen atoms and solvent molecules are omitted for clarity. R = Z||F0|-|FC||/Z|F0|. bwR2 = [Zw(F02-Fc2)2/Zw(F02)]1/2 3. 2. Electronic Absorption Spectra The UV-Vis absorption spectra of 2a-2b were recorded at room temperature in CH2Cl2 solutions, as shown in Fig. 2, and the data are summarized in Table 2. All of the Yang et al.: Cyclometalated Iridium(III) Complexes Containing ... Acta Chim. Slov. 2019, 66, 378-384 381 complexes exhibit intense absorption bands in the ultraviolet region at wavelengths below 310 nm, which are assigned to the spin-allowed n-n* transitions on the CUN main ligands and the NAN ancillary ligands. The weaker absorption bands in the range 350-450 nm are likely attributed to metal-to-ligand charge-transfer transitions (1MLCT and 3MLCT).21-22 Compared with complex 2a, complex 2b has a red-shifted, which may be caused by the ancillary ligand. This speculation will be confirmed by electrochemical analysis and DTF calculations. Wavelengte / nm Fig. 2. UV-Vis absorption spectra of 2a-2b, recorded in CH2Cl2 at room temperature. 3. 3. Emission Properties The photoluminescence emission spectra of iridium complexes 2a-2b in degassed CH2Cl2 solution at room temperature and corresponding data are described in Fig. 3 and Table 2, respectively. Complex 2a exhibits green phosphorescence with the broad emission maxima peak at 518 nm and a shoulder peak at 547 nm, whereas 2b is yellow emissive with the broad emission maxima peak at 529-552 nm. For their emission, the excited state of 2a is attributed to the mixing of 3MLCT and 3LC,23 while that of 2b is mainly attributed to 3MLCT.24 As expected, the emission band of 2b has red-shifted with respect to 2a due to different ancillary ligands, which is consistent with absorption analysis. In addition, the quantum yields of 2a and 2b in solution at room temperature were measured to Wavelength / nm Fig. 3. The emission spectra of 2a-2b in CH2Cl2 at room temperature. be 35.2% and 46.1% with reference to /ac-Ir(ppy)3 (0 = 0.40).25 3. 4. Theoretical Calculations Density functional theory (DFT) and time-dependent DFT (TD DFT) calculations have been performed on the complexes 2a-2b to obtain an insight into the lowest energy electron transition. The most representative molecular front orbital diagram of these complexes is shown in Fig. 4. The calculated spin-allowed electron transitions are provided in Table 3 and compared with the experimental absorption spectra data. The electron density distribution data are summarized in Table S2. As shown in Fig. 4, the HOMOs of these complexes are mainly located on the metal center and CAN ligands. Meanwhile, the LUMO of 2a is mostly dominated on CAN ligands, while LUMO of 2b is mainly located on the whole ancillary ligands. In addition, the LUMO+1s of these complexes are primarily centered on the CAN main ligands, while HOMO-1s are delocalized over the metal center, CAN ligands and ancillary ligands. The theoretical calculation of DFT shows that the lowest energy spin-allowed transitions of 2a-2b come from HOMO^LUMO/ LUMO+1 and HOMO^LUMO+1/HOMO-1^LUMO transitions (Table 3), and therefore attributed to met-al-to-ligand charge transfer transition and ligand-to-li-gand n-n* transition. These calculations support the pho-tophysical properties discussed above. Table 2. Photophysical and electrochemical data of complexes 2a-2b. Complex Absorption" Emission" 0 b E a ox HOMOc HOMOd Kbs (nm) \em (nm) (%) (V) (eV) (eV) 2a 229, 240, 295 518, 547(sh) 35.2 1.57 -6.37 -5.44 2b 228, 295, 308(sh) 529, 552 46.1 1.62 -6.42 -5.54 "Data were collected from degassed CH2Cl2 solutions at room temperature. bfac-Ir(ppy)3 as referenced standard (0.4).25 cHOMO energies are deduced from the equation HOMO = - (Eox+ 4.8 eV). ^Obtained from theoretical calculations. Yang et al.: Cyclometalated Iridium(III) Complexes Containing 382 Acta Chim. Slov. 2019, 66, 378-384 Table 3. Main experimental and calculated optical transitions for 2a-2b. Complex Orbital Excitations Transition Character Oscillation Strength Calcd (nm) Exptl (nm) 2a 2b HOMO ■ LUMO HOMO ■ LUMO+1 HOMO^LUMO+1 HOMO-1^LUMO MLCT/LLCT MLCT/LLCT MLCT/LLCT MLCT/LLCT dnIr/nbo^ n*bo dnIr/nbo/np 0.0802 0.0401 0.0242 0.0225 408 393 403 397 407 372 3. 5. Electrochemical Properties The electrochemical properties of 2a-2b were studied by cyclic voltammetry and shown in Fig. 5. The corre- -30 o 1.2 1.3 1.4 1.5 1.6 1.7 1.8 Potential / V Fig. 5. Cyclic voltammograms for 2a-2b in CH2Cl2 solution containing n-Bu4NClO4 (0.1 M) at a sweep rate of 100 mV/s. sponding electrochemical data and estimated HOMO energy levels are summarized in Table 2. The complexes 2a-2b exhibit quasi-reversible oxidation peaks at 1.57 V and 1.62 V, respectively. From DFT calculations (Table S2), HOMO is mainly located on Ir ions (47.77% for 2a and 46.90% for 2b) and CAN ligands (40.97% for 2a and 44.91% for 2b). Thus, their oxidation processes are assigned to Ir (III) to Ir (IV) and some contributions of the CAN ligands.26 Based on the oxidation potential, the HOMO energy is derived from the equation -Ehomo = -(Eox + 4.8 eV), and the trend is consistent with the theoretical calculations (Table 2). From these results, it can be seen that because of the different number of nitrogen atoms in the ancillary ligands, the HOMO level of 2b is more stable than that of the analogue 2a, and the oxidation process of 2b is more difficult than that of 2a. 4. Conclusions In summary, the syntheses, characterization, as well as electrochemical, spectroscopic and photophysical prop- Yang et al.: Cyclometalated Iridium(III) Complexes Containing ... Acta Chim. Slov. 2019, 66, 378-384 383 erties of two new bo-based iridium(III) complexes are reported. The room-temperature phosphorescence of these complexes is tunable from green to yellow depending on the different ancillary ligands. It was also found that as the number of nitrogen atoms increased on the ancillary ligands, the quantum yields became larger and emission became brighter. The DFT calculated results are in good agreement with the actual absorption spectra, indicating that the lowest absorption is assigned to the MLCT/LLCT transition. These results will facilitate the design of new bo-based iridium(III) complexes for highly efficient OLEDs. 5. Acknowledgments This work was supported by the Natural Science Foundation of Hainan Province (218QN236) and Program for Innovative Research Team in University (IRT-16R19). 6. Supplementary Material The selected bonds and angles of complexes 2a-2b, the frontier orbital energy and electron density distributions of complexes 2a-2b, as well as the FTIR spectra of complexes 2a-2b. Crystallographic data for the structural analyses have been deposited in the Cambridge Crystallographic Data Centre, CCDC reference number 1881007 (2a) and 1881008 (2b). Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336033; e-mail: deposit@ccdc.cam. ac.uk). 7. References 1. M. C. Gather, A. Kohnen, K. Meerholz, Adv. Mater. 2011, 23, 233-248. D01:10.1002/adma.201002636 2. C. Fan, C. Yang, Chem. Soc. Rev. 2014, 43, 6439-6469. D0I:10.1039/C4CS00110A 3. G. M. Farinola, R. Ragni, Chem. Soc. Rev. 2011, 40, 34673482. D0I:10.1039/c0cs00204f 4. L. Flamigni, A. Barbieri, C. Sabatini, B. Ventura, F. Barigelletti, Top. Curr. Chem. 2007, 281, 143-203. D0I:10.1007/128_2007_143 5. E. Baranoff, E. Orselli, L. Allouche, D. DiCenso, R. Scopelliti, M. Gr€atzel, M. K. Nazeeruddin, Chem. Commun. 2011, 47, 2799-2801. D0I:10.1039/c0cc05029f 6. M. A. Baldo, M. E. Thompson, S. R. Forrest, Nature. 2000, 403, 750-753. D0I:10.1038/35001541 7. E. Matteucci, A. Baschieri, A. Mazzanti, L. Sambri, J. Avila, A. Pertegas, H. J. Bolink, F. Monti, E. Leoni, N. Armaroli, Inorg. Chem. 2017, 56, 10584-10595. D01:10.1021/acs.inorgchem.7b01544 8. X. Li, X. T. Yu, H. J. Chi, Y. Dong, G. Y. Xiao, P. Lei, D. Y. Zhang, Z. Cui, Spectrochim. Acta, Part A. 2013, 116, 473-477. D0I:10.1016/j.saa.2013.07.075 9. X. Li, H. J. Chi, Y. Dong, G. Y. Xiao, P. Lei, D. Y. Zhang, Z. Cui, Opt. Mater. 2013, 36, 265-270. D0I:10.1016/j.optmat.2013.09.006 10. H. W. Hong, T. M. Chen, Mater. Chem. Phys. 2007, 101, 170176. D0I:10.1016/j.matchemphys.2006.03.011 11. Z.-G. Niu, T. Zheng, Y.-H. Su, P.-J. Wang, X.-Y. Li, F. Cui, J. Liang, G.-N. Li, New. J. Chem., 2015, 39(8), 6025-6033. D0I:10.1039/C5NJ00975H 12. G.-N. Li, C.-W. Gao, H.-H. Chen, T.-T. Chen, H. Xie, S. Lin, W. Sun, G.-Y. Chen, Z.-G. Niu, Inorg. Chim. Acta, 2016, 445, 22-27. D0I:10.1016/j.ica.2016.02.009 13. T. L. H. Doan, L. H. T. Nguyen, T. T. Nguyen, H. L. Nguyen, P. H. Tran, Catalysis Science & Technology, 2017, 7(19), 43464350. D0I:10.1039/C7CY01668A 14. X. Y. Yu, L. Deng, B. Zheng, B. R Zeng, P. Yi, X. Xu, Dalton Trans, 2013, 43(4), 1524-1533. D0I:10.1039/C3DT51986D 15. C.-H. Cheng, U.S. Pat. Appl. Publ. 20080217606, 11 Sep 2008. 16. CrysAlisPro Version 1.171.36.21; Agilent Technologies Inc., Santa Clara, CA, USA, 2012. 17. G. M. Sheldrick, Acta Cryst. 2008, A64 112-122. D0I:10.1107/S0108767307043930 18. O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, H. J. Puschmann, Appl. Crystallogr. 2009, 42, 339-341. D0I:10.1107/S0021889808042726 19. M. J. Frisch, G. W. Trucks, H. B. Schlegel, Gaussian 09, Revision A.01, Gaussian, Wallingford, Con, USA, 2009. 20. C. D. Sunesh, K. Shanmugasundaram, M. S. Subeesh, R. K. Chitumalla, J. Jang, Y. Choe, ACS Applied Materials & Interfaces, 2015, 7(14), 7741-7751. D0I:10.1021/acsami.5b00875 21. G. J. Zhou, Q. Wang, C. L. Ho, W. Y. Wong, D. G. Ma, L. X. Wang, Z. Y. Lin, Chem. Asian. J. 2008, 3, 1830-1841. D0I:10.1002/asia.200800074 22. N. M. Shavaleev, F. Monti, R. Scopelliti, A. Baschieri, L. Sam-bri, N. Armaroli, Organometallics, 2013, 32(2), 460-467. D0I:10.1021/om300894m 23. M. Y. Wong, G. Xie, C. Tourbillon, M. Sandroni, D. B. Cordes, A. M. Z. Slawin, I. D. W. Samuelb, E. Zysman-Colman, Dalton Trans. 2015, 44, 8419-8432. D0I:10.1039/C4DT03127J 24. V. Chandrasekhar, B. Mahanti, P. Bandipalli, K. Bhan-uprakash, Inorg. Chem. 2012, 51, 10536-10547. D0I:10.1021/ic300694m 25. K. A. King, P. J. Spellane, R. J. Watts, J. Am. Chem. Soc. 1985, 107, 1431-1432. D0I:10.1021/ja00291a064 26. G.-N. Li, S.-B. Dou, T. Zheng, X.-Q. Chen, X.-H. Yang, S. Wang, W. Sun, G.-Y. Chen, Z.-R. Mo, Z.-G. Niu, Organometallics, 2018, 37, 78-86. D0I:10.1021/acs.organomet.7b00740 Yang et al.: Cyclometalated Iridium(III) Complexes Containing 384 Acta Chim. Slov. 2019, 66, 378-384 Povzetek Sintetizirali in karakterizirali smo dve novi kompleksni spojini Ir(III): [(bo)2Ir(pzpy)] (2a) in [(bo)2Ir(pzpyz)] (2b) (bo = 2-fenilbenzo[d]oksazol, pzpy = 2-(1H-pyrazol-3-yl)piridin, pzpyz = 2-(1H-pirazol-3-yl)pirazine). Spojinama smo z rentgensko strukturno analizo monokristalov določili kristalni strukturi. DFT izračune smo uporabili za interpretacijo lastnosti spojin v povezavi z njunima strukturama. Kompleksa Ir(III) imata prehod iz vzbujenega stanja 3MLCT/3LLCT v zelenem in rumenem območju s kvantnima izkoristkoma v raztopini CH2Cl2 pri sobni temperaturi 35,2% in 46,1%. Rezultati teoretičnih izračunov in eksperimentalni podatki kažejo, da sta kompleksa Ir(III) (2a in 2b) obetajoča fosfo-rescenčna materiala. Yang et al.: Cyclometalated Iridium(III) Complexes Containing ... DOI: 10.17344/acsi.2018.4877 Acta Chim. Slov. 2019, 66, 385-394 ^creative ^commons Scientific paper A Vortex-Assisted Deep Eutectic Solvent-Based Liquid-Liquid Microextraction for the Analysis of Alkyl Gallates in Vegetable Oils Hasan Çabuk,* Yasemin Yilmaz and Elif Yildiz Zonguldak Bulent Ecevit University, Faculty of Arts and Sciences, Department of Chemistry, 67100, Zonguldak, Turkey * Corresponding author: E-mail: cabukhasan@hotmail.com Tel.: 00 90 372 291 1920; Fax: 00 90 372 257 41 81 Received: 11-27-2018 Abstract This study investigates the feasibility of using hydrophilic deep eutectic solvent (DES) as green and effective extractant for the extraction and preconcentration of alkyl gallates from vegetable oils. In a typical experiment, 120 |L of choline chloride:ethylene glycol DES was added to 1.0 g of oil sample which was previously diluted with 1 mL of «-hexane. The extraction was accelerated by vortex stirring of the two phases. At this stage, hydrogen bonding interactions between the phenyl hydroxyls of alkyl gallates and chloride anion of choline salt were likely the main forces driving the extraction. After extraction, the analytes in the DES phase were separated and determined by high-performance liquid chromatography with ultraviolet detection. The method detection limits for propyl gallate and octyl gallate were 2.1 and 4.6 |g kg-1, respectively. The precision of the method varied between 4.6-6.4% (intra-day) and 5.4-7.5% (inter-day). The recoveries (accuracies) obtained from spiked vegetable oil samples were in the range of 78-106%. Keywords: Deep eutectic solvent, alkyl gallates, vegetable oils, high-performance liquid chromatography 1. Introduction Synthetic antioxidants (SAs) are a class of compounds which are capable of preventing or slowing down the oxidation reactions of substrates in food, pharmaceuticals, and other consumer products.1 Common SAs used as food additives in many countries include butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, propyl gallate, octyl gallate and dodecyl gallate, due to their chemical stabilities, low costs and availabilities.2 Among SAs, al-kyl gallates have been widely used as food additives to slow down or prevent lipid oxidation in lipid-rich food.3 Contrary to their versatile and beneficial properties, the addition of excess gallates to food might cause different degrees of adverse and toxic effects on human health. They can induce DNA damage and mutagenicity, and their antioxidant potential can turn pro-oxidant under certain conditions.2,4 Therefore, the use of gallates as well as other SAs is strictly controlled in many countries worldwide. Generally, the allowed amounts of SAs in food range from 100 to 200 mg kg-1 in European Union, either singly or in combination.5 The determination of alkyl gallates in foodstuffs has been accomplished using specific electrochemical sen- sors6-8 and different separation techniques such as high-performance liquid chromatography (HPLC),5,9-12 micellar electrokinetic capillary chromatography13,14 and capillary electrophoresis.15,16 Electrochemical methods may offer relatively low operational costs and faster analysis. However, in the case of multiple analytes they require chemometric interpretation of the overlapping electrochemical signals for the estimation of individual analyte concentrations.17 Among the separation techniques, HPLC with ultraviolet (UV) detection has become the most widely used technique for the determination of alkyl gallates following the extraction by liquid-liquid extraction (LLE) of the sample. Acetonitrile, previously saturated with n-hexane, is the most preferred solvent for extracting alkyl gallates from oil samples.9,12 To a lesser extent, methods based on the direct injection of a diluted oil sample into the HPLC system have also been used.18,19 However, a pretreatment step for sample enrichment and cleanup is always desirable prior to any chromatographic analysis due to the complexity of the sample matrix. Recently, some novel microextraction procedures, such as cloud-point extraction (CPE)10 and dispersive liquid-liquid microextraction (DLLME)5,20 have also been devel- Çabuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based 386 Acta Chim. Slov. 2019, 66, 385-394 oped to detect alkyl gallates in oil samples as greener alternatives to classical LLE. In the last few years, the majority of the innovative studies on microextraction procedures are concerned with replacing toxic organic solvents with environmentally friendly ones, including supramolecular solvents, ionic liquids and deep eutectic solvents (DESs). DESs are regarded as new generation green solvents because of their attractive properties such as easy synthesis, low cost, low volatility, high biodegradability, and feasibility of structural design.21 A DES is typically prepared by mixing an appropriate amount of a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD). The resulting mixture has a lower melting point than that of each individual component, which makes it liquid at room temperature. The formation of hydrogen bonding between the DES components is responsible for the decrease in the melting point.22 The most common DESs are based on choline chloride (ChCl) paired with various HBDs, the most popular ones being urea, ethylene glycol, and glycer-ol, but other alcohols, amino acids, carboxylic acids, and sugars have also been used.23 Due to their unique physico-chemical characteristics, DESs have the potential to replace conventional organic solvents in separation and extraction processes. However, due to the hydrogen bonding ability of DESs, they are generally hydrophilic which restricts their application as extraction solvents in aqueous samples.24 Therefore, the major applications have focused on the extraction of naturally occurring compounds from organic liquid samples (mainly vegetable oils) and solids (plants and foods). Phenolic compounds,25,26 flavo-noids27,28 and terpenoids29 are the most commonly determined bioactive compounds, with extraction procedures based on the use of hydrophilic DESs. Very recently, hy-drophobic DESs combining quaternary ammonium salts, thymol or menthol with alkyl carboxylic acids have also been prepared and used for the extraction of pesticides,30 polycyclic aromatic hydrocarbons31 and benzophenone-type UV filters32 from water samples. DESs possess tunable solvent properties that make them highly efficient in extracting compounds from different chemical classes. With respect to SAs, tert-butylhydroquinone is the only analyte that has been determined in vegetable oils with the extraction procedures based on the use of ChCl:ascorbic acid,33 ChCl:sesamol34 and ChCl:ethylene glycol35 compositions. However, as far as we know, there is no DES-based extraction method reported in the literature for alkyl gallates. Therefore, the aim of this study was to investigate the feasibility of using a hydrophilic ChCl-based DES as an extraction solvent for the extraction and preconcentration of alkyl gallates including propyl gallate and octyl gallate (see their structures and some relevant data in Table 1) from vegetable oils. The dispersion of DES phase into the vegetable oil was achieved using vortex stirring, and then the resulting methodology was termed vortex-assisted DES-based liquid-liquid microextraction (VA-DES-LLME). Following the microextraction process, alkyl gal-lates in DES phase were separated and determined by HPLC-UV. 2. Experimental 2. 1. Reagents and Samples All of the reagents used in the experiments were of analytical grade (>98%). Ethylene glycol, glycerol, urea, choline chloride (ChCl), propyl gallate, octyl gallate and acetonitrile (ACN) were obtained from Sigma-Aldrich (Steinheim, Germany). Triflouroacetic acid (TFA) and M-hexane were purchased from Merck (Darmstadt, Germany). Isopropyl alcohol was provided from Kimetsan (Ankara, Turkey). Water was used after purification with a Milli-Q system (Millipore, Bedford, MA, USA). Stock standard solutions were prepared in acetonitrile at a concentration of 100 pg mL-1 and stored at 4 °C for one month. The working solutions at different concentrations were freshly prepared in isopropyl alcohol and used for the spiking of the oil samples. Spiked samples were mixed well for the complete dissolution of the antiox- Table 1. Chemical structure and properties of the selected alkyl gallates. Analyte Structure pKa36 LogKow37 ^abuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based ... Acta Chim. Slov. 2019, 66, 385-394 387 idants in the oil matrix before being extracted. The vegetable oil samples including sunflower oil, corn oil and hazelnut oil were collected from local supermarkets in Zonguldak, Turkey. The collected oil samples were packed in screw-cap glass tubes and stored at room temperature until analyzed. 2. 2. Instrumentation and Chromatographic Conditions The HPLC system (Thermo Finnigan, San Jose, USA) consisted of a P1000 pump, a AS3000 automatic injector system, and a UV1000 UV detector. The system was controlled by a Spectra System Controller SN 4000 and a software package ChromQuest 4.0. A Phenomenex C12 Max-RP column (250 x 4.6 mm i.d., 4.0 ^m) was used for separations. The mobile phase consisted of ACN and water with 0.1% TFA. The optimized elution program was set at 1.0 mL min-1, starting with 40% acetonitrile and then a linear gradient elution from 40 to 90% acetonitrile at 20 min. Next, 10 min was necessary for returning to the initial conditions. Before another injection, the system was permitted to stabilize for 5 min under the initial conditions. The UV monitoring wavelength for quantification was set to 280 nm. The injection volume was 20 ^L. A Per-kin Elmer Frontier Fourier transform infrared (FT-IR) spectrometer with a diamond attenuated total reflectance (ATR) attachment was used to determine the functional groups of DESs. A magnetic stirrer (RSM-01H, Phoenix, Garbsen, Germany), a vortex mixer (SA8, BioCote, UK), a centrifuge (NF 200, Nuve, Ankara, Turkey) were used in the sample preparation step. A Kern ABJ 220-4 M model analytical balance (Kern & Sohn, Germany) was used for weighing the samples and standard materials. Vortex stirring Figure 1. Schematic diagram of the preparation of ChCl:ethylene glycol DES and VA-DES- LLME method. Çabuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based 388 Acta Chim. Slov. 2019, 66, 385-394 2. 3. Preparation of DESs DESs were prepared by mixing ChCl with HBD (ethylene glycol, glycerol, or urea) at a constant molar ratio of 1:2 in screw-capped bottles. The mixture was stirred at a temperature of 80 °C until a clear liquid was formed. Figure 1 shows, as an example, the schematic of the preparation of ChCl:ethylene glycol DES. All DESs were used in the experiments without any further purification. 2. 4. VA-DES-LLME Procedure Figure 1 shows the schematic of the VA-DES-LLME procedure. Oil sample (1.0 g) was accurately weighed into a 12 mL glass test tube with conical bottom and diluted with 1.0 mL of M-hexane, to which 120 ^L of DES as extracting solvent was added. Afterwards, the mixture was vortex-stirred at 2500 rpm for 5 min and then centrifuged at 4000 rpm for 5 min. Finally, the DES extract was collected with a microsyringe and transferred to a glass vial for the HPLC-UV analysis. of the ChCl:ethylene glycol DES (37 cP) and ChCl:glycerol DES (259 cP) at 25 oC.38 Most DESs have higher viscosities (>100 cP) than other molecular solvents due to the presence of extensive hydrogen-bonding interactions between the components, which slows down the mass transfer of the analytes to the extraction media.23 Addition of water, in general, reduces the viscosity of these solvents, and therefore allows better mass transfer rates. However, at the same time the presence of water may reduce the extraction yields due to the weakening of the hydrogen bond interactions between the solvent and the target compounds.39 Due to the opposite effects of water on the extraction efficiency, it was not investigated in the present experiment. As shown in Figure 2, the extraction efficiencies of eth-ylene glycol and glycerol-based DESs were close to each other, meaning that each of DESs could be used as a solvent for the extraction of alkyl gallates. However, Ch-Cl:ethylene glycol DES provided some advantages in the solvent transfer and injection stages due to its lower viscosity; thus, it was selected as extraction solvent in the subsequent experiments. 3. Results and Discussion Optimization of VA-DES-LLME was carried out using 1.0 g of sunflower oil sample, spiked with propyl gallate and octyl gallate at a concentration of 1.25 ^g g-1 each. The type of DES, volume of diluent solvent, volume of DES and vortex stirring time were the variables investigated. The optimal conditions were selected based on the extraction recoveries (ERs) of the alkyl gallates. The ER was calculated based on the ratio of the amount of the analyte determined in the collected DES phase to the initial amount of analyte added in the sample. All the experiments were carried out in triplicate. 3. 1. Effect of the Types of DESs The composition of DESs determines their physico-chemical properties such as density, viscosity, and surface tension and consequently influences extraction efficiency of the target analytes. With the aim to select the most suitable DES for extraction of alkyl gallates from vegetable oils, three different ChCl-based DES, which contained eth-ylene glycol, glycerol, and urea as the HBDs at a constant molar ratio of 1:2 were tested. In all cases, aliquots of 1.0 g of sunflower oil sample were extracted with 100 ^L of DES under vortex stirring for 5 min. When ChCl:urea DES was used, it was not effectively dispersed into the oil sample with vortex stirring. In addition, it could not be withdrawn into the micro-injector prior to chromatographic analysis. On the contrary, these difficulties were not observed when ethylene glycol and glycerol-based DESs were tested. The results observed here could be attributed to the difference in their viscosities and other physical properties. The Ch-Cl:urea DES has much higher viscosity (750 cP) than those Figure 2. Effect of the type of DES. DES volume, 100 |iL; vortex time, 5 min. 3. 2. Characterization of ChCl:ethylene Glycol DES The formation of hydrogen bonding between the chloride anion of ChCl and ethylene glycol is the main force for the formation of DES. As shown in Figure 3, FT-IR spectra of ChCl, ethylene glycol, and ChCl:ethylene glycol DES were investigated. According to the FT-IR spectra, the broad band at 3294 cm-1 related to the stretching vibration of the O-H group in ethylene glycol shifted to 3303 cm-1 in ChCl:ethylene glycol DES. This shift towards higher wavenumber with the incorporation of ChCl and ethylene glycol was due to the decrease in the extent of intermolecular hydrogen bonding between ethylene glycol molecules.40 The spectrum of DES was dominated by eth-ylene glycol, however, an additional characteristic band at 953 cm-1 originating from ChCl was observed. This new band was attributed to the C-N+ stretching. In addition, DES presented vibrational bands at 2938 cm-1 and 2875 ^abuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based ... Acta Chim. Slov. 2019, 66, 385-394 389 cm-1 referring to C-H stretching, 1478 cm-1 to the CH2 bending of an alkyl group, and 1084 cm-1, 1036 cm-1, and 882 cm-1 to functional groups, namely C-O stretching, C-C-O asymmetric stretching, and C-C-O symmetric stretching.41 These results indicated the successful synthesis of ChCl:ethylene glycol DES. shown in Figure 4, the extraction efficiency of alkyl gal-lates increased slightly as the hexane volume was increased from 0 to 1 mL and remained nearly constant over this volume. The reason could be that the viscosity of oil sample reduced with the increase of the volume of hexane, which facilitated the mass transfer of the analytes from oil 3. 3. Effect of the Volume of Diluent Solvent The dilution of oil sample with an appropriate solvent has a significant effect on the extraction efficiency since it reduces the viscosity of oil and facilitates the formation of the cloudy emulsion. In addition, the fat and other impurities are easily dissolved in the diluent solvent which may be beneficial for the removal of impurities.42 Hexane is the most preferred solvent owing to its low tox-icity, low cost and high miscibility with the oil.43 In this work, «-hexane was selected as diluent solvent and the effect of its volume was studied in the range of 0-2 mL. Ali-quots of 1.0 g of sunflower oil sample were diluted with hexane and subjected to vortex stirring for 5 min with 100 ^L of ChCl:ethylene glycol DES as extraction solvent. As 0 0.5 1.0 1.5 2.0 Volume of diluent solvent (hexane) (mL) Figure 4. Effect of the volume of diluent solvent. DES, ChCl:ethyl-ene glycol; diluent solvent, n-hexane; DES volume, 100 |iL; vortex time, 5 min. Çabuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based 390 Acta Chim. Slov. 2019, 66, 385-394 to the DES phase. Therefore, the diluent solvent (n-hex-ane) volume was selected as 1 mL for all further experiments. 3. 4. Effect of the Volume of DES Selection of the optimum volume for the extraction solvent is another important step in the majority of microextraction methods. By the increase of extraction solvent volume, the final solvent volume collected after centrifuga-tion is increased, resulting in a decrease in sensitivity towards the analytes. In order to increase the sensitivity, it is essential to keep the volume of extraction solvent as low as possible. However, it is not always possible to reduce the solvent volume as desired, because a sufficient volume of solvent needs to be collected after centrifugation for the analysis. To examine the effect of the extraction solvent volume on the extraction efficiency, different volumes of ChCl:ethylene glycol DES from 60 to 140 ^L were investigated while the other experimental parameters, diluent solvent volume of 1 mL and vortex time of 5 min, were kept constant. The volumes less than 60 ^L were not examined because of the difficulty in getting a separated DES phase. The obtained results (Figure 5) showed that the extraction efficiency increased with the increase of DES volume up to 120 ^L and remained unchanged after that. Typically, a higher volume of solvent requires a longer mixing time to be broken up into fine droplets; here above 120 ^L, the extraction recovery did not further increase maybe due to the incomplete formation of fine droplets within a fixed vortex time. Therefore, 120 ^L was selected as the most suitable DES volume for subsequent experiments. Propyl gallate -■-Octyl gallate --1-* ____*_í 60 80 100 120 140 Volume of DES (ChCfethylene glycol) (nL) Figure 5. Effect of the volume of DES. DES, ChCl:ethylene glycol; diluent solvent, n-hexane; diluent solvent volume, 1 mL; vortex time, 5 min. 3. 5. Effect of Vortex Stirring Time The vortex stirring process facilitates the dispersion of the extraction solvent into the sample solution resulting in enhancement of extraction efficiency. The time required to reach extraction equilibrium under vortex stirring is an important parameter to be optimized. In this work, the vortex rotational speed was kept at maximum (2500 rpm) and different vortex times were investigated, from 1 to 15 min. The experimental conditions were fixed and included the use of 1 mL of diluent solvent (n-hexane) and 120 ^L of ChCl:ethylene glycol DES. The results (Figure 6) showed that the extraction efficiency increased with the increasing vortex time from 1 to 3 min, and then from 3 to 5 min, there was either a slight increase or no change, depending on the analyte. No significant change was observed at vortex times longer than 5 min. Therefore, 5 min was chosen as the optimum vortex time. According to these results, the optimal conditions of this method for the determination of alkyl gallates were as follows: the type of DES was ChCl:ethylene glycol with a molar ratio of 1:2; the volume of diluent solvent (hexane) was 1 mL; the volume of ChCl:ethylene glycol DES was 120 ^L; and the vortex stirring time was 5 min. Because the DES was not soluble in oil matrix, it could be collected after extraction without a significant loss in its volume. 1 3 5 8 15 Vortex time (min) Figure 6. Effect of the vortex time. DES, ChCl:ethylene glycol; diluent solvent, n-hexane; diluent solvent volume, 1 mL; DES volume, 120 |iL. Under the optimized conditions, the mean extraction recoveries for propyl gallate and octyl gallate were 93 and 73%, respectively. The enrichment factors were also calculated based on the ratio of the analyte concentration in the DES phase to the initial analyte concentration in the oil phase. Under the optimized conditions, the mean enrichment factors for propyl gallate and octyl gallate were 8.5 and 6.7, respectively. Both propyl gallate and octyl gallate have the same pKa value of about 7.9,36 meaning that their hydrogen bonding potentials are similar. The difference in the extraction efficiencies of alkyl gallates was then likely the result of their different hydrophobic characteristics. The octanol-water partition coefficients (logK^w) for propyl gallate and octyl gallate are 2.6 and 5.2, respectively.37 The hydrophobicity of alkyl gallates increases with the increasing alkyl chain length (see the structures in Table 1), making the hydrophobic interaction between octyl gallate and oil stronger, and thus decreasing the hydrogen bonding strength between the phenyl hydroxyls of octyl gallate and chloride anion of choline salt. ^abuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based ... Acta Chim. Slov. 2019, 66, 385-394 391 3. 6. Analytical Performance Linearity, limit of detection (LOD), limit of quantification (LOQ), and precision were investigated to verify the reliability of the method used in this study. The results are summarized in Table 2. Linearity was observed in the range of 0.01-5 ^g g-1, with the square of correlation coefficients (R2) higher than 0.9995. LODs, based on a signal-to-noise ratio (S/N) of 3, ranged from 2.1 to 4.6 ^g kg-1. The precision of the method was determined by analyzing the spiked samples at concentration level of 0.5 ^g g-1 on the same day and on three different days. The relative standard deviations (RSDs) were in the ranges of 4.6-6.4% and 5.4-7.5% for intra-day (n=5) and inter-day (n=3) precision, respectively, which indicates that the method is re-peatable. 3. 7. Real Samples Analysis The applicability of the proposed method was evaluated with three kinds of vegetable oil samples including sunflower oil, corn oil and hazelnut oil. The results indicated that the samples were free of alkyl gallates. To examine the applicability and accuracy of the method, all the samples were spiked at two concentration levels of 0.25 and 2.5 ^g g-1, and analyzed. The results are given in Table 3. The recoveries of alkyl gallates from oil samples spiked at low and high concentration levels were 78-93% and 87-106%, respectively. RSDs for these two levels were in the ranges 3.2-5.7% and 2.0-5.2%. These results demonstrated that the matrix had a negligible effect on the extraction. Figure 7 shows, as an example, the HPLC-UV chromatograms of the alkyl gallates extracted from a sunflower oil sample be- Table 2. Quantitative features of VA-DES-LLME method combined with LC-UV. Analyte Linear range R2 LOD LOQ Intra-day RSD Inter-day RSD (TO g-1) (re kg-1) (pg kg-1) (%, n=5) (%, n=3) Propyl gallate 0.01-5 0.9995 2.1 7.0 4.6 5.4 Octyl gallate 0.01-5 0.9996 4.6 15.3 6.4 7.5 Table 3. Spiked recoveries (accuracies of determination) of alkyl gallates in vegetable oil samples. Oil sample Added Propyl gallate Octyl gallate ^g g-1) RR (%) RSD (%, n=3) RR (%) RSD (%, n=3) Sunflower-1 0.25 83 4.8 84 5.7 2.50 96 2.4 98 3.2 Sunflower-2 0.25 78 3.8 93 4.2 2.50 98 2.7 100 2.2 Sunflower-3 0.25 90 4.3 87 4.6 2.50 99 4.0 106 5.2 Corn 0.25 86 5.4 79 5.5 2.50 90 2.5 87 2.0 Hazelnut 0.25 92 3.2 86 4.7 2.50 104 3.4 98 4.4 RR: Relative recovery Figure 7. HPLC-UV chromatograms, corresponding to (a) sunflower oil sample, and the same sample after spiking of the alkyl gallates at the (b) 0.25 |ig g-1 and (c) 2.5 |ig g-1 levels. 1: Propyl gallate, 2: Octyl gallate. Çabuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based 392 Acta Chim. Slov. 2019, 66, 385-394 Table 4. Comparison of the proposed method with other methods used in determination of alkyl gallates in vegetable oil samples. Method Analytes Sample Extraction Solvent Extraction RSD LOD Recovery Reference amount (g) solvent volume (mL) time (min) (%) (%) CPE-HPLC-DAD Propyl gallate 2 Triton X-114 5 30 <2.2 1.9 |g L-1 86-88 10 UAE-HPLC-UV Propyl gallate 10 Methanol + acetonitrile 45 15 <4.0 300 |g L-1 103-108 11 LLE-LC-TOF-MS Propyl gallate Octyl gallate 0.25 Acetonitrile + hexane 3 5 <6.3 20 |g kg-1 20 |g kg-1 82-96 12 DLLME- HPLC- DAD Propyl gallate Octyl gallate 0.025 Hexane + acetonitrile 1 2 <2.7 10 |g L-1 15 |g L-1 94-105 5 DLLME- MSPE- Propyl gallate 2 Heptanol 0.16 ~3 <6.7 1.2 |g L-1 90-92 20 HPLC-UV VA-DES- LLME- HPLC-UV Propyl gallate Octyl gallate 1 ChCl:ethylene glycol DES 0.12 5 <7.5 2.1 |g kg-1 4.6 |g kg-1 i [ 78-106 This study DAD: diode array detection; MSPE: magnetic solid-phase extraction; TOF-MS: time-of-flight-mass spectrometry; UAE: ultrasonic-assisted extraction fore and after spiking. No interfering peaks from the sample matrix or DES solvent were observed at the retention times of compounds of interest, which demonstrated the good applicability of the proposed method. Extraction and determination of alkyl gallates in vegetable oils by the proposed method was compared with those of other methods considering the performance parameters such as sample amount, type and volume of extraction solvent, extraction time, RSD, LOD, and relative recovery. The details of the comparison are summarized in Table 4. As shown, the proposed method offers lower consumption of sample amount and solvent volume, faster analyses, and lower detection limits than most of the existing methods while being comparable with respect to the precision and accuracy. More importantly, by using DES instead of conventional solvents, the main merits of the proposed method such as simplicity, low cost, and environmental benignity were enhanced. 4. Conclusions A green and efficient analytical methodology (VA-DES-LLME-HPLC-UV) was developed for the extraction and analysis of alkyl gallates (propyl gallate and octyl gallate) in vegetable oil samples. The ChCl:ethylene glycol DES proved to be an excellent solvent for alkyl gallates of low and medium polarity thanks to the hydrogen bonding interaction between the phenyl hydroxyl groups and anion of choline salt. Vortex stirring was employed to promote ^abuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based ... the dispersion of the DES solvent into the oil sample, which resulted in high extraction efficiency with the obtained extraction recoveries ranging from 73 to 93%. The optimized procedure displayed a good precision with RSDs <7.5% and reliable analytical results with spiked recoveries (accuracies) in the range of 78-106%. LODs were in the range of 2.1-4.6 ^g kg-1, which is better than or comparable with other reported approaches applied to the determination of alkyl gallates in vegetable oils. 5. Acknowledgements The authors would like to thank to Zonguldak Bulent Ecevit University for the opportunity and financial support. 6. References 1. D. W. Sin, Y. C. Wong, C. Y. Mak, S. T. Sze, W. Y. Yao, J. Food Compos. Anal. 2006, 19, 784-791. DOI:10.1016/j.jfca.2005.12.005 2. F. Shahidi, P. Ambigaipalan, J. Funct. Foods 2015, 18, 820-897. DOI:10.1016/j.jff.2015.06.018 3. Y. Hou, Z. Xie, H. Cui, Y. Lu, T. Zheng, S. Sang, L. Lv, Food Chem. 2018, 269, 396-403. DOI:10.1016/j.foodchem.2018.07.030 4. I. C. Silva, C. R. Polaquini, L. O. Regasini, H. Ferreira, F. R. Pavan, Food Chem. Toxicol. 2017, 105, 300-307. DOI:10.1016/j.fct.2017.04.033 Acta Chim. Slov. 2019, 66, 385-394 393 5. S. Xu, L. Liu, Y. Wang, D. Zhou, M. Kuang, D. Fang, W. Yang, S. Wei, A. Xiao, L. Ma, J. Sep. Sci. 2016, 39, 3205-3211. 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González-Barreiro, B. Cancho-Grande, J. Simal-Gándara, Food Chem. 2014, 150, 128-136. D0I:10.1016/j.foodchem.2013.10.157 Çabuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based 394 Acta Chim. Slov. 2019, 66, 385-394 Povzetek V tej študiji raziskujemo možnost uporabe hidrofilnih močnih evtektičnih topil (DES) kot zelenih in učinkovitih ek-straktantov za ekstrakcijo in predkoncentracijo alkil galatov iz rastlinskih olj. V tipičnem poskusu smo dodali 120 |L DES-a holin klorid:etilen glikol k 1,0 g vzorca olja, ki smo ga predhodno razredčili z 1 mL «-heksana. Ekstrakcijo smo pospešili z vrtinčastim mešanjem obeh faz. V tem koraku je bil verjetno glavni razlog za ekstrakcijo nastanek vodikovih vezi med fenil-hidroksilno skupino alkil galatov in kloridom iz holin klorida. Po ekstrakciji smo analite iz DES-faze ločili in določili z visokoločljivostno tekočinsko kromatografijo z ultravijolično detekcijo. Meje zaznave so bile 2,1 |g kg-1 za propil galat in 4,6 |g kg-1 za oktil galat. Natančnost metode je bila 4,6-6,4% (znotraj dneva) in 5,4-7,5% (med dnevi). Izkoristki (pravilnost) za vzorce rastlinskega olja z dodanimi analiti so bili v območju 78-106%. ^abuk et al.: A Vortex-Assisted Deep Eutectic Solvent-Based ... DOI: 10.17344/acsi.2018.4880 Acta Chim. Slov. 2019, 66, 395-401 ^creative ^commons Scientific paper Molecular Dynamics Simulations of p97 Including Covalent, Allosteric and ATP-Competitive Inhibitors Stefano Rendine,1,f Christian Orre^us,2^ Federico Dapiaggi,1,3 Stefano Pieraccini,1,3 Ilaria Motto,2 Roberto D'Alessio,2 Paola Magnaghi,2 Antonella Isacchi,2 Eduard Felder2 and Maurizio Sironi1,3^ 1 Dipartimento di Chimica, Universita degli Studi di Milano, Via Golgi 19, Milano, Italy 2 Business Unit Oncology, Nerviano Medical Sciences, 20014 Nerviano, Italy 3 Consorzio Interuniversitario Nazionale per la Scienza e Tecnologia dei Materiali (INSTM), UdR Milano f Syngenta Crop Protection AG, Crop Protection Research, Schaffhauserstrasse, CH-4332 Stein, Switzerland * Corresponding author: E-mail: christian.orrenius@nervianoms.com, maurizio.sironi@unimi.it Received: 11-28-2018 Abstract Binary (nucleotide-protein dimer and hexamer complexes) and ternary (nucleotide-protein-inhibitor complexes) p97 complexes were subjected to molecular dynamics simulations in an attempt to further our understanding of the p97 protein oligomer domain stability and, more importantly, of the recently reported diverse molecular mechanisms of inhibition including allosteric, ATP-competitive and covalent inhibitors. Analysis of stable states following equilibration phases indicated a higher intrinsic stability of the homohexamer as opposed to the dimer, and of N-D1 domains as opposed to the D2 domain. The molecular dynamics of the proposed allosteric binding model reproduced important molecular interactions identified experimentally with high frequency throughout the trajectory. Observed conformational changes occurring in the D2 nucleotide binding site provided a novel bind-rearrange-react hypothesis of stepwise molecular events involved in the specific covalent inhibitor mode of action. Keywords: Drug design; protein-protein interaction; mm-pbsa; molecular dynamics 1. Introduction The AAA+ (ATPases Associated with diverse cellular Activities) protein family carries out multiple functions, including chromatin decondensation, homotypic membrane fusion and ubiquitin dependent protein degradation by the proteasome. In particular, p97, or Valosine-contain-ing Protein (VCP), is a 97 kDa protein belonging to the AAA+ protein family. p97 includes two copies of a nucleo-tide-binding domain, called D1 and D2 which are interconnected by a 20 amino-acids long loop (the D1-D2 linker) and preceded by a non-nucleotide binding domain, the N-domain, which is oriented outward to permit interaction with adapter proteins.1 p97 regulates a number of processes involving ubiq-uitin, due to its ability to segregate ubiquitinated substrates from unmodified partners. In particular, p97 plays an essential role in the ubiquitin-dependent proteasomal degradation, including endoplasmic reticulum-associated degradation (ERAD),2,3 degradation of some cytosolic proteins by the ubiquitin-fusion domain (UFD) pathway4 and rapid degradation of nascent peptides during heat shock.5 Crystallographic studies of p976 testify that it functions as a homo-hexameric ring, formed by the D1 and D2 domains with the N-terminal domain oriented outward to permit interaction with adapter proteins. The formation of this complex molecular machinery is driven by protein-protein interactions (PPIs). In recent works,7-9 small molecules able to inhibit p97 were identified (Figure 1a). Different mechanisms of inhibition were proposed for these molecules. NMS-873 seems to act as an allosteric inhibitor of p97, binding in Rendine et al.: Molecular Dynamics Simulations 396 Acta Chim. Slov. 2019, 66, 395-401 close proximity to the N-terminal of the D1-D2 linker. NMS-322 is a ATP competitor and binds in the nucleotide binding site in D2. Finally, NMS-859 is a covalent inhibitor that binds to Cys522. However, there is a lack of structural models rationalizing these postulated binding modes. In this work we employ computational techniques to investigate the binding mode of these inhibitors both from the conformational and energetical point of view. Molecular dynamics simulations were carried out on these systems and a subsequent computational alanine scanning allowed us to identify the fundamental residues for the binding of these compounds. The aforementioned technique has been extensively used to map, from the energetic point of view, the interaction pattern of protein in complex with small ligands both of peptidic10 and non-peptidic11 nature. In the case of the allosteric inhibitor (NMS-873) the need of a good starting structure for molecular dynamics simulations prompted us to employ molecular docking. It is well proven in literature, indeed, that molecular docking studies on biological systems can successfully predict the structures of intermolecular complexes formed by ligands and their receptors (ranging from proteins12 to DNA13). 2. Materials and Methods The reference p97 structure was obtained from the Protein Data Bank (PDB ID code: 3CF1).The VCP hexam-er, the relevant biological assembly according to the authors of the original paper1 (as well as an analysis performed with the software PISA14), was derived from standard symmetry operations in PyMOL.15 The missing residues in the D2 domain were rebuilt using the software Modeller 9v7.16 Molecular dynamics simulations were carried out with Gromacs 4.5.317 using the Amber ff99SB18 force field for the protein and the Generalized Amber Force Field (GAFF)19 for the inhibitors and nucleotides. The complexes were solvated with TIP3P20 waters and neutralized with sodium ions. The molecular systems studied were first subjected to a steepest descent minimization run (10000 steps) with alpha carbons constrained by a force constant of 1000 kJ mol-1 nm-2. Subsequently, an equilibration of 200 ps was performed under NVT conditions at 310 K using the v-res-cale thermostat21 with a time constant of 0.1 ps. Electrostatic interactions were treated with the Particle Mesh Ewald (PME)22 algorithm using a 10 A cutoff. All bonds were constrained using the LINCS23 algorithm, allowing a 2 fs timestep. A further 200 ps equilibration run was performed under NPT conditions at 310 K and 1 atm using the Berendsen barostat method24 with a 0.5 ps correlation time. Alpha carbons were kept constrained as above. A 20 ns production run followed at constant temperature and pressure (1 atm, 310 K), releasing the constraints on the alpha carbons. In order to clarify relative importance of amino acid residues and their interactions, computational alanine scanning and free energy calculations were performed using the Molecular Mechanics Generalized Born Surface Area (MM-GBSA) approach25 on snapshots extracted at 20 ps intervals from the last 10 ns of the trajectories (500 snapshots in total) once the systems reached equilibrium. A 0.15 M NaCl solution was simulated as the solvent. The entropic contribution to the free energy data was neglected in the free energy calculations used in this study mainly due to the lack of reliability of results for the molecular systems considered here. An extended discussion on this topic can be found in literature.26-30 The starting structures of the complexes containing the inhibitor molecules NMS-873, NMS-322 and NMS-859 (non covalently bound, see discussion) were generated by molecular docking with QXP v0705 program31 setting the following parameters to the mcdock tool: in house modified QXP MacroModel forcefield, 1500 Monte Carlo steps saving 20 poses for visual inspection. The rather large number of poses saved enabled a procedure identifying orientations compatible with photo-affinity labeling experimental evidence reported by Magnaghi et al., 2013.7 Candidate binding sites were studied in order to identify pockets suitable for hosting inhibitor substituents. According to standard modeling procedures, binding site side chains were scrutinized in order to address protonation and 180° rotations of crystallographically equivalent groups and correct eventual atomic overlap. Subsequently, all structures were subject to the pre-simulation and simulation phases described above. The Cys522/NMS-859 covalently bound structure was prepared as follows. As the p97 crystal structure shows the side chain of Cys522 (covalent bond site) completely buried, the dynamical behaviour of the Walker A loop was evaluated with a 20 ns long MD simulation of the apo hex-amer protein to verify if a suitable conformation of Cys522 side chain exists for the reaction with NMS-859. The change in the surface accessible area of Cys522 was monitored and, interestingly, Cys522 was found to protrude into the ATP binding site in a solvent accessible conformation during the simulation. The protomer showing the highest solvent accessible area for Cys522 was extracted with its adjacent protomer and a relevant structural model of NMS-859 covalently bound to the Cys522 side chain was obtained building the Cys522/NMS-859 bond. A more energetically meaningful structure was sought for by subjecting this new model to 100 simulated annealing runs, raising the temperature to 600 K and then cooling down to 0 K. Even at high temperatures, NMS-859 did not undergo any major rearrangements within the binding site. A reference structure was obtained by performing a cluster analysis on the 100 final structures of the simulated annealing and choosing the central element of the most populated cluster. The structure resulting from the cluster analysis was used to generate the hexamer with a NMS-859 molecule bound in each of the D2 ATP binding sites. The hexamer was subsequently submitted to a 20 ns MD simulation. Rendine et al.: Molecular Dynamics Simulations Acta Chim. Slov. 2019, 66, 395-401 397 3. Results and Discussion A series of molecular dynamics simulations was performed on a p97 hexamer model in complex with NMS-873, NMS-322 and NMS-859 (Figure 1). Before doing so, we compared results from simulations performed on di-meric vs hexameric forms of p97 without inhibitors. The use of a dimeric in the place of the hexameric model constituted a significant advantage in terms of CPU-time required for these calculations. However, results from the test simulations excluded this possibility due to severe molecular instability observed with the dimer. Specifically, unlike the hexameric system, the p97 dimer suffered a gradual but complete detachment of the D2 domain from the D1 domain within the same protomer in the early stages of the production run. Such a dramatic rearrangement would interfere with the simulation of our set of ligands, which bind regions at the intra- and inter-protomer interfaces. The allosteric binding site and NMS-873. NMS-873 was originally docked into a putative binding site that was subsequently confirmed by photo-affinity labeling experiments. Depending on the position of reactive groups on NMS-873 analogues, Lys615 and Asn616 were specifically targeted as previously reported.11 The model was further refined according to the procedure in the material and methods section. The selected pose involved, among other pockets, the lateral tunnel leading to the central pore of the hexamer between the D1 and D2 domains of two adjacent protomers. On the basis of observed kinetics, a stoichiom-etry of 1:2 was applied with one molecule bound to the A-B, C-D and E-F interfaces, respectively. AG and alanine scanning. The three NMS-873 molecules showed a consistent behaviour throughout the molecular dynamics run, with a AG of binding of -69.2 ± 3.1 kcal/mol (A-B interface), -67.8 ± 2.7 kcal/mol (C-D interface) and -71.5 ± 2.9 kcal/mol (E-F interface). The coherent results for the three molecules are likely to be due to the tight packing and good fit of the molecule among residues and cavities at the D1 and D2 interface of the two adjacent protomers. In this case, the use of a hexameric model system turned out to be particularly important, since the simulation of the dimer would have led to a slacker binding site and a progressive weakening of the network of interactions due to protomer separation. Computational alanine scanning highlighted the most relevant protein residues involved in the binding of this inhibitor. Lys615 showed the highest AAG values on average (4.4 kcal/mol) with its contribution consisting of both hydrophobic interactions with the biphenyl moiety of NMS-873 and the hydrogen bond between the £-amino group and the pyridine moiety of the inhibitor. This particular hydrogen bond occurred in 20%, 34%, 71% of the snapshots in the A-B, C-D, E-F binding sites, respectively. The difference was due to the possible torsion of the pyri-dine moiety with respect to the triazole plane, allowing Figure 1.a) Structures of NMS-873, NMS-322 and NMS-859. b) Top view of the p97 hexamer: the single protomers are labelled with letters from A to F. c) Lateral view of the p97 hexamer coloured by domains and highlighting the NMS-873 binding site (red rectangle). Rendine et al.: Molecular Dynamics Simulations 398 Acta Chim. Slov. 2019, 66, 395-401 considerable variability in the making and breaking of this interaction. The aromatic pyridine ring was also found to effectively interact with Gln568 (1.3 kcal/mol) through van der Waals contacts. Gln215 (2.9 kcal/mol), Gln212 (1.0 kcal/mol) and Leu456 (1.6 kcal/mol) further stabilized the binding at the opposite ends of the molecule through hydrophobic contacts with the cyclopentyl and the methylsulphonyl group. Most relevant interactions are shown in Figure 2a. Other less frequent examples of amino acids that favourably took part in the binding with NMS-873, showing higher AAG values only in specific couples of protomers were Arg453 (1.3 kcal/mol, A-B interface) and His406 (2.0 kcal/mol, B-C interface). Both formed hydrogen bonds with the terminal p-methylsulphonyl moiety. Gln458 (1.2 kcal/mol) and Asn460 (1.7 kcal/mol) participated in stabilizing the biphenyl part of the molecule in the C-D couple. Further hydrophobic interactions involved Arg365 (2.0 kcal/mol, A-B couple) and Lys211 (2.0 kcal/mol, E-F couple), which make favourable contact with the pyridine and the cyclopentyl moieties, respectively. ATP-competitive inhibitors. NMS-322 belongs to the class of compounds sensitive to the ATP concentra-tion.13 Due to the complex and cooperative enzymatic mechanism of p97, it is impossible to distinguish between direct ATP competition and a mixed inhibitory mechanism where the binding of the compound affects the binding affinity of ATP in a different pocket of the hexamer. However, also in this case, there is experimental evidence of direct interactions within the nucleotide binding site in D2, namely the binding of Asp478 with a reactive azido analog of NMS-322 ("compound 35").13 AG and alanine scanning. Computational alanine scanning was performed to highlight the residues providing the principal interactions in NMS-322 binding. The results identified Thr525, Leu526 and Thr688 as the most important residues in all six protomers (Figure 2b). Leu526 showed the highest average AAG value (2.5 kcal/mol) establishing hydrophobic interactions with the nitroaniline and the cyclohexane moieties of NMS-322. Thr688 (average AAG of 1.1 kcal/mol) provided further hydrophobic interactions, whereas Thr525 (average AAG = 1.3 kcal/ mol) engaged hydrogen bonds with the carboxyl group of NMS-322, both through the backbone (44%) and the side-chain (52%). Other aminoacids displayed varying AAG values depending on the specific protomer, indicating some freedom of translation within this relatively large binding site. In particular, a group of residues in common for protomers A, B, D and E with cross-protomer interactions was identified. Only for these protomers, Arg635 showed average AAG values of 1.7 kcal/mol, providing interactions with the hydrophobic part of NMS-322 and hydrogen bonds with the carboxylic moiety. Hydrogen bonding was relatively often observed in protomers A (25% frequency) and E (15%). For the same group of NMS-322 molecules, the carboxylic substituent frequently made interactions with Lys524. The molecules bound to protomer C and F exhibited a slightly different behaviour, extending towards the bottom of the ATP pocket, rather than towards the adjacent protomer, resulting in null AAG values for Arg635, whereas Ile656 and Asn660 had higher AAG values in relation to the above mentioned protomers. Being Thr525, Leu526 and Thr688 constantly involved in the interaction with the molecule in all protomers, it can be pointed out that the shifting between the two poses occurred around Thr525, acting as a hinge, with NMS-322 sliding between Thr525 and Leu526, allowing a deeper interaction inside the pocket or with the residues from the adjacent protomer. In order to evaluate the NMS-322 displacement in the different protomers, the average RMSD on the molecule was calculated, after aligning the D2 domain backbone, with respect to the starting structure. The molecules bound to protomers A, B, D and E show an average RMSD over time of 3.82 A, 2.92 A, 3.45 A and 3.30 A, respectively, whereas the average RMSD rose to 5.93 A and 4.71 A for the molecules bound to protomers C and F, respectively. Despite the higher displacement for the molecule bound to protomer C, its AG of binding (-47.4 ± 3.8 kcal/mol) was consistent with the AG calculated for the molecules bound to protomers A (-44.4 ± 3.3 kcal/mol), B (-51.8 ± 3.2 kcal/mol), D (-46.2 ± 3.0 kcal/mol) and E (-53.3 ± 2.7 kcal/mol). In this case, NMS-322, as already highlighted by the alanine scanning results, could replace the original interactions with alternative ones, balancing the loss AG contributions. On the other hand, NMS-322 in protomer F was not found to efficiently rearrange to establish new binding interactions. Calculated AG of binding within protomer F and NMS-322 increased to -38.3 kcal/mol. Covalent and specific class of inhibitors. NMS-859 is an irreversible protein modifier that was found by mass spectroscopy to covalently and specifically bind VCP at an active site cysteine. The Cys522 residue belongs to the Walker A motif, which is the ATP phosphate binding loop of the D2 domain nucleotide binding site. This covalent modification has been experimentally shown to totally impair the ATPase activity of the enzyme. A possible mechanism of action would involve the binding of NMS-859 within the ATP binding site on the D2 domain, followed by the formation of a covalent bond with Cys522. In order to study the first step of the proposed binding process and evaluate the non-covalent fitting with the putative binding site, NMS-859 was docked in the D2 domain ATP pocket in the hexameric model (stoichiometry 1:1) and simulated for 20 ns. NMS-859 showed several similarities with NMS-322, primarily a significant contribution to the binding by hydrophobic contacts within the ATP site residues and the adjacent protomer. Computational alanine scanning performed on the last 10 ns of the trajectory highlighted Leu526, with an average AAG of 1.6 kcal/mol and Thr688 (AAG = 1.6 kcal/ mol) as hot-spots in all six protomers (Figure 2c). The two Rendine et al.: Molecular Dynamics Simulations Acta Chim. Slov. 2019, 66, 395-401 399 residues made up a hydrophobic cluster with the benzothi-azole and phenyl moieties of NMS-859. Ile656, which was identified as a possible partner of interaction for NMS-322, was also found to contribute to the binding with NMS-859 in all the protomers with an average AAG of 2.1 kcal/mol. The interaction with residues from the adjacent protomer also played a key role in the binding, in particular through Arg635, with an average AAG of 1.1 kcal/mol. NMS-859 adopted slightly different poses in the six protomers, resulting in AG values ranging from -34.4 ± 2.7 kcal/mol (protomer E) to -44.9 ± 2.0 kcal/mol (protomer A), as observed for NMS-322. NMS-859 enters the space between Leu526 and Thr688 allowing the molecule to interact more favourably with the hydrophobic residues located at the bottom of the ATP pocket or with Arg635 of the adjacent protomer (as observed for NMS-322). Overall, the ligand showed a good affinity for the ATP pocket with the chlorine atom preferentially oriented towards the Walker A loop, possibly representing a favourable molecular arrangement for the reaction with Cys522. The structural model of NMS-859 covalently bound to the Cys522 sidechain was obtained as described in the material and methods section. A qualitative analysis of the trajectory highlighted a binding pose comparable to non-covalently bound NMS-859. Obviously, an evaluation of the free energy of binding was excluded, given the covalent bonding with the protein. The analysis of residues in proximity of NMS-859 highlighted some of the same amino acids seen for the unbound molecule as possible partners of hydrophobic interactions. In particular, Leu526, Asn660 and Thr688 remained in close contact with the benzothiazole and phe-nyl moieties of the molecule throughout the entire length of simulations in all six protomers (Figure 2d). Ile656 and Ile479 also contributed in stabilizing the binding with the benzothiazole part of NMS-859. In a way similar to NMS-322, the Walker A motif provided a series of hydrogen bonds, in particular Lys524 and Thr525 backbone (41% and 48% of the snapshots, respectively) interacts with the amide group and the sulphur of NMS-859. A translation within the pocket, such as observed for the unbound molecule and for NMS-322, is also observed in the covalently bound NMS-859, in this case occurring by the pivoting of the ligand around Cys522. The molecules bound to protomers A and B preserved a pose close to the starting conformation, interact- Figure 2a) NMS-873 in complex with p97. b) NMS-322 in complex with p97. c) NMS-859 in complex with p97. d) NMS-859 covalently bound to residue Cys522 of p97. Interaction diagrams of these compounds can be found in Supporting information. Rendine et al.: Molecular Dynamics Simulations 400 Acta Chim. Slov. 2019, 66, 395-401 ing preferentially with residues belonging to the ATP pocket. The average RMSD (calculated on the molecule after the alignment of the D2 domain) resulted to be 1.04 A and 0.83 A, respectively. In these two cases, Thr688 was also favourably oriented to establish a hydrogen bond with the amine hydrogen of NMS-859, detected in 33% of the snapshots for the molecule bound to protomer A and in 35% of the snapshots for the molecule bound to protomer B. For the NMS-859 molecules bound to the protomers C to F, a preferential orientation towards Arg635 and Pro636 of the adjacent protomer was observed. Here the average RMSD with respect to the starting structure were 2.51 A in protomer C, 4.21 A in protomer D, 2.45 A in protomer E and 2.57 A in protomer F. In these cases, the loss of interactions with the inner residues of the ATP pocket was presumably balanced by a gain in hydrophobic interactions between Arg635 and Pro636 with the phenyl and thiomethylene moieties of NMS-859, as highlighted for NMS-322. 4. Conclusions The simulations reported in the study reproduce experimental observations made for what concerns both general aspects of molecular stability of the p97 protein and specific molecular interactions with inhibitors. The biologically relevant hexameric assembly was more stable than the dimer and the most unstable part of the single protomer was represented by the D2 domain as observed in crystallographic structures and in trypsin protein digestion experiments. Simulations performed on the different protein-inhibitor complexes showed good stability and reproducibility all around the hexamer. In particular, the postulated allosteric binding site and the binding mode of NMS-873 were surprisingly well conserved throughout the MD trajectory and consistently reproduce some of the most important interactions observed experimentally. Even though the ability to design more potent inhibitors remains to be proven, the approach adopted is certainly useful in providing a dynamic structural model able to rationalize a number of experimental observations regarding the mechanism of action of the different classes of p97 inhibitors reported to date. 5. References 1. B. DeLaBarre, A. T. Brunger, Nat. Struct. Mol. 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D01:10.1023/A:1007907728892 Povzetek Izvedli smo simulacije molekulske dinamike za binarne (kompleks nukleotid-dimer in heksamer proteina) in ternarne (kompleks nukleotid-inhibitor proteina) komplekse proteina p97, da bi razumeli stabilnost domene oligomera p97 in, kar je še bolj pomembno, molekularne mehanizme v inhibiciji, vključno z različnimi alosteričnimi, ATP-kompetitivnimi in kovalentnimi inhibitorji, o katerih so poročali kratkim. Analiza stabilnih stanj, ki sledijo uravnoteževalnim fazam, je pokazala, da je homoheksamer bolj stabilen v primerjavi z dimerom, in hkrati so domene N-D1 bolj stabilne v pirmerjavi z domeno D2. Molekulska dinamika predlaganega alosteričnega vezavnega modela reproducira pomembne molekulske interakcije, ki so bile ugotovljene eksperimentalno z visoko frekvenco vzdolž trajektorije. Opažene konformacijske spremembe, ki se pojavljajo v D2-vezavnem mestu nukleotidov, ponujajo novo hipotezo, imenovano bind-rearrange-react, o postopnih molekularnih dogodkih, ki so udeleženi pri specifičnem kovalentnem načinu delovanja inhibitorjev. Rendine et al.: Molecular Dynamics Simulations DOI: 10.17344/acsi.2018.4881 Acta Chim. Slov. 2019, 66, 402-413 ^creative ^commons Scientific paper Sandpaper Wastes as Adsorbent for the Removal of Brilliant Green and Malachite Green Dye Yasemin i§lek Co§kun,* Nur Aksuner and Jale Yanik Department of Chemistry, Faculty of Science, Ege University, 35100 Bornova, Izmir, Turkey * Corresponding author: E-mail: yasemin.islek@ege.edu.tr Telephone number: +90(232)3115447 Fax: +90(232)3888294 Received: 11-29-2018 Abstract Sandpaper wastes were used as adsorbent after pyrolysis at 500 °C and calcination at 800 °C for the removal of brilliant green and malachite green cationic dye from an aqueous solution. The effects of the pH, the adsorbent dose, the contact time, and the initial dye concentration on the removal efficiencies were investigated. The isotherm studies were conducted by using the Langmuir, Freundlich, and Dubinin-Radushkevich models, and thermodynamic studies were also performed. The adsorption of the Brilliant green and malachite green were found to comply with the Langmuir isotherm model and the Freundlich isotherm model, respectively. The thermodynamic studies showed that the adsorption of dyes were endothermic. The E values obtained from the Dubinin-Radushkevich isotherm showed that the adsorption mechanism was chemical in nature. Furthermore, the three kinetic models (pseudo first-order, pseudo second-order, and intraparticle diffusion) were investigated. It was found that the pseudo second-order kinetic model fitted well for adsorption of dyes. Keywords: Adsorption; brilliant green; malachite green; dye removal; sandpaper. 1. Introduction Brilliant green and malachite green are cationic (basic) dyes. Many industries such as paper, textile, furniture, and food industries use dyes for coloring purposes.1,2 Furthermore, in fish farming, brilliant green is used to protect fish from fungi, from parasites and from infections. However, the consumption of fish produced in this way is not recommended.2-5 Cationic dyes have toxic, carcinogenic and mutagenic properties.6 Significant risks arise after exposing these dyes to people. Therefore, the removal of the dyes prior to their discharge into the environment is crucial and essential. Physical, chemical and biological methods are widely used for the removal of dyes from water. Among these methods, adsorption, the physical method has advantages such as simplicity, low cost, and ease of application. Natural materials (raw or activated forms of clay minerals), synthesized materials, nanomaterial based adsorbents, agricultural wastes and by-products (raw or modified leaf based materials, coffee wastes, peels) and industrial wastes and by-products (fly ash, aluminum oxides), and activated carbon are the most used adsorbents for dye removal.1,7-11 Cost is an important parameter for choosing the adsorbent. Low-cost adsorbents include natural, agricultural and industrial by product wastes12. Furthermore, the waste materials have little or no economic value and usually present a disposal problem.7 The use of these waste materials for the purpose of wastewater treatment can play a significant role in solving the disposal problems. Numerous inexpensive and abundant biosorbents especially agro waste materials, as well as industrial and municipal wastes, have been proposed by several researchers for the removal of malachite green and brilliant green dyes from aqueous solution.13 The usage of waste as an adsorbent helps to reduce environmental pollution by recycling. In the literature some of the low-cost adsorbents used for dye removal were NaOH treated saw dust,14 waste rubber tire,15 white rice husk ash,16 Neem leaf powder,17 kaolin,2 peach stone,18 and medical cotton waste19 etc. Sandpaper is an abrasive used in the sanding process to correct the rough surfaces. It consists of sheets of paper or cloth with abrasive material glued with resin to one face. Formerly, sand and glass were used as abrasive surfaces, but nowadays materials such as aluminum oxide, zirconium oxide, and silicon carbide etc. are used.20 The storage and disposal of the sandpaper waste is a problem in terms of time, space, and cost. With this work, a useful area for sandpaper waste has been created, which will be beneficial for the environment and waste water remediation. Co^kun et al.: Sandpaper Wastes as Adsorbent for the Removal Acta Chim. Slov. 2019, 66, 402-413 403 The aim of the study is the removal of brilliant green and malachite green dyes from water by using sandpaper waste. Two adsorbents were prepared by applying pyrolysis and calcination process. To the best of our knowledge, it is the first study on brilliant green and malachite green removal using sandpaper waste based adsorbent. The important point of the study is that the pollution is reduced both by recycling of sandpaper waste, which is an industrial waste material and by removing the dyes from water. The effects of the experimental parameters such as the pH, the adsorbent dose, the contact time and the initial dye concentration were examined. The isothermal models (Langmuir, Freundlich and Dubinin-Radushkevich), thermodynamic and kinetic parameters (pseudo first order, pseudo second order and intraparticle model) were also evaluated. 2. Experimental 2. 1. Materials and Apparatus All the reactive used was of an analytical grade. Distilled water was used throughout the study. Cationic dye Brilliant Green (BG) (CI 42040, MW: 462.65), malachite green oxalate (MG) (CI 42000, MW: 927.01) hydrochloric acid, sodium hydroxide, acetic acid, and sodium acetate were obtained from Merck. The chemical structure of brilliant green and malachite green are presented in Figure 1. The working dye solution was prepared daily by diluting 1000 mg/L stock dye solution. The spectrophotometric measurements were carried out by TG 80+ model double beam UV/Vis spectrophotometer with PG Instruments. The pH was measured using a Mettler Toledo Five Go FG-2 pH meter. A Biosan 0S-10 orbital shaker at 350 rpm and Nuve ST-402 vibration water bath were used for the adsorption studies. The FTIR analyses of the adsorbents were carried out by using the Perkin Elmer 100 spectrum FT-IR spectrometer in the range of 4000-400 cm-1. The pore and surface morphology images were captured by using the Thermo Scientific Apreo S LoVac model scanning electron microscope (SEM). The chemical compositions of SW500 and SW800 were analyzed by X-ray Fluorescence spectrometer (Spectro Xepos, Ametec). BET analysis were executed by Quantachrome ASiQwin. The sandpaper was supplied by a fibre disk pad production company in Izmir, Turkey, as sheets. 2. 2. Preparation of Adsorbent from Sandpaper Wastes Two kind of adsorbent were obtained after pyrolysis and calcination processes. Pyrolysis and calcination processes were separately applied to the sandpaper sheets. The sandpaper sheets were cut into small pieces (< 2 cm) before the experiments. For pyrolysis, firstly, a quantity of 50 g of sandpaper was loaded into the reactor, and then the reactor was heated with a temperature rate of 7 °C per minute up to 500 °C and held at this temperature for 1 h. The reactor was continually purged with nitrogen at a flow rate of 25 mL/min. The nitrogen gas swept the volatile products from the reactor into the ice-cooled traps. The condensable volatiles, which were collected in the traps, were released. After pyrolysis, the furnace was cooled to room temperature in a nitrogen gas stream and the reactor content (carbonized residue) was withdrawn from the reactor.18 The obtained adsorbent was named SW500. Then, another part of the sandpaper sheets was calcined at 800 °C in a furnace up to 16 h and then stored in desiccators. The obtained adsorbent was named SW800. 2. 3. Adsorption Studies All of the adsorption studies were examined in batch mode. In order to find out the optimum experimental conditions, 25 mL of dye solutions were used. The initial concentration used was 20 mg/L. The contact time was 24 h unless otherwise stated. In the pH study, the adsorbent amounts were 10 mg for both adsorbents. The optimal sorbent doses were found to be 1.2 g/L of SW500 and 0.4 g/L of SW800 for the BG removal, while 2.4 g/L for SW500 and 0.6 g/L for SW800 for the MG removal after optimization study. The pH effect for the removal efficiencies was studied in the pH range between 3 and 10. The initial pH of the solutions was adjusted to the desired value using Na0H or HCl. The optimization studies, such as adsorbent dose (0.2, 0.4, 0.6, 1.2, 1.8 and 2.4 g/L), contact time (1, 5, 10, 15, 20, 40, 60, 120, 180, 240, and 1440 min), ini- a) HSO." b) H HO OH H -o OH — 2 Figure 1. The chemical structure of brilliant green (a) and malachite green (b) Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal 404 Acta Chim. Slov. 2019, 66, 402-413 tial dye concentrations (5, 10, 25, 50, 100, 200, and 400 mg/L), and temperature (298 K, 303 K, 313 K, and 323 K) were performed. The adsorption isotherms were evaluated in the range of 5-500 mg/L of dyes. Kinetic studies were investigated between 1-1440 min. The remaining BG and MG dye concentrations after sorption were measured at 624 nm and 617 nm by using UV-Vis spectrophotometer, respectively. All the experiments were conducted in triplicate. Before the spectrophotometric measurements, the pH of the dye solutions and standard solutions for the calibration were adjusted to 5.5 by using an acetic acid/acetate buffer. The removal efficiencies (R, %) and adsorbed dye amounts (q, mg/g) were calculated, respectively; (1) (2) Here, Ci and Ce are dye concentrations at an initial and equilibrium (mg/L), w is the amount of the adsorbent (g), and V is the volume of the dye solutions (L). 3. Results and Discussion 3. 1. Characterization of the Sandpaper Waste Adsorbent The morphology of the bare SW500 and SW800 are depicted in Figure 2a and 2b, respectively. The morphology of the SW500 surface was irregular and porous. In Figure 2b, it was seen that the particles of SW800 were spherical and aggregate. The higher number of pores increased the adsorption of the dyes. The FTIR spectra of the adsorbents before and after dye adsorption are shown in Figure 3a and 3b. In the spec- trum of SW800, the peak at 3454.78 cm-1 could indicate -OH stretching of the phenolic structure and crystal water. The intense peak at 1439.07 cm-1 was attributed to the aliphatic C-H stretching band. The peaks belonging to the crystal water and aliphatic C-H stretching band could also be seen in the other spectra. The two bands at 873.67 and 577.56 cm-1 could be Al-O vibration bands in Al2O3. After the adsorption for both the adsorbents, the C = C bands belonging to the aromatic ring of BG and MG appeared in the range of 1600-1700 cm-1. After BG adsorption on SW500, a peak assigning N-C band at 2969 cm-1 appeared. Besides, several adsorption peaks that emerged in the range of 1550-1380 cm-1 might be ascribed to the N-C groups after BG adsorption on SW500.13,14,21-26 The chemical compositions of SW500 and SW800 were determined by X-ray fluorimeter. It was found that SW500 contained 1.43% Al2O3, 0.31% SiO2, 0.72% P2O5, 0.16% SO3, 12.47% CaO, 0.68% TiO2, 0.74% Fe2O3, 0.03% CuO, 0.24% ZnO, 0.03% SrO and 0.05% ZrO2, while SW800 contained 3.6% Al2O3, 0.97% SiO2, 0.67% P2O5, 0.16% SO3, 44.06% CaO, 1.27% TiO2, 1.99% Fe2O3, 0.01% CuO, 0.03% ZnO, 0.08% SrO and 0.09% ZrO2. The amount of CaO in SW800, which was prepared by calcination, was found higher than that of SW500. Furthermore, higher dye removal efficiencies were obtained for SW800 in the adsorption studies. Therefore, that result could be a conclusion of existence of higher amount of CaO in SW800. Metal oxides containing calcium oxides are well known adsorbents for removal of various effluent gas streams. Because they have high adsorption capacity, high surface reactivity, low cost, and abundant.27 Calcium mineral is also efficiently used for the dye removal in the literature. Calcium rich biochar from crab shell showed highly efficient removal for Malachite Green and Congo Red although it showed low specific surface area and total pore volume.28 Jung et al. have synthesized an adsorbent using spent coffee grounds (SCG) calcium alginate beads for the removal of acid orange 7 and methylene blue. It was expressed that it was difficult to remove powdered SCG-based activated Figure 2. SEM images of bare adsorbents a) SW500 and b) SW800 Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal ... Acta Chim. Slov. 2019, 66, 402-413 405 a) b) Figure 3. FTIR spectra of adsorbents before and after dye adsorption a) SW500 and b) SW800 carbon from aqueous solution after adsorption. Therefore, to form porous hydrogels beads powdered SCG based activated carbon was entrapped in calcium-alginate beads. It was reported that such heterogeneous surface might be take part in removal of dyes.29 Basic Green 4 was successfully removed by using sea shell powder. It was reported the shell contained protein, calcite and calcium carbonate crystals, and the adsorbent had heterogeneous pores and cavities that gave large surface area for dye removal.30 In study of Xia et al., it was expressed that the various metal oxides for the adsorption capacities of Congo red dye were increased in the following order: NiO< MnO2 0). The value of AHo presents an idea about different physical forces being involved in the adsorption process such as van der Waals forces (4-10 kJ/mol), hydrophobic bond forces (5 kJ/mol), hydrogen bond forces (2-40 kJ/mol), coordination exchange (40 kJ/mol), dipole bond forces (2-29 kJ/mol), and for chemical forces (>60 kJ/ mol).39 Our results indicated that the forces affecting the adsorption of BG could be hydrogen bond forces and dipole bond forces because of AH° values belonging to BG being calculated in our study as 2.65 and 28.50 kJ/mol for SW800 and SW500, respectively. However, AH° values of MG were calculated to be 28.38 and 47.79 kJ/mol for SW800 and SW500, respectively. Hence, it was thought that the adsorption of MG could be affected by hydrogen bond forces. Table 1. Thermodynamic parameters for adsorption of BG and MG onto SW500 and SW800 (n = 3). T (K) AH° AS° AG° (kJ/mol) (J/mol.K) (kJ/mol) Brilliant Green SW800 298 303 313 323 SW500 298 303 313 323 Malachite Green SW800 298 303 313 323 298 303 313 323 , ,, AH0 AS0 InK, =----- L RT R AG0 = -RTlnKL AG0 = AH° - TAS° (3) (4) (5) SW500w 2.650 28.50 28.38 47.79 103.9 176.1 174.3 219.9 -13.10 -13.36 -13.90 -14.42 -8.972 -9.372 -10.17 -10.97 -8.361 -8.977 -10.21 -11.44 -17.74 -18.84 -21.04 -23.24 AG° is the free energy change (kJ/mol), R is the gas constant (8.314 J/mol K), KL is the Langmuir equilibrium constant (L/mol),36 and T is the temperature (K). KL values were found from the ratio of the adsorbed dye concentration (mg) and equilibrium dye concentration in the solution (mg/L). The parameters of AH and AS were obtained from the slope and the intercept of the Van't Hoff graph between lnKL and 1/T, respectively.14 The negative value of AGo indicates the adsorption is spontaneous and favorable. AHo values are positive whether the adsorption is endothermic, or vice versa. The positive ASo reveals that randomness increased at the solid-liquid interface. The positive AS° also indicates the affinity of the 3. 2. 6. Isotherm Studies The isotherm studies display the way of interactions between the dye molecules and the adsorbent, and also provide information about the nature of interactions. Experimental data was applied to the Langmuir,40 Freundlich and Dubinin Radushkevic (D-R) isotherm models.41 The Langmuir isotherm is based on the acceptance that the adsorption occurred at specific homogenous sites within the adsorbent while the Freundlich isotherm mentions the acceptance of a heterogeneous surface with a non-uniform distribution of heat of adsorption over the surface.38 D-R Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal ... Acta Chim. Slov. 2019, 66, 402-413 409 a) b) c) 100 200 300 C,(mg/L) 400 500 d) ■ BG SW800 aBGSWSOO • MGSW800 •MGSW500 logC2 Figure 8. Adsorbed amount of dye as a function of initial concentration (a), Langmuir isotherms (b), Freundlich isotherms (c) and DR isotherms (d) for adsorption BG and MG, (initial dye concentration range 5-500 mg/L, volume 25 mL, adsorbent dose of BG and MG: 0.6 and 0.4 g/L for SW800 and, 1.2 and 2.4 g/L for SW500, contact time 24 h, n = 3) Table 2. The isotherm parameters of Langmuir, Freundlich, and DR isotherms for BG and MG adsorption using SW500 and SW800 (n = 3). Brilliant Green SW800 SW500 Malachite Green SW800 SW500 Langmuir Isotherm Freundlich Isotherm logq = logK + — log C2 D-R Isotherm In Q = lnQm - ke2 E = (2k)" Qmax(mg/g) b (L/mg) R2 Separation factor 1/n K (mg/g)(L/mg)1/n R2 E (kJ/mol) Qm (mol/g) k (mol2/kJ2) R2 555.6 0.0833 0.9981 0.02-0.71 0.4271 59.01 0.9541 11.95 0.0041 0.0035 0.9741 294.1 0.0854 0.9963 0.02-0.07 0.7233 10.15 0.9289 9.535 0.0079 0.0055 0.8421 222.2 0.1372 0.9739 0.014-0.59 3.7805 0.3975 0.9882 10.43 0.0044 0.0046 0.9351 185.19 0.1013 0.9732 0.019-0.66 0.7041 1 4.3 0.9746 4.360 4.251x106 0.0263 0.95 C2 is the equilibrium concentration of the solution (mg/L), q is the amount of adsorbed dye/amount of adsorbent (mg/g), b is the Langmuir constant (L/mg), Qmax is the monolayer adsorption capacity (mg/g), K is the Freundlich constant ((mg/g)(L/mg)1/n), and 1/n is a dimensionless Freundlich constant for the intensity of the adsorbent, £ (Polanyi potential) is (RTln(1 + 1/C2)), Q is the amount of dye adsorbed per unit weight of adsorbent (mol/g), Qm is the adsorption capacity (mol/g), k is a constant related to adsorption energy (mol2/kJ2), R is the gas constant (kJ/mol K), and T is the absolute temperature (K). isotherm expresses the mechanism of adsorption onto a heterogeneous surface.42 In order to evaluate the adsorption isotherm, the used parameters were 25 mL of volume, an adsorbent dose of BG and MG 0.4 and 0.6 g/L for SW800 and 1.2 and 2.4 g/L for SW500, respectively and 24 h of contact time. Initial concentrations were in the range of 5-500 mg/L. Results and related equations were presented in Table 2 and Figure 8. The correlation coefficients were evaluated to find the best fit isotherm model for the system. As seen in Table Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal 410 Acta Chim. Slov. 2019, 66, 402-413 2, the highest correlation coefficient (R2) of BG was obtained for the Langmuir isotherm model while the highest R2 of MG was for Freundlich isotherm model. Thus, the adsorption of BG by SW500 and SW800 were monolayer on homogeneous sites. However, the adsorption of MG was a multilayer adsorption on a heterogeneous site. The maximum monolayer adsorption capacities of BG and MG were calculated to be 294.1 mg/g and 185.19 mg/g for SW500 and 555.6 mg/g and 222.3 mg/g for SW800. 1/n values indicate the adsorption intensity. The higher 1/n values mean the higher affinity between the dye molecules and adsorbent.38 The separation factor (RL) shows whether the adsorption is favorable (0 < RL < 1), unfavorable (RL > 1), linear (RL = 1) or irreversible (RL = 0).43 The separation factor is calculated by the equations given: (6) where C1 is the initial concentration and b is the Langmuir isotherm constant. Seeing that the RL values were in the range of 0-1, adsorption was favorable for both the adsorbents. The mechanism of adsorption can be determined by assessing E value (kJ/mol). The mean free energy of adsorption (E), which is defined as the free energy change when one mole of ion is transferred to the surface of a solid from the infinite space in the solution. Physical adsorption is val- id if the value is below 8 kJ/mol. When the E value is between 8 kJ/mol and 16 kJ/mol, chemisorption or ion exchange occurs.44 Since the values of BG (11.95; 9.54 kJ/mol for SW800; SW500) and MG (10.43; 4.36 kJ/mol for SW800; SW500) were between 8 and 16 kJ/mol, the presence of chemisorption or ion exchange could be mentioned. 3. 2. 7. Adsorption Kinetic Studies In this study, to understand the adsorption mechanism, three simplified kinetic models were elucidated: Lagergren pseudo first order, pseudo second order and intra-particle diffusion model.41 These models define the stages of the adsorption to be external film diffusion, adsorption, and intraparticle diffusion.45 Figure 9 presents the time effects on the adsorption, pseudo first-order kinetic model, pseudo second-order kinetic model, and intraparticle diffusion kinetic model for the adsorption of BG and MG onto SW500 and SW800 adsorbents. The calculated parameters belonging to the kinetic models are depicted in Table 3. As can be seen from Table 3, the closest R2 values to unity were obtained for the pseudo second order kinetic model for the studied dyes. The calculated (qe, cal) and experimental (qe,exp) values of adsorption capacities of BG and MG were very close to each other for the pseudo second order kinetic model. These findings indicated the adsorption fitted well with pseudo second order kinetic model and adsorption was chemisorption controlled.46 In the a) b) 100 150 t (min) 200 150 ' BG SW800 iBGSWSOO ' MG SW800 •MGSW500 t (miri) c) 50 Figure 9. Kinetic studies for BG and MG adsorption a) pseudo first-order kinetic model, b) pseudo second-order kinetic model, c) intraparticle diffusion kinetic model (initial dye concentration 20 mg/L, volume 25 mL, adsorbent dose of BG and MG: 0.4 and 0.6 g/L for SW800 and, 1.2 and 2.4 g/L for SW500, contact time 24 h, temperature 25 °C, n = 3) Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal ... Acta Chim. Slov. 2019, 66, 402-413 411 Table 3. The constants of the pseudo first-order, pseudo second-order kinetic models, and intraparticle kinetic model for BG and MG removal (n = 3). Kmetic Models Adsorbent SW800 SW500 qe, exp qe, cal k1 R2 qe, exp qe, cal k2 (mg/g) (mg/g) (1/min) (mg/g) (mg/g) (g/mgmin) R2 Pseudo first order ktt i°g(qe - qt) = '°s qe - BG 49.67 6.668 0.00184 0.1121 14.84 5.041 0.0205 0.7996 MG 33.33 6.637 0.00438 0.8687 8.33 3.472 0.0053 0.8469 Pseudo second order tit qt k2 qe BG 49.67 50 0.005076 0.9999 14.84 12.89 -0.00735 0.9990 MG 33.33 32.26 0.005128 0.9999 8.33 7.369 0.01363 0.998 Intraparticle diffusion model qt = kintt°5 + I R2 R2 I k I k (mg/g) (mg/gmin05) (mg/g) (mg/gmin05) BG Step 1 41.63 0.6047 0.8893 10.19 0.7498 0.7789 Step 2 49.483 0.0082 0.2363 14.522 0.0647 0.5396 MG Step 1 25.967 0.3118 0.8969 4.5719 0.1621 0.8509 Step 2 29.652 0.0654 0.9851 6.5568 0.0207 0.7878 qe and qt indicate the adsorption capacity at equilibrium (mg/g) and at time t; k1 and k2 are the pseudo first-order (1/min) and pseudo second-order rate constants (g/mg min); t is the contact time (min) and kint (mg/g min05) and I (mg/g) are the intraparticle diffusion constants. intraparticle diffusion model, the plot qt versus t05 gives k and I as slope and intercept, respectively. The intercept indicates the effect of the boundary layer thickness. The higher the intercept length, the more the adsorption is boundary layer controlled.46 Also, if the line passes through the origin (I = 0), the rate limiting mechanism is solely controlled by the intraparticle diffusion. Thus, it was concluded that the intraparticle diffusion was not the only rate limiting step. Since two separate regions were obtained for both the adsorbents, the adsorption process was affected by two or more steps. The initial region is ascribed to the bulk diffusion while the second to the intraparticle diffusion.38 4. Conclusion The adsorbents used in this study were obtained from the sandpaper wastes. Hazardous and toxic reagents were not used during the preparation of the adsorbents. Thus, environmentally friendly adsorbents were obtained. Furthermore, the removal of brilliant green from aqueous solutions was successfully carried out using both the adsorbents. Optimization studies (pH, adsorbent dose, contact time, and initial concentration etc.) were carried out to investigate the removal performance of both adsorbents. According to the pH study, SW800 provided a wider pH range than that of SW500 for both dyes. The optimum adsorbent doses of BG and MG were selected as 0.4 and 0.6 g/L for SW800 while 1.2 and 2.4 g/L for SW500, respectively. The removal efficiencies of BG and MG reached a plateau after 120 min and 180 min for SW500 and 180 min and 240 min for SW800, respectively. The adsorption kinetics of the dyes fitted well with the pseudo second-order kinetic model. The adsorption of the BG showed good agreement with the Langmuir isotherm model and indicated monolayer adsorption on homogeneous sites. However, it was found that the adsorption of the MG obeyed the Freundlich isotherm model. The values of E indicated the adsorption mechanism of dyes could be chemical or through ion exchange. The adsorption of BG and MG were found to be favorable for both SW500 and SW800. The thermodynam-ic studies indicated that the process was endothermic, spontaneous, and feasible. The comparison of the maximum BG and MG adsorption capacities with the reported adsorbents in the literature can be found in Table 4. By comparing the maximum adsorption capacities in Table 4, the highest capacity values belonged to the sandpaper waste. As a result, environmentally friendly adsorbents were developed that facilitate fast and efficient removal. Acknowledgments Authors would like to thank to Munevver Ozalp and Elif Cansu Tanriverdi for their help on laboratory study. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal 412 Acta Chim. Slov. 2019, 66, 402-413 Table 4 Comparison of the maximum adsorption capacities of BG and MG with the reported adsorbents in the literature. BG MG Adsorbent Capacity (mg/g) Isotherm Kinetic model Ref. Adsorbent Capacity (mg/g) Isotherm Kinetic model Ref. Tannin gel (TG) 8.55 Langmuir Pseudo second order 6 AMP clay 130.64 Langmuir Pseudo first order 1 Amine modified TG 2.41 Langmuir Pseudo second order 6 Coconot coir activated carbon 27.44 Langmuir Freundlich Pseudo second order 7 Acorn 2.01 Langmuir Pseudo second order 46 Rattan sawdust 62.71 Langmuir Pseudo first order 12 Peganum harmala-L seeds 35.97 Langmuir Pseudo second order 3 EM based compost 159.22 Sips Pseudo second order 13 Saklikent mud 1.18 Langmuir Pseudo second order 45 Potato peel 35.61 Redlich-Peterson Pseudo-nth order 50 White rice husk ash 85.56 - Pseudo second order 16 GO-Fe3O4 160.7 Langmuir Freundlich Pseudo second order 51 Red clay 125 Redlich-Peterson Pseudo second order 35 Citrus limetta peel 8.733 D-R Pseudo second order 22 Kaolin 65.42 Langmuir Pseudo second order 2 Zea mays cob 16.72 D-R Pseudo second order 22 NaOH treated saw dust 58.48 Redlich-Peterson and Temkin Pseudo second order 14 Organically modified hydroxyapatite 188.18 Langmuir 21 Ni/NixB nanoparticle-coated resin 147.1 Langmuir Pseudo second order 49 SW800 222.2 Pseudo second order This study SW800 555.6 Langmuir Pseudo second order This study SW500 185.19 Pseudo second order This study SW500 294.1 Langmuir Pseudo second order This study 5. 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DOI:10.1016/j.jwpe.2017.03.001 Povzetek Odpadke brusilnega papirja smo po pirolizi na 500 in kalcinaciji na 600 uporabili za odstranjevanje kationskih barvil briljantno zeleno in malahitno zeleno iz vodnih raztopin. Adsorpcijsko ravnotežje smo poskusili opisati z Langmuir-je-vo, Freundlich-ovo, in Dubinin-Radushkevich-ovo izotermo ter izvedli termodinamske študije. Adsorbcijo na briljantno zeleno smo najbolje opisali z Langmuir-jevo izotermo, adsorpcijo na malahitno zeleno pa z Freundlich-ovo. Izkazalo se je, da je adsorpcija endotermna. E vrednost pridobljena iz Dubinin-Radushkevich-ove izoterme je pokazala, da gre za kemijsko adsorpcijo. Kinetiko adsorpcije smo preučili s tremi kinetični modeli (psevdo prvi-red, psevdo drugi-red reakcije in modelom znotraj-delčne difuzije) in pokazali, da model psevdo-drugega reda najbolje opiše adsorpcijo na obe barvili. Coçkun et al.: Sandpaper Wastes as Adsorbent for the Removal DOI: 10.17344/acsi.2018.4885 Acta Chim. Slov. 2019, 66, 414-420 ^creative ^commons Scientific paper Preparation, Structure, Photoluminescent and Semiconductive Properties, and Theoretical Calculation of a Mononuclear Nickel Complex with 3-Hydroxy-2-Methylquinoline-4-Carboxylato Ligand Xiao-Niu Fang,1 Jia Li,1,3 Xiu-Guang Yi,1,2* Qi Luo,1 Jia-Yi Chen1 and Yong-Xiu Li2* 1 Institute of Applied Chemistry, School of Chemistry and Chemical Engineering, Jinggangshan University, Jian, Jiangxi, 343009, China 2 School of Materials Science and Engineering & Chemistry, Nanchang University, Nanchang, Jinagxi, 330031, China 3 State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China * Corresponding author: E-mail: jayxgggchem@163.com Tel: +86 (796)8100490; Fax: +86 (796)8100490 Received: 12-01-2018 Abstract A novel nickel complex with mixed ligands [Ni(L)2(EtOH)2(MeOH)2] (HL = 3-hydroxy-2-methylquinoline-4-carbox-ylic acid) has been synthesized through solvothermal reaction and its crystal structure was determined by single-crystal X-ray diffraction technique. Single-crystal X-ray diffraction analyses reveals that the title compound crystallizes in the triclinic system of the P-1 space group, and exists as isolated mononuclear complex. The intermolecular hydrogen bonds lead to the formation of chains, and the layered supramolecular structure is formed by the strong n—n stacking interactions. Solid-state photoluminescent characterization reveals that the title compound has an emission in the green region. Time-dependent density functional theory (TDDFT) calculation shows that the nature of the photoluminescence of the title compound originates from the ligand-to-ligand charge transfer (LLCT; from the HOMO of the p-orbital of ligand HMCA to the LUMO of the oxygen atoms). A wide optical band gap of 2.25 eV is found by the solid-state UV/vis diffuse reflectance spectrum. Keywords: Nickel; photoluminescence; semiconductor; TDDFT; LLCT 1. Introduction In recent years, metallo-organic coordination polymers have attracted considerable interest due to their diverse structures and potential applications in fluorescence, magnetic materials, gas adsorption, catalysis and medicine and so forth.1-6 The synthesis of coordination polymers with specific functions has gradually become a research hotspot in the field of material chemistry.7 From the perspective of crystal engineering, the most useful and facile way to construct coordination complexes is to adopt a suitable ligand to connect metal centers. The ligand is better to possess as much donor atoms as possible that enable it to bridge metal centers together to yield extended archi- tectures. The important feature of metallo-organic coordination polymers is the extension of low dimensional building blocks to high dimensional networks through weak intermolecular interactions, including weak van der Waals force, hydrogen bonding, n-n stacking, etc.8-10 Selecting a good ligand is very important in the design and preparation of complexes. Nitrogen heterocyclic compounds have attracted much attention due to their flexible coordination modes and easy coordination with metal ions. They are commonly used ligands for building complexes. A series of complexes based on these ligands have been reported.11-14 But carboxyl groups not only have many coordination modes such as monodentate coordination, symmetrical chelation coordination, asymmetrical Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive Acta Chim. Slov. 2019, 66, 414-420 415 chelation coordination, monooxy bridging coordination and so on, but also have strong coordination ability. They can coordinate with almost all metals to form complexes, and have a large number of unique structures and excellent properties. Various metal carboxylate complexes have been reported.15-20 If carboxyl groups are introduced into heterocyclic compounds, a series of heterocyclic carboxyl-ic acids with more abundant coordination sites and patterns can be obtained. For example, 4,5-imidazoledicarbo-xylic acid, 2-pyridine-4,5-imidazoledicarboxylic acid, and corresponding complexes. Up to now, there are few reports on the complexes of quinolinecarboxylates.21,22 Quinolinecarboxylate ligands, as ligands containing nitrogen atoms and carboxyl oxygen atoms, are easy to coordinate with metal ions. The coordination modes of car-boxyl oxygen atoms are diverse, and the degree of deproto-nation of carboxyl groups varies under different pH values, bringing more coordination modes. The coordination mode can exhibit various structures and unique properties. A new nickel coordination polymer (the title compound) was synthesized by hydrothermal method with 3-hydroxy-2-methyl-quinoline-4-carboxylic acid (HL) as ligand and nickel acetate. Its structure and properties were investigated with infrared spectrum, elemental analysis, single-crystal X-ray, solid-state diffuse reflectance spectrum, photoluminescent and theoretical calculations. 2. Experimental 2. 1. Materials and Instrumentation All reagents and chemicals were of reagent grade, commercially available and directly applied for the reaction. 1H NMR of the ligand were performed on Bruker Avance 400 MHz based on deuterium DMSO as solvent. Infrared spectra were obtained with a PE Spectrum-One FT-IR spectrometer using KBr discs. Elemental microanalyses of carbon, hydrogen and nitrogen were performed on an Elementar Vario EL elemental analyser. Solid-state UV/ Vis diffuse reflectance spectroscopy was acted on a computer-controlled TU1901 UV/Vis spectrometer, Finely-ground powder sample was coated on barium sulfate for a 100% reflectance. Photoluminescence characterization was performed on a F97XP photoluminescence spectrometer. Time-dependent density functional theory (TDDFT) calculation were carried out by virtue of the Gaussian09 suite of program packages. 2. 2. Synthesis of 3-hydroxy-2- methylquinoline-4-carboxylic acid (HL) Synthesis of isatin: indigo (262 g, 1.0 mol) and K2Cr2O7 (147 g, 0.50 mol) were added into 500 mL of water and stirred. After cooling, K2Cr2O7 (147 g, 0.50 mol), 300 mL of water and 500 mL of 10 % H2SO4 were added and kept stirring at 43 °C for 1.5 h. Then, the mixture was diluted with twice its volume of water, filtered off, dissolved in 10% KOH solution, filtered again, acidified with 10% HCl to pH = 7 and refiltered. Yield: 230 g (90%); m.p. 210 °C; HRMS m/z (ESI) calcd for C8H5NO2 ([M+H]+) 147.0320, found 147.0826. Synthesis of HL: isatin (147 g, 1.0 mol) and KOH (56 g, 1.0 mol) were dissolved into a sufficient amount of water and filtered. The filtrate and KOH (56 g, 1.0 mol) were added into chloroacetone (184 g, 2.0 mol), and hydrochloric acid was added dropwise to adjust pH = 7, then filtered. Yield: 193 g (95%); m.p. 225 °C. IR peaks (KBr, cm-1): 3433(vs), 3125(w), 3043(w), 2869(w), 2499 (m), 2040(m), 1621(m), 1553 (s), 1500 (m), 1462(m), 1410(m), 1242(vs), 1160(m), 1014(w), 906(m) and 686(s); HRMS m/z (ESI) calcd for C11H9NO3 ([M+H]+) 203.0582, found 203.0548; 1H NMR (400MHz, DMSO) 5 9.15 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 7.60-7.52 (m, 1H), 2.70 (s, 3H). 2. 3. Synthesis of Complex 1 0.124 g Ni(CH3COO)2-4H2O (0.50 mmol), 0.203 g HL (1.0 mmol), 5 mL methanol, 5 mL ethanol and 0.5 mL distilled water were added in turn into a 25 mL Teflon-lined stainless steel autoclave. The autoclave was heated to 100 °C in an oven and kept there for one week, then let to cool down to room temperature. Light yellow block crystals were obtained and used to collect the single-crystal X-ray data. Yield 0.434 g 1 (70% based on HL). IR (KBr, cm-1): 3421(s), 1610(s), 1558 (s), 1416(m), 1348(m), 1228(vs), 826(s), 659(w) and 636(s); Anal. Calcd for C28H36N2NiO10: C, 54.30; H, 5.86; N, 4.52; found: C, 54.39; H,5.81; N, 4.66%. 2. 4. X-ray Structure Determination The single-crystal data of the title complex were collected on the SuperNova CCD X-ray diffractometer equipped with a graphite monochromated Mo-Ka radiation source (0.71073 A) at 293(2) K. The reduction and empirical absorption correction of the diffraction data were carried out with CrystalClear software. The crystal structure was successfully solved by using the direct methods and Siemens SHELXTLTM Version 5 software package and refined with a full-matrix least-squares refinement on F2.23 All of the non-hydrogen atoms were generated based on the subsequent Fourier difference maps and refined anisotropically. The hydrogen atoms were located theoretically and ride on their parent atoms. Due to the problem of crystal quality and the week high-angle diffraction points, lead to the low completeness 0.806. Crystallo-graphic data and structural refinements for the title complex are summarized in Table 1. Selected bond lengths and bond angles for the crystal structure are displayed in Table 2. The hydrogen bonding interactions are presented in Table 3. Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive 416 Acta Chim. Slov. 2019, 66, 414-420 Table 1. Crystallographic data and structural analysis for the title compound Empirical formula C28H36N2NiO1o Mr 619.30 Color yellow Crystal system Triclinic Space group P-1 a (A) 7.8897(9) b (A) 8.8950(14) c (A) 10.4485(16) « ( °) 75.433(14) P ( °) 87.129(12) Y ( °) 70.233(12) V (A3) 667.38(17) Z 1 Reflections collected 5646 Independent, Observed reflections (Rint) 1895, 1686 (0.0464) dcalcd. (g/cm3) 1.541 ,«(mm-1) 0.791 F(000) 326 R1, wR2 0.0944, 0.2312 S 1.059 Largest and mean A/a 0,0 A/a(max, min)(e. A3) 1.147, -0.541 3. Results and Discussion The 3-hydroxy-2-methylquinoline-4-carboxylic acid (HL) was prepared by the reaction of isatin with chloroac-etone in alkali condition, and the isatin was obtained by the oxidation of indigo, as shown in Scheme 1. The experimental procedure was improved according to the basis of literature.24,25 Indigo Isatin CH3C0CH2C1 ^^ ,OH KÔH f I] I N ^ HL Scheme 1. Synthetic route of HL Table 2. Selected bond lengths (A) and bond angles (°) for the title compound Distance (A) Distance (A) Ni1-O1 2.044(5) Ni1-O3 2.088(6) Ni1-O4 2.052(5) O1-C11 1.298(10) O2-C11 1.248(10) O3-C14 1.393(14) O4-C13 1.412(10) O5-C8 1.346(12) N1-C9 1.307(10) N1-C1 1.362(10) Angle ( °) Angle ( °) O1-Ni1-O4 93.0(2) N1-C1-C6 121.3(6) O1-Ni1-O3 91.4(2) N1-C9-C10 117.5(7) O4-Ni1-O3 88.9(3) N1-C9-C8 122.7(7) C11-O1-Ni1 130.1(5) O2-C11-O1 122.4(6) C14-O3-Ni1 127.8(8) O2-C11-C7 120.2(7) C13-O4-Ni1 129.3(5) O1-C11-C7 117.5(7) C9-N1-C1 120.3(6) O4-C13-C12 83.4(7) Table 3. Hydrogen bonding interactions D-H-A D-H, A H-A, A D-A, A D-H-A, ° O4-H4B-N1i 0.93 1.94 2.728(7) 141 O5-H5B—O1 0.82 1.89 2.605(10) 145 C5-H5A-O2 0.93 1.97 2.647(10) 128 Symmetric code: (i) x, y, 1 + z. In the first step, the amount of oxidant and temperature control is the key factors to the success of the oxidation. In the second progress, the amount of KOH and the feeding mode of chloroacetone make the important effects on reactions. Single-crystal X-ray diffraction measurement revealed that the title compound crystallizes in the space group P-1 of the triclinic system. In the crystal structure of title compound 1, the metal Ni atom is sitting at the inversion center. The nickel (II) ion is hexacoordinated octahedron by the doubly deprotonated HL, two methanol and two ethanol molecules, yielding an octahedral geometry, as presented in Fig. 1. The bond distance of Ni-O1 is 2.044(5) Á, for is Niii-O3 2.088(6) Á, while that of Ni-O4 is 2.052(5) Á [ii = -x, -y, 1 - z]. These are comparable with that reported in the references.26,27 Quinolinecarboxylate (L-) acts as the monodentate ligand coordinated to the nickel metal center, and two such ligands occupy both axial positions. The four O atoms of methanol and ethanol are located in the equatorial plane with the good coplanar-ity. The intramolecular hydrogen bond can be found between the phenolic hydroxyl group and carboxylate group (O5-H5B—O1), and another week intramolecular hydrogen bond exist between the carbon atom and the carboxyl oxygen atom (C5-H5A—O2). The intermolecular hydrogen bond O4-H4B—NF can be found between ethanol oxygen and aromatic N atom of quinoline moiety, forming a one-dimensional supramolecular structure extending along the c-axis, as presented in Fig. 2. In the complex, there are strong offset face-to-face n—n stacking interactions between Cg1—Cg1m and Cg2—Cg1iv [Cg1 and Cg2 are Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive ... Acta Chim. Slov. 2019, 66, 414-420 417 N1/C1/C6-C9 and C1-C6 ring centroids; iii = -x, -y, -z; iv = 1 - x, -y, -z]. The centroid-centroid distance of Cg1-Cg1iii is 3.6528(4) A with the shift distance 1.3372(7) A and the twist angle of 0.00(4)°. The centroid-centroid distance of Cg2-Cg1iv is 3.5966(4) A with the shift distance 1.0197(8) A and the twist angle of 2.387(5)°. These n—rt stacking interactions yield the two-dimensional su-pramolecular layers along the ac-axis plane, then via van der Waals attraction complete a crystal packing as presented in Fig. 3. Figure 1. The molecular structure of the title compound. Hydrogen atoms not involved in the motif shown were removed for clarity. Figure 2. The 1-D chain structure of the title compound. Hydrogen atoms not involved in the motif shown were removed for clarity. The intramolecular hydrogen bond is shown as red stipple line, the intermolecular hydrogen bond is shown as blue stipple line. Figure 3. Packing diagram of the title compound viewed along the b axis. Hydrogen atoms not involved in the motif shown were removed for clarity. The intramolecular hydrogen bond is shown as red stipple line, the intermolecular hydrogen bond is shown as blue stipple line, and the magenta stipple line represent the n—n stacking interactions. In recent years, the photoluminescence properties of coordination complexes have gained increasing interest. Generally, coordination complexes containing lanthanide and transition elements can exhibit photoluminescence behavior because they possess rich 4/-orbit and 3/4d-orbit electron configurations. Many studies about the photoluminescence performance of lanthanide and transition compounds have been conducted so for.28-30 The title compounds contain Ni2+ ions; therefore, we deemed that nickel and HL complexes can possibly exhibit interesting photoluminescence performance. Based on the above considerations and in order to reveal its potential photoluminescent properties, we carried out the photoluminescence spectra with solid state samples at room temperature and the result is presented in Figs. 4. It is obvious that the pho-toluminesent spectrum of the title compound displays an effective energy absorption residing in the wavelength range of 350-450 nm. Upon the emission of 544 nm, the excitation spectrum shows a band at 408 nm. We further measured the corresponding photoluminescence emission spectrum of the title compound. Upon excitation at 408 nm, the emission spectrum is characterized by a sharp band at 544 nm in the blue region of the spectrum. The emission band of the title compound is located in the green light region with the CIE (Commission Internationale de I'Éclairage) Chromaticity coordinate (0.3551, 0.634) (Fig. 5). As a result, the title complex is a potential green photoluminescent material. Figure 4. Solid-state photoluminescence spectra of the title compound measured at room temperature (green curve: excitation; red curve: emission). In order to reveal the nature of the photoluminescence emission of the title compound, we truncated ground state geometry from its single-crystal X-ray diffraction data set (without optimization) and carried out its theoretical calculation in light of the time-dependent density functional theory (TDDFT) based on this ground state Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive 418 Acta Chim. Slov. 2019, 66, 414-420 Figure 5. The CIE chromaticity diagram and chromaticity coordinates of the emission spectrum of the title compound. could be assigned to the ligand-to-ligand charge transfer (LLCT; from the HOMO of the p-orbital of ligand HL to the LUMO of the oxygen atoms). To investigate the semiconductive properties of the title complex, the solid-state UV/vis diffuse reflectance spectra was measured on a powder sample at room temperature, using barium sulfate as the reference for 100% reflectivity, its surface was coated with finely-ground powder samples for measurement. After measuring, the data were treated with the Kubelka-Munk function which is known as a/S = (1 - R)2/2R. With regard to this function, the parameter a means the absorption coefficient, S means the scattering coefficient, and the R means the reflectance, which is actually wavelength independent when the size of the particle is larger than 5 ^m. From the a/S vs. energy gap diagram, we can obtain the value the optical band gap, which can be extrapolated from the linear portion of the absorption edges. In the way, the solid-state UV/vis diffuse reflectance spectrum reveals that the title compound has a wide optical energy band gap of 2.25 eV, as shown in Fig. 7. As a result, the title compound is a possible candidate for wide band gap semiconductors. The gentle slope of the optical absorption edge of title compound indicates that it must be an indirect transi-tion.34 The energy band gap of 2.25 eV is obviously larger than those of GaAs (1.4 eV), CdTe(1.5 eV) and CuInS2 geometry. The TDDFT calculations were performed using the B3LYP function31,32 and carried out by means of the Gaussian09 suite of programs,33 with SDD for Ni and 6-31G* basis for other atoms. The characteristics of HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) of the title compound is shown in Fig. 6. It is easy to find out that the electron-density distribution of HOMO is totally resided at the coordinating the p-orbital of ligand HL with an energy being of -0.21787 Hartrees; however, the electron-density population of LUMO is mainly distributed on the oxygen atoms and the energy of the LUMO is calculated to be -0.19871 Hartrees. The energy difference between LUMO and HOMO is 0.01916 Hartrees that is small enough to allow the charge transfer from HOMO to LUMO. In light of this observation, it is proposed that the essence of the photoluminescence of the title compound 2 3 4 5 E / eV 9 Figure 7. Solid-state UV/vis diffuse reflectance spectrum for the title compound. OMO LUMO Figure 6. B3LPY predicted frontier molecular orbital of the title compound. The isovalue of 0.04 is used for plotting isosurfaces. Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive ... Acta Chim. Slov. 2019, 66, 414-420 419 (1.55 ev),35'36 all of them are well known as highly efficient band gap photovoltaic materials. 4. Conclusions A novel nickel(II) complex [Ni(L)2(EtOH)2(MeOH)2] with the ligands of 3-hydroxy-2-methylquinoline-4-car-boxylic acid (HL), MeOH and EtOH was synthesized by solvothermal synthesis, its structure and properties were investigated with infrared spectrum, elemental analysis, single-crystal X-ray, solid-state diffuse reflectance spectrum, photoluminescent and theoretical calculations. The title compound crystallizes in the triclinic system of the P-1 space group as isolated mononuclear complex. The intermolecular hydrogen bonds lead to the formation of chains, and the layered supramolecular structure is formed by the offset face-to-face n—n stacking interactions. Solid-state photoluminescence spectrum reveals that it shows an emission in the green region of the light spectrum. Time-dependent density functional theory (TDDFT) calculations reveal that this emission can be attributed to li-gand-to-ligand charge transfer (LLCT). Solid-state diffuse reflectance data shows there is a narrow optical band gap of 2.25 eV. 5. Acknowledgements We gratefully acknowledge the financial support of the NSF of China (51363009), Jiangxi Provincial Department of Education's Item of Science and Technology (GJJ160745, GJJ170652), Jinagxi Provincial Department of Education's Item of higher education and teaching reform (JXJG-17-9-14), the Science and Technology Plan project Fund of Jiangxi Provincial Health Planning Commission (20194083), and Natural Science Foundation Project of Jinggangshan University (JZ09029). 6. Supplementary Material Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 1876986. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44 1223 336-033; e-mail: de-posit@ccdc.cam.ac.uk). 7. References 1. X. Liu, C. Manzur, N. 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Han, Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive 420 Acta Chim. Slov. 2019, 66, 414-420 E. Y. Shin, S. S. Kim, K. R. Kim, H. G. Cheon, J. K. Choi, Bull. Korean Chem. Soc. 2003, 24(10), 1455-1464. D01:10.5012/bkcs.2003.24.10.1455 25. J. W. Yu, L. N. Song, J. Taiyuan Normal University (Natural Science Edition) 2016, 15, 77-80. DOI: 10.1088/1475-7516/2016/04/019 26. Y. Hou, L. Xu, M. J. Cichon. S. Lense, K. I. Hardcastle, C. L. Hill, Inorg. Chem. 2010, 49, 4125-4132. D0I:10.1021/ic9024712 27. E. G Bajnoxzi, Z. Nemeth, G. Vanko, Inorg. Chem. 2017, 56, 14220-14226. D0I:10.1021/acs.inorgchem.7b02311 28. P. C. Ford, E. Cariati, J. Bourassa, Chem. Rev. 1999, 99, 36253648. D0I:10.1021/cr960109i 29. J. H. Zhang, J. L. Wang, L. Jia, Z. K Qu, Q. H. Kong, Adv. Mater. Res. 2011, 284-286, 2153-2156. D0I:10.4028/www.scientific.net/AMR.284-286.2153 30. F. Y. Liu, D. M. Zhou, Z. L. Zhao, J. F. Kou, Acta Cryst. 2017, 73, 382-392. D01:10.1107/S2053229617004697 31. A. D. Becke, J. Chem. Phys. 1993, 98, 5648-5652. DOI: 10.1063/1.464913 32. C. Lee, W. Yang, R. G. Parr, Phys. Rev. B. 1988, 37, 785-789. DOI: 10.1103/PhysRevB.37.785 33. M. J. Frisch et al., Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford CT, 2009. 34. F. Q. Huang, K. Mitchell, J. A. Ibers, Inorg. Chem. 2001, 40, 5123-5126. D0I:10.1021/ic0104353 35. W. Bensch, P. Dürichen, Eur. J. Solid State Inorg. Chem. 1996, 129, 1489-1492. D0I:10.1002/cber.19961291214 36. R. Tillinski, C. Rumpf, C. Näther, P. Duerichen, I. Jess, S. A. Schunk, W. Bensch, Z Anorg. Allg. Chem. 1998, 624, 12851290. D0I:10.1002/(SICI)1521-3749(199808)624:8<1285: :AID- ZAAC1285>3.0.C0;2-5 Povzetek Sintetizirali smo nov nikljev kompleks s prisotnimi različnimi ligandi [Ni(L)2(EtOH)2(MeOH)2] (HL = 3-hidrok-si-2-metilkinolin-4-karboksilna kislina) s solvotermalno tehniko in določili kristalno strukturo z monokristalno rentgensko difrakcijo. Monokristalna rentgenska analiza je razkrila, da spojina kristalizira v triklinskem sistemu v prostorski skupini P-1 kot izoliran enojedrni kompleks. Intermolekularne vodikove vezi sodelujejo pri tvorbi verig, plastovita su-pramolekularna struktura pa nastane zaradi močnih n—n interakcij. Fotoluminiscentne lastnosti v trdnem stanju kažejo, da ima spojina emisijo v zelenem območju. Izračuni na podlagi časovno odvisne teorije gostotnostnih funkcionalov (TDDFT) kažejo, da fotoluminiscenca spojine izvira v prenosu naboja ligand-ligand (LLCT; iz HOMO p-orbitale ligan-da L v LUMO kisikovega atoma). Široka razlika med optičnima pasovoma 2.25 eV je bila ugotovljena z UV/vis difuzno reflektanco v trdnem stanju. Fang et al.: Preparation, Structure, Photoluminescent and Semiconductive ... DOI: 10.17344/acsi.2018.4887 Acta Chim. Slov. 2019, 66, 421-426 ^creative ^commons Scientific paper Synthesis and in vivo Anti-inflammatory Evaluation of Piperazine Derivatives Containing 1,4-Benzodioxan Moiety Zhi-Ping Liu,t'1 Chang-Da Gong,tv2 Long-Yan Xie,2 Xiu-Li Du,2 Yang Li2 and Jie Qin*,:2 1 School of Medical, Pingdingshan University, Pingdingshan 467000, P. R. China 2 School of Life Sciences, Shandong University of Technology, Zibo 255049, P. R. China * Corresponding author: E-mail: qinjietutu@163.com Tel.: 0086-533-2780271; Fax: 0086-533-2781329 t These authors contributed equally to this work. Received: 12-01-2018 Abstract Six piperazine derivatives 6a-f containing 1,4-benzodioxan moiety have been synthesized and characterized by 1H NMR, ESI-MS and elemental analysis. The structure of 6d was further confirmed by single crystal X-ray diffraction. All these novel compounds were screened for their in vivo anti-inflammatory activity employing classical para-xylene-induced mice ear-swelling model. The results revealed that most of the target compounds showed significant anti-inflammatory activities, especially compound 6a with ortho-substituted methoxy group on the phenylpiperazine ring exhibited the best activity among the designed compounds. Keywords: Piperazine; 1,4-benzodioxan; crystal structure; anti-inflammatory activity 1. Introduction Inflammation, a biological process response to harmful stimuli or infection, has threatened human health seriously.1-3 Nonsteroidal anti-inflammatory drugs (NSAIDs) like diclofenac, indomethacin and naproxen are widely clinically used drugs for inflammatory disorders such as arthritis, peritendinitis and lupus erythemato-sus.4-7 However, long-term use of NSAIDs has been asso- Figure 1. The structure of piperazine derivatives reported in previous literature sources. ciated with adverse side effects, including gastrointestinal ulceration, and even fatal internal bleeding.8 The stomach damage from NSAIDs is generally attributed to their acidic character as most of them are weak acids with pKa values ranging from 3 to 5.8,9 Therefore, synthetic approaches based on design of non-acidic anti-inflammatory lead compounds have gained great interest nowadays.1,10-13 Piperazine group, as a known pharmacophore, is part of many important alkaline heterocyclic compounds displaying a broad range of biological activities, e.g. antibacterial,14 anti-tumor,15 antidiabetic,16 and anti-psychia-try.17 Continuous efforts have also been made to prepare anti-inflammatory therapeutic agents containing piperazine scaffold. Li et al. reported a series of chalcone derivatives containing aryl-piperazine or aryl-sulfonyl-pipera-zine fragment and found compound A (Figure 1) as a potential anti-inflammatory agent.18 Silva et al. evaluated the anti-inflammatory effect of 4-[(1-phenyl-1H-pyrazol-4-yl) methyl]-1-piperazinecarboxylic acid ethyl ester (B, Figure 1) and the involvement of the serotonergic pathway.3 Mao Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation 422 Acta Chim. Slov. 2019, 66, 421-426 et al. synthesized a series of piperazine substituted 3-ar-yl-5-furanyldihydropyrazole amide derivatives (C, Figure 1), the in vitro anti-inflamatary activity evaluation indicated that theses compounds showed good inhibitory effect on the generation of inflammatory factor, NO.19 A series of piperazine analogues bearing pyridine or thiophene moieties have been synthesized by Kumar et al. (D, Figure 1), they also showed favorable anti-inflammatory efficacy.20 Beyond that, it was reported that 1,4-benzodioxan afforded a new skeleton possessing anti-inflammatory activity.10,21,22 Potent anti-inflammatory activity of analogues containing 1,4-benzodioxan ring may be ascribed to the strong hydrogen-bonding interactions with amino acids in the active domain.10 Scheme 1. Synthetic route to 6a-f. As mentioned above, it is a worthwhile goal to synthesize compounds containing piperazine and 1,4-benzo-dioxan rings utilizing molecular hybridization approach. The presence of the two bioactive skeltons in a single molecular frame may lead to potent derivatives possessing good anti-inflammatory activity.10 With this in mind, we have synthesized six compounds (6a-f) possessing piperazine and 1,4-benzodioxan moieties (Scheme 1). Spectral analyses using ESI mass spectrometry and 1H NMR spectroscopy have been applied in order to affirm the structure of the synthesized molecules. Furthermore, the synthesized molecules have been subjected to in vivo anti-inflammatory evaluation. 2. Experimental 2. 1. Physical Measurements and Materials Reagents and solvents used in this study were analytical grade and purchased commercially from Aladdin Industrial Corporation (China). 1H NMR spectra were measured on a Bruker AM 500 spectrometer. Mass spectra were determined with an Autoflex II TM instrument for ESI-MS. Elemental analyses were performed on a Per- R kin-Elmer model 2400 analyzer. The intermediate 5-(2,3-dihydrobenzo[fr][1,4]dioxin-7-yl)-1,3,4-oxadi-azole-2-thiol (4) was synthesized according to the literature method.23,24 2. 2. General Procedure for the Synthesis of Target Compounds 6a-f To a solution of a substitued phenylpiperazines (6.0 mmol) in CH2Cl2 (30 mL) containing triethylamine (1 O O HO. HO MeOH HO. OH HO" OCH, co^N2H4* i VU Q n-N KOH CS2 -O O O M n^-7 cicoch2ci -n - à. -n Ö. n-n VNqA^ o o och3 R. R2 R3 -O«* 6a CI 6b 6c Ri Rs ""Ö R6 6d 6e 6f Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation Acta Chim. Slov. 2019, 66, 421-426 423 mL), chloroacetyl chloride (0.48 mL, 6.0 mmol) was added dropwise with stirring in ice water bath. Stirring was continued for 5 h and the reaction mixture was washed with 10% NaOH solution (2 x 5 mL). The organic layer was collected. After drying with anhydrous Na2SO4, the solvent was removed in vacuo to yield the intermediate 5a-f. Oxadiazole derivative 4 (0.24 g, 1.0 mmol) and KOH (0.056 g, 1.0 mmol) dissolved in CH3OH (20 mL) was treated with chloro derivatives 5a-f (1.0 mmol). The mixture was stirred at room temperature about 4 h until the solid was precipitated. The solids 6a-f were filtered and purified by recrystallization from MeOH. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1-(4-(2-methoxyphenyl)piperazin-1-yl)ethanone (6a). White powder, 0.354 g, yield 75.5%, mp: 171.0-172.5 °C. 1H NMR (400 MHz, CDCl3) 5 3.08 (t, 2H, CH2), 3.13 (t, 2H, CH2), 3.78 (t, 2H, CH2), 3.86 (t, 2H, CH2), 3.89 (s, 2H, OCH3), 4.29-4.33 (m, 4H, OCH-2CH2O), 4.43 (s, 2H, SCH2), 6.89-7.08 (m, 5H, ArH), 7.49-7.52 (m, 2H, ArH). ESI-MS: 959.33 ([2M+Na]+). Anal. Calcd for C23H24N4O5S: C, 58.96; H, 5.16; N, 11.96. Found: C, 59.15; H, 5.13; N, 12.01%. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1-(4-(3-methoxyphenyl)piperazin-1-yl)ethanone (6b). White powder, 0.367 g, yield 78.3%, mp: 158.1-159.4 °C. 1H NMR (400 MHz, CDCl3) 5 3.16 (t, 2H, CH2), 3.29 (t, 2H, CH2), 3.81 (s, 5H, CH2, OCH3), 3.88 (t, 2H, CH2), 4.29-4.34 (m, 4H, OCH2CH2O), 4.39 (s, 2H, SCH2), 6.50-6.59 (m, 3H, ArH), 6.95 (d, 1H, ArH), 7.21 (t, 1H, ArH), 7.48-7.52 (m, 2H, ArH). ESI-MS: 491.33 ([M+Na]+). Anal.Calcd for C23H24N4O5S: C, 58.96; H, 5.16; N, 11.96. Found: C, 58.78; H, 5.14; N, 11.98%. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1-(4-(4-methoxyphenyl)piperazin-1-yl)ethanone (6c). White powder, 0.358 g, yield 76.4%, mp: 194.2-196.3 °C. 1H NMR (400MHz, CDCl3) 5 3.07(t, 2H, CH2), 3.12 (t, 2H, CH2), 3.75 (t, 2H, CH2), 3.78(s, 3H, OCH3), 3.82 (t, 2H, CH2), 4.29-4.34 (m, 4H, OCH2CH2O), 4.41 (s, 2H, SCH2), 6.84-6.96 (m, 5H, ArH), 7.48-7.52 (m, 2H, ArH). ESI-MS: 491.25 ([M+Na]+). Anal. Calcd for C23H24N4O5S: C, 58.96; H, 5.16; N, 11.96. Found: C, 59.20; H, 5.14; N, 11.99%. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1-(4-(2-chlorophenyl)piperazin-1-yl) ethanone (6d). Yellow crystals suitable for X-ray diffraction were obtained from evaporation of a solution of 6d in dichloromethane/methanol solution. Brown powder, 0.308 g, yield 65.2%, mp: 173.1-174.6 °C. 1H NMR (400 MHz, CDCl3) 5 3.13 (t, 2H, CH2), 3.15 (t, 2H, CH2), 3.81 (t, Hz, 2H, CH2), 3.84 (t, 2H, CH2), 4.31-4.35 (m, 4H, OCH2CH2O), 4.44 (s, 2H, SCH2), 6.97 (d, 2H, ArH), 7.047.06 (m, 2H, ArH), 7.41 (dd, 1H, ArH), 7.51-7.54 (m, 2H, ArH). ESI-MS: 495.17 ([M+Na]+). Anal. Calcd for C22H21ClN4O4S: C, 55.87; H, 4.48; N, 11.85. Found: C, 56.09; H, 4.46; N, 11.90%. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1-(4-(3-chlorophenyl)piperazin-1-yl) ethanone (6e). White powder, 0.376 g, yield 79.5%, mp: 191.8-193.1 °C. 1H NMR (400 MHz, CDCl3) 5 3.22 (t, 2H, CH2), 3.34 (s, 2H, CH2), 3.89 (s, 2H, CH2), 3.84 (s, 2H, CH2), 4.32-4.36 (m, 4H, OCH2CH2O), 4.40 (s, 2H, SCH2), 6.94-7.01 (m, 4H, ArH), 7.24 (t, 1H, ArH), 7.24 (m, 2H, ArH). ESI-MS: 495.25 ([M+Na]+). Anal. Calcd for C22H21ClN4O4S: C, 55.87; H, 4.48; N, 11.85. Found: C, 56.03; H, 4.45; N, 11.91%. 2-(5-(2,3-Dihydrobenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxa-diazol-2-ylthio)-1 -(4-(4- chlorophenyl)piperazin-1 -yl) ethanone (6f). White powder, 0.359 g, yield 76.1%, mp: 278.2-279.6 °C. 1H NMR data for 6f is unattainable because of its low solubility. ESI-MS: 495.25 ([2M+Na]+). Anal. Calcd for C22H21ClN4O4S: C, 55.87; H, 4.48; N, 11.85. Found: C, 56.05; H, 4.46; N, 11.90%. 2. 3. Determination of Crystal Structure for 6d Crystal diffraction data for compound 6d were collected on a Bruker SMART APEX CCD-based diffractom-eter (Mo Ka radiation, A = 0.71073 Â) at 298 K. Multi-scan absorption corrections were applied by SADABS.25 The structures were solved by the directed method followed by Table 1. Crystallographic data for 6d. 6d Empirical formula C22H21ClN4O4S Mr 472.94 crystsyst Monoclinic Space group P2l/c a (Ä) 18.2038(19) b (Ä) 8.0092(8) c (Ä) 15.4024(16) « (°) 90.00 ß (°) 107.112(3) Y (°) 90.00 V (Ä3) 2146.2(4) Z 4 Pc (g cm-3) 1.464 F(000) 984 T / K 298(2) |i(Mo-Ka)/ mm-1 0.314 Data / param. / restr. 5257 /289 / 0 GOF (F2) 1.037 R1a, bwR2 (I>2o(I)) 0.0634 / 0.1475 Large diff. peak / hole (e Ä-3) 0.514 / -0.482 a Ri = ZjjCj - jFcjj/ZFoj. b wR2 = = [Ew(F02 - Fc2)2/£w(F02)]1/2 Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation 424 Acta Chim. Slov. 2019, 66, 421-426 Fourier syntheses. Structure refinement was performed by full-matrix least-squares procedures using SHELXL-97 program package.26 Non-hydrogen atoms were refined using anisotropic thermal parameters. Hydrogen atoms were placed in their geometrically idealized positions and constrained to ride on their parent atoms. Details of crystallo-graphic parameters, data collection, and refinements are summarized in Table 1. Relevant bond distances and bond angles are given in Table 2. 2. 4. In vivo Anti-Inflammatory Activities in Ear Edema The animal studies were conducted according to the guideline issued by the State Food and Drug Administration (SFDA) of China. All experimental protocols were approved by the Animal Ethics Committee of Pingding-shan University. All efforts were made to minimize animal suffering. The anti-inflammatory activity was evaluated using in vivo para-xylene-induced mice ear-swelling model on Kunming mice,18 35-40 days old and weighing 18-22 g, supplied by Jinan Pengyue Experimental Animal Breeding Company. Kunming mice were divided into eight groups of eight each. Group I, the control, comprised of animals treated with vehicle (0.5% CMC, 10 mL/kg), and groups II-VIII were dosage groups. Group II was treated with standard reference drug diclofenac (20 mg/kg), groups III-VIII were treated with target compounds 6a-f (30 mg/kg), respectively. One hour later, ear swelling was induced by smearing 50 ^L para-xylene to each side of the right ears, the left ear served as a control. After 1 hour, the mice were sacrificed, and both ears at the same position were removed using a 6 mm diameter punch. Swelling degree and swelling inhibition were calculated according to the following formula: Swelling degree (mg) = weight of right ear (mg) -weight of left ear (mg) Swelling inhibition (%) = (average swelling degree of control group - average swelling degree of dosage group) / average swelling degree of control group x 100% 3. Results and Discussion 3. 1. Chemistry of Compounds 6a-f Appropriate 2-chloro- 1-(4-phenylpiperazin- 1-yl) ethanone derivatives 5 were reacted with 5-(2,3-dihyd-robenzo[fo][1,4]dioxin-7-yl)-1,3,4-oxadiazole-2-thiol 4 in CH3OH/KOH solution via nucleophilic substitution reaction to furnish the respective target compounds 6a-f (Scheme 1). The structures of 6a-f were established by elemental and 1H NMR and electrospray ionization mass spectrometry. In the 1H NMR spectra of 6a-e, the protons of the piperazin ring resonated as four triplets appearing at S 3.07-3.88 ppm. The multiplet corresponding to four protons characteristic of the -OCH2CH2- group was observed at S 4.29-4.35 ppm. The -SCH2- protons appeared as a sharp singlet around S 4.39 ppm. The protons of the substituted phenyl ring resulted in the formation of the resonance signal at S 6.94-7.54 ppm. The methoxy protons of 6a-c appeared as a singlet at 3.78-3.89 ppm. The positive ion electrospray mass spectra of these compounds showed full abundance of the parent peak which corresponds to [M+Na)]+ or [2M+Na)]+. 3. 2. Crystal Structure Table 2. Selected bond distances (Â) and angles (°) for 6d. C(1)-O(1) 1.439(3) C(1)-C(2) 1.477(4) C(2)-O(2) 1.431(3) C(6)-C(9) 1.453(3) C(9)-N(1) 1.280(3) C(9)-O(3) 1.364(3) N(1)-N(2) 1.417(3) C(10)-N(2) 1.286(3) C(10)-0(3) 1.353(3) C(10)-S(1) 1.734(3) C(11)-S(1) 1.805(3) C(12)-N(3) 1.348(3) 0(1)-C(1)- C(2) 111.0(2) O(2)-C(2)-C(1) 110.6(2) C(3)-0(1)- C(1) 114.1(2) C(8)-O(2)-C(2) 113.0(2) N(1)-C(9)- C(6) 130.3(2) N(2)-C(10)-S(1) 131.8(2) C(10)-S(1) -C(11) 96.82(12) N(3)-C(12)-C(11) 117.6(2) Yellow crystals of 6d suitable for X-ray structure analysis were obtained by slow evaporation of a mixture of dichloromethane and methanol. X-ray single-crystal diffraction reveals that compound 6d crystallizes in mono-clinic P21/c space group. As shown in Figure 2, 6d has a nonplanar chair-like conformation, the dihedral angle between 2-chlorobenzene ring and piperazine ring being 49.710(78)°; meanwhile the dihedral angle between 2-chlorobenzene ring and oxadiazole ring is 75.512(86)°. The piperazine ring in 6d has a classical chair conformation. 3. 3. In vivo Anti-Inflammatory Activities The anti-inflammatory activities of the synthesized piperazine derivatives 6a-f were tested using in vivo pa-ra-xylene-induced mice ear-swelling model. All the target compounds were adminstered at a dose of 30 mg/kg and diclofenac (20 mg/kg) was used as the reference drugs. The results show that compounds 6a, 6c, 6d, and 6f exhibited significant anti-inflamatory activities (p < 0.01) (Table 3). In the present assay, compound 6a bearing methoxy substituent at the ortho position of the phenyl piperazine showed the most effective anti-inflamatory activity; it was 1.3 times more active than diclofenac (inhibition values 68.02 vs. 52.03%). Preliminary structure-activity relationship analysis revealed that: (i) the substituent at different positions led to different activity, and the potency order was ortho > para > meta; (ii) in view of the comparison of Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation Acta Chim. Slov. 2019, 66, 421-426 425 the structures of compounds 6a with 6d, 6b with 6e, and 6c with 6f, the in vivo anti-inflammatory activities were 6a > 6d, 6b > 6e, and 6c > 6f. These results indicate that substitution at phenyl-piperazine with electron-donating methoxy group shows advantage over electron-withdrawing chlorine group at the same position, thus improving compound's anti-inflammatory activity. Meanwhile compared with the chalcone derivatives containing aryl-piper-azine reported earlier, most of the compounds in the present assay were found to display enhanced in vivo anti-inflammatory activity.18 Table 3. Anti-inflammatory activities of the synthesized compounds 6a-f. Compound Swelling degree (mg) Inhibition (%) 6a 4.34 ± 0.24a 68.02 6b 6.81 ± 0.54a 49.82 6c 5.83 ± 0.74a 57.04 6d 6.36 ± 0.45a 59.15 6e 10.95 ± 0.31a 19.31 6f 7.12 ± 1.03a 52.47 Diclofenac 6.51 ± 0.95a 52.03 Control 13.57 ± 1.09 — a Multiple comparison test p < 0.01 as compared to control 4. Conclusions tions led to different activity. Most of the synthetic compounds emerged as effective in vivo anti-inflammatory agents, demonstrating swelling inhibition even better than the reference control, diclofenac. Compound 6a containing methoxy substituent at the ortho position of the phenyl piperazine displayed the most potent anti-inflammatory activity among the synthesized compounds. Our findings might provide information on developing potentially new and safe anti-inflammatory agents. 5. Supplementary Material Crystallographic data (excluding structure factors) for the structural analysis have been deposited with the Cambridge Crystallographic Data Center as supplementary publication Nos. CCDC 1882249 (6d). Copies of the data can be obtained free of charge via www.ccdc.ac.uk/ conts/retrieving.html (or from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK, Fax: +44-1223336-033. E-mail: deposit@ccdc.cam.ac.uk). 6. Acknowledgment This work was supported by the National Natural Science Foundation of China (21301108). To sum up, a series of six piperazine derivatives 6a-f containing 1,4-benzodioxan moiety were synthesized and screened primarily for in vivo anti-inflammatory activities potential in ear edema. The substituent at different posi- 7. References 1. L. Marin-Ocampo, L. A. Veloza, R. Abonia, J. C. Sepulve-da-Arias, Eur. J. Med. Chem. 2019, 162, 435-447. Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation 426 Acta Chim. Slov. 2019, 66, 421-426 DOI:10.1016/j.ejmech.2018.11.027 2. O. Kolomoets, O. Voskoboynik, O. Antypenko, G. Berest, I. Nosulenko, V. Palchikov, O. Karpenko, S. Kovalenko, Acta Chim. Slov. 2017, 64, 902-910. 3. D. P. B. Silva, I. F. 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Zhu, M. H. Wang, Y. H. Ye, Bioorg. Med. Chem. 2013, 21, 6763-6770. DOI:10.1016/j.bmc.2013.08.002 24. X. M. Zhang, M. Qiu, J. Sun, Y. B. Zhang, Y. S. Yang, X. L. Wang, J. F. Tang, H. L. Zhu, Bioorg. Med. Chem. 2011, 19, 6518-6524. DOI:10.1016/j.bmc.2011.08.013 25. G. M. Sheldrick, SADABS. Program for Empirical Absorption Correction of Area Detector, University of Gottingen, Germany, 1996. 26. G. M. Sheldrick, Acta Crystallogr. 2008, A64, 112-122. DOI:10.1107/S0108767307043930 Povzetek Sintetizirali smo šest piperazinskih derivatov 6a-f, ki vsebujejo 1,4-benzodioksanski fragment, ter jih karakterizirali z 'H NMR, ESI-MS in elementno analizo. Strukturo produkta 6d smo dodatno potrdili z rentgensko difrakcijsko analizo monokristala. Za vse nove spojine smo s pomočjo klasičnega para-ksilenskega testa otekanja ušes miši in vivo določili protivnetno aktivnost. Rezultati kažejo, da večina preiskovanih spojin izkazuje opazno protivnetno aktivnost, še posebej spojina 6a, ki vsebuje orto-metoksi skupino na fenilpiperazinskem obroču, saj kaže najboljšo aktivnost izmed vseh pripravljenih spojin. Liu et al.: Synthesis and in vivo Anti-inflammatory Evaluation DOI: 10.17344/acsi.2019.4890 Acta Chim. Slov. 2019, 66, 427-434 ^creative ^commons Scientific paper Growth of Silver Nanoparticles Using Polythiocyanatohydroquinone in Aqueous Solution Valentina A. Litvin,1'* Boris F. Minaev,1,2 Rostislav L. Galagan,1 Glib V. Baryshnikov1,2 and Hans Ägren2 1 Department of Chemistry, Bohdan Khmelnitsky National University, Cherkassy, 18031, Ukraine 2 Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal Institute of Technology, Stockholm, 10691, Sweden. * Corresponding author: E-mail: litvin_valentina@ukr.net Received: 12-01-2018 Abstract Motivated by evidence that silver nanoparticles have found numerous technological applications we have explored in this work utilization of polythiocyanatohydroquinone as a new efficient reducing and stabilizing agent for the preparation of such nanoparticles. The formation of silver nanoparticles has been confirmed by the UV-Vis spectroscopy, X-ray powder diffraction and by transmission electron microscopy. The potentiometric and spectroscopy kinetic measurements during the nanoparticles growth are also presented. Thermodynamic activation parameters for the silver nanoparticle formation have been determined from the reaction kinetic studies at variable temperatures. On the ground of observations using these techniques, a mechanism for silver nanoparticle growth has been proposed. The narrow size (20-40 nm) and spherical shape distribution of the fabricated nanoparticles together with the high stability of colloids for sedimentation provide a firm basis for applications of the polythiocyanatohydroquinone polymer as a reducing and stabilizing material for the metal nanoparticles preparation and storage. Keywords: Silver nanoparticles; polythiocyanatohydroquinone; potentiometric study; spectrophotometric study; kinetics 1. Introduction Silver nanoparticles (AgNPs) are of special interest because of their unique physicochemical properties, including optical, magnetic and electronic characteristics, catalytic activity and biological impact,1-3 which make these particles applicable in a variety of technological areas in medicine, agriculture, environment, and industry.4,5 The AgNPs have also a high potential as commercial nano-materials in cosmetics and as effective antimicrobial agents.1,6 There are several methods proposed for fabrication of AgNPs, including chemical and physical approaches.7-9 Among them, the chemical reduction of silver ions in the presence of a protecting agent, is the most common and useful approach. Special attention is often devoted to the choice of reducing and capping agents since the physico-chemical properties of the AgNPs are strongly dependent on the incorporated capping agent species.10-12 Organic polymers are in this context of especial interest because they can perform a reduction function while simultane- ously being adsorbed on the nanoparticles surfaces thereby stabilizing them.13-16 In this report we present the first attempt to synthesize AgNPs by using polythiocyanatohydroquinone (PTHQ)17 as a reducing and stabilizing agent with the aim to form the small size and highly distributed AgNPs. We present here kinetic investigations in order to predict the mechanism of the Ag+-ions reduction by PTHQ. 2. Experimental 2. 1. Materials All reagents (analytical-grade) were used as received from Sigma-Aldrich. De-ionized water (18 MO/cm) from a Millipore Milli-Q water purification system was used to prepare all aqueous solutions. The synthesis of PTHQ has been described previously.17 Briefly, the PTHQ was synthesized by the three-stage synthesis. The 1,4-benzoqui-none crystal was dissolved in the glacial acetic acid (GAA) and treated with the NH4SCN salt in the GGA medium. Litvin et al.: Growth of Silver Nanoparticles Using ... 428 Acta Chim. Slov. 2019, 66, 427-434 The forming benzene-1,4-diol and thiocyanogen at the same conditions produce next the ortho-substituted 2-thiocyanatobenzene-1,4-diol which polymerizes rapidly into the target PTHQ compound. The orange-brown PTHQ polymer was precipitated by water, filtered, washed and dried upon 80 °C. 2. 1. Preparation of silver nanoparticles using PTHQ In a typical experiment, a 19.6 mg of PTHQ was disolved in 1.1 ml 1M solution of NaOH and the volume was adjusted to 60 mL with distilled water. The flask with resulting mixed solution was placed in a thermostat water bath at 30 °C. Next, 3.0 mL 0.2 M AgNO3 was added under vigorous stirring. The final concentrations in the mixture were 1.67 mM PTHQ, 18.3 mM NaOH, and 10 mM AgNO3. Characterization. UV-Visible spectra were recorded as a function of reaction time on Lambda 35 Perkin Elmer UV-Visible spectrophotometer in the range of 320-520 nm. Aliquots of silver colloid (0.2 mL) were diluted to 25 ml for the UV-Visible experiments. The X-ray powder diffraction (XRD) analyses were performed on a DRON-2 X-ray diffractometer (LOMO, Russia) with Fe Ka radiation (X = 1.9360 A) at a scanning speed of 0.01°/s over the 20 range of 20-120°. Transmission electron microscopy (TEM) was carried out by using a JEOL microscope (JEM-200A, Japan) with the accelerating voltage 200 kV. Samples for TEM investigations were prepared by evaporation of products on the surface of carbon films. The TEM images were registered with the CE video camera. The size of the particle can be calculated by using the scale provided in the micrograph. Potentiometric study the process of Ag-NPs formation was carried out on ionomer EV-74 with electrode system containing a glass electrode of type ESL-63-07 and a silver electrode of type EVL-1M3. As a reference electrode was used silver-oxide half-cell. The FT-IR spectra of the samples were recorded on Perkin-Elmer spectrometer (SpectrumGX) with a resolution of 2 cm-1 over a scan range 4000-500 cm-1 using KBr pellet method. Computational details. The structure of PTHQ active site in different oxidized forms has been optimized as oligomer at the density functional theory (DFT) level using the B3LYP18,19 hybrid functional and the 6-31G(d)20 basis set. The edge vacancies within -(C=N)- chain of the PTHQ polymer have been terminated by methyl groups in the oligomer model. We have also calculated the IR spectra for the studied species. All vibration frequencies were found to be real, which indicates the global minimum finding on the potential energy hypersurface. For the correct estimation of ionization potential (IP) values the po-larizable continuum model (PCM)21 with water as a solvent has been used in the optimization procedure. The final IP values have been evaluated using Koopmans approach as the energy of the highest occupied molecular orbital (HOMO) with the opposite sign (IP = - s(HO-MO)). All the calculations have been carried out using the Gaussian- 16 program package.22 3. Results and Discussion Stable aqueous colloids of AgNPs were prepared by chemical reduction of Ag+ ions using the PTHQ in the presence of NaOH. Due to the presences of phenolic group in the PTHQ structure17 the former reduces the Ag+ ions to Ag0 atoms and are then adsorbed on the surface of the growing AgNPs. In the alkaline medium the chemosorbed PTHQ species and their partially oxidized products possess a large negative charge due to ionization of the hy-droxyl and carboxyl groups. The repulsive forces prevent the aggregation of nanoparticles and provide stability of the colloidal system. The final colloids are stable and can be stored more the one year without aggregation. X-ray diffraction (XRD) results. The formation of the nano crystalline AgNPs has been confirmed by the XRD analysis (Figure 1). Strong peaks observed at 48.6°, 56.8°, 84.5°, 104.1° and 110.9°, corresponding to the (111), (200), (220), (311) and (222) Bragg's reflections that determine the face-centered-cubic (fcc) crystal structure of AgNPs. The broadening of the Bragg's peaks indicates the formation of Ag NPs. The XRD pattern thus shows that the Fig. 1. XRD patterns of silver nanoparticles synthesized with PTHQ usage Ag NPs formed by the reduction of Ag+ ions by PTHQ polymer are of the crystalline nature. The average size of the Ag nanoparticles has been calculated from the XRD line width using the Debye-Scherrer equation23 and was found to be equal 20.8 nm. Transmission electron microscopy (TEM) study. TEM was used to determine the size and shape of the nanoparticles. The TEM images of the PTHQ, prepared Litvin et al.: Growth of Silver Nanoparticles Using ... Acta Chim. Slov. 2019, 66, 427-434 429 Fig. 2. The TEM images of PTHQ (a), AgNPs (b) and the corresponding size distribution histograms of AgNPs stabilized by PTHQ (c). The borders of the PTHQ globule are shown by the dotted line in part (b). AgNPs are shown in the Fig. 2. TEM images show that they have spherical shape. A particle size distribution histogram determined from TEM is shown in Fig. 2c. The average AgNPs size is 19.8 nm. The particle sizes calculated from the XRD pattern have been found to vary within 5% of those obtained from TEM studies. The main difference of these hydrosols from similar nanosystems received with the use of other reducers, in particular the humic substances,15 is that the metal nanoparticles are fixed on the globule of the polymer. Study of the silver nanoparticles formation by UV-Vis spectroscopy. The most popular method applied for the measurements of the kinetics of nanocluster formation in solution is UV-Visible spectroscopy since the sharp surface plasmon resonance band with maximum at 400 nm is a distinctive feature of the silver colloids.24,25 Evolution of the absorption spectrum of the silver colloidal solution during reduction of the Ag+ ions by PTHQ at 30 °C is shown in Figure 3 a. For the early stages of clusters formation, the broad absorption band near 480 nm appeared which indicates the formation of small Ag particles on the surface of the large Ag2O particles. That position of this band absorption is the result in electronic interactions be- tween the two types of materials because Ag2O has semiconducting properties. Research by the authors26 has explained this fact by electron density transfer from the pure metal to the metal oxide nanoparticles when the small silver particles are formed on the surface of the n-type Ag2O semiconductor. At longer reaction times, the absorption band shifts continuously to shorter wavelengths. The increase of absorption maximum intensity over the time is connected with continuation of the Ag+ ions reduction process and with the increase of the number of the colloidal particles. The final surface plasmon resonance band has been observed at 400 nm (Figure 3a). To investigate the reaction kinetics of the AgNPs formation, the absorbance at 400 nm has been plotted against the reaction time (Figure 3b). The visible absorbance of the product can be observed immediately after the mixing of the reagents and rises rapidly at the beginning of the reaction. Then absorbance reaches a plateau, which indicates the reaction completion (Figure 3b). Proposed mechanism of silver nanoparticles formation. Evidently, at the initial stage of the Ag+ cations reaction with the OH- anions in the presence of PTHQ the formation of an Ag2O microphase occurs. However, the Fig. 3. The UV-Visible spectra of silver nanoparticles as a function of time (a); Absorbance at 400 nm versus time plot for the silver nanoparticles formation (b) Litvin et al.: Growth of Silver Nanoparticles Using 430 Acta Chim. Slov. 2019, 66, 427-434 detection of Ag2O microphase in similar systems by X-ray diffraction is extremely difficult, no mater how fast the separation of the precipitate from the solution is.27 Huang et al.26 have shown that it might be possible to capture some Ag2O if the reaction was performed at a very low temperature (-45 °C). The polymer chemisorbed on the Ag20 particles in the alkaline environment has a negative charge due to ionization of the phenolic group, something that provides stabilization of nanoparticles. In the system "Ag2O - organic reducer" few simultaneous processes can take place. The first of them is a dissolution of Ag2O with the subsequent dissociation of AgOH: Ag20 + H,0 = 2Ag0H AgOH <=> Ag1 +OH (1) (2) The equilibrium within the Ag2O - H2O system can be considered as a metallo-buffer for the Ag+ ions, and where the concentration of the latter shows a hyperbolic function of the OH- ion concentration. Thus, in water suspension of the Ag2O species under intense stirring condition, the Ag+ ion concentration is expected to be constant and equal to 1.39 • 10-4 M (the solubility of Ag2O is 1.95 • 10-8). At the same time, the other important process of the NPs growth includes interaction of PTHQ with Ag+ and OH- ions, which results in the metal atom formation being aggregated into the nanoparticles. The major reducing groups in the structure of the PTHQ polymer are the phenolic groups, which are oxidized to the quinone structures. Thus, in situ-generated Ag2O nanocrystallites act as heterogeneous nucleation centers. The reduction of Ag+ ions proceeds according to the following scheme: The reduction reaction will proceed in the system until the whole Ag2O microphase is dissolved or the reductive possibilities of the organic reducer will be exhausted. However, during the synthesis the molar ratio "Ag+: elementary link PTHQ" (r) did not exceed 6. If r > 6 a formation of a black deposit already in the course of the nanosystem synthesis was observed. Apparently, at excess of the ratio, the polymer will not have enough phenolic groups for delivery of a stabilizing charge to the complex particles [Ag@PTOH]*- according to Eq. 4 (Scheme 1). From this equation, it is clear, that the reductive ability of the aromatic core was practically exhausted, and that there is no possibility of formation of a sufficient amount of ionized hydroxyl groups for the maintenance of a negative charge of the complex. However, from the scheme 1 the possibility of a stable nanosystem formation at r = 8 follows, but the real ratio r is much lower because of the presence of the ditiocianhydroquinone bridges in the PTHQ structure. Scheme 1. Proposed mechanism for the reduction of Ag+ ions by PTHQ in alkaline media Litvin et al.: Growth of Silver Nanoparticles Using ... Acta Chim. Slov. 2019, 66, 427-434 431 deactivation activation Scheme 2. The reductive ability of PTHQ active site upon the gradual rising of silver cluster In order to explain how the reductive ability of PTHQ changes with the AgNPs growing we have performed the quantum-chemical estimations for ionization potentials of the PTHQ active site in different oxidation states. As can be seen from Scheme 2 the ionization potential of the PTHQ active site increases strongly (5.66 vs. 6.63 eV) right after the first stage when two hydroxyl groups transform into carbonyl groups. Actually, this stage provides the deactivation of PTHQ for the next reduction activity. However, assuming the possible next oxidation steps of the qui-none ring in Scheme 1 we have found that the corresponding ionization potential (IP) decreases gradually with the appearance of OH groups (6.63 eV for quinone ring vs. 6.23 eV for trihydroxyquinone moiety), i.e. the reductive ability of PTHQ increases with the addition of new OH groups. We consider these stages as the activation of PTHQ for reductive activity. Finally, the oxidation of trihydroxy-quinone ring into the tetra-carbonyl form provides a complete deactivation of PTHQ active site due to its final IP increase and the complete electronic exhaustion. Concluding shortly, we should note that these computational findings additionally approve the proposed mechanism (scheme 1) for the reduction of Ag+ ions by PTHQ in alkaline media. Moreover, the tendency for the IP changes upon PTHQ oxidation sequence is in a good agreement with the experimental kinetic measurements data for the silver cation reduction. Potentiometric study of the kinetics and mechan-sim of the silver nanoparticles formation. For real-time control of the Ag+ ion reduction process, we have used a potentiometric method, as ion-selective electrodes (ISE) responds continuously and instantaneously to the changes of the silver ion concentration.28,29 The potentiometric method for studying the chemical reaction kinetic is based on measurements and interpretation of the time dependence of the redox potential for the indicator electrode reversible to the particles participating in the studied reaction. A use of this dependence in the kinetic studies is only possible if the rate of the establishment of the electrode equilibrium is higher than the rate of introduction of all others equilibriums in the system with the potential-deter- mined particles. As the Ag+/Ag system is one of the most prompt redox systems we can use the potentiometric method in our studies. Since the signal output of the ISE corresponds to the logarithm of the concentration of the solute ions, such a direct potentiometric control of the depleted metal ions provides additional information on the nanoparticle formation, which is not accessible by other techniques. In our work, we have used a simple silver electrode instead of ISE since the studied system does not contain any perturbing ions. The silver electrode is particularly useful for gaining information about the kinetic processes at later stages of the nanoparticle growth, in which case the solute ions become dilute and the concentration changes are much more easily discerned compared to other methods. The potentiometric curves of pAg as a function of time for the process of the AgNPs formation using PTHQ at different temperatures are shown in Fig. 4. The figure indicates a three-step growth process (ab; bc and cd site). Let us consider the correspondence of different regions on the potentiometric curve (Fig. 4) to the kinetic stages of the process proposed in Scheme 1. The decrease of the pAg curve (ab site) indicates the balance displace- PAg 5 _j—,—,—,—,—|—i—i—i—i—|—i—i—,—i— 0 50 100 150 Time, min Fig. 4. The pAg potentiometric curves as a function of time for silver nanoparticles formation process at different temperatures Litvin et al.: Growth of Silver Nanoparticles Using 432 Acta Chim. Slov. 2019, 66, 427-434 ment in Ag2O dissociation as a result of a decrease in the concentration of the OH- ions which are also the reagents of the given reaction. As the reaction of silver ion reduction occurs on the surface of the Ag2O particles (heterogeneous process), the rate of reaction does not depend on the Ag2O concentration in solution. Thus, this stage is described by a zero-order kinetics in respect to Ag2O and the rate constant (k0) of the AgNPs formation at the initial stage can be found from the equation: k0 = dn(Ag2O)ldt, where n(Ag2O) - is an amount of the Ag2O microphase (mole); t - time of the Ag2O microphase existence in the system (s); k0 - zero-order rate constant (mole ■ s-1). At the point b of the pAg curve the whole Ag2O microphase is dissolved and begins the growth of pAg values according to the linear law that specifies the first order reaction with respect to Ag+-ion. The rate constant at this stage can be found from the following equation: k1 = F / RT ■ (dE/dt)^ OH-, [Ag0], where k1 - is the first-order rate constant (s-1), F - is the Faraday constant (9.65 ■ 104 coulombl mole); R - the universal gas constant (8.314 JK-1mol-1), T - temperature (K); (dE/dt)t, oh-, [Ag0]- the change of the equilibrium potential E of the Ag-electrode during the time t. The value of the derivative (dE/ dt)t, oh-, [Ag0] was determined from the measurements of the slopes of the linear parts of the pAg plots versus time (bc site, Fig. 5 A), taking into account that E = 2.303'(RT7F) • ApAg. At a point c the pAg curve passes through a maximum. Some decrease of the pAg values (cd site) corresponds to an increase in the concentration of the Ag+ ions due to oxidation of the AgNPs by air oxygen30,31 according to the equation: 4Ag0 + O2 + 4H+ - 4Ag+ + 2H2O. (5) The process described by Eq. (5) was controlled by N2 barbotation through the reaction solvent. In this case the pAg potentiometric curve after the point c in Fig. 4 does not go down. This is because the AgNPs are quite small and very reactive (the surface/volume ratio is great) in comparison with the massive silver metal. On the ground of potentiometric curves it is possible to make a conclusion that the formation mechanism of the AgNPs with the use of PTHQ is similar to those mechanisms which use the synthetic humic substances;30 in particular: 1) a fast formation of the Ag2O microphase in the system after mixing of reagents; 2) reduction of silver ions on the surface of the Ag2O particles according to the zero-order kinetics; 3) growth of AgNPs due to reduction of silver ions from solution on a surface of already generated Table 1. Dependence of the rate constants at various temperatures for different stages of silver nanoparticles formation using PTHQ Initial stage zero-order rate constants k0 • 103 (mole • s1) Later stage first-order rate constants kj • 103 (s-1) 293 K 0.25 303 K 0.60 313 K 0.92 323 K 1.20 293 K 1.62 303 K 3.83 313 K 5.64 323 K 7.50 1 IT, K1 Fig. 5. The Arrhenius (A) and Eyring (B) plots of the silver nanoparticles formation for the initial (•) and later (▲) stages using the PTHQ reagent Litvin et al.: Growth of Silver Nanoparticles Using ... Acta Chim. Slov. 2019, 66, 427-434 433 nuclei of AgNPs according to the first order kinetics after the Ag2O microphase dissolution. The temperature influence on the reaction rate was also studied. Calculated values of the zero-order rate constant (k0) for the initial stage of nanoparticles formation (before full dissolution of Ag2O) and the first-order rate constants (k1) at the later stages of the nanoparticle growth for different temperature are presented in Table 1. It is possible to observe that the reaction rate increases with temperature. No extra yield is obtained at higher temperature. The data suggest that the process can easily be promoted by the thermal activation mechanism. Since the reduction of Ag+ ions occurs at the surface of Ag0 nuclei in the contact place of metal and silver oxide solid phases, the reaction rate does not depend on Ag2O concentration in the solvent. Thus, the reaction rate constant of the Ag2O depletion for the Ag NPs production at this initial stage is determined by the zero kinetic order according to equation k0 = dn(Ag2O)/dt Finally, from the Arrhenius Equation (k = Ae-Ea/RT) and the Eyring Equation plots (Fig. 5), the activation parameters, e.g. the activation energy (Ea), the enthalpy (H#) and entropy (S#) of activation, were calculated. The estimated values of these parameters for different stage of silver nanoparticle formation with the use of PTHQ as reducing and stabilizing agent are presented in Table 2. The Ag2O suspension is the source of the Ag+ ions at the initial stage. The rise of Ag+ ion concentration in Fig. 4 (ab site), being determined by the decrease in concentration of the OH- ions working also as reagents in the reaction of Ag+ with PTHQ polymer, is an important prerequisite for the upcoming activation process. The latter includes reduction of the ions with the simultaneous growth of nanoparti-cles. The organic polymer is a reservoir of electrons for the reduction processes, which is rather complicated; the Ag+ ions at the first stage represent large rising concentration. The activation energy spent for electron transfer from PTHQ polymer (all averaged processes presented in Scheme 1) includes C-O and O-H bond cleavage with simultaneous ionization and subsequent electron attachment. The similar processes occur at the later stage but in the limit of a decreased Ag+ ion concentration; the reaction strongly depends on this concentration (first order reaction). That is why the activation energies are quite similar for both stages (Table 2). The small difference can be connected with the fact that electronic resources are exhausted at the late step of the process. 4. Conclusions A facile approach for the synthesis of stable aqueous colloids of silver nanoparticles (AgNPs) is described in the present work using the polythiocyanatohydroquinone (PTHQ) polymer as a reducing and stabilizing reagent in water. The AgNPs were characterized by different techniques such as UV-vis spectroscopy, X-ray diffraction and transmission electron microscopy. The TEM experiments indicate that these nanoparticles are formed with spherical shapes. The X-ray diffraction pattern shows a high purity and face centered cubic structure of the Ag-NPs. On the basis of various observations a three-step mechanism is proposed for the reduction of the silver ions by PTHQ: 1) a fast formation of the Ag2O microphase in the system after mixing of reagents; 2) a reduction of the silver ions on the surface of the Ag2O particles according to the zero-order kinetics; 3) a growth of AgNPs due to reduction of silver ions from a solution on the surface of already generated nuclear of AgNPs according to the first order kinetics after the Ag2O microphase dissolution. The kinetic constants at various temperatures and for different stages were calculated and thermodynamic activation parameters were determined by variable temperature kinetic studies. 5. Acknowledgements The quantum-chemical calculations were performed with computational resources provided by the High Performance Computing Center North (HPC2N) which is a Swedish national center for Scientific and Parallel Computing through the project "Multiphysics Modeling of Molecular Materials" SNIC 2016-34-43. This work was supported by the Ministry of Education and Science of Ukraine (Project No. 0117U003908). The authors declare that they have no conflict of interest. 6. References 1. Yu. Krutyakov, A. A. Kudrinskiy, A. Yu. Olenin, G. V. Lisich-kin, Russ. Chem. Rev., 2008, 77, 233-251. DOI: 10.1070/RC2008v077n03ABEH003751 2. A. Polman, H. A. Atwater, Mater. Today, 2005, 8, 56. D0I:10.1016/S1369-7021(04)00685-6 Table 2. Activation parameters for different stages of silver nanoparticles formation using PTHQ. 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Nanodelce srebra smo karakterizirali z UV-Vis spektroskopijo, rentgensko praškovno difrakcijo in presevno elektronsko mikroskopijo (TEM). Predstavljene so tudi potenciometrične in spektrofotometrične kinetične meritve rasti nanodelcev srebra. Termodinamične aktivacijske parametre za tvorbo nanodelcev srebra smo določili iz reakcijskih kinetičnih študij pri različnih temperaturah. Predlagali smo mehanizem za nastanek nanodelcev srebra. Velikost (20-40 nm) in sferična porazdelitev nanodelcev skupaj z visoko stabilnostjo koloidov nam dajeta trdno osnovo za morebitno uporabo polimera kot redukcijskega in stabilizacijskega regenta za pripravo in shranjevanje kovinskih nanodelcev. Litvin et al.: Growth of Silver Nanoparticles Using ... DOI: 10.17344/acsi.2018.4897 Acta Chim. Slov. 2019, 66, 435-442 ^creative ^commons Scientific paper On Topological Indices of OT [m, n] Octagonal Tillings and TiO2 Nanotubes Hafiz Usman Afzal1 and Tahzeeb Fatima2 1 Department of Mathematics, GC University Lahore-54000, Pakistan 2 Department of Biochemistry, University of Otago, New Zealand * Corresponding author: E-mail: huafzal@gmail.com (coprresponding author), tahzeebfatima@gmail.com Received: 12-07-2018 Abstract Some well defined connectivity topological indices are Randic index, atom-bond connectivity index, geometric-arithmetic index and Shigehalli & Kanabur indices, brought into light by M. Randic, Estrada et al, Vukicevic et al and V. S. Shige-halli, in their respective research articles. Topological indices preserve the symmetry of molecular structures and provide a mathematical formulation to predict their properties like boiling points, viscosity and the radius of gyrations,1 mainly their study gets a cover under the category of physical chemistry. Due to its mathematical nature, this idea has caught the attention of many chemists. It has also been reported that these indices are useful in the study of anti- inflammatory activities of certain chemical instances. In this paper, we shall calculate these topological indices of an infinite class of octagonal tilling structures OT [m, n], which is a molecular graph of a semiconductor allotrope consisting of octagons and rectangles, for all possible values of the parameters m and n. We shall also calculate Shigehalli & Kanabur indices of infinite structure of the titania TiO2 nanotubes. Keywords: Randic index; atom-bond connectivity index; octagonal tilling OT [m, n]; Shigehalli & Kanabur indices; TiO2 nanotubes 1. Introduction Mathematical chemistry is a branch of theoretical chemistry in which we discuss the chemical structures by using various mathematical techniques. Chemical graph theory is a branch of mathematical chemistry in which we apply techniques of graph theoretic ideas to form the chemical phenomenon mathematically. This theory plays an enigmatic part in different fields of chemical sciences. Primarily, a molecular graph is a simple graph in which vertices denote the atoms and edges denote the chemical bonding in the underlying chemical structure. Let G be a molecular graph with vertex set V(G) = {v1, v2,..., vn} and edge set E(G). The order and size of G are denoted by p = \V (G)| and q = |E(G)|, respectively. Where, the order is defined to be number of vertices in G and size is defined to be number of edges in G. Also, an edge in E(G) with end vertices u and v is denoted by uv. A topological index is a molecular graph invariant which correlates the physic-chemical properties of a molecular graph with a number.2 The first such topological index was introduced by a chemist, Harold Wiener, in 1947 to derive the boiling points of paraffins. This mathematical representation of a molecular graph has shown to be very useful quantity in quantitative structure- property relationship abbreviated as QSPR.3 It has also many applications in communication, networking, coding theory and cryptography that are effectively modeled using a connected graph G under certain condi-tions.4 This index was originally derived for tree alike structures to correlate specific physic- chemical properties of alkanes, alcohols, amines and their compounds. H. Hosoya,5 defined the notion of Wiener index for any graph G as; A. Ashrafi et al,6 calculated the PI, Szeged and edge Szeged indices of some nanostar dendrimers. Recently, authors also investigated m- order connectivity indices of nanostar dendrimers.7 The atom-bond connectivity index Afzal and Fatima: On Topological Indices of OT [m, n] 436 Acta Chim. Slov. 2019, 66, 435-442 and geometric-arithmetic index of nanostar dendrimers and some polyomino- chains were studied by S. Hayat et al.8 The atom-bond connectivity index and geometric-arithmetic index of some fullerenes were studied by M. Baca et al.9 Rostami et al. studied the first kind of geometric- arithmetic index of some nanostar dendrimers.10 Ghorbani et al. 11 did their study on the nullity of an infinite class of nanostar dendrimers. The elemental two-dimensional 2D materials such as graphene, silicene, germanene, and black phosphorus have pulled considerable attention due to their fascinating physic- chemical attributes. Structurally, they possess the honeycomb, distorted honeycomb and continuous honeycomb lattices, which are composed of six atom rings. Recently,12 P. Li and W. Luo have studied a new structure of 2D allotropes of group V elements composed (a) Top View (b) Birds View (courtesy 12) Figure 1. General formation of the molecular octagonal tilling structure OT[m, n]. of eight-atom rings, which they termed as octagonal tilling structure, denoted generally by OT. These kinds of allo-tropes are comprehensively studied in materials sciences. Their findings indicated that these allotropes are dynamically stable and are also thermally stable at temperatures up to 600 K. They also showed, these allotropes are semiconductors with band gaps ranging from 0.3 to 2.0 eV, thus, they are potentially useful in near and mid- infrared devices. The molecular graph of these octagonal tilling structure, OT[m, n], is presented in Figure 1, in which, m denotes the number of octagons in an alternate row and n denotes the number of octagons in an alternate column of OT[m, n]. 2. The Randic, ABC and GA Indices of Molecular Octagonal Tilling Structure OT[m, n] Let H be a simple connected graph with vertex set V(H) and edge set E(H). The degree dv of a vertex v e V(H) is the number of edges incident on v and gu= ^d„ vcV(H) M. Randic defined the Randic index as follows,13 I X(H)= X m>eE(H) -Jdudv (1) E. Estrada et al,14 defined the atom-bond connectivity index, abbreviated as ABC-index, as: ABC(H)= X ave E(H) d„ + dv—2 d„dv (2) Another well used connectivity topological descriptor for the molecular graphs is geometric-arithmetic index (GA-index), introduced by Vukicevic and Furtula and is defined by,14 GA(H)= Y 2 4 d„d. du + dr (3) With each edge uv, we associate a pair (du, dv). The edge partition of octagonal tilling OT[m, n] with respect to the degrees of the end-vertices of edges is presented in Table 1. Table 1. The (du , dv )- type edge partition of octagonal tilling OT [m, n]. (du, dv)-Partition Edge Cardinality (2, 2) 2(m + n + 2) (2, 3) 4(m + n - 2) (3, 3) 4(3mn - 2m - 2n + 1) Afzal and Fatima: On Topological Indices of OT [m, n] ... Acta Chim. Slov. 2019, 66, 435-442 437 Theorem 1. For all m and n, the Randic index x of the octagonal tilling structure OT[m, n] is Proof. The (du, dv)-type edge partition of the graph OT[m, n] is shown in Table 1. We prove the desired result by using partition of Table 1 and the formula of Randic index given by Equation 1 as follows. = (m + n + 2) + (m + n - 2) + (3mn - 2m - 2n +1) — = m( 1 + 2V2 8 2V2 3 V3 8, „ 4V2 4, —) + 4 mn + (2--=r- + —) 3 V3 3 2V6 — 5 2V6-5 , 10 — 4-\/6. - -) + «(-) + 4/ww + (-) = Amn + ( 2V6-5 )(m + n — 2) _^ => /»,«]) = 4m« + (---)(m + n-2) Example 1. Consider a 2D structure of allotrope we are discussing called octagonal tilling OT [7, 8] consisting of 448 atoms and 642 chemical bonds, we obtain, ^(0717,8]) = 223.5632. Example 2. The Randic index of 2D allotrope, octagonal tilling OT [m, n], for m = 1, 2, . . ., 5 and n = 1, 2, . . ., 10 are given as follows OT[m, n] X(OT[m, n]) OT[1, 1] 4 OT[1, 2] 7.9664 OT[1, 3] 11.9328 OT[1, 4] 15.8992 OT[1, 5] 19.8656 OT[1, 6] 23.832 OT[1, 7] 27.7984 OT[1, 8] 31.7648 OT[1, 9] 35.7312 OT[1, 10] 39.6976 OT[2, 1] 7.9664 OT[2, 2] 15.9328 OT[2, 3] 23.8992 OT[2, 4] 31.8656 OT[2, 5] 39.832 OT[2, 6] 47.7984 OT[2, 7] 55.7648 OT[2, 8] 63.7312 OT[2, 9] 71.6976 OT[m, n] X(OT[m, n]) OT[2, 10] 79.664 OT[3, 1] 11.9328 OT[3, 2] 23.8992 OT[3, 3] 35.8656 OT[3, 4] 47.832 OT[3, 5] 59.7984 OT[3, 6] 71.7648 OT[3, 7] 83.7312 OT[3, 8] 95.6976 OT[3, 9] 107.664 OT[3, 10] 119.6304 OT[4, 1] 15.8992 OT[4, 2] 31.8656 OT[4, 3] 47.832 OT[4, 4] 63.7984 OT[4, 5] 79.7648 OT[4, 6] 95.7312 OT[4, 7] 111.6976 OT[4, 8] 127.664 OT[4, 9] 143.6304 OT[4, 10] 159.5968 OT[5, 1] 19.8656 OT[5, 2] 39.832 OT[5, 3] 59.7984 OT[5, 4] 79.7648 OT[5, 5] 99.7312 OT[5, 6] 119.6976 OT[5, 7] 139.664 OT[5, 8] 159.6304 OT[5, 9] 179.5968 OT[5, 10] 199.5632 Theorem 2. For all m and n, the atom-bond connectivity index of the octagonal tilling structure OT[m, n] is o _ o [j o [j _ 1 9 ABC(OT[m,»]) = 8 mn + (-¡¿-)(2 m + 2 n) + ( ). 3V2 3>/2 Proof. The (du, dv)-type edge partition of the graph OT[m, n] is shown in Table 1. We prove the desired result by using partition of Table 1 and the formula of atom bond connectivity index given by Equation 2 as: ABC(OT\m,n]) = 2(m + n + ^ + 4('n vn 2) i 1 4 = 2max values for adsorption of CR on Mg-Al-LDH nano-compound in section IIB (on 003 site) in water, 0.1 M NaCl and alkaline solutions at 308-328 K. Solvent T Temkin Langmuir c qe, max (K) ci c2 R2 qmon K R2 (mM) (mg g-1) Water 308 47.8 1.27 x 105 0.98 307.0 8.36 x 103 0.97 6.79 x 10- 2 100.0 318 37.6 2.77 x 105 0.99 153.0 3.60 x 104 0.99 5.66 x 10- 2 105.0 328 29.7 4.24 x 105 0.97 121.8 5.47 x 104 0.96 5.27 x 10- 2 86.8 0.1 M NaCl 318 36.3 4.18 x 105 0.99 158.0 4.76 x 104 0.99 5.84 x 10- 2 116.0 pH = 10 318 34.0 2.00 x 105 0.99 200.0 1.49 x 104 0.97 6.07 x 10- 2 81.6 pH = 11 318 27.3 1.23 x 105 0.99 120.5 1.37 x 104 0.99 8.71 x 10- 2 63.8 pH = 12 318 17.4 4.07 x 105 0.98 75.2 4.80 x 104 0.97 7.83 x 10- 2 60.4 pH = 13 318 19.2 2.67 x 105 0.97 76.9 3.76 x 104 0.98 5.17 x 10- 2 51.1 Dimension of qmon and Cj is in mg g '. Dimension of c2 and K is in M '. was clear that CR molecules orientation in section IIB was parallel to 003 layers, Fig. 8, and due to the negatively charged adsorbent surface, the adsorption interaction was weaker than that in section IIA. Also, it was seen from IR spectra in Figs. 2(a) and 2(b) that due to the interaction of CR molecule with adsorbent, peaks of -OH group and Al-O stretching mode of Mg-Al-LDH at 3532.9 and 617.1 cm-1 shifted to 3509.8 and 647.9 cm-1 at CR-adsorbed Mg-Al-LDH. Here, the interaction of CR molecule with -OH group of adsorbent decreased its wavenumber and by shifting electron density of O-H bond toward the oxygen atom increased wavenumber of Al-O stretching mode. Besides, as seen from Table S1, a large decrease in the rela- , r , max ~SSCB , , , tive magnitude of section IIB ( ) resulted in a disorder change of experimental maximum adsorption capacity (qe,max) with an increase in temperature, Table 3. 3. 2. Effects of pH and Ionic Strength on the Adsorption of CR on Mg-Al-LDH As shown in Tables 1-3, the increase in ionic strength in alkaline and 0.1 M NaCl solutions had different effects on the adsorption capacity of adsorbent and adsorption binding constants of adsorbate with MP (in region I and section IIA) and 003 (in section IIB) adsorption sites. The ionic atmosphere of Na+ ions surrounded interlayer carbonate ions of Mg-Al-LDH55 and CR molecules. The shielding effect of Na+ ions decreased the repulsion interaction between CR molecules and Mg-Al-LDH surface and consequently increased the adsorption binding constant values in region I and section IIA compared to those values in water at 318 K. But, the increase in pH of solutions and neutralization of more -OH groups of adsorbent increased its negative charge and finally resulted in a decrease in adsorption binding constant of the process at pH = 13 compared to that of neutral water at 318 K. Also, adsorption of CR molecules on the Mg-Al-LDH surface increased negative charge of adsorbent surface and finally decreased adsorption binding constant of CR on Mg-Al-LDH from region I to section IIB, Tables 1-3. On the other hand, the adsorption capacity of Mg-Al-LDH for CR molecules in region I, section IIA and section IIB and thus qe,max decreased with increase in pH of solution compared to those in water, Tables 1-3. As reported,55 the point of zero charge (pzc) of Mg-Al-LDH, i.e. the pH at which the surface charge is neutral, was at pH = 10 and the zeta potential of Mg-Al-LDH decreases with increase in pHs. Due to interaction of OH- ions Beyranvand et al.: Adsorption Mechanism of Congo Red on Mg-Al-layered ... Acta Chim. Slov. 2019, 66, 443-454 451 with a number of interlayer -OH groups of Mg-Al-LDH, Figs. 2(a), (c) and (d), -OH and the waveumbers of Al-O stretching modes of Mg-Al-LDH at 3532.9 and 617.1 cm-1 shifted to 3502.1 and 655.7 cm-1 in Mg-Al-LDH at pH = 10 solution and to 3478.9 and 655.7 cm-1 in Mg-Al-LDH at pH = 13 solution. It implied that in alkaline solution interaction of OH- ions with hydrogen atom of -OH groups of adsorbent surface decreased -OH wavenumber and by shifting electron density of O-H bond to its O atom caused an increase in wavenumber of Al-O vibration stretching mode. These evidence showed that an increase in pH value neutralized more -OH groups of adsorbent. In addition, there was a competition of OH- ions with CR molecules for interaction with adsorption sites which increased the repulsion interaction between Mg-Al-LDH surface and CR molecules and resulted in a decrease in the adsorption capacity of adsorbent. By comparison of results of measurement of the nitrogen-based BET specific surface areas of Mg-Al-LDH and its sample after being in a solution at pH = 13, it seems that the hydrogen bonding between -OH and -O- groups of adsorbent caused its structure shrinkage. As it is clear from Tables 1-3, a decrease in BET surface area and pore volume of Mg-Al-LDH in alkaline solutions could also result in a decrease in the adsorption capacity of adsorbent for CR molecules in alkaline solutions and also the random change in their adsorption binding constants, Tables 1-3. Because of dissolution of adsorbent and precipitation of congo red in acidic solutions, experiments didn't carry out in acidic pHs. 3. 3. Adsorption Kinetic Equations and Modeling The adsorption kinetic experiments were carried out in CR initial concentrations of 0.01, 0.06 and 0.09 mM, shaking rates of 30, 70 and 100 rpm, 0.1 M NaCl and alka- Table 4. TDs and TRAKs for adsorption of CR on Mg-Al-LDH nanocompound in water, 0.1 M NaCl and alkaline solutions at 308-328 K. Solvent T [CR]o rpm TD TRAXcrt TRAKc2C3 te qe (K) (mM) (min) (min) (min) (min) (mg g-1) Water 318 0.01 100 - - - 150 13.9 308 0.06 100 - 3-20 - 180 48.0 318 0.06 100 - 3-20 - 150 49.2 318 0.09 100 - 2-5 - 150 74.2 318 0.06 70 - 3-5 - 180 57.0 318 0.06 30 0-0.5 2-5 - 180 54.4 328 0.06 100 - 2-3 - 30 54.3 0.1M NaCl 318 0.06 100 - 1-3 90-120 210 60.1 pH = 10 318 0.06 100 - 0.75-5 30-50 180 41.5 pH = 12 318 0.06 100 0-2 10-40 - 120 30.0 pH = 13 318 0.06 100 0-5 20-50 - 240 26.0 TRAKc1-C2 and TRAKC C2 C are : 3 the observed TRAKs between the first and second kinetic < curves and between the second and third kinetic curves, respectively. te and qe are the time and adsorption capacity of starting plateau and reaching equilibrium. Beyranvand et al.: Adsorption Mechanism of Congo Red on Mg-Al-layered ... line (pHs of 10, 12 and 13) solutions at 308, 318 and 328 K. The adsorption of 0.01 mM CR initial concentration was in region II (section IIA) of the ARIAN thermodynamic model and included just MP adsorption sites. However, the adsorption of 0.06 and 0.09 mM CR initial concentration were in the region II (section IIB) of this model and included both MP and 003 sites. The results were analyzed by the KASRA model and ISO and intraparticle diffusion Figure 9. qt vs. ct for adsorption of CR on Mg-Al-LDH nanocompound (a) from CR solutions at different temperatures, shaking rates and initial concentrations and (b) from 0.06 mM CR in water, 0.1 M NaCl and alkaline solutions at 318 K and 100 rpm. 452 Acta Chim. Slov. 2019, 66, 443-454 equations, Figs. 9(a) and (b), Tables 4 and 5 and S2-S4. As shown in Table 4, there was a TD at the beginning of adsorption in 0.06 mM of CR at 30 rpm and 0.06 mM of CR at 100 rpm and pHs of 12 and 13. The observed TD at 30 rpm was due to the low rate of CR diffusion to the surface of adsorbent and the TDs at pHs of 12 and 13 were due to negatively-charged surface of adsorbent which was generated by the neutralization of a number of -OH groups of adsorbent. In this study, TRAKc1-c2 showed the time delay range between adsorption on MP and 003 sites and TRAKC2_C3 identified the time delay range between two steps of adsorption on 003 sites. These TRAKs happened because of repulsion interaction between negatively charged CR-adsorbed Mg-Al-LDH with CR molecules in the solution. The adsorption of 0.01 mM of CR initial concentration was in section IIA (based on the ARIAN thermodynamic model) and CR molecules were adsorbed on MP site. The analysis of kinetic data of 0.01 mM of CR initial concentration by the KASRA model showed that it was placed in regions 1 and 2 of its kinetic curves. But, the adsorptions using the initial concentrations of 0.06 and 0.09 mM CR were in section IIB (based on the ARIAN thermodynamic model) and CR molecules were adsorbed on MP (in region I and section IIA) and 003 sites (in section IIB). The kinetic study of both concentrations (0.06 and 0.09 mM) by the KASRA model showed that they were in region1 (adsorbed on MP sites) and region 2 (adsorbed on 003 sites), Tables 5 and S2. An increase in ionic strength in 0.1 M of NaCl and pH = 10 resulted in observing the third kinetic curves in these cases. Also the analysis of adsorption results of CR on MP sites of Mg-Al-LDH by the KASRA model and intraparti-cle diffusion equation showed that adsorption acceleration, velocity and kdif increased with the increase of experimental variables, such as initial concentration of CR, shaking rate, ionic strength and temperature. An opposite trend was observed to those kinetic parameters, when pH of solution was increased. This confirmed that adsorption kinetics on MP site was reaction-controlled, Tables 5 and S3. But, the adsorption acceleration and the velocity of adsorption of CR on 003 sites were approximately constant with increase in above-mentioned factors. The results verified that the adsorption kinetics was diffusion-controlled, Tables S2 and 5. As the CR concentration in the solution dropped during the course of adsorption, the adsorption acceleration, velocity and kdif values decreased from region 1 to region 2, Tables S2, S3 and 5. As seen in Table 5, in 0.1 M NaCl and pH of 10 solutions, adsorption was continued after the second TRAK and formed the third kinetic curves. This occurred because Na+ ionic atmosphere surrounding adsorbent surface and CR molecules decreased the repulsion interaction between them. As shown in Table 5, kdif values of these two processes were similar to kdif values of other adsorption processes in the second kinetic curves. Thus, in the third kinetic curve, the adsorption kinetics of CR molecules towards 003 sites was diffusion-controlled. On the other hand, a detailed study of kinetic data by the ISO equation showed that kI1 values for the adsorption of CR on MP sites (similar to trend of changes in the adsorption acceleration, velocity and kdif parameters) in- Table 5. Adsorption acceleration values, intraparticle diffusion constants and ISO rate constants for kinetics of CR adsorption on Mg-Al-LDH na-nocompound at different temperatures and in various shaking rates and initial CR concentrations. Solvent T [Cfi]0 (K) (mM) rpm KASRA region 1 (1st curve) KASRA region 2 (1st curve) ai kdif kii a2 kdif kI2a kI2b a3 kdif kI2b Corresponding to thermodynamic ARIAN region I (MP Site) Water 318 0.01 100 -0.45 1.85 6.34 x 104 ARIAN section IIA (MP Site) -0.001 1.0 3.05 x 104 6.81 x 104 - - - Corresponding to thermodynamic KASRA region 1 (1st curve) ARIAN region I and section II A (MP Site) KASRA region 2 (2nd curve) KASRA region 2 (3rd curve) ARIAN section IIB (003 Site) ARIAN section IIB (003 Site) Water 308 0.06 100 -3.54 4.36 1.75 x 105 -0.002 4.2 1.57 x 104 2.35 x 104 - - - 318 0.06 100 -5.70 15.5 5.01 x 105 -0.002 2.8 2.00 x 104 4.76 x 104 - - - 318 0.09 100 -20.40 21.7 1.22 x 106 -0.003 4.5 1.64 x 104 5.97 x 104 - - - 318 0.06 70 -3.52 11.4 3.18 x 105 -0.002 3.3 1.64 x 104 3.72 x 104 - - - 318* 0.06 30 -1.52 20.9 2.15 x 105 -0.002 3.8 1.13 x 104 3.69 x 104 - - - 328 0.06 100 -14.66 25.9 7.37 x 105 -0.05 4.5 1.13 x 105 6.33 x 105 - - - 0.1M NaCl 318 0.06 100 -11.69 8.6 3.65 x 105 -0.008 4.3 2.72 x 104 - -5.4 x 10- 4 4.9 2.93 x 104 pH = 10 318 0.06 100 -29.94 7.5 5.26 x 105 -0.034 2.8 5.33 x 104 - -0.004 4.7 2.16 x 104 pH = 12* 318 0.06 100 -0.25 8.0 5.67 x 104 -0.002 3.2 4.90 x 103 1.59 x 104 - - - pH = 13* 318 0.06 100 -0.06 2.8 1.77 x 104 -3.0 x 10-4 2.3 5.22 x 103 2.51 x 104 - - - Units of a1, a2 and a3 are in mg g 1 min CR, there is a TRAK between each two . Unit of kdf is in mg g 1 min 0 5. Units of kn , kI2a and kI2b are in mg g 1 M 1 min 1. Except for 0.01 mM of successive kinetic curves (Table 4). *There is a TD (time delay) before the first curve (Table 4). Beyranvand et al.: Adsorption Mechanism of Congo Red on Mg-Al-layered ... Acta Chim. Slov. 2019, 66, 443-454 453 creased with increase in initial concentration of CR, shaking rate, ionic strength and temperature and decreased with the increase in solution's pH values, Tables S4 and 5. An increase in pH increased the negative charge of adsorbent surface and thus increased the repulsion interaction between CR molecules and adsorbent. This consequently decreased kinetic parameters values. Results showed that kinetics of adsorption of CR on MP site was reaction-controlled51 and its Eact by using kI1 values at 308-328 K was 60.7 kJ mol-1. For the adsorption of CR on 003 sites, kI2a values (in part 2a), except at 328 K and pHs of 12 and 13, were similar to each other and kI2a values in the range of 308-328 K did not obey the Arrhenius equation. This showed that in part 2a the diffusion of CR molecules towards adsorbent surface was rate-controlling step, Table 5, which was similar with the adsorption of CR on GO/PAMAM.49 A decrease in kI2a values with the increase in pH was due to an increase in the repulsion interaction between the adsorbent surface and adsorbed CR molecules. Also, kI2b values did not obey from Arrhenius equation which showed that in part 2b (like part 2a) kinetics of CR adsorption was diffu-sion-controlled,49 Table 5. 4. Conclusions Adsorption of congo red (CR) on Mg-Al-LDH was carried out under different CR initial concentrations, alkaline pHs, temperatures, ionic strengths and shaking rates. Based on adsorbent structure and IR spectra it was clear that adsorption site of adsorbent were -OH groups attached to Al atoms of adsorbent layers. Analysis of adsorption isotherms by the ARIAN model showed that there were two types of adsorption sites on the adsorbent. These adsorption sites were -OH groups attached to Al atoms located in the surface of mesopores (named as MP sites) and micropores located in interlayer galleries between (003) layers (named as 003 sites) of adsorbent. Based on the ARIAN model, MP sites were in region I and section IIA and 003 sites were in section IIB. Adsorption in region I and sections IIA and IIB were endothermic, exothermic and endothermic, respectively. Kinetic data were analyzed by the KASRA model and ISO and intraparticle diffusion equations. It was verified that CR adsorption at first occurred on MP sites and its adsorption kinetics was reaction-controlled. 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DOI:10.1515/9781400853335 50. M. Rafi, B. Samiey and C.-H. Cheng, Materials, 2018, 11, 496. DOI: 10.3390/ma11040496 51. M. Ozacar, I.A. §engil, Colloids Surf. A, 2004, 242, 105-113. 52. B. Samiey and S. Farhadi, Acta Chim. Slov., 2013, 60, 763-773. 53. S. Nam, R. Slopek, B. D. Condon and P. Sawhney, TRJ, 2015, 85, 1221-1233. DOI:10.1177/0040517514561918 54. B. Samiey and A. Dadkhah Tehrani, JCCS, 2015, 62, 149-162. DOI: 10.1002/jccs.201400093 55. Q. Wang, Y. Gao, J. Luo, Z. Zhong, A. Borgna, Z. Guo and D. O'Hare, RSC Advances, 2013, 3, 3414-3420. DOI: 10.1039/c2ra22607c Povzetek V delu je opisana uporaba plastovitih Mg-Al- dvojnih hidroksidov (Mg-Al-LDH) z interkaliranim karbonatom kot ad-sorbentom za odstranjevanje kongo rdečega (CR). Šaržni adsorpcijski eksperimenti so bili izvedeni pri različnih temperaturah, ionski moči, začetnih koncentracijah CR, alkalnosti in stresanju. Maksimalne adsorpcijske kapacitete Mg-Al-LDH za CR so znašale 100 mg g-1 pri temperaturi 308 K, 105 pri 310 K in 86.6 pri 328 K. Adsorpcijska mesta na katere se je CR vezal so bile -OH skupine vezane na Al atome. Adsorpcijske izoterme so bile analizirane z modelom ARIAN. Izkazalo se je, da obstaja dva tipa adsorpcijskih mest na površini Mg-Al-LDH. Rezultati instrumentalne analize so pokazali, da gre v obeh primerih za -OH skupine, enkrat prisotne v mezoporah, poimenovane MP, in enkrat v mikroporah, poimenovane 003. Rezultati kinetičnih meritev so bili analizirani z modelom KASRA, ISO ter enačbami, ki opisujejo difuzijo v porah. Analiza je pokazala, da se CR molekule najprej vežejo na MP mesta, kjer predstavlja interakcija s površino limitni korak. Po drugi strani pa se je izkazalo, da je adsorpcija na 003 mesta difuzijsko omejena. Beyranvand et al.: Adsorption Mechanism of Congo Red on Mg-Al-layered ... DOI: 10.17344/acsi.2019.4933 Acta Chim. Slov. 2019, 66, 455-465 ^creative ^commons Scientific paper Preparation of Magnetite by Thermally Induced Decomposition of Ferrous Oxalate Dihydrate in the Combined Atmosphere Josef Kopp,1 Petr Novak,1^ Josef Kaslik2 and Jiri Pechousek1 1 Department of Experimental Physics, Faculty of Science, Palacky University, 17. listopadu 1192/12, 77146 Olomouc, Czech Republic 2 Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelü 27, 783 71 Olomouc, Czech Republic * Corresponding author: E-mail: petr.novak@upol.cz Received: 01-04-2019 Abstract This study presents an investigation of thermal decomposition of ferrous oxalate dihydrate in the combined atmosphere of inert and conversion gases to find an optimal route for a simple magnetite preparation. Homogenized precursor was isothermally treated inside the stainless-steel cells at 8 equidistant temperatures ranging from 300 to 650 °C for 1, 6, and 12 hours. The enclosure of samples inside the cells with the combined atmosphere eliminates the necessity of the inert gas to flow over the treated samples. Structural, magnetic, and morphological aspects of the prepared materials were examined by the combination of experimental techniques, such as Mossbauer spectroscopy, X-ray powder diffraction, and scanning electron microscopy. Keywords: Mossbauer spectroscopy; XRD; maghemite/magnetite mixture. 1.Introduction Magnetite is a form of an iron oxide, which finds numerous applications in the fields of materials science, information storage, medicine1,2 or catalysis (e.g. hydrogenation of CO2,3 and water gas shift reaction4). Since the boom of nanotechnology, the synthesis of magnetite in the form of nanoparticles have been discussed in many studies.5-11 Among the methods reported the thermally induced solid state synthesis represents a facile and cost effective method for preparation of iron oxides in the size of nanometres.12,13 Ferrous oxalate dihydrate is considered being a versatile precursor for iron oxides preparation. It is a metal organic mineral whose thermal decomposition leads to various forms of oxide, both pure or mixtures.14 The individual transformation routes of FeC2O4 • 2H2O are influenced by many experimental conditions, but the composition of reaction atmosphere, temperature and time of calcination process are among the most important ones. For example, Zhou et al.15 reported the use of different reaction atmospheres to prepare rod-like particles composed of different forms of iron oxides. The thermal decomposition of ferrous oxalate dihydrate in oxygen rich atmosphere (e.g., in air) has been reported in many studies.16-24 If the FeC2O4 • 2H2O is decomposed in air, the reaction is straight-forward and proceeds in two consecutive steps. It was shown that these two steps (endo + exothermal) partially overlap in differential thermal analysis (DTA) in the range up to 450 °C.19 Firstly, approximately at 180 °C the water evaporates from the crystals; and then the anhydrous ferrous oxalate decomposes to iron oxides. At the temperature of 210 °C the amorphous ferric oxide is formed.25,26 With increased temperature and prolonged calcination, the amorphous ferric oxide transforms to hematite, which is the final product of the reaction.25 It is also possible to obtain hematite in mixture with maghemite by employing specific reaction conditions, for example, changing the thickness of the precursor layer and the annealing temperature, which allows to control the hematite/magh-emite ratio.19 Nanoparticles prepared in a such simple way were reported to have the superior catalytic activity in degradation of hydrogen peroxide.16,27 In addition, Rao et al.28 reported an occurrence of the maghemite in the composition of the final decomposition product if the Kopp et al.: Preparation of Magnetite by Thermally Induced 456 Acta Chim. Slov. 2019, 66, 455-465 reaction atmosphere contained a significant amount of moisture. Another decomposition process was reported to take place under the inert atmosphere (e.g., nitrogen or argon).23,29-33 The differential thermal analysis (DTA) of this process generally show two major events in the range up to 450 °C, which are well separated and both of them are en-dothermic.29 The first event corresponds to the evaporation of water molecules (180 °C), while the other one, a large asymmetric peak that starts at approximately 320-350 °C, is attributed to the decomposition of oxalate group and the formation of oxides. One of the generally accepted decomposition mechanisms assumes that the anhydrous ferrous oxalate decomposes to wustite, which immediately transforms to magnetite and a-iron, due to its instability under 570 °C. 31 However, the experimental confirmation of this mechanism is still required, as the formation of wustite phase has never been observed directly. This mechanism was indirectly supported by the findings in studies31-33 which identified magnetite and a-iron as major and minor phases of the final product at the temperature up to 550 °C. Another accepted mechanism34 assumes that the anhydrous oxalate decomposes directly to ultra-fine magnetite particles with no wustite intermediate. The two Mossbauer spectroscopy investigations35,36 of thermal decomposition of ferrous oxalate dihydrate in the inert atmosphere did not exhibit any formation of a-iron, although the experiments as well as Mossbauer measurements were performed inside the glass tube, which prevented the oxidation of small particles of a-iron.36 Nevertheless, some authors15,37 reported completely different composition of the decomposition product in the inert atmosphere. For example, hematite was determined as a major phase of the decomposition in nitrogen at 440 °C in the study of Mohamed et al.37 In addition, Zhou et al.15 reported that the maghemite was the only decomposition product after treating the sample at 400 °C for 2 h in nitrogen atmosphere. A unique study of iron oxalate's decomposition in the atmosphere of its conversion gases was reported by Hermanek et al.,34 in which both Mossbauer spectroscopy and XRD were used to determine the composition of the samples. The magnetite was reported to be the main product of the thermal decomposition in the temperature range between 400-550 °C. In contrast to Hermanek et al.,34 the pure magnetite was obtained in three other studies,15,38,39 which reported the decomposition of ferrous oxalate dihy-drate in the atmosphere of its conversion gases as a very facile method of magnetite preparation. In each of these studies the ferrous oxalate was reported to be sealed inside a container and then calcined for a certain period. Being aware of the discrepancies concerning the possible contamination of magnetite by other iron phases, in this work we present a systematic study of thermal decomposition of ferrous oxalate in the combined atmosphere of inert and conversion gases to find an optimal synthetic route for magnetite preparation. The usage of conversion gases eliminates the necessity of the inert gas to flow through the furnace thus simplifying the magnetite synthesis process. All the samples were prepared inside the stainless-steel cells. To eliminate the possible effects of residual oxygen on the sample composition, the cells were sealed inside the box with nitrogen atmosphere. The composition and properties of the decomposition products were studied by Mossbauer spectroscopy and XRD. 2. Experimental Section 2. 1. Chemicals Ferrous Oxalate Dihydrate (500 g) supplied by Sigma Aldrich was utilized for all the experiments. 2. 2. Synthesis The ferrous oxalate dihydrate (Sigma Aldrich) was homogenized in the ceramic mortar for 5 minutes. This process was accompanied by a visible change in the material colour. Approximately 100 mg of homogenized material was enclosed inside each of the prepared sealable stainless-steel cells. The filling of the cells was conducted inside the box with a controlled atmosphere (nitrogen). Once filled and sealed, the cells were put inside the high-temperature laboratory furnace LAC LE/05 with HP40 controller. The decomposition of ferrous oxalate di-hydrate and the subsequent formation of magnetite were observed at 8 equidistant temperature steps from 300 °C to 650 °C (one step each 50 °C). The temperature range was chosen in accordance with the previous thermogravimet-ric analysis (TGA) or DTA studies performed on the ferrous oxalate dihydrate.23,28,31-34 In addition, the samples were calcinated during 3 different time intervals: 1, 6 and 12 hours, for each of the temperature steps, to study the influence of calcination time on properties of the prepared samples. Overall, twenty-four samples with the different temperature and time combinations were prepared. The temperature was measured by both in-built thermometer of the furnace and by an external thermocouple situated in the same place as the sample cell. 2. 3. Characterization Transmission 57Fe Mossbauer spectra of the studied samples were measured at room temperature using the MS2007 Mossbauer spectrometer based on virtual instrumentation technique.40,41 Constant acceleration mode and 57Co(Rh) source were employed. The Mossbauer spectra were processed by use of MossWinn software program.42 The isomer shift values were referred to the value of an a-Fe foil sample at room temperature. All the X-ray diffraction (XRD) patterns were measured by use of the X'PertPRO MPD diffractometer (Mal-vern Panalytical, Ltd) in the Bragg-Brentano geometry Kopp et al.: Preparation of Magnetite by Thermally Induced ... Acta Chim. Slov. 2019, 66, 455-465 457 equipped with a Co X-ray tube (iron filtered Co Ka radiation: X = 0.178901 nm), programmable divergence and diffracted beam anti-scatter slits and a fast X'Celerator detector. The patterns were recorded in the 29 range of 5-120° (20 resolution of 0.017°) at room temperature. Line positions determination and instrumental line broadening evaluation were carried out using commercially available standards SRM640 (Si) and SRM660 (LaB6) from the NIST (National Institute of Standard and Technologies). High Score Plus software in conjunction with the PDF-4+ and ICSD databases was employed for crystalline phase identification and Rietveld refinement. The Scanning electron microscopy (SEM) images were recorded by employing the Scanning electron microscope VEGA3 LMU equipped with Everhart-Thornley type secondary electron detector (TESCAN, Brno, Czech Republic). The accelerating voltage was set to 30 kV. The pressure inside the chamber during the measurements was decreased to 10-2 Pa using an in-built rotary vacuum pump. 3. Results and Discussion Before starting, the calcination of homogenized ferrous oxalate dihydrate precursor a portion of it was examined by room temperature Mossbauer spectroscopy. The acquired spectrum exhibited a symmetrical doublet with an isomer shift of S = 1.19 mm/s and quadrupole splitting of AEq = 1.72 mm/s. Both the spectrum shape and the parameter values are typical for ferrous oxalate dihydrate.23,34 The results obtained by XRD and Mossbauer spectroscopy of all 24 prepared samples are presented below. The individual samples are presented within the series, where each of them was prepared at different temperature (8 series, 3 samples each). All the samples were labelled TxHy, where x stands for temperature in °C and y stands for calcination time in hours. The complete list of obtained XRD and Mossbauer results is shown in Table 1. and Table 2., respectively. phous iron oxide or superparamagnetic magnetite. The similar doublet was also observed by Hermanek et al.,34 who identified the doublet as a superparamagnetic magnetite based on low temperature Mossbauer spectroscopy measurements in magnetic field. Due to relative similarity of the observed components we could suggest that the doublet was a superparamagnetic magnetite (SP). The relative area of the smaller doublet component was around 7 %. The other two samples of the series were comparable in the sample composition to the first one. The gradual increase up to 13 % in the relative area corresponding to the smaller doublet was observed in the second sample T300H6. Then, in the third sample, it dropped to 8 %. However, another component or components, which have the magnetic field, could be observed in a form of small distortions in the background (see Figure 1.) taking roughly 10 % of the relative area. This component or components could be small particles of crystalline magnetite, but they were too small to specify their nature. As it was expected, the calcination of ferrous oxalate at 300 °C did not result in a complete decomposition of the precursor, but some minor part of it was transformed. Figure 1. Mossbauer spectrum of T300H12. 3. 1. The T300 Series The calcination of the first series of samples was conducted at 300 °C (i.e., T300 series). The Mossbauer spectrum of the first sample T300H1 showed an asymmetrical doublet composed of two components. The first one, forming most of the spectrum, was a doublet with hyperfine parameters of 8 = 1.19 mm/s and AEq = 2.03 mm/s that could be assigned to anhydrous ferrous oxalate. In comparison with its hydrated counterpart, the quadrupole splitting was slightly higher, possibly because of the increased asymmetry of the electric field in the surroundings of the iron cations, which was caused by the missing water molecules. The ferrous oxalate doublet formed most of the MS spectrum. The other component was small, singlet resembling doublet with an isomer shift of 8 = 0.33 mm/s and quadrupole splitting of AEq = 0.45 mm/s. It possibly originated from the presence of amor- 3. 2. The T350 Series. The temperature 350 °C used for calcination of the samples of the second series T350 fits well inside the range of the second DTA peak as it was reported in previous works, e.g.31-33 The T350 series showed a gradual decomposition of anhydrous ferrous oxalate and formation of crystalline oxides. The Mossbauer spectra of all the three samples of the series exhibited the combination of a doublet and three sextets. The doublet could be again assigned to the remains of the precursor in the sample. Two new sextets were recognised to belong to two different cation sites (i.e., octahedral (O) and tetrahedral (T)) of a spinel structure of magnetite. The last sextet was ascribed to maghemite, whose presence in the spectra was probably the product of oxidation of nano-sized magnetite, which occurred after the sample cells were opened and the mate- Kopp et al.: Preparation of Magnetite by Thermally Induced 458 Acta Chim. Slov. 2019, 66, 455-465 rial located inside was exposed to ambient atmosphere. The presence of maghemite in the spectra explains the ratio between the two left-most spectral lines. To distinguish the mixture of magnetite and maghemite from non-stoi-chiometric magnetite based on Mossbauer spectra is generally very difficult.43 To fit all three sextet components, hyperfine parameters of magnetite sub-spectra were allowed to oscillate in a small interval around the fixed previously reported values.44 Generally, the maghemite spectra compose of two very similar sextets.44 To simplify the overall fit, maghemite was fitted by only one sextet whose hyperfine parameter values could belong only to an interval where the lower and upper limits were chosen to comply with the hyperfine parameters of both maghemite sub-spectra.44 Moreover, the ratio between the O and T sites of magnetite was firmly fixed at 2:1. The described approach was also employed for all the remaining series. The obtained hyperfine parameters of the samples are shown in Table 2. Compared with T300 series no superparamagnetic magnetite was detected in any of the T350 samples. In contrast with our results, Hermanek et al.34 reported that at 363 °C the doublet of superparamagnetic magnetite formed about 30 % of their Mossbauer spectrum area. Discrepancies could be explained by the absence of the prolonged calcination process in the study.34 The evaluation of diffraction patterns of the samples of both T300 and T350 series could be done from qualitative point of view. Although quantitative analysis was also employed for evaluation of all other diffraction patterns, the lack of suitable available patterns with different grade of iron oxalate dihydrate decomposition disabled quantification in this case. Nevertheless, the diffraction patterns displayed relatively broad peaks that could be qualitatively ascribed to anhydrous ferrous oxalate and magnetite. It was observed that the weight ratio of magnetite to oxalate increased with longer calcination and was higher for all the samples in the T350 series compared to the samples of T300 series. By evaluation of both Mossbauer and XRD data of the T350 series, the temperature 350 °C seemed sufficient for decomposition of the majority of ferrous oxalate and subsequent formation of iron oxides. However, even after 12 h of calcination a part of the precursor remained undecomposed. 3. 3. The T400 Series Regarding the third series, after heating up to 400 °C and treating the sample for 1 hour, i.e. sample T400H1, the product did not exhibit any traces of the precursor probably because the whole of the oxalate had been decomposed already. Employing the approach described above, Mossbauer spectrum of the sample T400H1 displayed three partially overlapping sextets that could be assigned to a contribution of Fe3+ and Fe2,5+ cations in tetrahedral (T) and octahedral (O) sites of magnetite and maghemite. Based on the XRD analysis four additional sextets were fitted in Mossbauer spectrum and ascribed to iron carbides. Three sextets were assigned the three different sites of iron nuclei in the structure of Hagg's carbide (Fe5C2) whereas the fourth sextet was assigned to cementite (Fe3C). Fitting such small sub-spectra into an overall fit the hyperfine parameters values of sextets corresponding to carbides were strictly fixed to the values previously reported by Malina et al.45 Although the inclusion of the carbides sub-spectra into the overall fit was based strictly on the XRD analysis, the carbide sub-spectra corresponded well with the distortions in the background of the spectra e.g. T400H1 spectrum (Figure 2.). The obtained XRD pattern of T400H1 agreed with the acquired Mossbauer data. Apart from the diffraction peaks that could be assigned to the spinel-structured iron oxide, a small amount of cementite (2.4 wt.%) and Hagg's carbide, (2.0 wt.%) were detected. Moreover, three additional small diffraction peaks could be observed at 17°, 28° and 31° 29 for the sample T400H1. Although some authors15 reported these peaks to be an unambiguous sign of maghemite, they might represent a reduction in crystal symmetry of cubic spinel structure driven by partial ordering of vacancies, which is also generated by magnetite non-stoichiometry. Figure 2. Mossbauer and XRD data of sample T400H1. Kopp et al.: Preparation of Magnetite by Thermally Induced ... Table 1. XRD parameters of the measured samples. Sample Magnetite/Maghemite a [A] w [%] MCL [nm] a-iron w [%] MCL [nm] Cementite w [%] MCL [nm] Hagg's carbide w [%] MCL [nm] Wüstite w [%] MCL [nm] « o T3 T3 si I A. K T300H1 T300H6 T300H12 T350H1 T350H6 T350H12 COULD NOT BE ACCURATELY QUANTIFIED* T400H1 8.390 95.6 41 - 2.0 60 2.4 26 - T400H6 8.393 98.0 44 - 1.4 59 0.6« 15 - T400H12 8.392 100.0 50 - - - - - - T450H1 8.396 100.0 51 - - - - - - T450H6 8.391 99.0 50 - - - 1 - - T450H12 8.395 97.9 55 - 1.0 58 1.1 26 - T500H1 8.396 98.2 53 - 0.8« 85 1.0 73 - T500H6 8.393 98.8 55 - 0.6« - 0.6« - - T500H12 8.394 98.0 56 - 2.0 85 - - - T550H1 8.394 100.0 56 - - - - - - T550H6 8.394 100.0 65 - - - - - - T550H12 8.396 100.0 162 - - - - - - T600H1 8.396 80.1 80 6.6 89 - - - 13.3 25 T600H6 8.393 86.3 76 2.5 86 - - - 11.2 31 T600H12 8.392 86.3 57 1.9 84 - - - 11.7 30 T650H1 8.398 41.0 64 0.3« - - - - 58.8 32 T650H6 8.396 44.5 69 4.6 458 - - - 50.9 32 T650H12 8.388 40.5 25 1.1 109 - - - 58.4«* 21(26) a - lattice parameter, w - weight ratio, MCL - mean coherence length * XRD patterns contain peaks belonging to anhydrous ferrous oxalate and magnetite, amount of magnetite gradually increases ** Quantification limit is 1 % *** Two different structures of wustite were observed in ratio of 15.5 % to 42.9 %. 460 Acta Chim. Slov. 2019, 66, 455-465 This idea was supported by values of acquired cell parameters (see Table 1.), which reflected the presence of non-stoi-chiometric magnetite instead of the mixture of magnetite and maghemite. On the other hand, Mossbauer spectrum suggested the presence of maghemite in the sample, as its absence in the spectrum would lead to highly unprobeable reversed magnetite stoichiometry of 1:2 (O:T). Similar results were obtained for the other two remaining spectra of the series T400. The presence of maghemite in the samples due to gradual oxidation of nano-sized magnetite could exTable 2. Mossbauer parameters of the measured samples. plain the discrepancies in the results of these two techniques as both measurements were carried out at different time. The crystallite size of iron oxide particles was determined by Rietveld refinement and varied for all the samples in the T400 series between 40 and 50 nm. 3. 4. The T450/T500 Series No major changes were observed in the results of the following two series, i.e. T450 and T500 series. The results Sample S ± 0.01 (mm/s) AEQ ± 0.01 (mm/s) H ± 0.3 (T) RA ± 2 (%) Site assignment T300H1 1.19 2.04 - 92 Ferrous oxalate 0.33 0.45 - 8 SP magnetite T300H6 1.17 2.21 - 87 Ferrous oxalate 0.33 0.45 - 13 SP magnetite T300H12 1.19 1.90 - 78 Ferrous oxalate 0.30 0.48 - 8 SP magnetite 0.18 - 43.3 14 Magnetite/Maghemite T350H1 1.19 2.20 - 78 Ferrous oxalate 0.25 - 48.9 5 Magnetite (site T) 0.65 - 45.7 11 Magnetite (site O) 0.23 0,03 49.7 5 Maghemite T350H6 1.16 2.24 - 12 Ferrous oxalate 0.26 - 48.9 12 Magnetite (site T) 0.65 - 45.7 24 Magnetite (site O) 0.33 0.02 49.7 52 Maghemite T350H12 1.19 2.20 - 12 Ferrous oxalate 0.25 - 48.9 17 Magnetite (site T) 0.65 - 45.7 34 Magnetite (site O) 0.29 0.03 49.7 37 Maghemite T400H1 0.28 - 49.1 14 Magnetite (site T) 0.65 - 46.1 28 Magnetite (site O) 0.32 0.02 50.0 46 Maghemite 0.24 0.09 21.5 2 Hagg's Carbide - I 0.17 0.06 18.2 3 Hagg's Carbide - II 0.22 0.13 10.9 3 Hagg's Carbide - III 0.20 0.03 19.9 4 Cementite T400H6 0.28 - 49.1 21 Magnetite (site T) 0.65 - 45.9 42 Magnetite (site O) 0.31 0.02 49.7 24 Maghemite 0.24 0.09 21.5 4 Hagg's Carbide - I 0.17 0.06 18.2 5 Hagg's Carbide - II 0.22 0.13 10.9 2 Hagg's Carbide - III 0.20 0.03 19.9 2 Cementite T400H12 0.28 - 48.9 27 Magnetite (site T) 0.68 - 45.7 54 Magnetite (site O) 0.32 0.02 49.7 19 Maghemite T450H1 0.27 - 48.9 12 Magnetite (site T) 0.65 - 45.7 25 Magnetite (site O) 0.34 0.02 49.7 63 Maghemite T450H6 0.28 - 48.9 27 Magnetite (site T) 0.67 - 45.7 54 Magnetite (site O) 0.35 0.03 49.7 19 Maghemite T450H12 0.28 - 48.9 24 Magnetite (site T) 0.67 - 45.9 50 Magnetite (site O) 0.31 0.03 49.7 12 Maghemite 0.24 0.09 21.5 2 Hagg's Carbide - I Kopp et al.: Preparation of Magnetite by Thermally Induced ... Acta Chim. Slov. 2019, 66, 455-465 461 Sample S ± 0.01 (mm/s) AEq ± 0.01 (mm/s) H ± 0.3 (T) RA ± 2 (%) Site assignment 0.17 0.06 18.2 2 Hagg's Carbide - II 0.22 0.13 10.9 2 Hagg's Carbide - III 0.20 0.03 19.9 6 Cementite T500H1 0.26 - 48.9 24 Magnetite (site T) 0.66 - 45.7 48 Magnetite (site O) 0.33 0.02 49.7 18 Maghemite 0.20 0.03 19.9 10 Cementite T500H6 0.27 - 49.2 24 Magnetite (site T) 0.66 - 46.1 48 Magnetite (site O) 0.32 0.02 49.8 18 Maghemite 0.24 0.09 21.5 3 Hagg's Carbide - I 0.17 0.06 18.2 3 Hagg's Carbide - II 0.22 0.13 10.9 2 Hagg's Carbide - III 0.20 0.03 19.9 2 Cementite T500H12 0.28 - 48.9 28 Magnetite (site T) 0.67 - 45.9 56 Magnetite (site O) 0.28 0.02 49.7 7 Maghemite 0.24 0.09 21.5 4 Hagg's Carbide - I 0.17 0.06 18.2 3 Hagg's Carbide - II 0.22 0.13 10.9 1 Hagg's Carbide - III 0.20 0.03 19.9 1 Cementite T550H1 0.28 - 49.1 23 Magnetite (site T) 0.67 - 46.1 46 Magnetite (site O) 0.30 0.02 - 49.7 31 Maghemite T550H6 0.28 - 48.9 22 Magnetite (site T) 0.66 - 45.9 43 Magnetite (site O) 0.32 0.02 49.7 35 Maghemite T550H12 0.28 - 49.0 27 Magnetite (site T) 0.67 - 45.9 54 Magnetite (site O) 0.31 0.02 49.7 18 Maghemite T600H1 0.28 - 48.9 20 Magnetite (site T) 0.67 - 45.9 41 Magnetite (site O) 0.26 0.02 49.7 5 Maghemite 0.00 - 33.0 17 a-Iron 1.04 0.05 - 16 Wustite T600H6 0.28 - 48.9 24 Magnetite (site T) 0.67 - 46.0 48 Magnetite (site O) 0.29 0.02 49.7 6 Maghemite 0.00 0.24 32.8 7 a-Iron 1.05 - - 14 Wustite T600H12 0.28 - 48.9 22 Magnetite (site T) 0.67 - 46.1 44 Magnetite (site O) 0.33 0.03 49.7 15 Maghemite 0.00 - 33.1 5 a-Iron 1.03 0.25 - 14 Wustite T650H1 0.27 - 49.1 14 Magnetite (site T) 0.65 - 46.0 29 Magnetite (site O) 0.00 - 32.8 8 a-Iron 1.06 0.12 - 49 Wustite T650H6 0.25 - 48.9 14 Magnetite (site T) 0.65 - 45.7 28 Magnetite (site O) 0.00 - 32.8 14 a-Iron 1.05 0.19 - 44 Wustite T650H12 0.25 - 48.9 12 Magnetite (site T) 0.65 - 47.0 24 Magnetite (site O) 0.00 - 32.8 5 a-Iron 1.04 0.27 - 40 Wustite 0.86 0.72 - 20 Wustite 5 - isomer shift, AEq - quadrupole splitting, H - hyperfine magnetic field, RA - relative area. The f-factor was considered equal to 1 for all reported phases. Kopp et al.: Preparation of Magnetite by Thermally Induced 462 Acta Chim. Slov. 2019, 66, 455-465 demonstrated similar composition of phases compared to the series T400 (see Table 1. and Table 2.). In both series, the crystallite size of iron oxide particles was slightly higher (50-56 nm) as excepted due to the increased calcination temperature. A slight rise in the crystallite size was also observed within individual series with prolonged calcination time. Mossbauer spectra displayed three overlapping sextets that could be ascribed to magnetite and maghemite. In some cases, according to XRD analysis, iron carbides were present in the samples and were fitted along in the Mössbauer spectra. However, there was no observed trend in the overall amount of iron carbides in individual samples and some samples of the series did not contain any carbides at all. The presence of carbides in the samples of T400, T450 and T500 series was probably caused by the reducing effect of CO molecule, which evolve during the decomposition of the oxalate anion. The molecules of CO then reacted with still un-decomposed ferrous oxalate to form iron carbides (1). 8 FeC204 + 6 CO ^ Fe3C + Fe5C2 + 19 C02. (1) Here, this reaction mechanism, firstly introduced by Hermanek et al.,34 is modified to accommodate the formation of Hagg's carbide as well. Hong et al.46 even used the thermal decomposition of ferrous oxalate in dynamic atmosphere of CO to prepare the microcubes of Hagg's carbide for their subsequent application in Fischer-Tropsch synthesis. Concerning the observed overall amounts of carbides, Hermanek34 reported that up to 20 % of iron ions belonged to cementite in Mössbauer spectra, that is higher compared to our results, where only up to 12 % of iron ions was ascribed to iron carbides. In contrast, no carbides were registered in other studies,15,38,39 where the conversion gases synthesis route was used for magnetite preparation. In addition, no carbides were also reported in the study by Angermann and Töpfer47 who decomposed ferrous oxalate in a combined atmosphere of CO/CO2. These discrepancies allowed us to conclude that the presence of iron carbides might depend on the partial pressure of CO inside the cells. The high amount of carbides in Hermanek's study34 could be explained by enclosing the sample into a relatively small container, which caused the high partial pressure of the formed CO. When the relatively larger containers were used15,38,39 the samples did not contain any iron carbides or their amounts were simply undetectable by the employed instruments. Some of the evolved CO could also react with the remaining oxygen to form CO2. On the basis of our experimental results, we could suggest that although the sealing of the sample cells prevented the intrusion of the oxygen into the cells, it was not sufficient to withstand the pressure evolved inside during decomposition of the ferrous oxalate. After reaching the critical level of pressure, a part of the gas leaked out and lowered the partial pressure of CO, which might explain the low amount of iron carbides or even their absence in the composition of the samples and the randomness in the carbide's occurrence. 3. 5. The T550 Series The next series was calcinated at 550 °C. All three samples of the T550 series were composed of spinel-structured iron oxides according to the data acquired from both Mossbauer spectroscopy and XRD (see Table 1., Table 2.). No signs of carbides or any other material were observed. Although the evaluation of the XRD patterns, especially the cell parameter, pointed to a non-stoichiometric magnetite, the relative areas of individual spectral lines in Mossbauer spectra suggest the presence of maghemite and magnetite mixture, see Figure 3. Compared with our results, Hermanek et al.34 also reported a significant amount of a-iron (around 20 % in Mossbauer spectrum) in the temperature range of 500-570 °C. They suggested that the a-iron was a product of iron carbides decomposition. The missing a-iron in our samples might be explained by relatively lower amount of iron carbides. In addition, the eventual traces of a-iron might have been 100 99 98 97 -10 iw'.' ... ... ...» rwwm ii Pi 9 f. I • Magnetite (T) - Magnetite (O) - Maghemite Fit Signal Fe.O. -5 0 5 Velocity (mm/s) Figure 3. Mossbauer and XRD data of sample T550H1. 10 10 20 30 40 50 60 70 20CoK(t n 80 90 100 Kopp et al.: Preparation of Magnetite by Thermally Induced ... Acta Chim. Slov. 2019, 66, 455-465 463 TP A I - * A . j * SIT- 1 J SEM HV: 30.0 kV WD: 5.35 mm | | | VEGA3 TESCAN SEM MAG: 140 kx Det: SE 1 pm Bl: 5.00 Date(m/d/y>: 07/15/18 KEF UPOL Figure 4. SEM images of samples T550H1 (left) and T550H12 (right). re-oxidised upon its exposure to the ambient atmosphere after the cells were open. The relatively high temperature of 550 °C is probably the cause of gradually increasing crystallite size within the series; from 56 nm to 162 nm for samples T550H1 and T550H12, respectively. The dramatic increase in particle size was also observed in SEM images of samples T500H1 and T550H12 (Figure 4.). 3. 6. The T600 Series After the samples of the seventh series had been calcinated at 600 °C, the significant change in Mossbauer spectra was observed. The two sextets which were ascribed to magnetite remained, but they were reduced in area and only a trace amount of maghemite was detected. Additionally, two new components appeared. Firstly, a relatively narrow sextet with hyperfine parameters 8 = 0.0 mm/s, H = 33.0 T was identified as a-iron. Secondly, a doublet with a low quadrupole splitting, which was assigned to non-stochiometric wustite. The exhibition of quadrupole splitting in Mossbauer spectra is a manifestation of the non-equivalent surroundings of iron cations and implies that the observed wustite phase is non-stoichiometric. The non-stoichiometry of wustite is generally caused by missing iron cations in its structure.44 On the other hand, in some cases it is also possible to fit wustite component with broad singlet, which suggests it being stochiometric.44 The detailed hyperfine parameters obtained for individual samples of the T600 series are listed in Table 2. The results obtained by Mossbauer spectroscopy were in good agreement to those obtained by XRD (Table 1.). All diffraction peaks could be ascribed to magnetite, wustite and a-iron. Such significant change in both Mossbauer spectra and XRD patterns was probably caused by the combination of increased temperature and reducing nature of conversion gases, which resulted in thermally induced reduction of magnetite to wustite and a-iron, as it was suggested by Hermanek et al.34 The increase in quadrupole splitting of wustite phase, which was observed in samples T600H6 and T600H12, was probably caused by the prolonged exposure of wustite to high temperature and it could be suggested that this rise was a result of wustite increasing non-stoichi-ometry. Similar trend was also observed in the T650 series. 3. 7. The T650 Series Evaluation of data acquired for samples of the last T650 series showed very similar composition in comparison to the samples of T600 series. Mossbauer spectra revealed the increased amount of wustite and decreased amount of a-iron and magnetite. An increase in a quadru-pole splitting of the wustite was observed with prolonged calcination, similarly to the previous series. Moreover, the large middle component in the spectrum of the sample T650H12 could not be fitted by only one doublet. Two doublets with following hyperfine parameters were thus ascribed to wustite in overall T650H12 spectrum fit, 8 = 1.04 mm/s, AEq = 0.27 mm/s for the first one of the two doublets and 8 = 0.86 mm/s, AEq = 0.72 mm/s for the other one. Concerning the XRD analysis of the sample T650H12, the acquired patterns were composed of two wustite structures with different cell parameters thus pointing to the presence of two different forms of wustite. The second wustite structure was probably the result of the increased non-stoichiometry after the wustite had been exposed to high temperatures for 12 h. In addition to Kopp et al.: Preparation of Magnetite by Thermally Induced 464 Acta Chim. Slov. 2019, 66, 455-465 Figure 5. Mössbauer and XRD data of sample T650H12. quantified phases, part of the a-Fe might have been re-oxidised back the most stable iron oxide, i.e., hematite, which was in a trace amount identified in the diffraction pattern of the sample T650H12 (Figure 5.). 4. Conclusion Ferrous oxalate dihydrate was enclosed inside stainless-steel cells under the nitrogen atmosphere. Thermal decomposition of the precursor was systematically carried out at 24 different temperature/time combinations (300650 °C; 1, 6 and 12 hours) to find the optimal route for magnetite preparation. The prepared materials were studied by Mossbauer spectroscopy and XRD. However, all decomposition routes resulted in obtaining the magnetite particles containing impurities. The remains of the precursor were observed in all the samples calcined below 400 °C, even after long calcination time (12 h). Almost spinel-structured iron oxide particles, magnetite and maghe-mite, (e.g. 90 %) were prepared in the temperature range of 400-500 °C. Maghemite was most probably formed by oxidation of small particles of magnetite, which is unstable in the air. Other phases consisted solely of iron carbides phases (i.e., Fe3C and Fe5C2). Mixture of magnetite and maghemite, free of carbides, was obtained at 550 °C. Above that temperature the formation of wustite and a-iron was observed. Both of them seemed to be products of thermally induced reduction of magnetite and maghemite by CO. The crystallite size of the obtained iron oxide particles was in the range of 41 to 162 nm, depending on the temperature and the calcination time of the preparation process. 5. Acknowledgment The authors gratefully acknowledge the financial support from the internal IGA grants of Palacky University (IGA_PrF_2019_002 and IGA_PrF_2019_023) and from the Czech-China mobility project Nr. 8H17065 of Ministry of Education, Youth and Sports of the Czech Republic. Authors would also like to thank Mgr. Ivo Medrik for sharing his expertise and offering a practical advice, Mgr. Tomas Ingr for SEM images and Helena Sedlackova for her help with the paper. 6. References 1. A. S. Teja, P. Y. Koh, Prog. Cryst. Growth Charact. Mater. 2009, 55, 22-45. DOI:10.1016/j.pcrysgrow.2008.08.003 2. A. Ito, M. Shinkai, H. Honda, T. Kobayashi, J. Biosci. Bioeng. 2005, 100, 1-11. DOI:10.1263/jbb.100.1 3. S. Saeidi, N. A. S. Amin, M. R. Rahimpour, J. CO2 Util. 2014, 5, 66-81. 4. M. Zhu, I. E. Wachs, ACS Catal. 2016, 6, 722-732. DOI:10.1021/acscatal.5b02594 5. J. M. Honig, Proc. Indian Acad. Sci. (Chemical Sci.) 1986, 96, 391-409. DOI:10.1007/BF02936294 6. X. Li, Z. Si, Y. Lei, J. Tang, S. 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Nucl. Chem. 1996, 210, 105-118. DOI:10.1007/BF02055410 43. G. M. Da Costa, E. De Grave, P. M. A. De Bakker, R. E. Vandenberghe, Clays Clay Miner. 1995, 43, 656-668. DOI:10.1346/CCMN.1995.0430602 44. R. M. Cornell, U. Schwertmann, The Iron Oxides, 2003. 45. O. Malina, P. Jakubec, J. Kaslik, J. Tucek, R. Zboril, Nanoscale 2017, 9, 10440-10446. DOI:10.1039/C7NR02383A 46. S. Y. Hong, D. H. Chun, J.-I. Yang, H. Jung, H.-T. Lee, S. Hong, S. Jang, J. T. Lim, C. S. Kim, J. C. Park, Nanoscale 2015, 7, 16616-16620. DOI:10.1039/C5NR04546K 47. A. Angermann, J. Töpfer, J. Mater. Sci. 2008, 43, 5123-5130. DOI:10.1007/s10853-008-2738-3 Povzetek V tej študiji predstavljamo raziskavo termičnega razkroja železovega oksalat dihidrata v kombinirani atmosferi inertnih in pretvorbenih plinov, z namenom iskanja optimalne poti za pripravo magnetita. Homogeniziran prekurzor je bil izoter-mično obdelan v celicah iz nerjavnega jekla pri različnih temperaturah v območju od 300 °C do 650 °C. Spreminjali smo tudi čas razkroja (1 uro, 6 ur in 12 ur). Vzorce smo zaprli v celice s kombinirano atmosfero in s tem odpravili potrebo po pretoku inertnega plina. Strukturne, magnetne in morfološke vidike pripravljenih materialov smo preučili s kombinacijo eksperimentalnih tehnik, kot so Mossbauerjeva spektroskopija, rentgenska praškovna difrakcija in vrstična elektronska mikroskopija (SEM). Kopp et al.: Preparation of Magnetite by Thermally Induced 466 DOI: I0.i7344/acsi.20i9.4988 Acta Chim. Slov. 2019, 66, 466-472 ©ccmmons Scientific paper Phase Equilibria in the MnGa2Te4-MnIn2Te4 System, Crystal Structure and Physical Properties of MnGaInTe4 Faig Mamedagha Mammadov,1 Imamaddin Rajabali Amiraslanov,2 Yegana Rasul Aliyeva,2 Sadiyar Sultan Ragimov,2 Leyla Farkhad Mashadiyeva1 and Mahammad Baba Babanly1^ 1 Institute of Catalysis and Inorganic Chemistry, Azerbaijan National Academy of Science, 113, H.Javid. ave., AZ-1143, Baku, Azerbaijan, 2 Institute of Physics, Azerbaijan National Academy of Science, 131, H.Javid. ave., AZ-1143, Baku, Azerbaijan * Corresponding author: E-mail: babanlymb@gmail.com Received: 03-01-2019 Abstract The phase equilibria in the MnGa2Te4-MnIn2Te4 system were experimentally investigated by means of differential thermal analysis and powder X-ray diffraction technique. It was found that this system is quasi-binary and characterized by dystectic and eutectic equilibria and the formation of a wide area of solid solutions based on the starting compounds. The crystal structures of the MnGaInTe4 and MnIn2Te4 were refined by the Rietveld method using powder X-ray diffraction data. It was established, that both phases crystallize in the tetragonal system (Space group I-42m). Electron paramagnetic resonance and Raman spectra, as well as the temperature dependences of the electrical conductivity and the Hall effect for the MnGaInTe4 crystal, were studied. Keywords: MnGa2Te4-MnIn2Te4 system; phase diagram; solid solutions; MnGaInTe4 crystal structure; rietveld method; EPR spectroscopy; raman spectroscopy 1. Introduction Complex metal chalcogenides are essential functional materials possessing optical, photoelectric, thermoelectric, magnetic and other properties.1-7 Recent studies have shown that some of these phases are topological insulators and are considered promising for use in spintronics and quantum computing.8-11 Among the chalcogenide materials, magnetic semiconductors of the type MB2X4 (where M - Mn, Fe, Co, Ni; B - Ga, In; X - S, Se, Te) and phases on their basis are very promising for use in the manufacture of lasers, light modulators, photodetectors and other electronic devices controlled by a magnetic field.12-17 Search and development of methods for the directed synthesis of new multicomponent phases and materials requires the study of phase equilibria in the relevant sys-tems.18,19 For this case, the systems including compounds which are structural or formula analogs are the greatest interest since the formation of broad areas of substitutional solid solutions can be expected in them.20-24 Herein, the phase equilibria in the MnGa2Te4-Mn-In2Te4 system are studied, the crystal structures of the MnGaInTe4 and MnIn2Te4 are refined, as well as the EPR and Raman spectra for the MnGaInTe4 crystals are measured, and the temperature dependences of the electrical conductivity and the Hall Effect of the MnGaInTe4 are investigated. The starting compounds MnGa2Te4 and MnIn2Te4 were studied in detail. According to authors,25 the Mn-Ga2Te4 compound melts congruently at 1118 K (at 1093±10 K,26) and has a homogeneity region of 49.8-50.2 mol% MnTe over the MnTe-Ga2Te3 section. The MnIn2Te4 compound also melts congruently at 1013 K,27 (at 1040 K according to reference,28). The crystal structure of the MnGa2Te4 was studied in a number papers.26, 29, 30 The authors,29 showed that this compound has a pseudo-tetragonal monoclinic unit cell with parameters: a = b = 0.847 nm; c = 4.83 nm; a = ~ 90°; Z = 16. Close parameters were obtained: a = b = 0.8486 Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 Acta Chim. Slov. 2019, 66, 466-472 467 nm; c = 4.840 nm; a = p = y ~ 90°; Z = 16.26 The crystal structure of MnGa2Te4 was studied by the Laue method.30 It was shown that this compound crystallizes in the mono-clinic structure (Sp. gr. C2/c) with parameters: a = 1.1999(3); b = 1.1999(3); c = 2.4922(6) nm; p = 104.01(2)°; Z = 16. The MnIn2Te4 compound crystallizes in a tetragonal structure (Sp. Group I 42m) with the parameters: a = 0.6191(2) nm; c = 1.2382(3) nm; Z = 2.31 Mn-00003 2. The Experimental Part 2. 1. Synthesis For research ternary compounds MnGa2Te4 and Mn-In2Te4 were synthesized. Simple substances from the company EVOCHEM ADVANCED MATERIALS GMBH (Germany) of high purity were used for the synthesis: gallium ingots (Ga-00009; 99.999%), indium in granules (In-00005; 99.999%), manganese pieces (Mn-00003; 99.98%), tellurium pieces (Te-00005; 99.9999%). The synthesis was carried out by melting of elemental components in stoichiometric ratios in evacuated to about 10-2 Pa quartz ampoules at temperatures ~50 K higher than their melting points followed by slow cooling in the furnace off mode to room temperature. In order to prevent the interaction of quartz with manganese, the synthesis of compounds and alloys of the studied system was carried out in graphitized ampoules. The individuality of the synthesized compounds was controlled by differential-thermal analysis (DTA) and powder X-ray diffraction technique (PXRD). According to the DTA data, the MnGa2Te4 and MnIn2Te4 compounds melt congruently correspondingly at 1075 ± 3 K and 1021 ± 3 K, which is somewhat different from the literature data.25-28 Analysis of the powder X-ray diffraction patterns confirmed the single-phase of both compounds. The MnGa2Te4-MnIn2Te4 alloys were prepared from the starting ternary compounds also by vacuum alloying in graphitized quartz ampoules. All alloys were subjected to thermal annealing at 900 K for 500 hours in order to achieve complete homogenization and then slowly cooled in the furnace off. 2. 2. Methods Phase equilibria in the MnGa2Te4-MnIn2Te4 system were investigated by means of DTA and PXRD methods. DTA of the equilibrated alloys was carried out using a NETZSCH 404 F1 Pegasus system. The measurement was performed between room temperature and ~1300 K with a heating and cooling rate of 5 K • min-1 under the inert gas (Ar) flow. Temperatures of thermal effects were determined from the heating curves with an accuracy of ± 2 K. NETZSCH Proteus Software was used for measuring and evaluating data. Scanning Electron Microscopy combined with Energy-Dispersive X-ray spectroscopy (SEM/EDXS) was used for elemental analysis of samples. SEM/EDXS was performed using a JEOL JSM 6610-LV Scanning Electron Microscope. The PXRD analysis was performed using a "D2 Phaser" diffractometer with CuKa1 radiation (5°<2-the-ta<120°). The solution and refinement of the crystal structure were done by the Rietveld method using the TO-PAS-4.2 Software (Bruker). Electron Paramagnetic Resonance (EPR) spectroscopy of MnGaInTe4 crystals was carried out with the EL-EXSYS E 580 spectrometer (Bruker), and the Raman spectra were obtained using the "Nanofinder 30" 3D Laser Raman Microspectroscopy System (Tokyo Instruments). The temperature dependences of the electrical conductivity and the Hall effect for the MnGaInTe4 were studied in the temperature range 100-400 K on the "HL5500 PC-Hall effect measurement system" (Nanometric). 3. Results and Discussion 3. 1. Phase equilibria in the MnGa2Te4-MnIn2Te4 system From the DTA data (Table 1) and PXRD studying of annealed MnGa2Te4-MnIn2Te4 alloys, it is established that this system is characterized by the dystectic (D) and eutec-tic (e) equilibria (Fig. 1a). The P-phase of the MnGaInTe4 composition melts with an open maximum at 1028 K. The eutectic has a composition of 30 mol% MnIn2Te4 and crystallizes at 1012 K. Wide regions of solid substitutional solutions based on starting compounds are formed. At the Table 1. Experimental DTA and PXRD data for the MnGa2Te4-Mn-In2Te4 system Composition, The thermal Parameters of the mol% MnIn2Te4 effect, K tetragonal lattice, nm a c 0 (MnGa2Te4) 1083 10 1030-1062 15 1013-1050 20 1012-1034 0.60816(3) 1.21308(8) 25 1012 30 1011-1016 0.60812(3) 1.21310(8) 35 1013-1020 40 1018-1024 0.60852(3) 1.21403(8) 45 1026 50 1030 0.610293 (7) 1.21766(2) 60 1013-1023 0.61207(3) 1.22232(7) 70 1004-1011 80 1000 0.61553(3) 1.23026(7) 90 1005-1012 100 (MnIn2Te4) 1022 0.61949(5) 1.23956(2) Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 468 Acta Chim. Slov. 2019, 66, 466-472 Fig. 1. The phase diagram of the MnGa2Te4-MnIn2Te4 system and the concentration dependences of the crystal lattice parameters of the p-phase eutectic temperature the solubility based on MnGa2Te4 (a-phase) is 15 mol%, and on the basis of MnIn2Te4 is 65 mol% (P-phase). On the liquidus and solidus curves of the P-phase there is a minimum point (M) at 1000 K. The congruent melting of both starting compounds, the presence of the invariant three-phase eutectic equilibrium L -o a + P and the extreme points D and M, as well as the absence of multiphase areas on the phase diagram, show the quasi-binary nature of this system. PXRD results confirmed the formation of broad areas of solid solutions in the system. Figure 2 shows that powder diffraction images of the alloys from 40-100 mol% MnIn2Te4 composition area are qualitatively identical with the diffraction pattern for pure MnIn2Te4 and characterized by a certain shift of reflection lines with composition changing. The PXRD pattern of the alloy with composition 20 mol% MnIn2Te4 consists of reflection lines of both phases MnGa2Te4 (a-phase) and Mnln-2Te4 (P-phase). I A.n .... ([ ^ MnGa2Te4 (B) 1 ^ 20%A + 80%B ( A 40%A+ 60%B 1 50%A+S0%B j 60%A + 40%B * J ul j . k 'i [ 80%A + 20%B 1 t 1 1 MnIn2Tet(A) f 10 20 30 40 50 2-theta - 60 The concentration dependence of the parameters of the crystal lattice (Table 1, Fig. 1b) of the p-phase has linear character and allows refining the region of its homogeneity at room temperature (38 mol% MnIn2Te4). 3. 2. Crystal structure of MnIn2Te4 and MnGaInTe4 Based on the PXRD data, we determined the crystal-lographic characteristics of the MnIn2Te4 and MnGaInTe4 samples (Table 2). PXRD patterns for MnIn2Te4 and Mn-GaInTe4 and the intensities differences between the experimental and calculated by Rietveld method data are shown in Fig.3. The obtained results show good agreement of symmetry and cell parameters of these samples with the corresponding characteristics.31 The indexing of the diffraction patterns based on obtained crystallographic parameters showed that the studied samples are single-phase. Comparison of the unit cell parameters of MnIn2Te4 and MnGaInTe4 shows a noticeable difference in their values (Aa ~ 0.0092 nm; Ac ~ 0.0219 nm), which exceeds the value of errors many times. This clearly confirms the entry of Ga atoms into the structure. Despite solving the crystal structure of MnIn2Te4 on the basis of single crystals data,31 we also used their results to refine the structure of this compound, but on the basis of powder data and the Riet-veld method. The above results were also used to refine the structure of the compound MnGaInTe4 by the Rietveld method. The Mn and In atoms in the structure are located in identical positions statistically, where the ratio of the number of Mn atoms to In is 1:2.31 This structure belongs to the defective type of chalcopyrite and all metals are located in the tetrahedron of tellurium atoms. In order to refine the crystal structures of MnIn2Te4 and MnGaInTe4, first, for both compounds, the Lebail and Pawley methods were used to approximate the profiles of the diffraction peaks and to refine the unit cell parameters. For both methods, the obtained results turned out to be almost identical. Later, on the basis of the obtained diffraction data, the crystal structures of the noted compounds were refined. The final results are presented in Tables 2-4. Figure 4 shows a three-dimensional presentation of the crystal structure of these phases. Table 2. Refined structure parameters for MnIn2Te4 and Mn- GaInTe4 Fig. 2. PXRD patterns for some alloys of the MnGa2Te4-MnIn2Te4 system Structure parameters MnIn2Te4 MnGaInTe4 Space group I-42m I-42m Cell parameters: a (nm) 0.619490(5) 0.610293 (7) c (nm) 1.23956(2) 1.21766(2) The cell volume (nm3) 0.47583(3) 0.453528(13) Density (g/cm3) 5.56 (2) 5.50 (2) R-LeBail (%) 0.406 0.155 R-Pawley (%) 0.435 0.171 R-Bragg (%) 1.898 0.235 Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 Acta Chim. Slov. 2019, 66, 466-472 469 Fig. 3. PXRD patterns of MnIn2Te4 and MnGaInTe4. The difference between the experimentally obtained and calculated by the Rietveld method intensities is given under the X-ray diffraction spectrum The results of the elemental analysis (Table 5) and X-ray fluorescence spectrum of MnGaInTe4 crystals (Fig. 5) are in good agreement with the chemical formula. Table 5. Elemental analysis results for MnGaInTe4 Element Weight % Atomic % Mn K 7.03 13.76 Ga K 9.26 14.27 In L 15.74 14.73 Te L 67.97 57.24 Total 100.00 1 00.0 Fig. 4. Occupancy of metal positions in MnIn2Te4 and MnGaInTe4 crystal structures Fig. 5. The XRF spectrum for the MnGaInTe4 crystal Table 3. Atomic positional parameters in the MnIn2Te4 crystal Atoms Multiplicity and Wyckoff letter y Atom type and relative occupation Beq, (nm2) Mj(In+Mn) In+3 0.67(1) M2(In+Mn) Te Interatomic distances (nm) 2a 4d 0 0.5 0 0.25 Mn+2 0.33(1) 8i 0.2756(2) 0.2756(2) M1(In1, Mn1) - Te = 0.2772(1) Mn+2 0.33(1) In+3 0.67(1) 0.1142(1) Te 1 M2(In2,Mn2) - Te = 0.2800(1) 0.005 (1) 0.005 (1) 0.009 (1) 0.009 (1) 1.001(1) x z Table 4. Atomic positional parameters in the MnGaInTe4 crystal Atoms Multiplicity and Wyckoff letter x y z Atom type and relative occupation Beq, (nm2) Ga 2a 0 0 0 Ga3+ 1.018(26) 0.006(3) M(In+Mn) 4d 0 0.5 0.25 In3+ 0.495(22) 0.007(1) Mn2+ 0.505(22) 0.007(1) Te 8i 0.2651(4) 0.2651(4) 0.1129(2) Te 1 0.008(1) Interatomic 0.2669(3) M(In, Mn) - - Te = 0.2731(1) distances (nm) Ga - Te = Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 470 Acta Chim. Slov. 2019, 66, 466-472 As can be seen from Tables 3 and 4, and also from Fig. 4, the crystal lattices of MnIn2Te4 and MnGaInTe4 differ significantly in the occupancy of crystallographic positions. In the MnIn2Te4 structure, the 2a and 4d positions are occupied in ratio 1:2 by the Mn and In atoms. In the structure, positions 2a are completely occupied by gallium atoms, and the Mn and In atoms are statistically located in 4d positions with a 1:1 ratio. The localization of Ga atoms in position 2a in MnGaInTe4 is apparently due to the fact that the ionic radius of Ga3+ is noticeably smaller (~ 0.018 nm) of the ionic radius of In3+. Such occupancy of crystal-lographic positions in the MnGaInTe4 phase allows us to characterize it as an ordered solid solution based on the MnIn2Te4 compound. This is in good agreement with the phase diagram (Fig. 1a), according to which the stoichiometric composition of MnGaInTe4 corresponds to the dys-tectic point D. 3. 3. Physical Properties of the MnGaInTe4 3. 3. 1. EPR Spectrum of MnGaInTe4 Crystal In fig. 6 showing the EPR spectrum of MnGaInTe4 crystals, where only one broad peak is observed. Based on the composition of this phase, it is clear that this peak refers to the resonant frequencies of manganese. If in the structure the smallest distances between Mn atoms are Fig. 6. EPR spectrum for the MnGaInTe4 crystal more than 1 nm, then their EPR spectra are characterized by a fine structure consisting of six nearby lines. However, the decrease in the distances between these paramagnetic ions leads to the merging of these fine lines to a single broad peak.32 In our case, the distance between the Mn atoms is equal to the translational parameter of the cell a (a = 0.610293 nm), which is significantly less than 1 nm. Consequently, the EPR spectrum should consist of one broad peak, which was confirmed experimentally. 3. 3. 2. Raman Spectrum of MnGaInTe4 Crystal The Raman spectrum for the MnGaInTe4 crystal was represented in Fig.7. As can be seen three peaks at 96, 119 and 139 cm-1 are observed in the low-frequency region of the spectrum. Raman spectra of CdIn2Te4, ZnIn2Te4, and MnIn2Te4 were studied by authors of,.33 All these compounds crystallize in the tetragonal system and their structures such as MnGaInTe4 belong to the defective type of chalcopyrite.34 This closeness is well reflected in their Raman spectra (Table 6). As is seen from Table 6 the overall characteristics of the spectra of MnGaInTe4 are the same as its ternary analogs. Some distinction between the frequencies of the Raman-active modes of these materials is due to the difference in their chemical composition. Table 6. Raman spectra data for MnGaInTe4 and some of its structural analogs Phase Raman peaks, (cm 1) MnGaInTe4 96 119 139 MnIn2Te4 33 97 123 153 CdIn2Te4 33 102 128 159 ZnIn2Te4 33 100 123 143; 155 Fig. 7. Raman spectrum for the MnGaInTe4 crystal 3. 3. 3. Conductivity and Hall Effect of the MnGaInTe4 Crystal The temperature dependences of the electrical conductivity (Fig. 8a) and the Hall effect (Fig. 8b) for the Mn-GaInTe4 in the temperature range 100-400 K were studied. The value of electrical conductivity increases with increasing temperature. However, above 300 K, the electrical conductivity begins to increase more sharply. The sign of the Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 Acta Chim. Slov. 2019, 66, 466-472 471 a) 3 I 0.05, Fig. 8. The temperature dependences of the electrical conductivity (a) and the Hall Effect (b) for the MnGaInTe4 crystal Hall coefficient indicates the hole type of conductivity in the entire range of temperatures of 100-400 K. The hole concentration at room temperature calculated from measurements of the Hall coefficient is p = 1.25 • 1015 cm-3. The dependence RHan (T) passes through a maximum at the 140 K and then decreases with increasing temperature (Fig. 8b). In the literature, we did not find any information about the electrical conductivity and Hall Effect in Mn-Ga2Te4 and MnGaInTe4 compounds. Only,35 reports the energy gap width of MnGa2Te4 Eg is 1.52 eV at 300K, which is determined from optical measurements. The determination of Eg for MnGaInTe4 compounds from our measurements is impossible since its expected temperature region of intrinsic conductivity is much higher than 400 K. The presence of a maximum on the temperature dependence of the Hall coefficient for MnGaInTe4 may be explained by the impurity band existence. At temperatures above Tmax, charge transfer is performed by holes in the valence band. Below the maximum temperature, the transfer is carried primarily by thermally activated jumps between the acceptors. The decrease in the value of the Hall coefficient above Tmax with increasing temperature indicates an increase in the concentration of charge carriers. As a result, an increase in the value of electrical conductivity is observed (Fiq.8a). With increasing temperature, the activated electrons from the valence band are captured at these levels and become increasing of concentration of holes. The activation energy Ea has estimated from the temperature dependences of electrical conductivity was about 28 meV. 4. Conclusion The MnGa2Te4-MnIn2Te4 quasi-binary system is characterized by a phase diagram with dystectic and eutec-tic equilibria and the formation of broad regions of solid solutions with a monoclinic structure (0-12 mol% Mnln- 2Te4) and a defect structure of chalcopyrite (38-100 mol% MnIn2Te4). Using powder X-ray diffraction data by means of the Rietveld method the crystal structures of the Mn-GaInTe4 and MnIn2Te4 were refined. It was established, that both phases crystallize in the tetragonal system (Space group I-42m), but they differ significantly in the occupancy of the crystallographic positions. This allows characterizing MnGaInTe4 as an individual chemical compound, which is in accordance with the phase diagram. Comparison of Raman peaks of MnGaInTe4 with isostructural compounds MIn2Te4 (M = Zn, Mn, Cd) showed that the overall characteristics of the spectra are the same and differ only in values of the peaks frequencies. The observed EPR signal for MnGaInTe4, consisting of one broad peak, indicates the presence of long-range ordering in the arrangement of Mn atoms, which is in accordance with the crystallographic data. The Hall effect study was used to determine the type of conductivity and the concentration of holes in MnGaInTe4 crystals. 5. Acknowledgment This work was supported by the Science Development Foundation under the President of the Republic of Azerbaijan - Grant N EiF-BGM-4-RFTF-1/2017-21/11/ 4-M-12 6. References 1. G. K. Ahluwalia (Ed.), Applications of Chalcogenides: S, Se, and Te, Springer, 2016. 2. A. V. Kolobov, J. 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Ugotovili smo, da je ta sistem kvazi-binaren in ima karakteristična distetična in evtektična ravnotežja ter široka območja trdnih raztopin na osnovi izhodnih spojin. Kristalni strukturi MnGaInTe4 in MnIn2Te4 smo določili z Rietveldovo metodo z uporabo podatkov rentgenske praškovne difrakcije. Ugotovili smo, da obe fazi kristalizirata v tetragonalnem kristalnem sistemu (prostorska skupina I-42m). Kristale MnGaInTe4 smo preučevali tudi z elektronsko paramagnetno resonanco (EPR), ramansko spektroskopijo in meritvami električne prevodnosti v odvisnosti od temperature. Mammadov et al.: Phase Equilibria in the MnGa2Te4-MnIn2Te4 DOI: 10.17344/acsi.2019.5011 Acta Chim. Slov. 2019, 66, 473-783 ^creative ^commons Scientific paper Subcritical Water for Recovery of Polyphenols from Comfrey Root and Biological Activities of Extracts Jelena Vladic,1 Natasa Nastic,1 Tatjana Stanojkovic,2 Zeljko Zizak,2 Jelena Cakarevic,1 Ljiljana Popovic1 and Senka Vidovic1^ 1 Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia 2 Institute of Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia * Corresponding author: E-mail: senka.curcin@yahoo.com Received: 02-01-2019 Abstract In the present study, subcritical water was used for extraction of bioactive compounds of Symphytum officinale root. Temperature (120-200 °C), extraction time (10-30 min) and HCl concentration in extraction solvent (0-1.5%) were investigated as independent variables in order to obtain the optimal conditions for extraction and to maximize the yield of total phenols, flavonoids and antioxidant activity of obtained extracts. The application of optimal conditions (200 °C, 25.6 min and 0.0075%) provided extracts rich in total phenols and flavonoids and high antioxidant activity. Results also demonstrated that subcritical water extraction showed significant advantages for recovery of comfrey root bioactive compounds comparing to maceration and ultrasound-assisted extraction techniques. In addition, subcritical water extracts of S. officinale root are the promising sources of compounds with antioxidant, ACE inhibition, and antiproliferative properties and could potentially be used for production of new pharmacologically-active formulations. Keywords: Symphytum officinale root; Subcritical water extraction; Antioxidant activity; ACE inhibition activity; Anti-proliferative activity 1. Introduction Comfrey (Symphytum officinale L.), a member of the genus Boraginacae, is a well-known medicinal plant with large hairy leaves and small purple bell-shaped flowers, found predominantly in moist habitats especially beside river. Native in Europe and Asia, the plant has a striking record of medicinal use. The root and leaf have been used externally to treat a wide variety of ailments as well as internally as a decoction for oral and pharyngeal gargle or as a part of dietary supplements.1 Comfrey root is used by both traditional and modern herbalists for closing up joint inflammations, musculoskeletal injuries, inflamed breasts and wounds.2-4 Its pharmacological properties and clinical efficacy are ascribed mainly to allantoin, ros-marinic acid, mucopolysaccharides, triterpenoids and tannins.5 Allantoin is responsible for the stimulation of cell proliferation. It protects tissues and accelerates the healing process, with abundant growth of healthy granulation tissue in slowly healing suppurative wounds. The root of comfrey is also a good source of bioactive compounds including antioxidant polyphe- nols, A, B and C vitamins, steroidal saponins, proteins, inulin, calcium, potassium, copper, sulfur and selenium.6 Extraction of bioactive compounds from comfrey root has been investigated in the last few years focusing mainly on conventional solid/liquid extraction.7,8 Conventional extraction techniques, however, are quite laborious, time- and solvent-consuming.9 They present low efficiency, insufficient selectivity, and use of toxic solvents which further requires additional purification and consequently increases costs. The rising interest in the bioactive compounds and products on basis of herbal materials has resulted in increased demand of the industry to implement contemporary extraction technologies. These technologies aim to provide high yield and standardized quality of products, in a short amount of time. Therefore, a great deal of effort is invested into development of modern extraction technologies, such as ultrasound-assisted extraction (UAE) and subcritical water extraction (SWE), in order to overcome aforementioned disadvantages of classical technologies. Application of innovative technologies and optimization of Vladic et al.: Subcritical Water for Recovery of Polyphenols 474 Acta Chim. Slov. 2019, 66, 473-783 production processes to produce natural products has been in the focus of numerous scientific studies.10-14 Ultrasound-assisted extraction has been demonstrated as an advisable tool to recover polyphenols from plant materials, due to its cheapness and maintenance cost.15 Report by Wang et al. indicates that UAE reduces processing time, accelerates heat and mass transfer and requires a simple and easy to operate apparatus which helps in easy extraction of thermolabile compounds, without any degradation.16 The enhancement of extraction process by ultrasounds is attributed to the cavitation phenomenon and its consequent thermal and mechanical effects which can result in disruption of the cell walls, reduction of the particle size and enhanced mass transfer across cell membrane caused by the collapse of the numerous tiny bubbles. From the literature review, subcritical water extraction, as a potential alternative to conventional extraction techniques, has been successfully applied to extract target compounds from various matrices mainly due to its safety and compatibility with consecutive applications, environmental concerns and solvent recovery operations.17-21 SWE is based on the use of water at temperatures ranging from boiling point to the critical temperature and pressures high enough to keep the water in a liquid state throughout the extraction process. The change of the dielectric constant of water with temperature results in the possibility of tuning its polarity, for high-selectivity ex-tractions.22 The aim of this study was to optimize SWE of polyphenol antioxidants from S. officinale root. Response surface methodology and Box-Behnken experimental design were employed to investigate the effect of three process parameters (temperature, time and amount of added acidifier) on the extraction of targeted compounds. Their effect on total phenols and total flavonoids content, as well as on antioxidant activity, was evaluated. Investigated responses were compared in extracts obtained by conventional and novel extraction techniques (UAE, SWE). In addition, ACE inhibition and antiproliferative activities of extracts obtained by using contemporary SWE and UAE, and classical maceration extraction of S. officinale root were determined. 2. Materials and Methods 2. 1. Chemicals and Reagents 1,1 -Diphenyl-2-picryl-hydrazyl-hydrate was purchased from Sigma-Aldrich (Sternheim, Germany; CAS Number 1898-66-4). Folin-Ciocalteu reagent and gallic acid (CAS Number 149-91-7) were purchased from Merck (Germany). All other reagents used were either analytical or HPLC grade. 2. 2. Plant Sample All analysis was performed by using commercial dry S. officinale root supplied by a producer of medicinal plants (Chamomilla, Banatski Karlovac, Vojvodina, Serbia). 2. 3. Preparation of Extracts 2. 3. 1. Maceration Conventional solid-liquid extraction was performed with two different extraction solvents: methanol and ethanol solution (50%, w/w). All the extractions were performed by macerating 5.0 g of dry sample with 50 mL of extraction solvent at room temperature (25 °C) for 48 h in a shaker with temperature control (KS 4000i, IKA, Germany) at 150 rpm. After extraction, obtained extracts were filtrated through filter paper. Extracts were collected into glass vials and stored at 4 °C prior analysis. 2. 3. 2. Ultrasound-Assisted Extraction Ultrasound-assisted extraction was performed in sonication water bath (EUP540A, Euinstruments, France). The bath consisted of a rectangular container with frequency fixed at 40 kHz. The samples of dried powdered S. officinale root (5 g) were placed in a flask (250 mL) and mixed with 50 mL of selected solvent (methanol and 50% ethanol (w/w)). The sonication was performed at temperature of 30 °C and ultrasonic power of 60 W/L for 40 min. Flasks with condensers were always positioned in the same distance from the transducer in order to provide constant ultrasonic power. After extraction, extracts were filtered through filter paper under vacuum, collected into glass vials and stored in a dark place at 4 °C prior analysis. 2. 3. 3. Subcritical Water Extraction Subcritical water extraction (SWE) was carried out in a batch-type high-pressure extractor (Parr Instrument Company, USA) using the same experimental process previously described by Zekovic et al.23 For each run, the extraction cell was filled with 5 g of dried ground S. officinale root and 50 mL of double-distilled water. All SFE extractions were performed at 30 bar with different combinations of temperature (120-200 °C), extraction time (10-30 min) and concentration of HCl (0-1.5%). Obtained extracts were filtrated through filter paper under vacuum, collected into glass vials and stored in a dark place at 4 °C prior analysis. 2. 4. Analyses of Extracts 2. 4. 1. Determination of Total Phenols Content and Flavonoids Content Vladic et al.: Subcritical Water for Recovery of Polyphenols ... Acta Chim. Slov. 2019, 66, 473-783 475 The total phenolics (TP) content in obtained S. officinale extracts was determined spectrophotometri-cally using standard Folin-Ciocalteu procedure.24 Ab-sorbances of the samples were measured at 750 nm (6300 Spectrophotometer, Jenway, UK). The results are expressed as g of gallic acid equivalents (GAE) per 100 g dry weight (DW). The total flavonoids (TF) content in S. officinale extracts was determined using aluminium chloride colorimetric assay, as reported by Markham.25 The ab-sorbance for all tested extracts was measured at 510 nm. The content of total flavonoids was expressed as g of catechin equivalents (CE) per 100 g DW. 2. 4 .2. Determination of Antioxidant Activity-DPPH Assay Free radical scavenging activities of S. officinale subcritical water extracts were evaluated following the procedure previously reported by Espin et al.26 Antioxidant activity were expressed as IC50 value, defined as the inhibition concentration of the sample required to inhibit 50% of radical scavenging capacity (mg/ mL). 2. 4. 3. Analysis of Angiotensin-I-converting Enzyme Inhibition Activity Angiotensin-I-converting enzyme (ACE) inhibitory activity was measured following the assay described by Yoshie-Stark et al.27 The IC50 value of samples was defined as the inhibition concentration of the sample (in ^g/mL) required to inhibit 50% of ACE activity. 2. 4. 4. Cell Lines - Treatment Malignantly cell lines used in this assay (human cervix adenocarcinoma HeLa, breast carcinoma MDA-MB-453, human myelogenous leukemia K562 and human fetal lung fibroblasts MRC-5 cells) were obtained from the American Type Culture Collection (Manassas, VA, USA). All cancer cells were kept in the RPMI-1640 medium supplemented with 10% heat-inactivated (56 °C) fetal bovine serum (FBS), l-glu-tamine (3 mmol/L), streptomycin (100 mg/mL), penicillin (100 IU/mL) and 25 mM HEPES, and adjusted to pH 7.2 by bicarbonate solution. Upon reaching confluence, cells were cultured in an atmosphere of 5% CO2 and 95% relative humidity at 37 °C. 200 mg/ ml of Stock solutions of compounds were prepared in dimethylsulfoxide (DMSO), and afterwards diluted in RPMI-1640 medium. HeLa cells (2000 cells per well), MDA-MB-453 cells (3000 cells per well), and MRC-5 cells (5000 cells per well) were distributed in sterile 96-well microtiter plates, and were cultured for 20 h. Thereafter, the cells were treated with investigated extracts of various concentrations. The concentrations tested were in the range of 0.0625 to 2 mg/mL. In control wells, only nutrient medium was added to the cells. The investigated extracts were added to a suspension of K562 cells (5000 cells per well) 2 h after cell seeding, in the same final concentrations. The cultures were incubated for 72 h at 37 °C in a humidified incubator with 5% CO2. Determination of cell survival (MTT test) The influence of S. officinale extracts on the viability of malignantly transformed cell lines was determined by MTT (microculture tetrazolium test) following the procedure described by Mosmann28 with slight modification by Ohno and Abe,29 72 hours after the investigated extract was added. Briefly, 20 ^L of MTT solution (5 mg/mL in phosphate buffered saline (PBS)) was added to each well and plates were incubated for a further 4 h at 37 °C in 5% CO2 humidified atmosphere. After incubation, the MTT formazan crystals were dissolved by adding 100 ^l of 10% SDS to each well. The number of viable cells was measured by detecting absorbance in an ELISA microplate reader at a wavelength of 570 nm 24 h later. The cell survival (%) was calculated as absorbance of treated cells divided with absorbance of control cells and multiplied with 100. Cell growth inhibitions were expressed as IC50 values, defined as the concentrations of sample required to inhibit 50% of cell survival. All experiments were done in triplicate. 2. 5. Design of Experiments and Statistical Analysis Response surface methodology was employed to study the effect of independent variables temperature (X1, 120-200 °C), extraction time (X2, 10-30 min) and HCl concentration (X3, 0-1.5%) on the total phenols content, total flavonoids content and antioxidant activity parameters as responses. The Box-Behnken experimental design (BBD) was selected to propose the model for the investigated responses. The experimental design consisted of fifteen trials in random order, including three replicates at the central point. Table 1 shows the three independent variables encoded to three levels (-1, 0 and 1). Table 1. Box-Behnken experimental design with natural and coded levels of SWE parameters Independent variable Factor levels (-1) (0) (1) Temperature (°C) 120 160 200 Extraction time (min) 10 20 30 Concentration HCl (%) 0 0.75 1.5 Vladic et al.: Subcritical Water for Recovery of Polyphenols 476 Acta Chim. Slov. 2019, 66, 473-783 A second-order polynomial model (Eq. (3)) was fitted to results in order to correlate the relationship of each factor to the response:30 Y = ßo +Z ßiXi + ZbiiXii2 + ZbijXjXj (1) where Y represents the response variable, Xi, Xii and XiXj represent the linear, quadratic, and interactive terms of the coded independent variables, respectively, and (0, pi, (ii, and (3ij are the regression coefficients for intercept, linearity, quadratic, and interaction intercept terms, respectively. Analysis of variance (ANOVA) was carried out to identify the adequacy of the developed model with the significance levels of 0.05 (significant) and 0.10 (moderately significant). The coefficient of multiple determination (R2), coefficient of variance (CV) and p-values for the model and lack of fit coefficient were obtained from analysis of variance. The statistical software Design-Expert v.7 Trial (Stat-Ease, Minneapolis, Minnesota, USA) was employed for the all computation and graphics in this study. 3. Results and Discussion 3. 1. Statistical Analysis and Model Fitting In this study, a Box-Behnken design was carried out to optimize the procedure for the extraction of phenolic compounds using SWE. The investigated response values (TP and TF content, and antioxidant activity) for different combination of SWE parameters (extraction temperature, extraction time and HCl concentration) are presented in Table 2. The experimental results of SWE were fitted to a second-order polynomial model (Eq. (3)). Multiple regression coefficients for linear, interaction and quadratic terms were achieved using method of least square (MLS) (Table 3). Table 3. Estimated coefficients of second-order polynomial models for investigated responses Regression coefficient TP Response TF IC5 Intercept Po +3.99696 +1.43103 +0.016078 Linear Pi +2.02431* +0.60021* -9.04385 x 10-3* P2 +0.44419 +0.12192 -4.92939 x 10-4 P3 -0.13947 -0.39902* +2.44993 x 10-3* Interaction P12 +0.082186 +0.080163 +4.22203 x 10-4 Pl3 -0.63150 -0.41380* -9.44828 x 10-5 P23 -0.60841 -0.10725 +5.20550 x10-4 Quadratic P11 +0.21706 +0.14668 + 1.96614 x 10-3 P22 +0.22050 +0.27633* +3.11321 x 10-3 P33 -0.45832 +0.15653 -1.53053 x 10-3 R2a 0.9654 0.9738 0.9569 * Significant at 0.05 level a Coefficient of multiple determination Analysis of variance (ANOVA) was employed to check adequacy of the applied models and the results are summarized in Table 4. Statistical analysis suggested that the experimental values correlate well with the models. Good model fitness has been confirmed by highly significant p-values (p < 0.05) for all models. Table 2. Natural values of independent variables for Box-Behnken design and experimentally observed responses (TP, TF, IC50) in obtained extracts Run Independent variables Investigated responses order T (°C) t (min) c HCl (%) TP TF IC50 (g GAE/100 g) (g CE/100 g) (mg/mL) 1 160 10 1.5 3.24 1.33 0.0191 2 120 20 0 1.11 0.97 0.0203 3 120 10 0.75 2.16 1.23 0.0329 4 160 20 0.75 3.90 1.39 0.0160 5 160 20 0.75 3.91 1.49 0.0175 6 200 10 0.75 6.34 2.23 0.0105 7 200 30 0.75 6.87 2.64 0.0103 8 160 20 0.75 4.19 1.41 0.0147 9 200 20 0 6.13 3.04 0.0059 10 200 20 1.5 5.14 1.67 0.0125 11 160 30 0 5.49 2.62 0.0152 12 160 10 0 2.87 2.17 0.0171 13 120 20 1.5 2.65 1.26 0.0273 14 160 30 1.5 3.44 1.34 0.0192 15 120 30 0.75 2.37 1.32 0.0310 Vladic et al.: Subcritical Water for Recovery of Polyphenols ... Acta Chim. Slov. 2019, 66, 473-783 477 For all three investigated responses, mathematical models proved to be statistically acceptable due to insignificant lack of fit (p > 0.05). Validity of the models has been also confirmed by significant regression (p < 0.05). Furthermore, considering particularly high determination coefficients (R2 > 0.90) for the TP content, TF content, and IC50 values (0.965, 0.974 and 0.957, respectively), applied second-order polynomial model is well adjusted to the experimental results. Considering the obtained values, these data were used for creation of response surface 3D plots for each experimental model. Table 4. Analysis of variance (ANOVA) of the fitted second-order polynomial model for TP content, TF content and IC50 value Sum of squares DF Mean Square F-value p-value TP content Model 38.84 9 4.32 15.51 0.0038 T 32.78 1 32.78 117.84 0.0001 t 1.58 1 1.58 5.67 0.0630 c 0.16 1 0.16 0.56 0.4882 T x t 0.027 1 0.027 0.097 0.7679 T x c 1.60 1 1.60 5.73 0.0620 t x c 1.48 1 1.48 5.32 0.0692 T2 0.17 1 0.17 0.63 0.4649 t2 0.18 1 0.18 0.65 0.4583 c2 0.78 1 0.78 2.79 0.1558 Residual 1.39 5 0.28 Lack of Fit 1.34 3 0.45 16.28 0.0584 Pure Error 0.055 2 0.027 Cor Total 40.23 14 TF content Model 5.43 9 0.60 20.65 0.0019 T 2.88 1 2.88 98.63 0.002 t 0.12 1 0.12 4.07 0.0997 c 1.27 1 1.27 43.59 0.0012 T x t 0.026 1 0.026 0.88 0.3914 T x c 0.68 1 0.68 23.44 0.0047 t x c 0.046 1 0.046 1.57 0.2650 T2 0.079 1 0.079 2.72 0.1601 t2 0.28 1 0.28 9.65 0.0267 c2 0.090 1 0.090 3.10 0.1388 Residual 0.15 5 0.029 Lack of Fit 0.14 3 0.047 15.83 0.0600 Pure Error 5.905 x 10-3 2 2.952 x 10 3 Cor Total 5.58 14 IC50 value Model 7.664 x 10-4 9 8.515 x 10 5 12.30 0.0065 T 6.543 x 10-4 1 6.543 x 10 4 94.49 0.0002 t 1.944 x 10-6 1 1.944 x 10 -6 0.28 0.6189 c 4.802 x 10-5 1 4.802 x 10 5 6.93 0.0463 T x t 7.130 x 10-7 1 7.130 x 10 -7 0.10 0.7613 T x c 3.571 x 10-8 1 3.571 x 10 8 5.157 x 10-30.9455 t x c 1.084 x10-6 1 1.084 x 10 6 0.16 0.7087 T2 1.427 x 10-5 1 1.427 x 10 5 2.06 0.2106 t2 3.579 x 10-5 1 3.579 x 10 5 5.17 0.0721 c2 8.649 x10-6 1 8.649 x 10 6 1.25 0.3145 Residual 3.462 x 10-5 5 6.925 x 10 6 Lack of Fit 3.053 x 10-5 3 1.018 x 10 5 4.97 0.1721 Pure Error 4.097 x 10-5 2 2.049 x 10 6 Cor Total 8.010 x 10-4 14 Vladic et al.: Subcritical Water for Recovery of Polyphenols 478 Acta Chim. Slov. 2019, 66, 473-783 3. 2. Influence of Independent Variables on the Investigated Dependent Variables Total phenolic content observed in S. officinale root extracts obtained under different SWE conditions ranged from 1.11 to 6.87 g GAE/100 g DW. The highest TP content was observed at temperature of 200 °C, extraction time of 30 min and HCl concentration of 0.75%, while the lowest TP content was obtained at temperature of 120 °C and extraction time of 20 min without added acid modifier (Table 2). All SWE extracts obtained at 200 °C had significantly higher TP content comparing to extracts obtained at 120 and 160 °C. According to ANOVA analysis, the linear temperature terms had an important influence (p < 0.05), while other effects were not statistically important (Table 4). Based on the sum of squares, the importance of the independent variables on TP content was insignificant, as well as their interactions. Determination of regression coefficients using the least squares method provided the predictive model equation for TP extraction depicted in the Table 5. ed highly significant influence on TF content, while linear influence of extraction time was insignificant. Extraction temperature expressed the most significant positive effect on TF meaning that TF will significantly increase with increasing the temperature, which is in accordance with experimental results obtained for TP content. Linear term of HCl concentration exhibited negative influence on TF content, which imply that comfrey root flavonoids were better extracted if reduced acidifier concentration in extraction solvent were applied. Also, the interaction term between temperature and HCl concentration interaction affected TP content negatively. This means that elevated temperature and increased HCl concentration caused reduction in TF content, probably due to thermal degradation of flavonoid compounds on these severe extraction conditions. Quadratic term of extraction time exhibited significant positive influence on TF content. This suggested that an increase of extraction time increased the TF content of S. officinale extract. The response surface plots describing influences of SWE parameters on TF content of S. officinale root ex- Table 5. Model equations which are able to predict response values within investigated experimental domain Response Second-order polynomial equation TP content TF content IC50 value TP = 3.9969 + 2.0243 Xj TF = 1.43103 + 0.60021 Xj - 0.39902 X3 - 0.41380 Xj X3 + 0.27633 X22 IC50 = 0.0161 - 9.04385 x 10-3 Xj + 2.44993x 10-3 X3 The combined effect of the extraction parameters on the TP content of S. officinale root extracts obtained by SWE is presented in Figure 1a. The TP content increased linearly with temperature, while the extraction time did not influence this investigated response. The HCl concentration effect on phenols extraction of obtained extracts was rather insignificant. Similar findings for SWE of phenolic compounds were previously reported for other plant species.23,31 In subcritical water extraction, temperature is one of the most prominent operational parameter that affect the efficiency and selectivity of the process. Water at high temperatures breaks bonds between phenolics and cell wall releasing them more efficiently.32 Elevated temperatures enhance also the diffusion rate and desorption kinetics, resulting in a decrease in its viscosity and surface tension.22 A decrease in solvent surface tension allows better pore filling, thus permitting analytes to be better solubilised. Concerning the total flavonoids content, predictive second-order polynomial model equation, which is able to describe influence of SWE parameters on investigated response, is presented on Table 5. The linear terms of temperature and HCl concentration exhibit- tracts are shown in Figure 1b. TF content of extracts ranged from 0.97 to 3.04 g CE/100 g DW. The highest TF value was measured in the samples obtained at a temperature of 200 °C and extraction time of 20 min, but with the absence of HCl. Extracts obtained at 120 °C, for 20 min, without using acid modifier, exhibited the lowest TF content. Experimentally obtained values of antioxidant activity observed in S. officinale root extracts obtained by SWE are presented in Tables 2 and 4. The antioxidant activity of comfrey root extracts is not affected by extraction time, while the temperature and HCl concentration had a significant influence on this property, as it was the case with TF content. Linear terms of temperature displayed highly significant negative influence on investigated response (IC50 value). On the other hand, HCl concentration exhibited significant positive influence on IC50 value, which means that pure, or slightly acidified water should be used as extraction solvent. The final equation which could predict the behaviour of the investigated response is shown in Table 5. Depending on the extraction parameters antioxidant activity of extracts varied from 0.0059 to 0.0329 mg/ Vladic et al.: Subcritical Water for Recovery of Polyphenols ... Acta Chim. Slov. 2019, 66, 473-783 479 Figure 1. Response surface of TP content (a), TF content (b) and IC50 value (c) as simultaneous function of temperature, extraction time and HCl concentration mL. The highest antioxidant activity was measured in the extracts obtained at a temperature of 200 °C and extraction time of 20 min without using HCl modifier. On the other hand, extracts obtained at 120 °C for 10 min using 0.75% HCl as extraction solvent, showed the lowest antioxidant activity. Antioxidant activity significantly increased with the increase in extraction temperatures from 120 to 200 °C being in accordance with the content of polyphenols and fla-vonoids. These findings were in agreement with previously published studies on using SWE for the isolation of natural antioxidants where antioxidant activity increased with temperature up to 200 °C in SWE of deodorized thyme.33 He et al.34 also examined the effects of temperature on the antioxidant activity of pomegranate seed residues extracts. The highest antioxidant activities were seen when extraction was performed at temperature between 180 and 240 °C. The high antioxidant activity of extracts acquired at such relatively high temperature may have been related to the formation of new bioactive compounds during the extraction process via Maillard reactions.33 Visual representation of extraction parameters influence on antioxidant activity in comfrey root extracts is given in Figure 1c. Moreover, moderate correlation between IC50, TP and TF can be noticed, proving that polyphenols were the most important compounds responsible for antioxidant activity of S. officinale root. Regarding all these aspects, the efficiency of extraction of phenolic compounds and antioxidative activity of SWE extracts depend on temperature directly. Extraction time had a negligible effect on all three investigated variables, while HCl concentration affected TF content and antioxidant activity. Other authors that dealt with subcritical water extraction came to similar conclusion.17, 31 35 However, in order to gain complete information about extraction parameters effect on investigated responses during SWE of S. officinale root, further studies including chemical characterization of the extracts and the identification of responsible bioactive compounds are required. 3. 3. Optimization of Process Parameters Optimization of SWE process was the primary subject of this work, which allowed setting process parameters that maximized the value of all investigated Vladic et al.: Subcritical Water for Recovery of Polyphenols 480 Acta Chim. Slov. 2019, 66, 473-783 responses. Optimized extraction conditions for maximized yields of TP, TF, and minimized IC50 value, i.e. maximized antioxidant activity are presented in Table 6. By applying the response surface methodology and the multiple response optimization in the experimental domain investigated, the optimal process parameters of all three responses simultaneously of S. officinale root were determined to be as follows: temperature of 200 °C, extraction time of 25.6 min and 0.0075% HCl concentration in extraction solvent. Predicted values of investigated responses on these conditions were 7.26 g GAE/100 g DW, 3.40 g CE/100 g DW and 0.0058 ^g/mL, for TP, TF and IC50, respectively. Determination of optimal conditions and predicted values was based on desirability function, D = 1. The results showed that there was no significant difference observed between the predicted values and experimental ones, indicating that the model was adequate for reflecting the expected optimization. In addition, the extract obtained under optimal conditions was used for further analysis. Table 6. Optimized extraction conditions for maximized yields of TP and TF and minimized IC50 value Investigated variable TP TF IC50 Optimal Temperature 200 197.60 200 extraction Extraction time 29.2 29.9 21.1 condition c (HCl) 0.3375 0.075 0.0225 Optimal value 7.50 3.52 0.00527 3. 4. Comparison of Extraction Techniques Currently, literature data concerning S. officinale root extracts is very limited. In the present study, sub-critical water extracts of S. officinale root obtained under optimal conditions (200 °C, 25.6 min, and 0.0075% HCl) were compared to those obtained by conventional extraction and ultrasound-assisted extraction, where Figure 2. Total phenolic, total flavonoid contents of comfrey root extracts obtained by different extraction techniques ethanol (50%) and methanol were used as extraction solvents (Figure 2; Supplementary data, Table 1). SWE was found to be much more efficient for extraction of TP and TF from S. officinale root in comparison to maceration and UAE. Lower TP and TF contents were observed by Alkan et al.36 in methanolic and aqueous extracts of comfrey root obtained by Soxhlet extraction and decoction method. Furthermore, antioxidant activity obtained by SWE was significantly higher comparing to that obtained by maceration and UAE with ethanol and methanol. Moderately high antioxidant activity of S. officinale ethanolic extract (0.0397 mg/mL) was reported by Al-kan et al.36 According to the results from Figure 2, addition of methanol showed the lowest recovering capacity for bioactive compounds from comfrey root. Addition of ethanol to the water improved her capabilities to extract these bioactive compounds. But, by application of increased pressure and temperature, and transformation of water into the subcritical state, these capabilities were much more improved. These observations are related to dielectric constant of extraction solvent. In subcritical conditions, as the temperature of water is increased, the polarity of water decreases. That allows dissolving compounds of intermediate or low polarity. The dielectric constant of water decreases with the temperature (e.g., from e = 80 at ambient temperature to e = 27 at 250 °C) and becomes close to that of methanol (e = 33.6 at 25 °C) and ethanol (e = 24.5 at 25 °C).37 In addition, SWE process only needed 25.6 min to reach significantly higher values of investigated responses, while extraction time for maceration was 48 h and for UAE 40 min. This might be due to the dissolving properties of subcritical water, thus providing a much faster mass transfer. Given the advantages of used solvent, including its safety, selectivity and low cost, functionality, good yields of target compounds and reduced energy consumption, make this technique favorable for potential industrial applications. 3. 5. ACE Inhibition Activity Angiotensin-I-converting enzyme (ACE) is a glycosylated zinc dipeptidyl-carboxypeptidase whose main function is to regulate arterial blood pressure by converting angiotensin I to angiotensin II, a potent vasoconstrictor.38 Thus, inhibition of ACE has been considered as important in the chronic treatment of various cardiovascular diseases. A series of synthetic ACE inhibitors, which have been clinically used as antihypertensive drugs, cause some adverse side effects in humans.39 Last years of the twentieth century were marked with increased worldwide interest in herbal medicine and the development of new drugs based on naturally occurring biologically active compounds. Vladic et al.: Subcritical Water for Recovery of Polyphenols ... Acta Chim. Slov. 2019, 66, 473-783 481 Within this context, in the present study the extracts obtained by maceration, UAE, and SWE extract obtained under optimal conditions (temperature of 200 °C, extraction time of 25.6 min and HCl concentration of 0.0075%) were assayed for their ACE inhibitory activity. The results of the ACE inhibition activity of S. officinale extracts are depicted in Figure 3 (Supplementary data, Table 1). Figure 3. Antioxidant and ACE inhibition activity of comfrey root extracts obtained by different extraction techniques All samples exhibited ACE inhibition. The sub-critical water extract of comfrey root showed the highest ACE inhibitory with the IC50 being 0.18 ^g/mL. The lowest inhibitory activity was recorded for methanol extracts obtained by maceration (5.24 ^g/mL). Ethanol fractions showed higher ACE inhibition activities than did methanol fractions. This is the first report with ACE inhibition evaluation from this plant. Previous studies on the ACE inhibition of some other plant extracts obtained by different extraction techniques indicated their strong activity.40,41 According to Wagner et al.,42 the in vitro activity of some flavonoids was due to the formation of chelate complexes within the active centre of ACE. This chelate complex could be formed between a heterocyclic oxygen and a phenolic hydroxyl group in its vicinity. In studies reported by other authors, phenolic acids inhibited ACE via interaction with the zinc ion and this interaction was stabilized by other interactions with amino acids in the active site of ACE.31 Early works with S. officinale root extract indicated presence of some phenolic compounds such as rosmarinic acid, chlorogenic acid, caf-feic acid, rutin, kaempferol, apigenin, quercetin.7, 8 Some of these compounds such as caffeic acid,31 ru- 45 were tin,43 quercetin,44 apigenin and kaempferol, evaluated for their potency for ACE inhibition. The screening results of several plant methanolic extracts showed a low ACE inhibition activity of the isolated chlorogenic acid.32 Thus, based on the reported previous and present studies, it is feasible to suppose that the ACE inhibition activity of S. officinale root extracts may be due to its rich content of phenolic compounds. However, further research is needed to provide structural information of the ACE-inhibitory compounds from S. officinale root. In addition, the present findings indicated the efficiency and potential use of subcritical water extracts for the development of antihypertensive functional foods and nutraceuticals formulation that would be economical and a natural alternative therapy to commercial synthetic drugs. 3. 6. Antiproliferative Activity Research for new anticancer drugs with improved pharmacological profiles is a very active domain. Natural products represent an important source of new drugs. A number of studies have demonstrated the anticancerogenic effects of bioactive compounds isolated from various plants. In the current research, the influence of the extraction techniques on antiproliferative activity of comfrey root extracts was evaluated. MTT assay was used in the analysis of subcritical water extract obtained at optimal conditions and extracts obtained by maceration and UAE. Antiproliferative effects of extracts were determined for three human cancer cell lines: the human cervix carcinoma (HeLa), breast cancer cell line (MDA-MB-453) and chronic myelogenous leukemia cell line (K562), as well as toward normal human lung fibroblast (MRC-5). Calculated activities of the tested extracts are summarized in Table 7. Table 7. Antiproliferative activity of comfrey root extracts Extraction HeLa IC50 (mg/mL) MDA-MB-453 K562 MRC-5 Maceration Ethanol (50%) >2 >2 >2 >2 Methanol >2 >2 >2 >2 UAE Ethanol (50%) 0.719±0.028 >2 0.578±0.054 >2 Methanol >2 >2 >2 >2 SWE 0.337±0.005 0.319±0.028 0.209±0.027 0.483±0.005 Values are expressed as average ± standard deviation Vladic et al.: Subcritical Water for Recovery of Polyphenols 482 Acta Chim. Slov. 2019, 66, 473-783 Regarding antiproliferative effects, subcritical water extract of S. officinale root was the most toxic of all extracts screened (Table 7). The viability of cell lines was minimally affected by the extract obtained by maceration. The extract prepared by the SWE was the most active against K562 cell line (IC50 = 0.209 mg/mL). Moderate IC50 values could be explained by the presence or synergistic activities of polyphenolic compounds in the extracts since the number of studies have suggested that anticarcinogenic activity is closely associated with this group of secondary metabolites.46, 47 Qualitative phytochemical screening of S. officinale root have identified the presence of allantoin, ros-marinic acid, ellagic acid, caffeic acid, rutin, tannin, which are believed to contribute to the antiproliferative activity.7,8,36 Antiproliferative activity of plant extracts is also closely related to the extraction technique and the operational parameters applied during the process. Namely, subcritical water has much better selectivity in comparison to methanol and ethanol, being able to simultaneously extract different chemical classes. Therefore, by selecting the appropriate extraction parameters of subcritical water extraction, it is possible to influence the antiproliferative activity of extracts. 4. Conclusions The results of the present study demonstrate that subcritical water extracts of S. officinale root are the promising sources of compounds with antioxidant, ACE inhibition and antiproliferative properties. Applied second-order polynomial model provided adequate mathematical description of SWE of investigated responses: TP, TF and antioxidant activity. Optimization of extraction conditions, in order to provide maximum yields for the observed responses, was successfully performed. Numerical optimization determined the optimum extraction conditions to be temperature of 200 °C, extraction time of 26.5 min and HCl concentration of 0.0075%. Predicted values of investigated responses on these conditions were 7.26 g GAE/100 g DW, 3.40 g CE/100 g DW and 0.0058 ^g/mL, for TP, TF and IC50, respectively. Extraction temperature had notable influence on each investigated response, followed by effect of HCl concentration in extraction solvent. When compared to other extraction techniques, SWE indicated better bioactivity. According to the results, subcritical water extracts of S. officinale root can find application in the development and production of new safe, pharmacologically-active formulations. Taking into consideration the efficiency, functionality, and safety of the SWE process, present investigation also demonstrates the potency of subcritical water application in food and pharmaceutical technology. 5. References 1. F. Stickel, H. K. Seitz, Public Health Nutr. 2000, 3, 501-508. 2. M. Kucera, J. Kalal, Z. Polesna, Adv. Ther. 2000, 17, 204-211. D0I:10.1007/BF02850297 3. J. A. Duke, Chemicals and their biological activities, in: Symphytum officinale L. (Boraginaceae)-Comfrey, Phytochemical Database, USDA-ARS-NGRL, Beltsville Agricultural Research Center, Beltsville, MD, USA, 2002. 4. H. G. Predel, B. Giannetti, R. Koll, M. Bulitta, C. Staiger, Phy-tomed. 2005, 12, 707-714. D0I:10.1016/j.phymed.2005.06.001 5. R. 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Basilio Heredia, D. Sepulveda, E. M. Yahia, G. A. Gonzalez-Aguilar, Compr Rev Food Sci Food Saf. 2013, 12, 5-23. D01:10.1111/1541-4337.12005 14. R. G. Maroun, H. N. Rajha, N. El Darra, S. El Kantar, S. Cha-car, E. Debs, E. Vorobie, N. Louka, 2018. In Polyphenols: Properties, Recovery, and Applications (pp. 265-293). 15. H. F. Zhang, X. H. Yang, L. D. Zhao, Y. Wang, Innov. Food Sci. & Emer. Technol. 2009, 10, 54-60. D0I:10.1016/j.ifset.2008.09.007 16. J. Wang, Y. M. Zhao, Y. T. Tian, C. L. Yan, C. Y. Guo, Scientific World Journal, 2013. 17. J. Vladic, O. Canli, B. Pavlic, Z. Zekovic, S. Vidovic, M. Kaplan, J. Supercrit. Fluid. 2017, 120, 86-94. 18. M. Munir, H. Kheirkhah, S. Baroutian, S. Y. Quek, B. R. Young, J Clean Prod. 2018, 183, 487-494. D0I:10.1016/j.jclepro.2018.02.166 19. S. Er^an, O. G. Ustundag, R. Carle, R. M. Schweiggert, Food Chem, 2018, 253, 46-54. D0I:10.1016/j.foodchem.2018.01.116 20. J. H. Lee, M. J. Ko, M. S. Chung, J. Supercrit. Fluid. 2018, 133, 177-183. 21. K. S. Duba, A. A. Casazza, H. B. Mohamed, P. Perego, L. Fiori, Food Bioprod. Process. 2015, 94, 29-38. D0I:10.1016/j.fbp.2015.01.001 Vladic et al.: Subcritical Water for Recovery of Polyphenols ... Acta Chim. Slov. 2019, 66, 473-783 483 22. M. Herrero, A. Cifuentes, E. Ibañez, Food Chem. 2006, 98, 136-148. DOI:10.1016/j.foodchem.2005.05.058 23. Z. Zekovic, S. Vidovic, J. Vladic, R. Radosavljevic, A. Cvejin, M. A. Elgndi, B. Pavlic, J. Supercrit. Fluid. 2014, 95, 560-566. 24. M. P. Kähkönen, A. I. Hopia, H. J. Vuorela, J. P. Rauha, K. Pi-hlaja, T. S. Kujala M. Heinonen, J. Agric. Food Chem. 1999, 47, 3954-3962. DOI:10.1021/jf990146l 25. K. R. Markham, in: Harborne, J. B., Dey, P.M. (Eds.), Methods in Plant Biochemistry, Academic Press, London, 1989, pp. 193-237 26. J. C. Espín, C. Soler-Rivas, H. J. Wichers, J. Agric. Food Chem. 2000, 48, 648-656. DOI:10.1021/jf9908188 27. Y. Yoshie-Stark, Y. Wada, A. Wäsche, Food Chem. 2008, 107, 32-39. DOI:10.1016/j.foodchem.2007.07.061 28. T. Mosmann, J. Immunol. Methods. 1983, 65, 55-63. DOI: 10.1016/0022-1759(83)90303-4 29. M. Ohno, T. Abe, J. Immunol. Methods. 1991, 145, 199-203. DOI:10.1016/0022-1759(91)90327-C 30. M. A. Bezerra, R. E. Santelli, E. P. Oliveira, L. S. Villar, L. A. Escaleira, Talanta. 2008, 76, 965-977. DOI:10.1016/j.talanta.2008.05.019 31. A. Tomsik, B. Pavlic, J. Vladic, M. Cindric, P. Jovanov, M. Sakac, A. Mandic, S. Vidovic, J. Supercrit. Fluid. 2017, 128, 79-88. 32. R. K. Toor, G.P. Savage, Food Chem. 2006, 94, 90-97. DOI:10.1016/j.foodchem.2004.10.054 33. J. R. Vergara-Salinas, J. Pérez-Jiménez, J. L. Torres, E. Agosin, J. R. Pérez-Correa, J. Agric. Food Chem. 2012, 60, 1092010929. 34. L. He, X. Zhang, H. Xu, C. Xu, F. Yuan, Z. Knez, Z. Novak, Y. Gao, Food Bioprod. Process. 2012, 90, 215-223. DOI:10.1016/j.fbp.2011.03.003 35. A. Naffati, J. Vladic, B. Pavlic, R. Radosavljevic, A. Gavaric, S. Vidovic, J. Supercrit. Fluid. 2017, 121, 1-9. 36. F. U. Alkan, C. Anlas, O. Ustuner, T. Bakirel, A. B. Sari, Asian J. Plant Sci. Res. 2014, 4, 62-68. 37. M. Uematsu, E. U. Frank, J. Phys. Chem. Ref. Data. 1980, 9, 1291-1306. DOI:10.1063/1.555632 38. G. H. Li, G. W. Le, Y. H. Shi, S. Shrestha, Nutr. Res. 2004, 24, 469-486. DOI:10.1016/S0271-5317(04)00058-2 39. L. Paiva, E. Lima, A. I. Neto, J. Baptista, J. Funct. Foods. 2016, 26, 65-76. DOI:10.1016/j.jff.2016.07.006 40. N. Al Shukor, J. Van Camp, G.B. Gonzales, D. Staljanssens, K. Struijs, M. J. Zotti, K. Raes, G. Smagghe, J. Agric. Food Chem. 2013, 61, 11832-11839. DOI:10.1021/jf404641v 41. M. A. Lacaille-Dubois, U. Franck, H. Wagner, Phytomed. 2001, 8, 47-52. DOI:10.1078/0944-7113-00003 42. H. Wagner, G. Elbl, H. Lotter, M. Guinea, Pharm. Pharmacol. Lett. 1991, 1, 15-18. 43. H. M. Shaw, J. L. Wu, M. S. Wang, J. Funct. Foods. 2017, 35, 68-73. DOI:10.1016/j.jff.2017.05.033 44. H. Wagner, ACS Symposium Series. 1998, 691, 46-61. 45. L. Guerrero, J. Castillo, M. Quiñones, S. Garcia-Vallvé, L. Arola, G. Pujadas, B. Muguerza, PLOS one. 2012, 7, e49493. DOI:10.1371/journal.pone.0049493 46. S. Vuorela, K. Kreander, M. Karonen, R. Nieminen, M. Hämäläinen, A. Galkin, L. Laitinen, J.P. Salminen, E. Moila-nen, K. Pihlaja, H. Vuorela, J. Agric. Food Chem. 2005, 53, 5922-5931. DOI:10.1021/jf050554r 47. K. A. Lee, K. T. Kim, P. S. Chang, H. D. Paik. J. Ginseng Res. 2014, 38, 289-292. DOI:10.1016/j.jgr.2014.05.009 Povzetek Študija preučuje možnost uporabe vode v subkritičnih pogojih za ekstrakcijo bioaktivnih substanc iz korenine Symphytum officinale (navadni gabez). Preverili smo vpliv temperature (120-200 °C), časa ekstrakcije (10-30 min) in koncentracije HCl v ekstrakcijskem topilu (0-1.5 %), da bi določili optimalne pogoje ekstrakcije ter maksimirali izkoristek skupnih fenolov, flavonoidov ter antioksidativne aktivnosti ekstrakta. Z uporabo optimalnih pogojev (200 °C, 25.6 min in 0.0075 %) smo dobili ekstrakt bogat s skupnimi fenoli in flavonoidi ter visoko antioksidativno aktivnostjo. Rezultati kažejo izrazite prednosti uporabe subkritične vode za pridobivanje biokativnih substance iz korenin gabeza v primerjavi z maceracijo in ultrazvočno ekstrakcijo. Poleg tega pa ekstrakti korenine S. officinale pridobljeni z ekstrakcijo z vodo v subkritičnih pogojih predstavljajo obetaven vir snovi z antioksidativnimi, ACE inhibitornimi in antiproliferativnimi lastnostmi in bi lahko predstavljale potencialen vir za proizvodnjo novih farmakološko aktivnih formulacij. Vladic et al.: Subcritical Water for Recovery of Polyphenols DOI: 10.17344/acsi.2019.5019 Acta Chim. Slov. 2019, 66, 484-489 ¿^creative ^commons Scientific paper Synthesis, Crystal Structure and Antimicrobial Activity of a Linear Trinuclear Nickel(II) Complex with Schiff Base Ligand Cui-Lin Zhang,1 Xiao-Yang Qiu,1,2,* Shu-Juan Liu1 1 College of Science & Technology, Ningbo University, Ningbo, 315212, P. R. China 2 State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, P. R. China * Corresponding author: E-mail: xiaoyang_qiu@126.com Received: 02-03-2019 Abstract A new linear trinuclear Schiff base nickel(II) complex, [NijNiL^-n^^-OAcXOH^J-H^, where L is the dianionic form of N,N'-bis(5-chloro-2-hydroxybenzylidene)-1,3-propanediamine (H2L), was synthesized and characterized by elemental analyses, IR spectroscopy, and X-ray single-crystal determination. There are three bridges across the Ni-Ni atom pairs, involving two phenolate O atoms of a Schiff base ligand, and an O-C-O moiety of a ^2-rjl:rjl-OAc group. The Ni atoms have octahedral coordination. The acetate bridges linking the central and terminal nickel atoms are mutually trans. The adjacent Ni—Ni distances are 3.047(1) A. The complex was evaluated for its antibacterial (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) and antifungal (Candida albicans and Aspergillus niger) activities by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. Keywords: Nickel(II) complex; Trinuclear complex; Schiff base; Crystal structure; Antibacterial activity 1. Introduction Schiff bases are biological active compounds bearing the -N=CH- functional groups, which can be prepared by the condensation reactions of carbonyl-containing compounds with primary amines. The compounds have been attracted considerable attention for their wide range of biological activities, such as antibacterial,1 antifungal,2 antitumor,3 anti-inflammatory,4 and cytotoxic.5 Metal-organic complexes containing Schiff bases and bridging ligands are of current interest because of their interesting molecular topologies as well as the fact that they may be designed with specific functionalities.6 Among the bridging groups, acetate anion is highly flexible, and can coordinate to metal atoms in a variety of coordination modes such as mono-dentate, chelating, bidentate bridging, monoatomic bridging, and chelating bridging (Scheme 1).7 Bis-Schiff bases Monodentate Chelating Bidentate bridging Monoatomic bridging Chelating bridging ^1-nV ^2-n1:n1 2 0 ^2-n-n Scheme 1. Various coordination modes of acetate anion Zhang et al.: Synthesis, Crystal Structure and Antimicrobial ... Acta Chim. Slov. 2019, 66, 484-489 485 derived from salicylaldehyde and its derivatives with various diamines usually coordinate to metal atoms through NNOO donor atoms,8 thus, it is possible for the introduction of bridging groups. It was reported that Schiff bases bearing electron-withdrawing groups can improve their antimicrobial activities.9 Rai and co-workers reported a series of fluoro, chloro, bromo, and iodo-substituted compounds, and found that they have significant antimicrobial activities.10 A number of metal complexes have been reported with the bis-Schiff base N,N'-bis(5-chloro-2-hy-droxybenzylidene)-1,3-propanediamine (H2L; Scheme 2).11 As a continuation of work on the exploration of new antimicrobial agents, in this paper, a new trinuclear nickel complex was obtained. Scheme 2. H2L 2. Experimental 2. 1. General Methods and Materials 5-Chlorosalicylaldehyde and 1,3-propanediamine with AR grade were purchased from Sigma-Aldrich. All other chemicals (reagent grade) used were commercially available. Elemental analyses for C, H and N were performed on a Perkin-Elmer 240C elemental analyzer. IR spectra were recorded on a Nicolet AVATAR 360 spectrometer as KBr pellets in the 4000-400 cm-1 region. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance 300 spectrometer operating at 300 MHz for 1H NMR and 75.5 MHz for 13C NMR. Molar conductivity value was measured with a Shanghai DDS-11A conductome-ter. 2. 2. Synthesis of H2L 5-Chlorosalicylaldehyde (2.0 mmol, 0.31 g) was dissolved in methanol (20 mL), to which a methanol solution (10 mL) of 1,3-propanediamine (1.0 mmol, 0.074 g) was added dropwise with stirring at room temperature. The mixture was stirred at room temperature for 30 min, and most of the solvent was removed by distillation. The yellow crystalline product was obtained by filtration. Yield: 93% (0.33 g). IR data (cm-1, KBr): 3353w, 1645s, 1585s, 1487s, 1382w, 1329w, 1258s, 1213m, 1127w, 1033m, 985w, 865m, 831m, 772m. 1H NMR (300 MHz, DMSO) 5 12.33 (s, 2H, OH), 8.54 (s, 2H, CH=N), 7.58 (s, 1H, ArH), 7.37 (d, 1H, ArH), 6.92 (d, 1H, ArH), 3.73 (t, 4H, CH2), 1.99 (m, 2H, CH2). 13C NMR (75 MHz, DMSO) 6 158.77, 156.32, 134.57, 131.26, 124.81, 121.05, 117.73, 61.78, 32.50. Elemental analysis found: C, 58.32; H, 4.67; N, 7.91%, C17H16Cl2N2O2 calcd: C, 58.13; H, 4.59; N, 7.98%. 2. 3. Synthesis of the Complex H2L (0.2 mmol, 0.70 g) was dissolved in methanol (20 mL), to which a methanolic solution (20 mL) of nickel acetate tetrahydrate (0.3 mmol, 0.75 g) was added with stirring. The mixture was stirred at room temperature for 30 min to give a green solution, which was kept still to slow evaporate of the solvents. Green block-like single-crystals suitable for X-ray diffraction were formed. Yield: 45% (0.16 g). IR data (cm-1, KBr): 3451, 2910, 2862, 1637, 1580, 1464, 1416, 1385, 1306, 1164, 1075, 948, 859, 712, 549, 517, 465. Elemental analysis found: C, 43.77; H, 3.98; N, 5.27%, C38H40Cl4N4Ni3O11 calcd: C, 43.61; H, 3.85; N, 5.35%. 2. 4. X-ray Crystallography X-ray diffraction was carried out at a Bruker SMART 1000 CCD area diffractometer equipped with MoKa radiation (A = 0.71073 Â). The collected data were collected with SMART and reduced with SAINT,12 and multi-scan absorption correction was performed using SADABS.13 The structure of the complex was solved by direct method, and refined against F2 by full-matrix least-squares method using SHELXTL.14 All of the non-hydrogen atoms were refined anisotropically. The H5A and H5B atoms attached to O5 was located in a difference Fourier map and refined Table 1. Crystallographic information for the complex Parameter Value Formula C^C^N^On Fw 1046.67 T, K 298(2) Crystal system Monoclinic Space group P2Jc a, A 11.049(2) b, A 13.148(2) c, A 14.988(2) b, ° 90.971(1) V, A 3 2177.0(6) Z 2 Dc, g cm-3 1.597 F(000) 1072 Measured reflections 11692 Observed reflections [7 3 2s(7)] 4052 Data/restraints/parameters 2008/3/284 Goodness-of-fit on F 1.049 R„ wR2 [7 3 2s(7)]a 0.0642, 0.1684 Ru wR2 (all data)a 0.1429, 0.2024 a R1 = k\\Fo\ - \Fc\\/â\Fo\, wR2 = [kw(Fo2 - Fc2)2/âw(Fo2)2]1/2, w = [a2(Fo)2 + (0.091(Fo2 + 2Fc2)/3)2 + 0.5732(Fo2 + 2Fc2)/3]-1. Zhang et al.: Synthesis, Crystal Structure and Antimicrobial ... 486 Acta Chim. Slov. 2019, 66, 484-489 isotropically, with O-H and H—H distances restrained to 0.85(1) and 1.37(2) A, respectively. The remaining H atoms were placed in geometrically ideal positions and constrained to ride on their parent atoms. The O6 atom is disordered and its hydrogen atoms cannot be added reasonably. The crystallographic data for the complex are summarized in Table 1. Selected bond lengths and angles are given in Table 2. Table 2. Selected bond lengths (Â) and angles (°) for the complexes with estimated standard deviations (e.s.d.s) in parentheses Nil -Ol l.985(5) Nil-O2 2.0l8(4) Nil -Nl 2.009(5) Nil-N2 2.000(6) Nil -O3 2.05l(6) Nil-O5 2.248(8) Ni2 -O2 2.092(5) Ni2-Ol 2.ll7(4) Ni2 -O4 2.l2l(7) Ol- -Nil- N2 l70.4(2) Ol-Nil-Nl 9l.2(2) N2- -Nil- -Nl 96.l(2) Ol-Nil-O2 83.l3(l8) N2- -Nil- O2 89.0(2) Nl-Nil-O2 l72.0(2) Ol- -Nil- O3 92.2(3) N2-Nil-O3 94.0(2) Nl- -Nil- O3 90.3(2) O2-Nil-O3 95.5(2) Ol- -Nil- -O5 82.8(3) N2-Nil-O5 9l.5(3) Nl- -Nil- -O5 85.0(3) O2-Nil-O5 88.7(3) O3- -Nil- -O5 l73.l(2) O2-Ni2-OlA l0l.73(l8) O2- -Ni2- -Ol 78.27(l7) O2-Ni2-O4A 92.0(2) Ol- -Ni2- -O4A 90.4(2) O2-Ni2-O4 88.0(2) Ol- -Ni2- O4 89.6(2) Symmetry code for A: 1 - x, - y, - z. 2. 5. Antimicrobial Assay The antibacterial activity of the compounds was tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa using LB medium. The antifungal activities of the compounds were tested against Candida albicans and Aspergillus niger using RPMI-1640 medium. The IC50 (half inhibitory concentration) of the test compounds were determined by a colori-metric method using the dye MTT (3-(4,5-di-meth-ylth-iazol-2-yl)-2,5-diphenyltetrazolium bromide). A stock solution of the synthesized compound (1000 ^g mL-1) in DMSO was prepared and graded quantities of the test compounds were incorporated in specified quantity of sterilized liquid LB medium. Suspension of the microorganism was prepared and applied to 96-well assay plate with serially diluted compounds to be tested. 10 ^L of tested samples at pre-set concentrations were added to wells with penicillin G as a positive reference, the solvent control (5% DMSO) in medium and incubated at 37 °C for 24 h. After 24 h exposure, 10 ^L of PBS (phosphate buffered saline 0.01 mol L-1, pH = 7.4) containing 4 mg mL-1 of MTT was added to each well. After 4 h, the medium was replaced by 150 ^L DMSO to dissolve the purple formazan crystals produced. The absorbance at 492 nm of each well was measured with an ELISA plate reader. The IC50 value was defined as the concentration at which 50% of the bacterial strain could survive. 3. Results and Discussion The Schiff base ligand H2L was readily prepared by the condensation reaction of 5-chlorosalicylaldehyde with 1,3-propanediamine in 2:1 molar ratio. Recently, two mononuclear nickel complexes were prepared in a 1:1 molar ratio of nickel acetate and H2L under reflux in methanol.15 And also an acetate and phenolate bridged trinucle-ar nickel complex was prepared by nickel acetate with H2L under solvothermal condition.16 Interestingly, the present complex was prepared by the reaction of the Schiff base ligand with nickel acetate in a 2:3 molar ratio under ambient condition. Crystals of the complex are stable in air at room temperature, soluble in DMF, DMSO, MeOH, EtOH and MeCN, insoluble in water. The elemental analyses of the complex agree well with the component determined by X-ray analysis. The molar conductivity of the complex measured in DMSO/water at concentration of 10-3 mol L-1 is 25 O-1 cm2 mol-1, indicating the non-electrolytic nature of the complex in solution.17 3. 1. Crystal Structure Description of the Complex The molecular structure of the complex is shown in Fig. 1. The asymmetric unit of the compound contains half of a trinuclear complex. The molecule of the complex possesses crystallographic inversion center symmetry, with the inversion center located at the site of Ni2 atom. There are three bridges across the Ni—Ni atom pairs, involving two phenolate O atoms of a Schiff base ligand, and an O-C-O moiety of a ^2-^1:^1-OAc group. The acetate bridges linking the central and terminal nickel atoms are mutually trans. The trinuclear nickel complex molecule consists of two NiL units connected to each other by a completely encapsulated third metal atom, Ni2. The adjacent Ni1-Ni2 distance is 3.047(1) A. The cage of Ni2 is formed by phenolate bridges, O1 and O2, from the Schiff base ligands, and by two O atoms from two ^2-n1:n1-OAc ligands that furthermore connect the central metal with the two outer metal atoms resulting in an octahedral environment. The coordination around Ni2 atom displays only slight distortion. The bond distances Ni-O are relatively similar and range from 1.985(5) to 2.121(7) A. The greatest deviation of the bond angles from those expected for an ideal octahedral geometry is found for O1-Ni2-O2 with 78.3(2)°, and O1-Ni2-O2A with 101.7(2)°. The remaining bond angles are close to the ideal values for the octahedral coordination. The coordination around the inversion-related terminal Ni atoms is also octahedral, with two imino N and two phenolate O atoms from a Schiff base ligand defining Zhang et al.: Synthesis, Crystal Structure and Antimicrobial Acta Chim. Slov. 2019, 66, 484-489 487 the equatorial plane, and with two O atoms respectively from a methanol and a ^-n^^-OAc ligands occupying the axial positions. The coordination around the terminal metal atoms also displays slight distortion. The greatest deviation of the bond angles from those expected for an ideal octahedral geometry is O1-Ni1-O2 (81.9(2)°), which is Fig. 1. Molecular structure of the complex. Displacement ellipsoids are drawn at the 30% probability level and H atoms are omitted for clarity. Atoms labeled with the suffix A or unlabeled are related to the symmetry position 1 - x, - y, - z. caused by the strain created by the four-membered chelate ring Ni1-O1-Ni2-O2. The NiL units in the complex are butterfly-shaped, with the dihedral angles formed by the two benzene rings of the Schiff base ligands of 53.2(5)°. In the crystal structure of the complex, the water hydrate molecules are linked to the nickel complex molecules through O5-H5A—O6i, O5-H5B—Cl2" and C18-H18B-O6m hydrogen bonds [O5-H5A = 0.86 Â, H5A^^^O6i = 1.66 Â, O5-W = 2.518(5) Â, O5-H5A—O6i = 173°; O5-H5B = 0.85 Â, H5B-Cl2" = 2.48 Â, O5-Cl2ii = 3.251(5) Â, O5-H5B— Cl2ii = 151°; C18-H18B = 0.96 Â, H18B-O6iu = 2.36 Â, C18—O6iii = 3.207(5) Â, C18-H18B-O6m = 147°; symmetry codes: i: x, V - y, V + z; ii: 1 - x, -y, 1 - z; iii: 1 - x, V + y, V - z], forming chains along the b axis (Fig. 2). 3. 2. Infrared Spectra The IR spectra of the free Schiff base ligand and the nickel complex provide information about the metal-ligand bonding. The assignments are based on the typical group frequencies. The broad absorptions centered at 3353 cm-1 for H2L and 3451 cm-1 for the complex are generated by the v(O-H) of the hydroxy groups or methanol molecules. The strong absorption band at 1645 cm-1 for H2L is assigned to the azomethine groups, v(C=N). The band is shifted to 1637 cm-1 in the spectrum of the complex, what can be attributed to the coordination of the nitrogen atoms of the azomethine groups to the metal atoms. The phenolic v(Ar-O) for the free ligand exhibits medium band at 1213 cm-1.18 However, in the complex, the band appears at 1164 cm-1. This may be assigned to the skeletal vibrations related to the phenolic oxygen of the Schiff base ligands, and the bands are known to shift to lower frequency when the phenolic oxygen coordinates to metal atoms.19 There exhibit typical acetate vibrations v (OAc) at 1580 cm-1 and v (OAc) at 1464 cm-1.20 Fig. 2. Molecular packing diagram of the complex, viewed along the a axis. Hydrogen bonds are drawn as dashed lines. 3. 3. Antibacterial Activity The complex and the free Schiff base were screened for antibacterial activity against two Gram (+) bacterial strains (B. subtilis and S. aureus) and two Gram (-) bacterial strains (E. coli and P. aeruginosa) by MTT method. The IC50 values of the compounds against four bacteria are listed in Table 3. Penicillin G was used as the standard drug. The Schiff base H2L showed medium activity against the bacteria B. subtilis, S. aureus, and E. coli, while no activity against P. aeruginosa. The complex has strong activity against B. subtilis, medium activity against S. aureus, while no activity against the other two bacteria. From the results, it is difficult to give a definite conclusion about which one is good for the antibacterial activities of the free Schiff base and the complex. For example, the complex has stronger activities against B. subtilis and S. aureus than the Schiff base. However, as for E. coli, the Schiff base has stronger activity than the complex. The particular interest is that Zhang et al.: Synthesis, Crystal Structure and Antimicrobial ... 488 Acta Chim. Slov. 2019, 66, 484-489 Table 3. IC50 values (|ig mL ') of the tested material Tested material B. subtilis S. aureus E. coli P. aeruginosa C. albicans A. niger h2l 15.50 29.11 12.35 > 50 > 50 > 50 Complex 2.16 13.33 > 50 > 50 17.72 > 50 Penicillin G 2.38 0.71 18.23 16.31 > 50 > 50 Ketoconazole > 50 > 50 > 50 > 50 2.45 9.97 the complex showed the most effective activity against B. subtilis, which is even more effective than penicillin G. The antifungal activities of the complex and the free Schiff base were also evaluated against two fungal strains (C. albicans and A. niger) by MTT method. Ketoconazole was used as a reference drug. It is interesting that the complex has effective activity against C. albicans, with IC50 value of 17.72 ^g mL-1. 4. Conclusion In summary, a new linear trinuclear Schiff base nick-el(II) complex derived from N,N'-bis(5-chloro-2-hydroxy-benzylidene)-1,3-propanediamine was presented. Structure of the complex was confirmed by single-crystal X-ray determination. The Ni atoms are in octahedral coordination. The complex has interesting antibacterial activities against B. subtilis and S. aureus, and antifungal activity against C. albicans. 5. Supplementary Material CCDC-1507164 contain the crystallographic data for the complex. The data can be obtained at https://www. ccdc.cam.ac.uk or from the Cambridge Crystallographic Data Centre (CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033 or e-mail: deposit@ ccdc.cam.ac.uk. 6. Acknowledgments This work was financially supported by K.C. Wong Magna Fund in Ningbo University, Ningbo natural science fund (Project No. 201701HJ-B01019), State Key Laboratory Development Fund of Structural Chemistry and Ningbo Education Research Project (Project No. 2017YZD001). 7. References 1. (a) P. J. Low, Coord. Chem. Rev. 2013, 257, 1507-1532; DOI:10.1016/j.ccr.2012.08.008 (b) R. T. Acha, E. L. Gavey, J. Wang, J. M. Rawson, M. Pilking- ton, Polyhedron 2014, 76, 122-127; DOI:10.1016/j.poly.2014.04.001 (c) D.-L. Peng, N. Sun, Acta Chim. Slov. 2018, 65, 895-901; DOI:10.17344/acsi.2018.4543 (d) X. W. Zhu, Russ. J. Coord. Chem. 2018, 44, 335-339; (e) H. Y. Qian, X. W. Zhu, Russ. J. Coord. Chem. 2018, 44, 32-38. DOI:10.3103/S1068367418040213 2. A. Bienka, R. Kruszynski, D. Bienko, Polyhedron 2014, 75, 1-8. DOI:10.1016/j.poly.2014.02.045 3. (a) U. Kumar, J. Thomas, N. Thirupathi, Inorg. Chem. 2010, 49, 62-72; DOI:10.1021/ic901100z (b) R. Biswas, S. Mukherjee, P. Kar, A. Ghosh, Inorg. Chem. 2012, 51, 8150-8160. DOI:10.1021/ic300547w 4. (a) C. Biswas, M. G. B. Drew, E. Ruiz, M. Estrader, C. Diaz, A. Ghosh, Dalton Trans. 2010, 39, 7474-7484; DOI:10.1039/c0dt00331j (b) B. Cristovao, J. Klak, B. Miroslaw, Polyhedron 2012, 43, 47-54. DOI:10.1016/j.poly.2012.05.045 5. (a) F. Cimpoesu, F. Dahan, S. Ladeira, M. Ferbinteanu, J.-P. Costes, Inorg. Chem. 2012, 51, 11279-11293; DOI:10.1021/ic3001784 (b) M. L. Mejia, K. 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Bruker AXS Inc, Madison, 2002. 13. G. M. Sheldrick, SADABS. University of Göttingen, Germany, 1996. 14. G. M. Sheldrick, SHELXTL V5.1, Software Reference Manual, Bruker AXS Inc, Madison, 1997. 15. A. Elmali, C. T. Zeyrek, Y. Elerman, T. N. Durlu, J. Chem. Crystallogr. 2000, 30, 167-171. D0I:10.1023/A:1009526913565 16. Z.-L. You, M. Zhang, D.-M. Xian, Dalton Trans. 2012, 41, 2515-2524. D0I:10.1039/c1dt11566a 17. W. J. Geary, Coord. Chem. Rev. 1971, 7, 81-122. D0I:10.1016/S0010-8545(00)80009-0 18. G. C. Percy, D. A. Thornton, J. Inorg. Nucl. Chem. 1972, 34, 3357-3362. D0I:10.1016/0022-1902(72)80230-6 19. C. Fukuhara, E. Asato, T. Shimoji, K. Katsura, M. Mori, K. Matsumoto, S. Ooi, J. Chem. Soc. Dalton Trans. 1987, 13051311. 20. B.-H. Ye, X.-Y. Li, I. D. Williams, X.-M. Chen, Inorg. Chem. 2002, 41, 6426-6431. D0I:10.1021/ic025806+ Povzetek Sintetizirali smo nov linearni trijedrni nikljev(II) kompleks s Schiffovo bazo, [Ni{NiL(^2-^1:^1-OAc)(OH2)}2]-H2O, kjer je L dianionska oblika N,N'-bis(5-kloro-2-hidroksibenziliden)-1,3-propandiamina (H2L), in ga okarakterizirali z ele-mentno analizo, IR spektroskopijo in rentgensko monokristalno analizo. Trije mostovi povezujejo Ni-Ni atomske pare, in sicer preko dveh fenolatnih O atomov Schiffove baze in O-C-O skupine ^2-^1:^1-OAc liganda. Nikljevi atomi imajo oktaedrično koordinacijo. Acetatna mostova, ki povezujeta sredinski in terminalna nikljeva atoma, sta v medsebojnem trans položaju. Ni—Ni razdalja je 3.047(1) A. Kompleksu smo določili antibakterijske (Bacillus subtilis, Staphylococcus aureus, Escherichia coli in Pseudomonas aeruginosa) in antimikotične (Candida albicans in Aspergillus niger) lastnosti z uporabo MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolijev bromid) metode. Zhang et al.: Synthesis, Crystal Structure and Antimicrobial ... DOI: 10.17344/acsi.2019.5066 Acta Chim. Slov. 2019, 66, 490-500 ^creative ^commons Scientific paper Synthesis, X-Ray Structure Determination and Related Physical Properties of Thiazolidinone Derivative by DFT Quantum Chemical Method Youcef Megrouss,^* Fayssal Triki Baara,2 Nourdine Boukabcha,1 Abdelkader Chouaih,1 Antonis Hatzidimitriou,3 Ayada Djafri,2 Fodil Hamzaoui4 1 LTPS Laboratory, University Abdelhamid Ibn Badis - Mostaganem, 27000 Mostaganem, Algeria 2 Laboratory of Applied Organic Synthesis (LSOA), Department of Chemistry, Faculty of Sciences, University of Oran-1 Ahmed Ben Bella, 31000 Oran, Algeria 3 Department of General and Inorganic Chemistry, Faculty of Chemistry, Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece 4 LPFM Académie de Montpellier, France * Corresponding author: E-mail: youmeg@hotmail.fr Received: 02-20-2019 Abstract In this paper we report the synthesis and characterization of the (.Z)-3-N-(ethyl)-2-W-((3-methoxyphenyl)imino)thiazo-lidine-4-one by means of FT-IR, 'H and 13C NMR and by single crystal X-ray diffraction. The experimental determination of the crystal structure of the compound has been achieved using X-ray diffraction data. The important characteristic of the structure is the existence of a dihedral angle formed by the benzene and thiazolidinone rings being equal to 86.0° indicating an absence of n-n stacking as well as that the structure is non planar. In the crystal, the molecules are linked by C-H—O and C-H—N hydrogen bonds, these bonds being responsible for the three-dimensional molecular structure packing. In order to compare the experimental results with those of the theoretical calculation, quantum chemical DFT calculations were carried out using B3LYP/6-311G(d,p) basis set. In this context, the molecular electrostatic potential around the molecule and HOMO-LUMO energy levels were also computed. The dipole moment orientations were determined in order to understand the nature of inter- and intramolecular charge transfer. Finally, the stability of the title compound was confirmed throughout the calculation of the chemical reactivity descriptors. Keywords: X-ray diffraction; single crystal; DFT calculations; spectroscopy; FT-IR; 'H and 13C NMR. 1. Introduction Heterocyclic compounds are the major family of organic compounds; in medicinal chemistry thiazole derivatives are of great importance for their chemical and pharmacological properties, consequently thiazole derivatives have an extended range of pharmacological applications. Over the years, interesting biological activities were combined with thiazole derivatives.1-2 These materials are extremely necessary with wide range of synthetic, pharmaceutical and industrial applications and are famous for their biological activities.3,4 Recently, in drug development the application of thiazoles was required for the treatment of allergies,5 hypertension,6 inflammation,7 schizophrenia,8 bacterial9 and HIV infections,10 as hypnotics11 and more recently for pain treatment,12 as fibrinogen receptor antagonists with antithrombotic activity.13 In addition, thiazolid-inones and thiazoles present a very powerful activity as anti mycobacterium in tuberculosis.14 In this context, we have tried to realize the synthesis of the title compound, to characterize and perform its structural analysis as well as the theoretical density functional theory (DFT) calculations. In this work we present the synthesis, single crystal structure, IR and NMR spectroscopic characterizations as well as DFT calculations of this new thiazole derivative compound, namely the (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imi- Megrouss et al.: Synthesis, X-Ray Structure Determination and Acta Chim. Slov. 2019, 66, 490-500 491 no)thiazolidine-4-one. Furthermore, theoretical vibrational frequencies and and 13C NMR chemical shifts were calculated and compared to the experimental values. Additional parameters as molecular orbitals and chemical reactivity descriptors are evaluated in order to confirm the stability of the title compound. Finally, the molecular electrostatic potential was computed to determine electrophilic and nucleophilic regions of the title molecule. 2. Experimental 2. 1. Synthesis and Crystallization An equimolar solution of N-ethyl-3-W-(3-methoxy-phenyl)thiourea and ethyl bromoacetate in absolute etha-nol in the presence of sodium acetate was refluxed for 6 h. The solvent was removed by vacuum distillation and the residue was isolated, washed with cold water, filtered, dried and crystallized from ethanol to yield (Z)-3-N-(ethyl)-2-W-((3-methoxyphenyl)imino)thiazolidin-4-one as presented in the scheme 1. Scheme 1. Reaction sequence for the title compound synthesis. 2. 2. Spectral Data Measurements Infrared (IR) spectrum of the molecule (Z)-3-N-(ethyl)-2-N '-((3-methoxyphenyl)imino)thiazoli-dine-4-one was recorded in the range 500-4000cm-1 on a Nicolet FT-IR 6700 spectrometer using sample prepared as KBr pellets. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra of the molecule using CDCl3 as the solvent were recorded on Bruker AC250 spectrometer at 298 K. 2. 3. X-Ray Data Collection and Processing A transparent-yellowish parallelepiped crystal was selected and separated from the mother liquor, immediately cooled to 130 K and mounted on a Bruker Kappa APEX 2 diffractometer, equipped with a Triumph mono-chromator using MoKa radiation. The crystal presented no decay during the data collection. The frames collected (running ^ and « scans) were integrated with the Bruker SAINT Software package,15 using a narrow-frame algorithm. Data were corrected using the SADABS program.16 The structure was solved by the SUPERFLIP package.17 Crystals program package version 14.4018 has been used for the refinement and all the rest subsequent calculations through full-matrix least-squares on F2. All non-hydrogen atoms have been refined anisotropically. Hydrogen atoms were found at their expected positions and refined using proper riding constraints to the pivot atoms. Molecular illustrations were made through the MoPro-viewer crystal-lographic program.19 Crystallographic and experimental details are summarized in Table 1. Table 1. Crystallographic and experimental details. Crystal data Chemical formula C12H14N2O2S Mr 250.32 Crystal system Monoclinic Space group C2/c a (À) 23.7067 (19) b (À) 6.8884 (6) c (À) 15.7244 (12) P (°) 111.6504 (17) V (À3) 2386.66 (18) Z 8 || (mm-1) 0.26 Crystal size (mm) 0.31 x 0.28 x 0.19 Diffractometer Bruker Kappa Apex2 Absorption correction Numerical Analytical Absorption (De Meulenaer & Tompa, 1965) T ■ T 1 min 1 max 0.93, 0.95 Radiation type, X (À) Mo Ka, 0.71073 Temperature (K) 130 Measured 17268 Independent Observed [I > 2.0a(I)] 3006 2843 Rint 0.015 R[F2 > 2a(F2)] 0.029 wR(F2) 0.066 S 1.00 No. of reflections 2843 No. of parameters 154 APmax Apmin (e À~3) 0.33, 0.21 3. DFT Calculations In this theoretical study, the hybrid functional B3LYP with the 6-311G (d, p)20 basis set were used in all calculations by using the Gaussian 09 program,21 the X-ray structure was used as starting geometry to optimize a molecular structure of the investigation compound. Vibrational frequencies were calculated and the Gauss-view molecular visualization program22 and VEDA software23 were used for the assignment. Additionally, 1H and 13C NMR chemical shifts were calculated using the same level of theory. The theoretical calculation also allowed us to compute the energy values of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Furthermore, the molecular electrostatic Megrouss et al.: Synthesis, X-Ray Structure Determination and ... 492 Acta Chim. Slov. 2019, 66, 490-500 potential was calculated with B3LYP/6-311G(d, p) to highlight the electrophilic and nucleophulic attack sites. 4. Results and Discussion 4. 1. Structure Description The molecular geometry of the title compound is defined by the presence of two fragments, methoxyphenyl and thiazolidine rings forming a dihedral angle of 86°. Detailed results containing atomic positions and thermal parameters are given in the CIF file. Selected bond lengths, bond and torsion angles for all atoms by X-ray diffraction and theoretical calculations are listed in Tables 2, 3 and 4, respectively. An ORTEPIII diagram24 of the title compound showing the X-ray structure with thermal ellipsoids of the different atoms and the theoretical structure are given in Figure 1. In our study we employed full geometry optimization for the molecule without symmetry constraint. The results of our calculations showed that S1-C5, S1-C1, C1-N1, N1-C2, N1-C4, C6-N2 and O 2-C8 bonds exhibit single bond characteristics, while C4-O2 (1.23 A) and N2-C1 (1.26 A) bonds show typical double bond characteristics.25 The amine N2 atom exhibits a geometry that is typical for an sp2 rather than an sp3 atom hybridisation. In addition, the difference in the thiazole ring bonds between theoretical calculation and experimental values does not exceed 0.13 A. Bond angles C6-N2-C1, C2-N1-C4 and C1-N1-C4 are also near 120° (119.56°, 121.08° and 116.72°, respectively).26 Figure 1. Experimental (a) and theoretical (b) structure of (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imino)thiazolidine-4-one. The bond angles centered on C1 are all between 109° and 129°. The C1 atom is of sp2 hybridization type because the total adds up to 360° at B3LYP/6-311G(d,p) level. The corresponding theoretical values of this angle is 64° (C7-C6-N2-C1). The C8-O2-C12 angle is 116.98°, and the C2-N1-C4-O1 fragment is approximately planar (Table 4). Generally, the observed difference between experimental and calculated geometrical parameters does not seem very large. Table 2. Experimental and optimized bond lengths (Ä). Bond lengths X-ray B3LYP/6-311G(d,p) S1-C5 1.811(1) 1.887 S1-C1 1.767(1) 1.893 O1-C4 1.216(1) 1.233 N1-C2 1.469(1) 1.485 N1-C1 1.399(1) 1.392 N1-C4 1.371(1) 1.388 N2-C6 1.425(1) 1.407 N2-C1 1.263(1) 1.264 O2-C8 1.367(1) 1.386 O2-C12 1.431(1) 1.459 C6-C7 1.389(1) 1.398 C6-C11 1.402(1) 1.408 C7-C8 1.398(1) 1.401 C5-C4 1.509(1) 1.527 C2-C3 1.518(1) 1.537 C10-C9 1.393(1) 1.399 C10-C11 1.386(1) 1.387 C8-C9 1.395(1) 1.397 Table 3. Experimental and optimized bond angles (°). Bond angles X-ray B3LYP/6-311G(d,p) C5-S1-C1 92.24 90.13 C2-N1-C1 122.15 120.13 C2-N1-C4 121.08 119.58 C1-N1-C4 116.72 29.83 C6-N2-C1 119.56 129.18 C8-O2-C12 116.98 117.97 N2-C6-C7 119.43 124.07 N2-C6-C11 119.94 116.82 C7-C6-C11 120.34 119.08 C6-C7-C8 119.87 120.72 S1-C5-C4 107.51 108.27 N1-C2-C3 112.36 111.42 S1-C1-N1 110.92 109.07 S1-C1-N2 127.32 129.12 N1-C1-N2 121.76 121.81 O1-C4-N1 123.71 123.73 O1-C4-C5 123.91 124.08 N1-C4-C5 112.37 112.18 C9-C10-C11 121.52 120.98 O2-C8-C7 115.23 115.22 O2-C8-C9 124.48 124.87 C7-C8-C9 120.27 119.91 C10-C9-C8 119.01 119.34 C6-C11-C10 118.98 119.96 Megrouss et al.: Synthesis, X-Ray Structure Determination and Acta Chim. Slov. 2019, 66, 490-500 493 Table 4. Experimental and optimized torsion angles (°). a) Torsion angles X-ray B3LYP/6-311G(d,p) C1-S1-C5-C4 3.6 2.4 C5-S1-C1-N1 -4.7 -2.4 C5-S1-C1-N2 174.5 176.7 C1-N1-C2-C3 -99.6 -86.2 C4-N1-C2-C3 77.9 92.2 C2-N1-C1-S1 -177.7 -179.7 C2-N1-C1-N2 3.1 1.1 C4-N1-C1-S1 4.7 1.8 C4-N1-C1-N2 -174.6 -177.3 C2-N1-C4-O1 -0.2 1.7 C2-N1-C4-C5 -179.5 -178.4 C1-N1-C4-O1 177.4 -179.9 C1-N1-C4-C5 -1.9 0.1 C1-N2-C6-C7 86.0 64.5 C1-N2-C6-C11 -100.2 -119.3 C6-N2-C1-S1 1.6 4.4 C6-N2-C1-N1 -179.2 -176.7 C12-O2-C8-C7 -170.8 179.8 C12-O2-C8-C9 10.9 -0.1 N2-C6-C7-C8 172.3 178.5 C11-C6-C7-C8 -1.5 0.6 N2-C6-C11-C10 -173.1 -179.4 C7-C6-C11-C10 0.6 -1.3 C6-C7-C8-O2 -177.2 -179.5 C6-C7-C8-C9 1.3 0.4 S1-C5-C4-O1 178.9 178.06 S1-C5-C4-N1 -1.8 -1.9 C11-C10-C9-C8 -0.8 -0.1 C9-C10-C11-C6 0.5 1.1 O2-C8-C9-C10 178.2 179.2 C7-C8-C9-C10 -0.1 -0.7 b) 4. 2. Hydrogen Bonding Hydrogen bonds join chains of molecules to stabilize the crystal structure of the title compound. Along the b axis in the unit cell, the translation of equivalent molecules allows the linking of the almost linear hydrogen bonding. C-H—O and C-H—N intra- and intermolecular interactions are present in the crystal structure. The molecular conformation is in part influenced by the formation of two weak intramolecular C2-H22—O1 and C2-H21—N1 hydrogen bonds that enclose S(5) rings (Figure 2 and Table 5). Figure 2. Hydrogen bonding view in the crystal showing: (A) intermolecular interactions, (B) C2-H21—N2 and C2-H22—O1 intramolecular interactions. These interactions are responsible for the stability of the molecular packing, as the C2, C3, C5, C12 and C7 carbon atoms act as donor groups and both oxygen and nitrogen atoms play the acceptor role. Hydrogen bond interactions are presented in Table 5. Figure 2 shows all interaction types in the crystal. The molecular stacking that has been provided by the different existing hydrogen bonds in the Table 5. Geometry of the C-H—O and C-H—N hydrogen bonds in (2)-3-N-(ethyl)-2-N'-((3-methoxyphe-nyl)imino)thiazolidine-4-one crystal by X-ray diffraction. D-H-A D-H (Á) D-A (Á) H-A (Á) D-H-A (°) Bond type C2-H21-N2 0.981 2.852 2.453 103.88 Intramolecular C2-H22-O1 0.962 2.817 2.582 93.97 Intramolecular C5-H51-O1(i) 0.987 3.380 2.489 149.92 Intermolecular C7-H7-O1(íí) 0.947 3.432 2.508 164.98 Intermolecular C5-H52-N2fiiiJ 0.955 3.370 2.514 149.26 Intermolecular Symmetry codes: (i) -x, y, -z + 1/2; (ii) -x, -y, - -z; (iii) x, -y, z + 1/2 Megrouss et al.: Synthesis, X-Ray Structure Determination and ... 494 Acta Chim. Slov. 2019, 66, 490-500 Figure 3. View of the crystal packing along the b axis of (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imino)thiazolidine-4-one molecule. 4. 3. 2. C=O and C=N Vibrations Usually, the C=O stretching vibration mode can be easily observed as a strong band in the region 1850-1550 cm-1.30 The n-n bonding between carbon and oxygen is responsible for forming a double bond between the carbon and oxygen atoms. The electronic distribution in this link is not equal because these atoms have different electronegativities. The lone pair of electrons on oxygen is responsible for the polar nature of the carbonyl group. In the present study, the single C=O stretching vibration mode was observed as a high-intense peak in FT-IR at 1716 cm-1 while the calculated value shows at 1726 cm-1 with a PED (potential energy distribution) of 79%. The stretching frequency of the double bond C=N is perfectly observed at the high absorption 1640 cm-1. DFT/B3LYP functional with 6-311G(d,p) basis set gives exactly the same value of 1640 cm-1 for the C=N vibration. crystal is shown in Figure 3. This figure shows the existence of eight molecules in the unit cell which is in good agreement with the multiplicity of the space group C2/c. 4. 3. Vibrational Frequencies Assignments Using analytic second derivatives to validate the convergence to minima on the potential energy surface the fundamental frequencies of the studied molecule have been calculated to understand the nature of these modes of vibration IR absorption spectroscopy based on DFT calculation. A theoretical analysis has been realized using B3LY-P/6-311G(d,p) level of theory in gas phase. The probable assignments were performed by means of VEDA 4 pro-gram.23 The vibrational frequencies obtained from B3LYP functional calculations have been scaled by a factor of 0.967.27 Table 6 shows the calculated (unscaled and scaled) and experimental frequencies of the title compound. Simulated and experimental IR spectra of (Z)-3-N-(ethyl)-2-W-((3-methoxyphenyl)imino)thiazolidine-4-one are shown in Figure 4. As can be seen in Figure 4, the experimental fundamentals are in better agreement with the scaled fundamentals. 4. 3. 1. Carbon-Hydrogen Vibrations In the aromatic compounds, multiple weak bands are exhibited in the region of 3100-3000 cm-1.28 They appear in this range of like multiple weak bands due to the stretching vibrations of C-H.29 In the present work, the carbon-hydrogen of aromatic ring (C-H) stretching (vCH modes) were found in a range of 3000 and 3100 cm-1 using the B3LYP/6-311G(d,p) calculations. These modes involve exact contribution of >91% suggesting that they are pure stretching modes. The C-H stretching of aromatic ring showed symmetric modes at 3001, 3070 and 3080 cm-1 while asymmetric stretching mode was observed at 2989 cm-1. 4. 3. 3. Thiazolidine Ring Vibrations The C-S stretching vibration presents average bands in the region 1020-1010 cm-1.31 In this paper C-S vibrations were observed at 704, 647 and 526 cm-1. The examination of the theoretical results gives us the following values with their PED contribution: 720 (21%), 642 (10%), 522 (12%) and 482 (23%) respectively, which shows a good agreement between theoretical and experimental ones as shown in the Table 6. C-N vibration is a difficult task to identify since the appearance of several bands is possible in the region, Gunasekaran et al?2 have observed C-N stretching band at 1312 cm-1,33 and C-N assigned stretching vibration in the region 1350-1000 cm-1 for amines. In thiazolidine, C-N stretching band is found to be present at 1382 and 1307 cm-1. The C-N stretching vibrations are expected to occur in the region 1200-1130 cm-1.34 In our present work of the title molecule FTIR bands were observed at 896 cm-1 and 1234 cm-1, the theoretically calculated bands at 940, 1405, 1357, 1301, 1257, 1131 are shifted Figure 4. Comparison of FT-IR and calculated IR Spectra for (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imino)thiazolidine-4-one. Megrouss et al.: Synthesis, X-Ray Structure Determination and 495 Acta Chim. Slov. 2019, 66, 490-500 Table 6. Comparison of the calculated harmonic frequencies using B3LYP method 6-311G(d,p) basis set and experimental (FT-IR) wavenumbers (cm-1) for (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imino)thiazolidine-4-one. No Experi- B3LYP/6-311G(d,p) Assignments with PED>10% mental Unscaled Scaled Iir 87 3209 3080.64 8.75 v CH (91%) Ar ring 86 3198 3070.08 0.77 v CH (92%) Ar ring 85 3127 3001.92 9.61 v CH (100%) Ar ring 83 3139 3013.44 9.25 v CH (79%) + vas CH (11%) 81 3127 3001.92 0.33 v CH (100%) 80 2971 3114 2989.44 13.61 vas CH (92%) 74 3001 2880.96 61.94 v CH (92%) 73 1716 1798 1726.08 182.70 v C=O (79%) 72 1640 1709 1640.64 943.72 v N=C (73%) 71 1640 1574.4 329.64 v CCasy (63%) Ar ring +5 HCC (14%) Ar ring 70 1617 1552.32 93.33 v CC (51%)asy Ar ring+ 5 HCC (11%) Ar ring+ 5 CCC (12%) Ar ring 69 1482 1518 1457.28 72.82 5 HCC asy (44%) Ar ring+ 5 CCC (19%) Ar ring 68 1508 1447.68 33.99 5 HCH (67%)+ 67 1506 1445.76 3.59 5 HCH(71%)+ rHCCNasy (22%) Th ring 66 1498 1438.08 8.56 5 HCH(71%)+ rHCCNasy (24%) Th ring 65 1492 1432.32 7.71 5 HCHasy(69%)+ rHCOCasy(24%) Ar ring 64 1482 1422.72 0.82 5 HCH(48%) 62 1465 1406.4 40.95 v CCasy (31%) Ar ring+ 5 HCC (31%) Ar ring+ 5 HCCasy (16%) 61 1462 1403.52 8.14 5 HCH (88%) 60 1391 1420 1363.2 18.06 5 HCC (13%)+ 5 HCH (15%)+ tHCNC (46%) Th ring 59 1405 1348.8 110.90 v NC(13%) Th ring+ 5 HCC (10%)+ 5 HCH (39%)+ rHCNCasy (10%) Th ring 58 1401 1344.96 12.94 5 HCC (56%)+ 5 HCH (14%) 57 1357 1302.72 247.21 v NC(46%) Th ring 56 1284 1350 1296 24.41 v CCasy (59%) Ar ring+ 5 HCC (23%) 55 1312 1259.52 111.70 v COasy(79%) Ar ring+ 5 HCC (37%) Ar ring 54 1234 1301 1248.96 13.79 v CC (10%) Ar ring+ v NQh(12%) Ar ring+ 5 HCC (11%) Ar ring+ 5 HCHasy (14%) 53 1257 1206.72 43.11 v NCasy (16%)+ Th ring +v CO(13%) Ar ring+ tHCCN (12%) Th ringasy 52 1253 1202.88 25.58 tHCSC asy(84%) Th ring 51 1212 1163.52 40.44 5 HCHasy(15%)+ tHCOC(44%) Ar ring 50 1148 1194 1146.24 22.35 v CC (10%) Ar ring+ 5 HCC (38%) Ar ring+ rHCOCasy(14%) Ar ring 49 1172 1125.12 45.03 5 HCH(21%)+ tHCOC(35%) Ar ring 48 1171 1124.16 101.55 5 HCCasy (13%) Ar ring+ rHCOCasy(17%) Ar ring 47 1148 1102.08 3.08 5 HCSasy (52%) Th ring+ tHCSC(28%) Th ring 46 1131 1085.76 146.72 v NCasy (15%) Th ring+ v CC(11%) Th ring 45 1111 1066.56 17.22 5 HCHasy(12%)+ 5 CCNasy (13%) Th ring+ tHCCN (36%) Th ringasy 44 1109 1064.64 12.00 v CC(14%) Ar ring+ 5 HCC(33%) ar ring+ 43 1046 1075 1032 63.45 v OC(55%) Ar ring+ 5 HCC(17%) Ar ring 41 1007 966.72 0.82 v CCasy (52%) Ar ring+ 5 CCCasy(39%) Ar ring 40 973 934.08 4.63 v CCasy (33%) Th ring 39 927 968 929.28 0.03 tHCCC (77%) Ar ring 38 896 940 902.4 27.82 v NC(10%) Ar ring+ v OCasy(10%) Ar ring+ v CCasy (27%) Th ring 37 922 885.12 7.21 5 HCS(45%) Th ring+ tHCSC (21%) Th ring+ TCCNCasy(19%) Th ring+ Y OCNCasy(15%) 36 891 855.36 29.40 v CCasy (12%) Th ring+ 5 NCNasy (10%)+ tHCCC (12%) Ar ring 35 872 837.12 7.58 tHCCC (68%) Ar ring 34 863 828.48 5.52 tHCCC (68%) Ar ring 33 775 791 759.36 5.21 5 NCNasy (32%) Th ring+ tHCNC (33%) Th ring 32 782 750.72 30.71 tHCCC (17%) Ar ring+ THCCCasy (17%) Ar ring+TCCCCasy(29%) Ar ring 31 740 779 747.84 20.41 5 CNC (27%) Th ring 30 704 751 720.96 8.75 v CCasy (10%) Ar ring+ v CC(10%) Th ring+ v SC(21%) Th ring 29 705 676.8 22.45 THCCCasy (56%) Ar ring+ TCCCCasy(10%) Ar ring 28 647 669 642.24 0.28 v SC(10%) Th ring+ THCCCasy (12%) Ar ring+ tCCCC(43%) ar ring 27 609 643 617.28 4.95 v CC(28%) Th ring+ 5 CCCasy(11%) Ar ring Megrouss et al.: Synthesis, X-Ray Structure Determination and ... 496 Acta Chim. Slov. 2019, 66, 490-500 26 617 592.32 4.44 ySNNC asy(54%) Ar ring 25 584 610 585.6 5.88 5 CNCasy (11%) Th ring 24 593 569.28 17.41 5 CCCasy (14%) Ar ring+ yOCNC(19%) Th ring 23 581 557.76 1.53 rHCSCasy (16%) Th ring+ yOCNCasy (30%) Th ring 22 526 544 522.24 3.20 v SC(12%) Th ring+ 5 CCC (25%) Ar ring+ tCCCC (12%) Ar ring 21 503 482.88 23.21 v SCasy (23%) Th ring+ 5 CCNasy(14%) Ar ring 20 484 464.64 0.63 5 CNCasy(10%) Ar ring+ 5 CCOasy(32%) Ar ring 19 471 452.16 16.37 5 CNCasy(19%) Th ring+ 5 CCNasy(16%) Th ring+ 5 CCOasy(12%) Th ring 18 454 435.84 1.08 5 CCOasy(11%) Ar ring+ rCCCCasy (36%) Ar ring 17 403 386.88 2.62 5 CCC(12%) Ar ring+ 5 COCasy (21%) Ar ring v: stretching(elongation); sy: symmetric; asy: asymmetric; in plane bending; y: out-of-plane bending, w: wagging; t: twisting; ô: bending; p: rocking; vibrational modes are based on potential energy distribution (PED) and only contributions over 10% are given; scaled frequencies are in unit of cm-1; IIR infrared intensities are in unit of km mol-1. down by scaling the previous values to 902 (10%), 1348 (13%), 1302 (46%), 1248 (12%), 1206 (16%), 1085 (15%), respectively with the percentage of the PED contribution indicated between brackets. 4. 4. 1H and 13C NMR Calculations To calculate isotropic chemical shifts (5) with respect to tetramethylsilane (TMS): 5xiso = aTMSiso - a xiso,34 isotropic shielding values 184.2796 and 32.2899 ppm of aTMSiso were used for C and H NMR spectra, respectively. It is known that the range of 13C NMR chemical shifts for analogous organic molecules usually are >100 ppm.35,36 Methanol solvent has been used for chemical shift measurements. The atoms were labeled according to the numbering presented in Figure 1 (B). Calculated and experimental values for 1H and 13C NMR are collected in Table 7. In this research, aromatic carbons give signals in overlapped areas of the spectrum with chemical shift values from 106 to 170 ppm while experimental chemical shift values of aromatic carbon atoms are in the range 107 to 160 ppm as they would be expected (Table 7). Carbon atoms (C1, C4 and C6) attached to the N atom have larger chemical shifts due to the more electronegative property of the N atom which polarizes the electron distribution in its binding to the atom adjacent carbon and reduces the value of the chemical shifts. Usually, the chemical shifts obtained and calculated for the 1H atoms of the methyl groups are quite weak. The maximum chemical shift value for all the hydrogen atoms is 7.24 ppm. 4. 5. Frontier Molecular Orbitals (FMOs) The highest occupied molecular orbitals (HOMO) and the lowest unoccupied molecular orbitals (LUMO) are named as frontier molecular orbitals (FMO). The HOMO represents the ability to donate an electron, whereas LUMO as an electron acceptor represents the ability to obtain an electron.37 The calculation of these parameters is very important because it allowed us to verify a lot of properties, such as the kinetic stability and chem- Table 7. Experimental and calculated 1H and 13C NMR data for (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl)imino)thiazolidine-4-one. Chemical shifts 'H Experimental B3LYP/6-311G(d) H51 3.78 4.03 H52 3.78 3.94 H21 3.90 4.49 H22 3.90 4.25 H31 1.29 1.02 H32 1.29 1.92 H33 1.29 1.19 H9 6.52 6.26 H7 6.67 6.71 H11 6.56 6.64 H10 7.24 7.42 H121 3.79 4.32 H122 3.79 3.74 H123 3.79 3.73 13C C1 154.23 158.27 C2 32.76 37.78 C3 12.52 15.06 C4 171.55 176.61 C5 38.26 37.57 C6 149.32 158.15 C7 110.39 113.89 C8 160.37 170.89 C9 107.22 106.29 C10 129.99 136.22 C11 113.20 120.10 C12 55.25 58.70 ical reactivity. Figure 5 shows the atomic orbital HOMO-LUMO plot of the frontier molecular orbitals computed at B3LYP/6-311G(d,p) level for the title compound. As shown in Figure 5, in HOMO density, electrons are mainly located on the methoxyphenyl group. However, when an electron transition occurs, the electron enters into the LUMO, and then the electron will mainly be localized on thiazole ring and carboxylic group. The positive phase is red and the negative one is green coloured. The HOMO- Megrouss et al.: Synthesis, X-Ray Structure Determination and ... Acta Chim. Slov. 2019, 66, 490-500 497 m^V j-0.245 eV V LUMP A-0.892 eV 5.273 eV ▼ -6.165 eV HOMO -6.493 eV fca HOMO 1 Figure 5. Electron distribution of the HOMO-1, HOMO, LUMO and LUMO+1 energy levels for the title molecule. LUMO energy gap of our molecule was calculated at the B3LYP/6-311G (d,p) level with HOMO energy -6.165 eV, LUMO energy -0.892 eV and HOMO-LUMO energy gap 5.273 eV. The second highest occupied MO (HOMO-1) and the second lowest unoccupied MO (LUMO+1) were calculated using the same level of theory. 3D plots of the HOMO-1, HOMO, LUMO and LUMO+1 orbitals of the studied molecule are drawn in Figure 4. 4. 6. Chemical Reactivity Global chemical reactivity descriptors (GCRD) parameters are a good indication to highlight the relationship between chemical reactivity and strength of structure. As are mentioned in literature, GCRD parameters can be obtained by using the following equations: r/ = \(elumo-ehomoY, 1 = ~Ehomo■ The electron affinity (A) and ionization potential (I) can be evaluated as and . The calculated values of GCRD parameters for the title molecule are summarized in Table 8. The chemical hardness (r|) value is 2.636 eV indicating that the charge transfer occurs within the molecule. From Table 8, the electrophilic behaviour of the molecule is confirmed by the global electrophilicity index («) which has a greater value of 2.361 eV. On the other hand, the chemical stability of the title molecule is explained by the chemical potential (p) value which is -3.528 eV. Table 8. Calculated energy values of the title compound by B3LY-P/6-311G(d,p) method. Parameters Calculated energies ehomo -6.165 elumo -0.892 Energy gap (AE) 5.273 Ionization potential (I) 6.165 Electron affinity (A) 0.892 Electronegativity (x) 3.528 Chemical potential (p) -3.528 Chemical hardness (r|) 2.636 Chemical softness (s) 0.189 Electrophilicity index («) 2.361 4. 7. Electrostatic Potential In a crystal, the location of positive and negative charges allowed us to define very interesting physical properties such as the molecular electrostatic potential (MEP).38 Nowadays, theoretical and experimental MEP surfaces are determined using quantum chemistry and X-ray diffraction.39-42 The MEP around the title molecule was calculated from the total density (TD) for the title compound, the two maps (TD and MEP) are represented in the Figure 6. From the figure it can be seen that the negative MEP is related to the electronegativity and partial charges of oxygen atoms O1 and O2. The blue color in Figure 6B gives the maximum positive region as the preferred Megrouss et al.: Synthesis, X-Ray Structure Determination and 498 Acta Chim. Slov. 2019, 66, 490-500 -4.059e-4 I I 4.059e-4 a) -4.686e-2 b) 4.686c-2 Figure 6. MEP plots for (Z)-3-N-(ethyl)-2-N'-((3-methoxyphenyl) imino)thiazolidine-4-one: (a) total electron density, (b) MEP surface. site for nucleophilic attack. In terms of color, the MEP plot lies in the fact that it simultaneously displays molecular size, shape as well as positive, negative and neutral electrostatic potential regions (Figure 6). The MEP indicate well the relationship between the molecular structure and these physico-chemical properties.43-47 Among them, the molecular dipole moment can be calculated. The orientation of the molecular dipole moment for the title compound is represented in Figure 7. This orientation confirms the elec- Figure 7. Orientation of the molecular dipole moment from DFT calculation. trostatic distribution previously defined. The calculated dipole moment value is 2.24 D. 5. Conclusion In this study, (Z)-3-N-(ethyl)-2-N'-((3-methoxyphe-nyl)imino)thiazolidine-4-one was synthesized and characterized by IR, and 13C NMR and X-ray single-crystal diffraction techniques. The crystal structure determination shows that the title compound crystallizes in mono-clinic system with space group C2/c. Theoretical calculations of the molecular structures, wavenumbers and NMR spectra of the title compound have been carried out using DFT-B3LYP/6-311G(d,p). As result, the experimental and the optimized crystal structures of the title compound were slightly different. Most of the experimental bond lengths and bond angles are slightly larger than the optimized values. These differences are due to that the theoretical calculations are performed for isolated molecule in gaseous phase and the experimental results are for a molecule in the crystalline environment. The geometry of the solid-state structure is subject to intermolecular forces, such as Van der Waals interactions and crystal packing contacts. Comparison between the chemical shifts and the experimental data shows a very good agreement for both 1H and 13C NMR shift values. The HOMO-LUMO gap and chemical reactivity descriptors indicate that the compound is more stable. The general conclusion from the estimation of the dipolar moment (2.24 D) and the electrostatic potential of ((Z)-3-N-(ethyl)-2-N'-((3-methoxyphe-nyl)imino)thiazolidine-4-one molecule is that the region of the thiazolidinone group is electronegative and the methyl and hydrogen atoms connected to the benzene ring are electropositive. 6. Supplementary Material Crystallographic data for the structure reported in this article have been deposited with Cambridge Crystallographic Data Center, CCDC 1871013. Copies of this information may be obtained free of charge from the Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK. Facsimile (44) 01223 336 033, E-mail: deposit@ccdc.cam. ac.uk or http//www.ccdc.com.ac.uk/deposit. 7. References 1. J. Quiroga, P. Hernandez, B. Insuasty, R. Abonia, J. Cobo, A. Sanchez, M. Nogueras, J. Chem. Soc. Perkin Trans. 1 2002, 4, 555-559. D01:10.1039/b109676a 2. I. Hutchinson, S. A. Jennings, B. R. Vishnuvajjala, A. D. Westwell, M. F. G. Stevens, J. Med. Chem. 2002, 45, 744-747. D0I:10.1021/jm011025r Megrouss et al.: Synthesis, X-Ray Structure Determination and ... Acta Chim. Slov. 2019, 66, 490-500 499 3. R. I. Bahoussi, A. Djafri, A. Chouaih, A. Djafri, F. Hamzaoui, Acta Cryst. 2017, E73, 173-176. DOI:10.1107/S205698901700041X 4. A. Srinivas, M. Sunitha, P. Karthik, K. V. Reddy, Acta Chim. 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Sundius, Spectrochim. Acta A Mol. Biomol. Spectrosc. 2013, 108, 307-318. D0I:10.1016/j.saa.2013.02.022 35. G. Socrates, Infrared Characteristic Group Frequencies, Wiley Inter Science Publication, 1980. 36. G. Varsanyi, Vibrational Spectra of Benzene Derivates, Academic Press, New York, 1969. 37. K. Toubal, N. Boukabcha, Ö. Tamer, N. Benhalima, S. Altürk, D. Avci, A. Chouaih, Y. Atalay, A. Djafri, F. Hamzaoui, J. Mol. Struct. 2017, 1147, 569-581. D0I:10.1016/j.molstruc.2017.06.102 38. R. Rahmani, N. Boukabcha, A. Chouaih, F. Hamzaoui, S. Goumri, J. Mol. Struct. 2018,1155, 484-495. D0I:10.1016/j.molstruc.2017.11.033 39. H. Benaissi, M. Drissi, S. Yahiaoui, Y. Megrouss, A. Chouaih, F. Hamzaoui, J. Optoelectron. Biomed. M. 2018, 10, 73-82 40. N. Boukabcha, A. Feddag, R. Rahmani, A. Chouaih, F. Hamzaoui, J. Optoelectron. Adv. M. 2018, 20, 140-148 41. N. Boubegra, Y. Megrouss, N. Boukabcha, A. Chouaih, F. Hamzaoui, Rasayan. J. Chem. 2016, 9, 751-761. 42. M. Drissi, N. Benhalima, Y. Megrouss, R. Rahmani, A. Chouaih, F. Hamzaoui, Molecules 2015, 20, 4042-4045. D01:10.3390/molecules 43. J. S. Murray, K. Sen, Molecular Electrostatic Potentials, Concepts and 399 Applications, Elsevier, Amsterdam, 1996. 44. E. Scrocco, J. Tomasi, in: P. Lowdin (Ed) Advances in Quantum Chemistry, Academic Press, New York. 1978. 402. 45. F. J. Luque, M. Orozco, P. K. Bhadane, S. R. Gadre, J. Phys. Megrouss et al.: Synthesis, X-Ray Structure Determination and 500 Acta Chim. Slov. 2019, 66, 490-500 Chem. 1993, 97, 9380-9384. D01:10.1021/j100139a021 46. J. Sponer, P. Hobza, Int. J. Quant. Chem. 1996, 57, 959-970. D0I:10.1002/(SICI) 1097-461X(1996)57:5<959::AID-QUA16>3.0.C0;2-S 47. M. Govindarajan, M. Karabacak, Spectrochim. Acta A Mol. Biomol. Spectrosc. 2012, 96, 421-435. D0I:10.1016/j.saa.2012.05.067 Povzetek V prispevku poročamo o sintezi in karakterizaciji (Z)-3-N-(etil)-2-W-((3-metoksifenil)imino)tiazolidin-4-ona z FT-IR, 'H in 13C NMR ter z rentgensko difrakcijo monokristala. Eksperimentalna potrditev kristalne strukture temelji na pridobljenih rentgenskih difrakcijskih podatkih. Pomembna značilnost strukture je obstoj dihedralnega kota, ki ga tvorita ravnini benzenskega in tiazolidinonskega obroča, v vrednosti 86.0°, kar kaže na odsotnost n-n interakcij, hkrati pa nakazuje na neplanarno strukturo. V kristalu so molekule povezane s C-H-O in C-H—N vodikovimi vezmi, ki so odgovorne za trodimenzionalno molekulsko pakiranje v strukturi. Da bi lahko eksperimentalne rezultate primerjali s teoretično izračunanimi, smo izvedli kvantno kemijske DFT izračune s pomočjo B3LYP/6-311G(d,p) baznega seta. Ob tem smo izračunali še elektrostatski potencial okoli molekule ter HOMO in LUMO energijske nivoje. Ugotovili smo orientacije dipolnih momentov in s tem razkrili naravno inter- in intramolekularnih prenosov naboja. Nazadnje smo s pomočjo izračuna deskriptorjev kemijske reaktivnosti potrdili še stabilnost opisane spojine. Megrouss et al.: Synthesis, X-Ray Structure Determination and ... DOI: 10.17344/acsi.2019.5192 Acta Chim. Slov. 2019, 66, 501-509 ^creative ^commons Scientific paper Feedback Regulation of Cathepsin C by the Propeptide Dipeptides of Granzymes A and B Janja Božič1 and Iztok Dolenc^* 1 Department of Biochemistry and Molecular and Structural Biology, Jozef Stefan Institute, Jamova 39, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: iztok.dolenc@ijs.si Tel.: +386 1 477 3776 Fax: +386 1 477 3984 Received: 04-11-2019 Abstract Granzymes A and B are activated by proteolytic removal of their N-terminal dipeptides by cathepsin C (dipeptidyl-pep-tidase I). However, the possible physiological role of the cleaved dipeptides Glu-Lys and Gly-Glu is not yet understood. In this study, adding either of the two dipeptides to NK-92 cells, resulted in enhanced cytotoxicity toward the targeted K562 cells and increased death rate of the target cells. Cathepsin C is known to generate cytotoxic polymers from various dipeptides, however, in the case of the dipeptides Glu-Lys and Gly-Glu, cathepsin C was unable to polymerize them. Unexpectedly the dipeptides were found to be inhibitors of the transferase activity of cathepsin C (IC50 < 20 mM), and weak competitive inhibitors of the peptidase activity with Ki values in the millimolar range. This suggests that the dipeptides can play role in a feedback loop that controls transferase and proteolytic activities of cathepsin C in various biological processes. Keywords: Cathepsin C; granzyme; HPLC; dipeptides; transferase activity; product inhibition 1. Introduction Natural killer cells (NK cells) are a subpopulation of lymphocytes, and as a part of the innate immune response mediate cytotoxic activity against cancer cells and virally infected cells.1 A finely controlled mechanism is necessary to eliminate harmful cells from spreading in the body. Recognition of the infected and MHC class I-deficient cells triggers different cascades of the cytotoxic process.2 One of the most important processes is the granzyme's route, including the movement of granules toward the immunological synapse.2 Granzymes A and B are the most abundant granzymes and thought to play a leading role in gran-zyme-mediated cytotoxicity.3 They are synthesized in the form of inactive zymogens, and during the activation of NK cells, cysteine cathepsin C (dipeptidyl-peptidase I) activates granzymes by removing the N-terminal dipepti-de.4 Following the removal of dipeptide, conformational rearrangements of the newly formed N-terminal occur.5 The conserved N-terminal tail of the mature enzyme is further inserted in the interior of the protein, forming an active granzyme.6 Afterward, these granzymes are vectori-ally released into the submicroscopic intercellular cleft between the NK cell and the target cell.2 From this immu- nological synapse, granzymes, with the support of perforin, translocate into the cytoplasm of the target cells, where they cleave and activate multiple critical protein substrates, resulting in the death of the target cell.7 The activating protease, cathepsin C,4 is a cysteine protease and member of the papain family,8 inhibited by cystatins.9 The enzyme is ubiquitously expressed in various tissues,10,11 having several important functions.12-14 The inherited mutation in the cathepsin C gene, generating an inactive enzyme, causes autosomal-recessive disease Papil-lon-Lefevre syndrome.15 Opposite, deficiency of cathepsin C lighten severity of acute pancreatitis by reduction of neutrophil elastase activation and cleavage of E-cadherin.16 Cathepsin C is synthesized as a single chain preproen-zyme.17 During activation, the tetrameric form of the mature enzyme is assembled.18 From the crystal structure of the mature enzyme it is evident that the monomer consists of N-terminal fragment of 119 residues from Asp1 to Gly119, termed "exclusion domain", and 233 residues of the papain-like structure.18 However, the enzyme is functionally active only as tetramer composed of four identical subunits,19,20 with four active site clefts positioned at the tetrahedral corners of the molecule.18 At the end of each active site cleft is positioned the N-terminus of the exclusi- Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide 502 Acta Chim. Slov. 2019, 66, 501-509 on domain, which determines dipeptidyl peptidase activity.18 The carboxylic group of Aspl side chain controls the entry into the S2 binding pocket by recognizing the N-ter-minal amino group of the substrate.18 At the bottom of the pocket are chloride ions which provide additional negative charges and are required for cathepsin C activity.18 The enzyme is optimally proteolytically active at slightly acidic pH17 and is relatively unspecific. It cleaves dipeptides from proteins and peptidyl substrates until the N-terminus is no more available or a stop sequence of proline is reached.21 Relatively unspecific cleavage patterns exclude cleavages of substrates with positively charged amino acids (Arg and Lys) on the N-terminal part of proteins (P2 position).22 At neutral pH and above, the enzyme exhibits dipeptidyl transferase activity.23 Its perhaps the best known example is the lysosomotropic detergent LeuLeuOMe, which accumulates in the lysosomes.24 Once polymerized, it triggers the lysosomal pathway of cell death.25,26 In this study, we found that the dipeptides Glu-Lys and Gly-Glu, released during activation of granzymes A and B, promote cell death of target cells when incubated together with NK cells, but cannot be polymerized by cathepsin C. Instead, they act as weak competitive inhibitors of the enzyme, blocking both the proteolytic and transferase activities of the enzyme. 2. Experimental 2. 1. Materials Acetonitrile (ACN) and pentafluoropropionic acid (PFPA) were supplied by Sigma (Germany). Ultrapure water was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). Dipeptides Glu-Lys and Gly-Glu, their amides, and the fluorogenic substrate Gly-Phe-AMC were obtained from Bachem (Switzerland). The human NK cell line was derived from blood, mononuclear cells, NK-92, and human hematopoietic malignant cell line K562, were kindly provided by E. Vivier (Marseille, France). The growth medium was RPMI-1640 (Sigma-Aldrich). 2. 2. Cytotoxicity Assay A fluorescence-activated cell sorter (FACS) was used to perform the NK cell cytotoxicity assay, as described previously.27 The following day, the target K562 cell (105 cells) were added at the desired ratio, 10:1, and the mixture was incubated for 4 h at 37 °C and 5% CO2 . 0.5 ^M propidium iodide (Sigma-Aldrich) was used to evaluate cell viability. The analysis was performed using a FACSCalibur flow cytometer (Becton-Dickinson, USA), and the data acquisition was performed using Cellquest software. The cells were incubated overnight with a supplement of 1 mM di-peptides Glu-Lys or Gly-Glu. The obtained results are presented as mean values with standard deviations of at least three replicates. 2. 3. Cathepsin C expression, Activation, and Purification Human recombinant procathepsin C was expressed, activated and purified, essentially as described earlier with some minor modifications.28 Briefly, activation by cathepsin L was performed overnight at 4 °C in an activating buffer (250 mM sodium acetate pH 5.5, 150 mM NaCl, 10 mM DTT). Afterward, the protein mixture was loaded into a Sepharose 12 size-exclusion column, using fast protein liquid chromatography (AKTA system, GE Healthcare, Sweden), equilibrated with 50 mM of sodium acetate, 1mM EDTA, and 300 mM of NaCl, at a pH level of 5.5, and a flow rate of 0.5 ml/min. The protein peak at 200 kDa was collected, concentrated to concentration 1 mg/ ml, and analyzed using SDS-PAGE. 2. 4. High-performance Liquid Chromatography (HPLC) Analysis of Dipeptide Polymerization The dipeptides were dissolved in double distilled water to 1 M concentration and further used in the reaction with a final concentration of 40 mM. The final concentration of cathepsin C was 75 nM (an active concentration, determined as earlier described18) and the buffer in the reaction was PBS (10 mM NaH2PO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.4), with 5 mM DTT. Next, polymerization reaction was performed in 100 ^l volume for four hours at 37 °C, and then an aliquot of 20 ^l was applied on an Ascentis Express Peptide ES-C18 reversed phase column (150 mm x 2.1 mm, 2.7 ^m) (Sigma, Germany). The analysis was completed on an HPLC system (Waters, Milford, MA, USA) using the Waters M600 solvent delivery module and the Waters M2489 detector system. The analysis of the reaction mixture was optimized for the separation of dipeptides, using 0.1% PFPA as anionic ion-pairing reagents for reversed-phase highperformance liquid chromatography (RP-HPLC).29 A gradient elution from water/0.1% PFPA (phase A) to ace-tonitrile/water 60%/ 40% (v/v) with 0.1% PFPA (phase B) was used to separate the products. 2. 5. Determination of Inhibition Constants for Glu-Lys and Gly-Glu The active site titration of cathepsin C, the determination of the inhibition type, and the inhibition constant (Ki) for the interaction of dipeptides with cathepsin C were carried out as described earlier.18 Briefly, measurements were performed in 50 mM sodium acetate, 1 mM EDTA, and 300 mM NaCl, pH 5.5. Five hundred ^l of the reaction mixture contained 0.02 nM cathepsin C and increased concentration of the dipeptides Glu-Lys or Gly-Glu. In addition, these experiments were performed at different concentrations of the substrate Gly-Phe-AMC to Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide Acta Chim. Slov. 2019, 66, 501-509 503 identify the inhibition type. The release of the product at 25 °C was monitored with a Perkin-Elmer spectrofluori-meter. 2. 6. Molecular Docking UCSF Chimera 1.13rc30 has been used to prepare the structure of cathepsin C (PDB: 1K3B) for docking. Docking simulations of ligands were performed using the Autodock Vina Plugin31 and the results were examined with UCSF Chimera. Dipeptides were docked with the enzyme's structure giving a Vina score, which is the predicted affinity of the molecule to bind to the PDB structure, calculated in kcal mol-1. 2. 7. Molecular Modeling of Progranzyme B The three-dimensional model of human granzyme B was generated with the online server program I-TASSER,32 using the progranzyme K crystal structure (PDB entry 1MZA)5 as the primary template. Structures were visualized with UCSF Chimera 1.13rc.30 Figure 1. Dipeptides Glu-Lys (EK) or Gly-Glu (GE) enhanced the cytotoxic effect ofNK-92 cells toward K562 target cells. NK-92 cells were incubated overnight with 1 mM dipeptide supplement, and a number of dead target cells was compared to the control samples. The impact of the dipeptides on the target cells is seen as their enhanced death. The data are expressed as mean ± standard deviations (SD) of at least three independent experiments performed in duplicate. A t-test is used to compare the mean of two given samples. P value (»»») is below 0.001. a) Gly-Glu + catC Gly-Glu b) c) Gly-Glu-NI-h + catC, Gly-Glu-NHz 20 30 t [min] t [min] e) Gly-Tyr-NH2 + catC lil --—-(i Gly-Tyr-NHz polymerization products u UL. LL 10 20 30 40 t [mini Figure 2. Dipeptides Gly-Glu, Glu-Lys or their amide forms are not polymerized by cathepsin C. Reverse phase HPLC using 0.1% pentafluoropro-pionic acid (PFPA) as anionic ion-pairing reagents for separation, following a four-hour reaction with cathepsin C, in comparison to the control samples (a) Gly-Glu, (b) Glu-Lys, (c) Gly-Glu-NH2, (d) Glu-Lys-NH2. (e) Polymerization of Gly-Tyr-NH2 by cathepsin C as a control. For clarity purposes, the upper chromatograms are slightly shifted. Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide ... 504 Acta Chim. Slov. 2019, 66, 501-509 3. Results 3. 1. Dipeptides Glu-Lys and Gly-Glu Enhance the Cytotoxicity of NK-92 Cells In order to test, whether the released granzyme propeptides Glu-Lys and Gly-Glu, have an additional role in NK cell-mediated cytotoxicity, we incubated the dipeptides overnight with the target K562 cells, prior to addition of NK-92 cells. The reaction resulted in a substantially enhanced cytotoxicity, which was almost 1.5-fold in case of Gly-Glu dipeptide (Figure 1). 3. 2. Cathepsin C is unable to Polymerize Dipeptides Gly-Glu and Glu-Lys, Nor their Amide Forms As the increased cytotoxicity could have resulted from cathepsin C-mediated polymerization of the dipepti-des, we next tested whether the enzyme was able to polymerize both dipeptides. Therefore, we incubated the reaction mixture containing the dipeptides Gly-Glu or Glu-Lys, for four hours with cathepsin C. However, no larger peptides were found after separation of the mixture on a reverse phase C18 column of the HPLC system (Figure 2A and Figure 2B). As cathepsin C is known to prefer dipepti-de-amides, we next used the amide forms of the two dipeptides, Gly-Glu-NH2 and Glu-Lys-NH2. To our surprise, the amide dipeptides were not polymerized, similar to the free dipeptides (Figure 2C and Figure 2D). In contrast, cathepsin C polymerized the dipeptide-amide Gly-Tyr--NH2, which was already reported to be a transferase substrate of cathepsin C33 (Figure 3). 3. 3. Dipeptides Glu-Lys and Gly-Glu are Inhibitors of Cathepsin C Finally, we investigated, whether the two dipeptides, Glu-Lys and Gly-Glu, have any effect on cathepsin C activi- a) polymerization products b) c) LA. Gly-Tyr-NH2 JLLa- JLl Gly-Tyr-NH2 + catC Gly-Tyr-NH2 + Gly-Glu + catC d) J IL Gly-Tyr-NH2 + ^ I^ Glu-Lys + catC 10 20 30 40 t [min] Figure 3. Gly-Glu, the prodipeptide from granzyme B, inhibits the transferase and the peptidase activity of cathepsin C. (a) The dipeptide Gly-Tyr-NH2 as a control sample. (b) Polymerization of Gly-Tyr-NH2 by cathepsin C (catC) using a reverse phase HPLC. The products of polymerization are eluted with a retention time 15-20 minutes. Dipeptides Gly-Glu (b) and Glu-Lys (c) inhibit polymerization reaction of Gly-Tyr-NH2 by catC. ties. Both dipeptides were found to inhibit the transferase activity of cathepsin C, monitored by the polymerization of the Gly-Tyr-NH2 substrate. The separation of products on a reverse phase C18 column of HPLC system, obtained from the polymerization reaction of Gly-Tyr-NH2 with cathepsin C is illustrated (Figure 3). The formation of the polymerization product was completely inhibited by both dipeptides at 40 mM concentration, indicating an inhibition of the cathepsin C transferase activity at neutral pH (pH 7.4; Fi- I [mM] I [mM] Figure 4. Proteolytic inhibition of cathepsin C by dipeptides Glu-Lys and Gly-Glu. K values for inhibition of cathepsin C by dipeptides (a) Glu-Lys (20 mM) and (b) Gly-Glu (2.5 mM) were determined by secondary plots. Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide Acta Chim. Slov. 2019, 66, 501-509 505 gure 3). Moreover, both dipeptides inhibited also the peptidase activity of cathepsin C as monitored by the hydrolysis of fluorogenic substrate Gly-Phe-AMC at the more acidic pH 5.5 (Figure 4). The dipeptides Glu-Lys and Gly-Glu inhibited cathepsin C with Ki values of 20 mM and 2.5 mM, respectively. The results revealed that both dipeptides are weak competitive inhibitors of cathepsin C. 3. 4. Molecular Docking of Dipeptides In order to understand the potential mechanism of dipeptide binding at the structural level, we modelled the dipeptides into the active site of human cathepsin C. The crystal structure of human cathepsin C (PDB: 1K3B)18 was used to assess the fitness and orientation of the docked dipeptides. As Glu-Lys and Gly-Glu shown to be competitive inhibitors of cathepsin C, suggesting a binding into the active site of the enzyme, we limited our docking in the area of a substrate binding. Despite the similarities in docking sites, some differences were apparent (Figure 5). All representative docking predictions with a high score of probability fitted into the S1 position of the active cleft. The best fit was calculated for the Gly-Tyr-NH2 dipeptide, the substrate which was successfully polymerized (Figure 5A), and can be compared with the position of the inhibitor Ser-Tyr-CN (Figure 5B). Docking of Gly-Tyr-NH2 into the 3D structure of cathepsin C placed the dipeptide in the position stabilized by the amino acid residues Gly232 and Glu275 of the S1 binding site, and Asp1 at the entrance of the S2 pocket. The amide group was facing towards the S1' site, interacting with Asn380. Docking studies for dipeptides Glu-Lys and Gly-Glu suggested representative positions with similar scores. Their top-ranked positions were placed in the active site cleft of cathepsin C, where both dipeptides were well stabilized. The dipeptide Glu-Lys interacts with Trp405, Asn380, Thr379, and Asp1, expanding the side chain towards S2 (Figure 5C). Opposite, Gly-Glu stretches between S1 and S1, and is stabilized with Val352, Asn380, Trp405, Gln228, Gly232, and Cys234 (Figure 5D). However, the predicted positions do not favor catalysis by the enzyme. Namely, the docking studies predicted their binding to the S1 and S1' positions, with peptide bond in a position that cannot be cleaved. a) substrate Gly-Tyr-NH b) irreversible inhibitor Ser-Tyr-CN His381 TrP4°5 "c;S2 c) reversible inhibitor Glu-Lys d) reversible inhibitor Gly-Glu Figure 5. The active site of cathepsin C with selected predicted positions of dipeptides using docking predictions in comparison to the experimentally determined position of the inhibitor Ser-Tyr-CN in the crystal structure of cathepsin C.22 Docking simulations of ligands were performed using the Autodock Vina Plugin31 and the results were visualized with UCSF Chimera 1.13rc.30 (a) The predicted accommodation of dipeptide-amide Gly-Tyr-NH2, a substrate for polymerization by cathepsin C; (b) the covalently bound irreversible inhibitor Ser-Tyr-CN in the crystal structure of cathepsin C. (c) The best predicted binding of the inhibitory dipeptide Glu-Lys, the prodipeptide of granzyme A. (d) The best predicted position of the inhibitory dipeptide Gly-Glu, the prodipeptide of granzyme B. Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide ... 506 Acta Chim. Slov. 2019, 66, 501-509 3. 5. Comparison of the Putative Structure of Progranzyme B with Granzyme B To further investigate the inhibitory role of N-termi-nal dipeptides from granzymes, we performed sequence alignment of the N-terminal parts of granzymes A and B in comparison to selected serine proteases with dipeptide proregions (Figure 6A). This similarity enabled the modeling of the structure of progranzyme B, using the crystal structure of progranzyme K as a template. The obtained result, with the C-score value of 1.52, signifies an accurate model with high confidence. Comparing both 3D structures, the model structure of progranzyme B, and the crystal structure of granzyme B, revealed exposure of the N-ter-minal prodipeptide (Figure 6B), which is removed by cathepsin C. The N-terminus of the mature granzyme B is buried inside the protein (Figure 6C). 4. Discussion Activation of neutrophil serine proteases, gran-zymes, and cathepsin G is achieved by a rapid truncation of the N-terminal dipeptide by cathepsin C.4 Possible involvement of these dipeptides in the cellular protection system is supported by the immunomodulatory effect of dipeptidyl derivatives, described earlier.34 Therefore, the cytotoxic activity of the NK-92 cells towards the target K562 cells was evaluated in the presence of prodipeptides from granzyme A (Glu-Lys) or granzyme B (Gly-Glu). The presence of either dipeptide enhanced the cytotoxic response and cell death of the target cells (Figure 1). One possible explanation for this increased cytotoxicity would be polymerization of the released dipeptides by cathepsin C into membranolytic forms, as seen for Leu-Leu-OMe,24 acting as substance with cytotoxic activity.35 However, cathepsin C failed to polymerize any of the two N-termi-nal dipeptides of granzymes A and B (Figure 2). Moreover, cathepsin C even failed to polymerize the amide forms of the two dipeptides, i.e. Glu-Lys-NH2 and Gly-Glu-NH2, although the enzyme preferentially polymerizes dipeptidyl esters or amides,21,33 such as also seen for Gly-Tyr-NH2 we used for the positive control.33 Furthermore, the two granzymes-originating dipeptides Glu-Lys and Gly-Glu inhibited transferase and peptidase activities of cathepsin C in the millimolar range. Similarly, it has been shown that some dipeptides inhibit dipeptidyl pepti-dase IV in mM range and have potential influence on of the enzyme's function in diabetes.36,37 Our observation is in agreement with previous findings, where some dipepti- a) grA grB catG grH grK chy nHvtI phsrpy phsrpy phsrpy ¡VLB -drkti tgck| phsrpy layfflmn-wdqkslkr BayJq -qspagqsr «Iaf qf-lqeksrkr ]as| qy----gghhv ¡ayJeJvtsngpskf! Figure 6. Prodipeptides of granzymes A and B, together with highly similar N-terminal parts are preventing the activity of the granzymes. Following the removal of prodipeptide, the truncated enzyme undergoes structural rearrangement yielding an active enzyme. (a) Sequence alignment of N-ter-minal of human granzymes A, B, H, and K (grA, grB, grH, grK, respectively), cathepsin G (catG), and chymase (chy). Prodipeptides are marked by the box. Identical amino acids have a black background, and similar amino acids have a grey background. The alignment was performed using Clustal Omega, and shading was done using the BoxShade online service. (b, c) A comparison of modeled progranzyme B (b) with the structure of mature granzyme B (c) (PDB entry 1IAU). N-terminal residues of both molecules are marked with thicker lines. The prodipeptide of granzyme B, pointed out and accessible to cathepsin C, is marked by light gray. The N-terminal part of the reposition of the mature granzyme is perceived. Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide Acta Chim. Slov. 2019, 66, 501-509 507 des competitively inhibited cathepsin C with the K values in the same range.21 However, it seems that the P1' position of the substrate is also important for hydrolysis by cathepsin C. Comparing the interaction of several dipepti-de-amides21 and dipeptidyl-fluorogenic or protein substrates21 with cathepsin C, seems that a bulky group like ^-naphthylamide in Gly-Phe-^-naphthylamide or AMC in Gly-Glu-AMC facilitate hydrolysis,21,22 whereas an amide group or a free N-terminus such as in Gly-Phe-NH2,21 Gly--Glu and Gly-Glu-NH2 in some cases facilitate inhibition of the enzyme. To gain insight in the inhibitory nature of prodipep-tides we performed molecular docking predictions. The calculated prediction for the dipeptide amide Gly-Tyr--NH2, a substrate which is polymerized by cathepsin C, on S2 and S1 positions corresponds to the experimentally positioned inhibitor Ser-Tyr-CN, bound into cathepsin C cleft,22 as well to the predicted position of the tetrapeptide Glu-Arg-Ile-Ile.18 However, the inhibitory dipeptides bind differently. In contrast to Gly-Tyr-NH2, docking predictions for dipeptides Glu-Lys and Gly-Glu show non-productive binding to the active site of cathepsin C. The inhibitory dipeptides bind to the enzyme on positions in the area of S1 and S1', and are stabilized by surrounding amino acids of cathepsin C. Thus, by binding to the active site of the enzyme, dipeptides interfere the interaction of cathepsin C with substrates. Knowing that the proregion dipeptides of granzyme A and B can not polymerize, but are weak inhibitors of cathepsin C, raises the question regarding the physiological importance of the prodipepti-des. The model of progranzyme B structure revealed very different N-termini positions of progranzyme B and granzyme B.38 Only the exposed N-terminus of progranzyme B is accessible to cathepsin C. As observed in granzyme C39 and K,5 the truncation of the polypeptide chain for two amino acids with cathepsin C results in a rapid allosteric reorientation, remodeling of the activation domains, and activation. We suggest that the released propeptide, together with the newly formed N-terminus Ile-Ile,40 may at least partially block the activities of cathepsin C within the granules, especially at high local concentrations. The importance of native sequence of granzymes was revealed by mutation studies. Namely, introduction of any mutation in the region of the six N-terminal amino acid residues resulted in lower stability or in an incorrect activation pro-cess.41,42 Expressing granzyme B without the N-terminal dipeptide yielded a low level of active enzyme which was unstable.41 Moreover, a Glu residue at the P1 position of granzyme K was shown to be critical, as any mutations resulted in expression of inactive enzyme.42 Little is known about the regulation of cathepsin C activity. Basically, the enzyme activity is regulated by the endogenous protein inhibitors to prevent a granzyme maturation to the active form. Based on the size of granules,43 we can estimate that mM concentration is reached by 2000 dipeptide molecules, when granules have diameter of 200 nm. The concentration of the released propeptide dipeptides may be too low to inhibit cathepsin C. However, granzymes and other serine proteases listed in the Figure 6A have similar structure of propeptides. Therefore, we suggest that all the released dipeptides from granule serine proteases may have effect on cathepsin C. The development of cathepsin C inhibitors might serve as effective therapeutics for the treatment of diseases, such as inflammatory diseases including rheumatoid arthritis, cystic fibrosis and pulmonary disease. The current therapeutic strategy is based on inhibitors derived from the dipeptidyl substrates, which contain an electrop-hilic "warhead", such as diazoketones, vinyl sulfones, nitri-les, and cyanamides, forming reversible or irreversible co-valent bonds with the enzyme active site Cys234.44 Their effective inhibition is at concentration of ^M range. However, only few data show biological effects in vivo. Currently, a variety of cathepsin C inhibitors are used in preclinical and clinical studies.12 5. Conclusion In the present study we suggest that the proregion dipeptides of granzymes A and B represent a feedback loop to control the transferase and peptidase activities of cathepsin C. This insight sheds light on the regulation of cathepsin C activity in biological processes and broadens the knowledge about the role of dipeptides in the mechanisms of cytotoxicity of NK cells. Moreover, the studied dipeptides show immunomodulatory effect. In conclusion, an increase in cytotoxicity is not the result of potentially toxic polymers produced by cathepsin C, but is rather due to modulation of NK cell activity as shown for Glu--Tyr.45 This study might be important for possible design of immunosuppressive dipeptide-like drugs. 6. Acknowledgments The authors wish to thank Dr. Vito Turk for critical comments on the manuscript, Dr. Boris Turk for discussion, advice and continuous support. I am grateful to Dr. Dusan Turk for his critical discussion about structural aspects, and to Andreja Sekirnik for expression of recombinant cathepsin C. This study was supported by grants from the Slovenian Research Agency research program P1-0140 lead by Dr. Boris Turk. 7. References 1. E. Vivier, E. Tomasello, M. Baratin, T. Walzer and S. Ugolini, Nat. Immunol. 2008, 9, 503-10. DOI:10.1038/ni1582 2. L. Martinet and M. J. Smyth, Nat. Rev. Immunol. 2015, 15, 243-54. 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D0I:10.1074/jbc.274.38.27331 Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide Acta Chim. Slov. 2019, 66, 501- 509 509 43. J. P. Goodridge, B. Jacobs, M. L. Saetersmoen, D. Clement, Q. Hammer, T. Clancy, E. Skarpen, A. Brech, J. Landskron and C. Grimm, Nat. commun. 2019, 10, 514. D01:10.1038/s41467-019-08384-x 44. D. I. Laine and J. Busch-Petersen, Expert Opin. Ther. Pat. 2010, 20, 497-506. D01:10.1517/13543771003657172 45. D. L. Smith, J. Cai, S. Zhu, W. Wei, J. Fukumoto, S. Sharma, R. Masood and P. S. Gill, Int. J. Cancer. 2003, 106, 528-33. D0I:10.1002/ijc.11253 Povzetek Katepsin C (dipeptidil peptidaza I) aktivira grancima A in B s proteolitično odstranitvijo N-terminalnih dipeptidov. Možna fiziološka vloga odcepljenih dipeptidov Glu-Lys in Gly-Glu ni znana. V naši študiji smo pokazali, da je dodatek omenjenih dipeptidov k celicam NK-92 povzročil njihovo povečano citotoksičnost in s tem hitrejšo smrt tarčnih celic K562. Katepsin C lahko tvori citotoksične polimere iz različnih dipeptidov, vendar dipeptidov Glu-Lys in Gly-Glu encim ni sposoben polimerizirati. Pri tem smo ugotovili, da sta dipeptida šibka inhibitorja transferazne aktivnosti katepsina C (IC50 <20 mM) in kompetitivna inhibitorja njegove proteolitične aktivnosti, z vrednostmi Ki v milimolarnem območju. Rezultati odkrivajo potencialno vlogo teh dveh dipeptidov, ki lahko s povratno regulacijo vplivajo na transferazno in proteolitično aktivnost katepsina C v različnih bioloških procesih. Božič and Dolenc: Feedback Regulation of Cathepsin C by the Propeptide ... S10 Acta Chim. Slov. 2019, 66, (2), Supplement DRUŠTVENE VESTI IN DRUGE AKTIVNOSTI SOCIETY NEWS, ANNOUNCEMENTS, ACTIVITIES Vsebina Doktorska in magistrska dela, diplome v letu 2018.............................................................. S3 Koledar važnejših znanstvenih srečanj s področja kemije, kemijske tehnologije in kemijskega inženirstva.................................................................. S37 Navodila za avtorje.................................................................................................................. S40 Contents Doctoral theses, master degree theses, and diplomas in 2018............................................. S3 Scientific meetings - chemistry, chemical technology and chemical engineering......... S37 Instructions for authors.......................................................................................................... S40 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement UNIVERZA V LJUBLJANI FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar - 31. december 2018 DOKTORATI DOKTORSKI ŠTUDIJSKI PROGRAM KEMIJSKE ZNANOSTI KEMIJA TANJA ZIDARIČ RAZVOJ IN ŠTUDIJ UPORABE BIZMUTOVIH IN DRUGIH MODIFICIRANIH ELEKTROD V SODOBNI ELEKTROANALIZI Mentor: znan. svet. dr. Samo Hočevar Somentor: prof. dr. Marjan Veber Datum zagovora: 12. 01. 2018 BARBARA VOLARIČ KONVERZIJSKE PREVLEKE NA OSNOVI LANTANOIDNIH SOLI ZA ZAŠČITO ALUMINIJEVIH ZLITIN Mentorica: znan. svet. dr. Ingrid Milošev Somentor: prof. dr. Miran Gaberšček Datum zagovora: 2. 02. 2018 EVA KRANJC DOLOČANJE METABOLITOV NAVADNEGA VOLČJEGA JABOLKA (PHYSALIS ALKEKENGI L. ) S KROMATOGRAFSKIMI IN SKLOPLJENIMI TEHNIKAMI Mentorica: viš. znan. sod. dr. Irena Vovk Somentorica: prof. dr. Helena Prosen Datum zagovora: 30. 03. 2018 URŠA TOMAŽIN PRIPRAVA, KARAKTERIZACIJA IN UPORABA ENAMINONSKIH LIGANDOV Mentor: prof. dr. 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Bogdan Štefane Datum zagovora: 22. 06. 2018 KLARA RETKO RAZVOJ NOVIH SUBSTRATOV ZA PREISKAVE BARVNIH SLOJEV NA UMETNIŠKIH DELIH S POVRŠINSKO OJAČENO RAMANSKO SPEKTROSKOPIJO Mentor: izr. prof. dr. Romana Cerc Korošec Somentorica: znan. sod. dr. Angelja Kjara Surca Datum zagovora: 12. 10. 2018 MARTIN GLADOVIC MOLEKULARNO MODELIRANJE ALKILACIJE DNA Mentor: izr. prof. dr. Urban Bren Somentor: prof. dr. Tomaž Urbič Datum zagovora: 12. 10. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement BIOKEMIJA JASNA BRČIC STRUKTURNE ŠTUDIJE Z GVANINI IN CITOZINI BOGATIH OLIGONUKLEOTIDOV POVEZANIH Z NEVRODEGENERATIVNIMI OBOLENJI Mentor: prof. dr. Janez Plavec Datum zagovora: 13. 04. 2018 MATEVŽ KORENČ MEHANIZMI INTERAKCIJ MED CISTEINSKO PEPTIDAZO KATEPSINOM K IN KOLAGENOM NA MOLEKULSKEM NIVOJU Mentorica: prof. dr. Brigita Lenarčič Datum zagovora: 24. 10. 2018 ALJAŽ GABER OLIGOMERIZACIJA PROTEINA EPCAM IN NJEGOVA VLOGA PRI CELIČNI ADHEZIJI Mentorica: prof. dr. Brigita Lenarčič Datum zagovora: 9. 11. 2018 KEMIJSKO INŽENIRSTVO NATAŠA MILOŽIČ ENCIMSKO KATALIZIRANA TRANSAMINACIJA V MIKROREAKTORSKIH SISTEMIH Mentor: prof. dr. Igor Plazl Datum zagovora: 19. 04. 2018 ŽIGA ŠTIRN NAČRTOVANJE IN UPORABA POLIMERNIH MATERIALOV S SPOSOBNOSTJO SAMOCELJENJA NA OSNOVI DIELS-ALDER REAKCIJE Mentor: prof. dr. Matjaž Krajnc Datum zagovora: 1. 06. 2018 SARA DRVARIČ TALIAN ŠTUDIJ VPLIVA IZBRANIH PROCESOV IN PARAMETROV NA NOTRANJO UPORNOST LITIJ-ŽVEPLOVIH AKUMULATORJEV Mentor: izr. prof. dr. Robert Dominko Datum zagovora: 9. 10. 2018 MAJA PIVKO DVOSTOPENJSKI POSTOPEK ZA PRIPRAVO LIMNPO4 KOT KATODNEGA MATERIALA V LITIJ IONSKIH AKUMULATORJIH Mentor: prof. dr. Miran Gaberšček Datum zagovora: 12. 10. 2018 ANA BJELIC KATALITSKE PRETVORBE DERIVATOV LIGNINA IN NJIHOVA REAKCIJSKA KINETIKA Mentor: doc. dr. Blaž Likozar Datum zagovora: 8. 11. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement MAGISTRSKI ŠTUDIJ - 2. STOPNJA KONDA, MOJCA PREUČEVANJE VKLJUČEVANJA VSEBIN O ALTERNATIVNIH GORIVIH V POUK KEMIJE V SPLOŠNIH GIMNAZIJAH Mentor: Vesna Ferk Savec Datum zagovora: 16. 1. 2018 BANIČ, MOJCA VALIDACIJA OBETAVNIH BIO OZNAČEVALCEV GLIOBLASTOMA IN OPREDELITEV UČINKA ZANJE SPECIFIČNIH NANO-TELES NA IZBRANIH CELIČNIH LINIJAH Mentor: prof. dr. Radovan Komel Datum zagovora: 22. 1. 2018 KUŠAR, KAJA ANODNI MATERIALI NA OSNOVI KOMPLEKSNIH KOVINSKIH OKSIDOV ZA KERAMIČNE GORIVNE CELICE Mentor: doc. dr. Klementina Zupan Datum zagovora: 24. 1. 2018 ROZMARIČ, TOMAŽ PRIMERJAVA METILACIJSKIH VZORCEV DNA PRI ČEZMERNO PREHRANJENIH OTROCIH Z IN BREZ INZULINSKE REZISTENCE Mentor: doc. dr. Primož Kotnik Somentor: prof. dr. Simon Horvat Datum zagovora: 24. 1. 2018 MEŠKO, TOMAŽ KARAKTERIZACIJA IN OCENA VISOKO-KONCENTRIRANIH ELEKTROLITOV Mentor: prof. dr. Miran Gaberšček Datum zagovora: 31. 1. 2018 JOŠIC, DANIJELA PRIPRAVA IN KARAKTERIZACIJA LASTNOSTI KULTURE ASTROCITOV Z ZVEZDASTO OBLIKO TER IZRAŽANJE MOLEKULSKIH OZNAČEVALCEV PODTIPOV ASTROCITOV V PRIMARNI KULTURI PODGANE Mentor: Matjaž Stenovec Somentor: prof. dr. Robert Zorec Datum zagovora: 1. 2. 2018 MOHAR, MAŠA SINTEZA DERIVATOV KURKUMINA, MODIFICIRANIH S HIDROFILNIMI SKUPINAMI Mentor: doc. dr. Martin Gazvoda Datum zagovora: 12. 2. 2018 ŠKRJANC, MONIKA IZRAŽANJE GENOV IN GENSKI POLIMORFIZMI POVEZANI Z ODPORNOSTJO NA MASTITIS PRI KOZAH Mentor: prof. dr. Peter Dovč Datum zagovora: 14. 2. 2018 KMETIČ, MIRJAM UPORABA TEHNOLOGIJE ROGFP ZA DOLOČANJE CELIČNEGA REDOKS STANJA PRI KROMPIRJU Mentor: prof. dr. Kristina Gruden Somentor: izr. prof. dr. Marko Dolinar Datum zagovora: 15. 2. 2018 ZAKOTNIK, SAMO IZDELAVA PROMOTORSKE KNJIŽNICE Z MUTAGENEZO PROMOTORJA PTEF1 KVASOVKE SACCHAROMYCES CEREVISIAE Mentor: izr. prof. dr. Uroš Petrovič Datum zagovora: 16. 2. 2018 ŽAGAR, JAKA PRIPRAVA BIOOGLJA IZ STABILIZIRANEGA AKTIVNEGA BLATA IN NJEGOVA UPORABA ZA ČIŠČENJE ODPADNIH VOD Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 14. 3. 2018 CEZAR, KLEMEN HIDROLIZA ODPADNEGA BLATA IZ BIOLOŠKE ČISTILNE NAPRAVE PAPIRNIŠKE INDUSTRIJE Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 28. 3. 2018 PRAZNIK, MONIKA ULTRASTRUKTURA APIKALNEGA IN BAZALNEGA LABIRINTA V ČREVESNIH CELICAH TER DIFERENCIACIJA MED RAZVOJEM EMBIJEV IN LIČINK PRI NEVRETENČARSKEM MODELNEM ORGANIZMU Mentor: doc. dr. Nada Žnidaršič Datum zagovora: 29. 3. 2018 KALAN, MARCEL ŠIRJENJE OGLJIKOVEGA MONOKSIDA IN OPTIMALNA NAMESTITEV DETEKTORJA V PROSTORU Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 19. 4. 2018 PREZELJ, PETER MOLEKULARNI IN SEKRETORNI PROFIL MIŠIČNE IN NE-MIŠIČNE CELIČNE KULTURE Mentor: prof. dr. Tomaž Marš Datum zagovora: 19. 4. 2018 PUCELJ, KATARINA IMOBILIZACIJA BIOKATALIZATORJA V MIKROREAKTORJU Z NANOVZMETMI Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 19. 4. 2018 DUŠAK, BARBARA VLOGA GENOV STRBOHA IN STRBOHD1 PRI ODZIVU KROMPIRJA NA VIRUS PVY Mentor: prof. dr. Kristina Gruden Somentor: izr. prof. dr. Marko Dolinar Datum zagovora: 23. 4. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement ROJC, AJDA AKTIVNOST KATEPSINOV C, L, B IN S V MODELU DIFERENCIRANIH ČREVESNIH EPITELIJSKIH CELIC CACO-2 Mentor: prof. dr. Boris Turk Somentor: doc. dr. Tina Zavašnik Bergant Datum zagovora: 17. 5. 2018 SAVANDIC, PETRA VPLIV KALIBRACIJE NA DOLOČANJE MIKROKOMPONENT Mentor: izr. prof. dr. Nataša Gros Datum zagovora: 28. 5. 2018 MEGLEN, ANŽE ALKALNO KARBONATNA REAKCIJA V BETONU Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 30. 5. 2018 MLINARIC, KATARINA SINTEZA IN KARAKTERIZACIJA NEKATERIH IZBRANIH BIOPOLIMERNIH MATERIALOV Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 7. 6. 2018 ŠIVEC, ROK VPLIV NAČINA SEGREVANJA, DIMENZIJ CELICE IN UPORABLJENIH MATERIALOV NA NASTALE MEHANSKE NAPETOSTI V TROSLOJNEM SOFC SISTEMU Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 12. 6. 2018 ANDREJČIČ, MIHA PH-PREHODI IN DEBELINA ADSORPCIJE NA MONOLITNIH KROMATOGRAFSKIH NOSILCIH Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 14. 6. 2018 BOŽIČ, DARJA EVALVACIJA METOD DINAMIČNEGA IN STATIČNEGA SIPANJA SVETLOBE ZA KARAKTERIZACIJO ZUNAJCELIČNIH MEŠIČKOV V BIOLOŠKIH VZORCIH Mentor: prof. dr. Ksenija Kogej Datum zagovora: 15. 6. 2018 MAJC, BERNARDA PRIPRAVA IN KARAKTERIZACIJA PROTEINA C9ORF72 Mentor: doc. dr. Vera Zupunski Datum zagovora: 19. 6. 2018 ŠUŠTERŠIČ, MOJCA OCENA BIODOSTOPNOSTI POTENCIALNO STRUPENIH ELEMENTOV V HIŠNIH PRAHOVIH Mentor: doc. dr. Marija Zupančič Datum zagovora: 20. 6. 2018 HORVAT, MONIKA SINTEZA TRIFLUOROMETILTIOLIRANIH AROMATOV IN NJIHOVA OKSIDACIJA Z VODIKOVIM PEROKSIDOM Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 20. 6. 2018 HORVAT, MARJETA METILACIJA DNA IN SAMOMORILNO VEDENJE Mentor: doc. dr. Alja Videtič Paska Datum zagovora: 27. 6. 2018 KAPŠ, URŠA SOODVISNOST MED IZRAŽANJEM UROPLAKINOV IN KERATINA 20 TER VEZAVO LEKTINOV PRI PAPILARNIH UROTELIJSKIH KARCINOMIH Mentor: doc. dr. Daša Zupančič Datum zagovora: 3. 7. 2018 BENCIK, TIM PREUČEVANJE STABILNOSTI DERIVATIZIRANE CELULOZE Z VELIKOSTNO IZKLJUČITVENO KROMATOGRAFIJO Mentor: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 4. 7. 2018 JUDEŽ, EVA SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN PREHODNIH KOVIN Z LIGANDI, KI VSEBUJEJO SALICILNO SKUPINO Mentor: doc. dr. Nives Kitanovski Datum zagovora: 5. 7. 2018 PERŠIČ, ŠPELA ORGANORUTENIJEVI KOMPLEKSI Z ENOVEZNIM DUŠIKOVIM LIGANDOM LETROZOLOM Mentor: prof. dr. Iztok Turel Datum zagovora: 6. 7. 2018 STUPAR, UROŠ PRIPRAVA, RENATURACIJA IN KARAKTERIZACIJA ČLOVEŠKEGA REKOMBINANTNEGA PROTEINA GILT Mentor: prof. ddr. Boris Turk Datum zagovora: 10. 7. 2018 ČAMIČ, ŽIGA SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN BAKRA(II) IN KOBALTA(II) S TIOCIANATNIM LIGANDOM IN HIDROKSIPIRIDINSKIMI LIGANDI Mentor: doc. dr. Nives Kitanovski Datum zagovora: 10. 7. 2018 VOLAVŠEK, JANEZ SINTEZA IN KARAKTERIZACIJA HIBRIDNIH ORGANSKO- ANORGANSKIH ELEKTROLITOV ZA SONČNE CELICE GRATZELOVEGA TIPA Mentor: izr. prof. dr. Miha Lukšič Somentor: doc. dr. Ivan Jerman Datum zagovora: 11. 7. 2018 ORAŽEM, TILEN DETEKCIJA NITROAROMATSKIH SPOJIN Z MODIFICIRANO ELEKTRODO IZ STEKLASTEGA OGLJIKA Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 11. 7. 2018 GRŠIČ, MARIJA SINTEZA CILJNIH AMIDOV IN NJIHOVA AKTIVNOST NA HBCHE IN MACHE Mentor: doc. dr. Uroš Grošelj Datum zagovora: 11. 7. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement KLOFUTAR, DOMEN KARAKTERIZACIJA MEHANIZMOV DELOVANJA NEKATERIH ALOSTERIČNIH REGULATORJEV IN HEPARINA NA KATEPSIN B Mentor: izr. prof. dr. Marko Novinec Datum zagovora: 11. 7. 2018 KERT, DOMINIK PRETVORBA ŠARŽNEGA PROCESA Z DOHRANJEVANJEM V PERFUZIJSKI PROCES NA PRIMERU BIOPROCESA S SESALSKO CELIČNO KULTURO Mentor: izr. prof. dr. Uroš Petrovič Datum zagovora: 13. 7. 2018 LAVRENČIČ, JAKOB GAŠPER KARAKTERIZACIJA STR-LOKUSA FH2054 PRI PSIH Mentor: prof. dr. Peter Dovč Somentor: Jernej Ogorevc Datum zagovora: 13. 7. 2018 VIDMAR, VITA VPLIV CISTEINSKIH OSTANKOV NA NEKROPTOTIČNO AKTIVNOST ČLOVEŠKEGA PROTEINA MLKL Mentor: doc. dr. Gregor Gunčar Datum zagovora: 21. 8. 2018 NAROBE, ROK KLASIČNO IN FOTOKATALITSKO OKSIDATIVNO JODIRANJE ARENOV Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 24. 8. 2018 VAH, LUKA NAČRTOVANJE, SINTEZA IN IN VITRO TESTIRANJE PIRAZOLNIH INHIBITORJEV DIHIDROOROTATA DEHIDROGENAZE PRI PLASMODIUM FALCIPARUM Mentor: prof. dr. Jurij Svete Datum zagovora: 24. 8. 2018 PLUT, EVA KIRALNI SKVARAMIDNI IN (TIO)SEČNINSKI ORGANOKATALIZATORJI PRI ENANTIOSELEKTIVNI TVORBI OGLJIK-OGLJIK VEZI Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 24. 8. 2018 IVANČIČ, ANŽE UPORABA 5-SUBSTITUIRANIH PIROLONOV V REAKCIJAH, KATALIZIRANIH Z UVELJAVLJENIMI IN NOVIMI BIFUNKCIONALNIMI KATALIZATORJI S KAFRINIM SKELETOM Mentor: doc. dr. Uroš Grošelj Datum zagovora: 24. 8. 2018 KONČAN, LARA SINTEZA SPOJIN ELEMENTOV PRVE VRSTE PREHODA S 4,6-DIHIDROKSIPIRIMIDINOM Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 29. 8. 2018 KOŠIČEK, MARTIN OSMOZNI KOEFICIENTI NATRIJEVIH SOLI POLI(A-ALKILKARBOKSILNIH KISLIN) Mentor: prof. dr. Ksenija Kogej Datum zagovora: 29. 8. 2018 ZUPANC, ANŽE SINTEZA NOVIH ENAMINONSKIH POLIMEROV Mentor: prof. dr. Jurij Svete Datum zagovora: 29. 8. 2018 BREČKO, ŽIVA ČIŠČENJE R-FIKOERITRINA IZ RDEČIH MAKROALG V SISTEMU MIKROFLUIDNIH NAPRAV Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 30. 8. 2018 STRAŠEK, NIKA SINTEZA IN TESTIRANJE POTENCIALNIH INHIBITORJEV PLASMODIUM FALCIPARUM DIHIDROOROTAT DEHIDROGENAZE Mentor: prof. dr. Jurij Svete Datum zagovora: 31. 8. 2018 HOIVIK, ANDREJ SINTEZA DVO- IN VEČVEZNIH LIGANDOV IZ SUBSTITUIRANIH BICIKLO[2. 2. 2]OKTENOV IN NJIHOVIH KOORDINACIJSKIH SPOJIN Mentor: doc. dr. Krištof Kranjc Datum zagovora: 31. 8. 2018 PUCIHAR, URH FUNKCIONALIZACIJA KROMATOGRAFSKIH NOSILCEV Z IONSKO IZMENJEVALNIMI SKUPINAMI Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 31. 8. 2018 HROVAT, SARA VEČKRATNA KATALITSKA C-H FUNKCIONALIZACIJA DIHIDROPIRAZINSKEGA DERIVATA V VODI Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 31. 8. 2018 ŠPINDLER, VIDA NAČTOVANJE VELIKOSTNE PORAZDELITVE IN OBLIKE KRISTALOV Z OSCILACIJO TEMPERATURE SUSPENZIJE IN UPORABO MOKREGA MLETJA Mentor: doc. dr. Blaž Likozar Somentor: prof. dr. Igor Plazl Datum zagovora: 4. 9. 2018 KOPAČ, TILEN KINETIČNA ANALIZA TERMIČNEGA RAZPADA HIDRATIZIRANEGA TITANOVEGA DIOKSIDA Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 4. 9. 2018 DLOUHY, MATJAŽ REAKTIVNOST IN STABILNOST HIDROTRIOKSI RADIKALA Mentor: prof. dr. Tomaž Urbič Datum zagovora: 5. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement SIMONČIČ, MATJAŽ NASTANEK BIOKEMIJSKIH PREKURZORJEV V MEDZVEZDNEM PROSTORU Mentor: prof. dr. Tomaž Urbič Datum zagovora: 5. 9. 2018 KOBAL, TJAŽ KINETIKA DIELS-ALDER REAKCIJE MED N-FENILMALEIMID OM IN BENZOKSAZINOM NA OSNOVI FURANA V TALINI Mentor: prof. dr. Urška Šebenik Datum zagovora: 6. 9. 2018 NOSAN, MIHA PRIMERJAVA HPLC IN REFLEKTOMETRIČNIH METOD ZA DOLOČANJE FORMALDEHIDA IN 5-(HIDROKSIMETIL) FURFURALA Mentor: izr. prof. dr. Zdenko Časar Somentor: prof. dr. Helena Prosen Datum zagovora: 6. 9. 2018 GAČNIK, JAN RAZVOJ IN OPTIMIZACIJA METOD DOLOČANJA 90SR V VODNIH VZORCIH Mentor: doc. Marko Štrok Somentor: prof. dr. Helena Prosen Datum zagovora: 6. 9. 2018 GRIČAR, EMA ELEKTROKEMIJSKE LASTNOSTI IN DOLOČANJE FEROCENSKIH DERIVATOV AMINOFOSFONATOV Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 6. 9. 2018 PALJK, TINA ODLAGANJE FILMA LITIJEVEGA SULFIDA V RAZLIČNIH ELEKTROLITIH V LI-S AKUMULATORJIH Mentor: izr. prof. dr. Robert Dominko Datum zagovora: 6. 9. 2018 MENEGATTI, TADEJ IMOBILIZACIJA BIOKATALIZATORJA V HIDROGELU IN NJEGOVA UPORABA V MIKROREAKTORJU Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 7. 9. 2018 OBLAK, DOMEN VPLIV DODANE SOLI NA KONFORMACIJSKE SPREMEMBE DNA ČLOVEŠKEGA TELOMERNEGA ZAPOREDJA 22GT Mentor: prof. dr. Jurij Lah Datum zagovora: 7. 9. 2018 HLADNIK, LUCIJA SAMOCELJENJE BIO-OSNOVANIH BENZOKSAZINOV NA PRINCIPU SUPRAMOLEKULARNIH INTERAKCIJ Mentor: prof. dr. Matjaž Krajnc Datum zagovora: 7. 9. 2018 PAVLIN, ANŽE TVORBA HALOGENSKIH VEZI V SOLEH DIATRIZOIČNE KISLINE Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 7. 9. 2018 ŽOS, EVA LUMINISCENČNE LASTNOSTI CINKOVIH KOORDINACIJSKIH SPOJIN S PIRIDIN-2,6-DIKARBOKSILNO KISLINO Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 7. 9. 2018 ŽAGAR, TOMAŽ KARAKTERIZACIJA OLIGOMERNIH STANJ PROTEINA EPCAM IN VIVO Mentor: prof. dr. Brigita Lenarčič Datum zagovora: 7. 9. 2018 MRAVLJAK, ROK PRIPRAVA, FUNKCIONALIZACIJA IN KARAKTERIZACIJA GMA/EGDMA KOPOLIMERA IZ EMULZIJE Z VISOKO VSEBNOSTJO DISPERGIRANE FAZE Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 7. 9. 2018 ŠTEMBERGER, ROK PROFIL IZRAŽANJA MIŠIČNO-SPECIFIČNIH PROTEINOV V ČLOVEŠKI SKELETNI MIŠICI IN VITRO Mentor: prof. dr. Tomaž Marš Datum zagovora: 7. 9. 2018 HRIBAR, UROŠ SINTEZA PEROVSKITNE KERAMIKE V SISTEMU NA0. 5BI0. 5TIO3-BATIO3-K0. 5NA0. 5NBO3 -MIKROSTRUKTURNE IN ELEKTRIČNE LASTNOSTI Mentor: prof. dr. Danilo Suvorov Datum zagovora: 10. 9. 2018 TAVČAR, PETRA VPLIV AKTIVACIJE Z G-PROTEINI SKLOPLJENIH RECEPTORJEV NA BIOGENEZO LIPIDNIH KAPLJIC V ASTROCITIH V ORGANOTIPSKIH KULTURAH MOŽGANSKIH REZIN ODRASLIH PODGAN Mentor: doc. dr. Nina Vardjan Datum zagovora: 11. 9. 2018 CIGOJ, MATEJA OPTIMIZACIJA METODE PRENOSA WESTERN ZA DOLOČANJE RECEPTORJEV ZA ESTROGEN IN RECEPTORJEV ZA ERITROPOETIN V CELIČNIH LINIJAH RAKA DOJKE Mentor: izr. prof. dr. Nataša Debeljak Datum zagovora: 11. 9. 2018 PAPEŽ, PETRA HIDRATACIJA NIŽJIH ALKOHOLOV Z UPORABO TEORETIČNIH METOD Mentor: prof. dr. Tomaž Urbič Somentor: izr. prof. Franci Merzel Datum zagovora: 11. 9. 2018 LORBER, KRISTIJAN RAZVOJ FOTOKATALIZATORJA BIZMUT-TITANOV DIOKSID IN FOTOKATALITSKA REAKCIJA BISFENOLA A V ŠARŽNEM IN PRETOČNEM SISTEMU Z RECIKLOM Mentor: prof. dr. Igor Plazl Somentor: Petar Djinovic Datum zagovora: 12. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement JUTERŠEK, MOJCA PRIPRAVA IN PREVERJANJE UPORABNOSTI SINTEZNOBIOLOŠKEGA PLAZMIDA PMJC01 TER REPORTERSKEGA PROTEINA CISTATINA ZA DELO S CIANOBAKTERIJO SYNECHOCYSTIS SP. PCC 6803 Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 12. 9. 2018 REBERC, MATEJ KOVINSKO-ORGANSKI MATERIALI NA OSNOVI IMIDAZOLA IN TETRAHIDRIDOBORATOV LAHKIH KOVIN Mentor: prof. dr. Anton Meden Somentor: Jernej Stare Datum zagovora: 13. 9. 2018 PODJED, NINA ŠTUDIJ DIKETONATNIH LIGANDOV IN PRIPADAJOČIH KOVINSKIH KOMPLEKSOV Mentor: prof. dr. Iztok Turel Datum zagovora: 13. 9. 2018 PETRIČ, BOŠTJAN PRIPRAVA FLUORESCENČNIH NANOTELES ZA DETEKCIJO KOFEINA Mentor: doc. dr. Gregor Gunčar Datum zagovora: 14. 9. 2018 ZALOKAR, MATEJA AEROSOLI V KMETIJSKI DEJAVNOSTI Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 14. 9. 2018 PRELOŽNIK, SIMON TVORBA HALOGENSKIH VEZI V SOLEH HALOGENIRANIH DERIVATOV BENZOJSKE KISLINE Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 14. 9. 2018 SVETLIČIČ, MAJA NAČRTOVANJE IN OVREDNOTENJE SISTEMOV SESALSKIH CELIC, KI OMOGOČAJO PRESKOK PREZGODNJIH TERMINACIJSKIH KODONOV Mentor: znan. sod. dr. Iva Hafner Bratkovič Somentor: doc. dr. Gregor Gunčar Datum zagovora: 14. 9. 2018 POTOČNIK, KIM STANDARDIZACIJA JEMANJA PERITONEALNIH VZORCEV PRI KONTROLNIH BOLNICAH ZA RAK JAJČNIKOV IN NORMALIZACIJA MERITEV KONCENTRACIJE TUMORSKEGA OZNAČEVALCA GLEDE NA KOLIČINO PROTEINOV Mentor: Katarina Černe Somentor: doc. dr. Borut Kobal Datum zagovora: 14. 9. 2018 JAZBEC, VID OPTIMIZACIJA IZRAŽANJA REKOMBINANTNEGA PROTEINA RANKL IN TESTIRANJE MOŽNIH INHIBITORJEV OLIGOMERIZACIJE Mentor: izr. prof. dr. Marko Novinec Datum zagovora: 14. 9. 2018 ZEKIČ, JURE RAZVOJ METODE ZA DOLOČANJE ANTRAKINONA V KARTONU Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 14. 9. 2018 TRATNIK, BLAŽ UPORABA POLIMERNE IONSKE TEKOČINE V SODOBNIH BATERIJSKIH SISTEMIH Mentor: prof. dr. Miran Gaberšček Datum zagovora: 17. 9. 2018 BENSA, TJAŠA VLOGA PREPISOVALNIH DEJAVNIKOV ICER PRI URAVNAVANJU CIRKADIANE URE Mentor: prof. dr. Damjana Rozman Datum zagovora: 17. 9. 2018 ČERNOŠA, TANJA POŽARNA ODPORNOST VITKIH SOVPREŽNIH STEBROV Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 17. 9. 2018 KOSTANJEVEC, MOJCA REKONSTITUCIJA PROTEINA EPCAM V FOSFOLIPIDNEM DVOSLOJU Mentor: prof. dr. Brigita Lenarčič Datum zagovora: 18. 9. 2018 ZGONC, ALJA DOLOČANJE LOKUSOV KVANTITATIVNE LASTNOSTI POVEČANE VSEBNOSTI NEVTRALNIH LIPIDOV PRI KVASOVKI SACCHAROMYCES CEREVISIAE Mentor: izr. prof. dr. Uroš Petrovič Datum zagovora: 18. 9. 2018 BERKOPEC, KAJA BIOLOŠKI VPLIV FLIKERJA Mentor: prof. dr. Grega Bizjak Datum zagovora: 18. 9. 2018 SVETELJ, JURE PRIMERJAVA ŠARŽNE IN PRETOČNE PROIZVODNJE AKTIVNIH FARMACEVTSKIH UČINKOVIN Mentor: prof. dr. Igor Plazl Datum zagovora: 20. 9. 2018 VELIKONJA, MELITA IZDELAVA EKSPERIMENTA IN E-UČNE ENOTE O ETERIČNIH OLJIH KRAŠKIH RASTLIN ZA POUK KEMIJE V GIMNAZIJI Mentor: prof. dr. Bojana Boh Podgornik Datum zagovora: 21. 9. 2018 CUZNAR, KRISTINA TADEJA VPLIV KISLINSKEGA LUŽENJA NA POVRŠINO ŽELEZO-NIKELJ-KROMOVE ZLITINE Mentor: doc. dr. Barbara Novosel Datum zagovora: 21. 9. 2018 ŽUGELJ, LIDIJA PREGLED 14. SKUPINE PERIODNEGA SISTEMA Mentor: doc. dr. Nives Kitanovski Datum zagovora: 21. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement LAPANJA, TJAŠA OPTIMIZACIJA METOD ZA AKTIVACIJO GENOV, VKLJUČENIH V DIFERENCIACIJO REGULATORNIH CELIC T (TREG), Z UPORABO NAČRTOVANIH TRANSKRIPCIJSKIH FAKTORJEV IN SISTEMA CRISPR/ CAS9 Mentor: prof. dr. Simon Horvat Datum zagovora: 24. 9. 2018 TRDIN, EVA RAZVOJ METODE ZA DOLOČEVANJE RAZLIČNIH ZVRSTI OGLJIKA V SEDIMENTU Mentor: prof. dr. Matevž Pompe Datum zagovora: 24. 9. 2018 FRANTAR, ANJA KATALITSKO HIDROAMINIRANJE ACETILENOV S PD-NHC KOMPLEKSOM Mentor: prof. dr. Janez Košmrlj Datum zagovora: 24. 9. 2018 AVBELJ, JUDITA VLOGA STEFINA A PRI VNETJIH IN OKSIDATIVNEM STRESU Mentor: Nataša Kopitar-Jerala Somentor: prof. dr. Boris Turk Datum zagovora: 24. 9. 2018 POTOKAR, KARIN SINTEZA IN KARAKTERIZACIJA KOORDINACIJSKIH SPOJIN BAKRA(II) Z NIKOTINAMIDOM IN ANIONI DIKARBOKSILNIH KISLIN Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 24. 9. 2018 ČEBULJ, ANŽE SINTEZA IN KARAKTERIZACIJA SPOJIN PREHODNIH ELEMENTOV Z ANIONOM 4-HIDROKSIBENZOJSKE KISLINE Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 25. 9. 2018 KOVAČIČ, MATIC DOLOČANJE STRUKTUR G-KVADRUPLEKSOV STABILIZIRANIH S PIRENSKIMI SKUPINAMI Mentor: prof. dr. Janez Plavec Datum zagovora: 25. 9. 2018 JELENC, PETRA ANALIZA EVAKUACIJE IZ VISOKIH STAVB Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 26. 9. 2018 KUHAR, TINA DETEKCIJA PROTEINA MLKL V RAZLIČNIH CELIČNIH LINIJAH IN NEKROPTOZNA AKTIVNOST PROTEINSKEGA KONSTRUKTA MLKL N-154 D144K Z ZUNANJE STRANI CELIČNE MEMBRANE Mentor: prof. dr. Peter Veranič Somentor: doc. dr. Mojca Pavlin Datum zagovora: 26. 9. 2018 STRAŽAR, PETRA ELEKTROKEMIJSKO RAZTAPLJANJE RODIJA NA OGLJIKU V RAZREDČENI KISLINI Z UPORABO REAKTIVNIH PLINOV Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 26. 9. 2018 ČRNIGOJ, TOMAŽ DOLOČANJE EKSPLOZIJSKIH PARAMETROV HIBRIDNIH ZMESI Mentor: doc. dr. Barbara Novosel Datum zagovora: 26. 9. 2018 TEKAVEC, SIMONA PRIPRAVA MINIATURNIH PRETOČNIH METAKRILATNIH MONOLITNIH SISTEMOV NA PLASTIČNIH NOSILCIH Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 27. 9. 2018 ŽIŽEK, MATEJ OCENJEVANJE VPLIVA VIBRACIJ CELEGA TELESA NA UPRAVLJALCA VILIČARJA V LOGISTIKI TRGOVSKE DEJAVNOSTI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 27. 9. 2018 KORPAR, TANJA RAZPOREDITEV PROTEINOV MATRIKSA GOLGIJEVEGA APARATA V DEŽNIKASTIH CELICAH UROTELIJA SEČNEGA MEHURJA Mentor: prof. dr. Rok Romih Datum zagovora: 28. 9. 2018 BENČIČ, ALEKSANDER PRIPRAVA SAMOSESTAVLJIVIH PROTEINSKIH KOMPLEKSOV Z UPORABO FUZIJSKEGA PROTEINA Z DIMERIZIRAJOČO IN TRIMERIZIRAJOČO DOMENO Mentor: doc. dr. Gregor Gunčar Datum zagovora: 5. 10. 2018 SMOLE, JANJA VPLIV ORGANIZACIJSKIH, ČLOVEŠKIH IN KOMUNIKACIJSKIH DEJAVNIKOV NA VARNOST Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 5. 10. 2018 GOGNJAVEC, TJAŠA FOTOKEMIJSKE PRETVORBE SULFIDOV Z VODIKOVIM PEROKSIDOM V VODI Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 8. 10. 2018 ŠVAGELJ, MIHA VZPOSTAVLJANJE ORIGINALNE PROSOJNOSTI MATERIALOV Z ZAPOLNJENJEM MIKRORAZPOK Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 11. 10. 2018 ŽALIK, MIHA UPORABA IN ZANESLJIVOST JAVLJALNIKOV CO TER EMISIJE CO PRI GORENJU KADIL Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 15. 10. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S11 BIRKELBACH, JOŽICA PRENOS ROČNIH EKSTRAKCIJ V PRETOČNI SISTEM Mentor: izr. prof. dr. Nataša Gros Datum zagovora: 18. 10. 2018 PIRMAN, TOMAŽ MIKROKINETIKA PARCIALNE FOTOKATALITSKE OKSIDACIJE GLICEROLA PREKO TIO2 KATALIZATORJA DO PRODUKTOV Z VIŠJO DODANO VREDNOSTJO Mentor: izr. prof. dr. Aleš Podgornik Somentor: doc. dr. Blaž Likozar Datum zagovora: 19. 10. 2018 HODNIK, BLAŽ RAZVOJ IN VALIDACIJA KROMATOGRAFSKE METODE ZA DOLOČANJE MIKROKRISTALINIČNE CELULOZE V TABLETAH Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 25. 10. 2018 KUMAR, VANJA VPLIV LINEARNE GOSTOTE NABOJA NA POLIIONU NA ENTALPIJE MEŠANJA RAZTOPIN POLIELEKTROLITOV Z RAZTOPINAMI ENOSTAVNIH SOLI Mentor: izr. prof. dr. Janez Cerar Datum zagovora: 26. 10. 2018 DEBELJAK, NATAŠA IZRAŽANJE GLIKOZIDAZ IN NANOTELESA NA POVRŠINI KVASOVKE Mentor: izr. prof. dr. Uroš Petrovič Datum zagovora: 30. 10. 2018 VIDMAR, HELENA OBIČAJNA IN POSEBNA DVIGALA ZA PREVOZ OSEB Mentor: doc. dr. Boris Jerman Datum zagovora: 12. 11. 2018 KOTNIK, ROBERT SINTEZA AKRILATNIH NA PRITISK OBČUTLJIVIH LEPIL PO POSTOPKU RAZTOPINSKE POLIMERIZACIJE Mentor: doc. dr. Jernej Kajtna Somentor: doc. dr. Aleš Ručigaj Datum zagovora: 13. 11. 2018 KISILAK, MARIJA ŠTUDIJ VPLIVA NEKATERIH KOVINSKIH IONOV IN KOORDINACIJSKIH SPOJIN RUTENIJA NA AKTIVNOST KATEPSINA K Mentor: prof. dr. Iztok Turel Datum zagovora: 15. 11. 2018 CEROVIC, ROBERT SINTEZA IN KARAKTERIZACIJA NOVIH KOVINSKO-ORGANSKIH HIBRIDNIH MATERIALOV Mentor: viš. znan. sod. dr. Matjaž Mazaj Somentor: prof. dr. Anton Meden Datum zagovora: 19. 11. 2018 REGENT, LANA SINTEZA IN KARAKTERIZACIJA NOVIH FLUORIDOOKSIDOVANADATOV Mentor: prof. dr. Alojz Demšar Somentor: doc. dr. Andrej Pevec Datum zagovora: 28. 11. 2018 FURAR, URŠKA DOLOČANJE AKTIVNOSTI IN KONCENTRACIJE TIMIDIN KINAZE V SERUMU BOLNIC Z RAKOM DOJKE Mentor: prof. dr. Joško Osredkar Datum zagovora: 30. 11. 2018 MIHOVEC, ROK OKSIDACIJA ANTIBIOTIKA TIAMULIN FUMARATA Z OZONIRANJEM Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 4. 12. 2018 KONDA, MATEJA UPORABA LIGNINOLITIČNIH ENCIMOV GLIVE DICHOMITUS SQUALENS ZA RAZGRADNJO ODPADNEGA MATERIALA Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 4. 12. 2018 KARADŽA, MATEJA SINTEZA ELEKTROKATALIZATORJA ZA OKSIDACIJO VODE NA OSNOVI KOMPOZITA OGLJIKA IN Z DUŠIKOM DOPIRANEGA TITANOVEGA DIOKSIDA Mentor: prof. dr. Miran Gaberšček Datum zagovora: 17. 12. 2018 LAPORNIK, KATARINA SPREMLJANJE DELOVANJA IN OPTIMIZACIJA MALE ČISTILNE NAPRAVE PLANINSKE KOČE VALVASORJEV DOM POD STOLOM Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 18. 12. 2018 SEDMINEK, ANJA KOORDINACIJSKE SPOJINE NIKLJA S TIOSEMIKARBAZIDI IN TIOSEMIKARBAZONI Mentor: doc. dr. Andrej Pevec Datum zagovora: 18. 12. 2018 STRNAD, KATJA VZORČENJE IN ANALIZA HLAPOV V ZRAKU Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 19. 12. 2018 LUKAN, TANJA VPLIV POROZNOSTI IN POR KONVEKTIVNIH NOSILCEV NA HIDRODINAMSKE LASTNOSTI Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 20. 12. 2018 ŠAVRIČ, ALEŠ VLOGA AKTIVNEGA OGLJA IN NEČISTOČ PRI DEHIDROGENACIJI 5,6-DIFENIL-2,3-DIHIDROPIRAZINA Mentor: doc. dr. Krištof Kranjc Datum zagovora: 21. 12. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement DIPLOME - UNIVERZITETNI ŠTUDIJ UNIVERZITETNI ŠTUDIJ - 1. STOPNJA ŽIBERNA, KATARINA SINTEZA PALADIJEVIH PRODUKTOV OKSIDATIVNE ADICIJE Mentor: doc. dr. Martin Gazvoda Datum zagovora: 6. 2. 2018 MIKUŽ, ALENKA PROGNOSTIČNA VREDNOST CELOKUPNIH PROTEINOV V SERUMU IN ASCITESU Mentor: Katarina Černe Datum zagovora: 13. 2. 2018 KRANJC, DOMEN TITRACIJA Z AVTOMATSKIM TITRATORJEM Mentor: izr. prof. dr. Nataša Gros Datum zagovora:13. 2. 2018 AVGUŠTIN, NASTJA PRIMERJAVA REAKTIVNOSTI ORGANSKIH SPOJIN PRI RAZLIČNIH POGOJIH Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 14. 2. 2018 JELŠEVAR, MARUŠA FUNKCIONALIZACIJA SPOJIN PRI KLASIČNIH IN ALTERNATIVNIH POGOJIH Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 14. 2. 2018 POLES, PETER GRAFENSKI MATERIALI ZA NAPREDNE TEKSTILNE MATERIALE Mentor: doc. dr. Boštjan Genorio Datum zagovora: 19. 2. 2018 MORAN, ANJA MERITVE IN SANACIJA HRUPA V LESNI INDUSTRIJI Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 28. 2. 2018 LONČAR, ANJA SINTEZA IN KARAKTERIZACIJA KOMPLEKSA MANGANOVEGA(II) KLORIDA S PIRIDIN-2-ONOM Mentor: doc. dr. Saša Petriček Datum zagovora: 28. 2. 2018 UZAR, ALEKSANDRA PRIPRAVA REKOMBINANTNIH ANTITOKSINOV IPF_1062 IN IPF_1067 CIANOBAKTERIJE MICROCYSTIS AERUGINOSA PCC 7806 V BAKTERIJI ESCHERICHIA COLI Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 1. 3. 2018 KRELJ, ŽIGA KATALITSKA HIDRO(DEOKSI)GENACIJA LK IN GVL V REAKTORJU Z GOŠČO Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora:2. 3. 2018 TOMAN, ŽIGA NOVI MATERIALI ZA VISOKOTEMPERATURNE GORIVNE CELICE S TRDNIM OKSIDNIM ELEKTROLITOM Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 12. 3. 2018 KASTELIC, KRISTINA PREPREČEVANJE NEZGOD PRI UPORABI NEVARNIH KEMIKALIJ Mentor: doc. dr. Barbara Novosel Datum zagovora: 20. 3. 2018 KUKOVIČIČ, KARMEN UPORABA SAMODEJNE GASILNE AMPULE IN GRANATE BONPET PRI GAŠENJU ZAČETNIH POŽAROV Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 21. 3. 2018 BLAGOJEVIC, ALEKSANDAR ALKALNO-KARBONATNA REAKCIJA V HIDRAVLIČNIH VEZIVIH Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 26. 3. 2018 GAŠPERŠIČ, EMA IZRAŽANJE IN KARAKTERIZACIJA MAKROFAGNE KOLONIJE STIMULIRAJOČEGA FAKTORJA (M-CSF) Mentor: prof. dr. Boris Turk Datum zagovora: 29. 3. 2018 GREGORC, JURE SINTEZA METIL-4-OKSO-1-AZASPIRO[4. 4]NON-2-EN-3- KARBOKSILATA Mentor: prof. dr. Jurij Svete Datum zagovora: 11. 4. 2018 GOLTNIK, TJAŠA NIZKOTEMPERATURNA SINTEZA IN SAMOČISTILNA UČINKOVITOST TANKIH PLASTI TIO2-ZRO2 NA STEKLENIH IN KOVINSKIH PODLAGAH Mentor: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 20. 4. 2018 KOPAČ, URŠA PARALELNE HETERODIMERNE OBVITE VIJAČNICE, NAČRTOVANE NA OSNOVI TROPOMIOZINA Mentor: prof. dr. Simon Horvat Datum zagovora: 24. 4. 2018 SUŠNIK, BLAŽ ZDRAV ŽIVLJENJSKI SLOG ZAPOSLENIH NA OSNOVNI ŠOLI Mentor: prof. dr. Marjan Bilban Datum zagovora: 24. 4. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S13 ŽIGANTE, NIKA UPORABA POLI(METIL METAKRILATA) V KOZMETIKI Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 25. 4. 2018 CIRIKOVIC, DAVID PROUČEVANJE ENTALPIJSKIH SPREMEMB PRI MEŠANJU RAZTOPIN MODELNIH ELEKTROLITOV S TEORIJO OZ/ MSA Mentor: izr. prof. dr. Miha Lukšič Datum zagovora: 26. 4. 2018 TEKAVEC, SARA VLOGA SIGNALNE POTI ERK PRI UČINKIH VLDL NA KARDIOMIOCITE Mentor: doc. dr. Tadeja Režen Datum zagovora: 26. 4. 2018 STRADOVNIK, ADISA DIMERIZACIJA TERMINALNIH ACETILENOV Mentor: doc. dr. Martin Gazvoda Datum zagovora: 8. 5. 2018 ČERNE, ANA VPLIV DETERGENTA NA OMOČENJE VLAKEN Mentor: izr. prof. dr. Marko Hočevar Datum zagovora: 15. 5. 2018 JOŠT, GAŠPER VREDNOTENJE PORAZDELITVE ALKALNE ZALOGE V RAZKISLINJENIH KNJIGAH Mentor: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 16. 5. 2018 ŽGANK, ANDREJ DELNA PRIPRAVA AMINOMALEIMIDA KOT OSNOVNE MOLEKULE PRI PRIPRAVI RAZLIČNIH MALEIMIDOBENZOKSAZINOV Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 28. 5. 2018 GEORGIEVSKI, ANA TEMPERATURNO ODZIVNI POLIMERNI MATERIALI Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 28. 5. 2018 SMRDELJ, BENJAMIN MODELIRANJE KEMIJSKIH REAKCIJ Z UPORABO MREŽNE BOLTZMANNOVE METODE IN DISIPATIVNE DINAMIKE DELCEV Mentor: prof. dr. Igor Plazl Datum zagovora: 1. 6. 2018 JAKLIN, MATEJ PREGLED PLASTOVITIH MATERIALOV IN NJIHOVA UPORABA Mentor: doc. dr. Boštjan Genorio Datum zagovora: 7. 6. 2018 ALEKSIČ, SIMON VPLIV OKSIDACIJE GVANINA NA STRUKTURO IN ZVITJE TELOMERNIH G-KVADRUPLEKSOV Mentor: prof. dr. Janez Plavec Datum zagovora: 11. 6. 2018 RUS, AJDA ZAMENJAVA IZOCIANATOV V DVOKOMPONENTNIH POLIURETANSKIH PREMAZIH Mentor: prof. dr. Urška Šebenik Datum zagovora: 15. 6. 2018 FAJS, ALENKA UPORABA MALE VODNE LEČE ZA FITOREMEDIACIJO ODPADNIH VODA Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 19. 6. 2018 KEJŽAR, NEJC STRUKTURNE ZNAČILNOSTI KLAVDINSKE OLIGOMERIZACIJE Mentor: doc. dr. Miha Pavšič Datum zagovora: 26. 6. 2018 BAKAČ, ALJOŠA DOLOČEVANJE ŽELEZOVIH IONOV V TLEH Z MOLEKULSKO ABSORPCIJSKO SPEKTROMETRIJO Mentor: prof. dr. Helena Prosen Datum zagovora: 28. 6. 2018 COTMAN, KLEMEN PIRIMIDINSKI DERIVATI KOT SUBSTRATI V REAKCIJAH C-H AKTIVACIJE Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 2. 7. 2018 FERJAN, ŠPELA FOTOKATALITIČNA PRODUKCIJA VODIKA Z CU/TIO2 KATALIZATORJEM Mentor: prof. dr. Miran Gaberšček Datum zagovora: 2. 7. 2018 RATAJ, JAN ANALIZA TEKOČIH PRODUKTOV HIDROGENIRANJA LEVULINSKE KISLINE Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 3. 7. 2018 KRIŽNIČ, KLARA ŠKODLJIVI UČINKI HRUPA NA ZAPOSLENE V ODPRTIH PISARNAH Mentor: doc. dr. Klementina Zupan Datum zagovora: 4. 7. 2018 MARINIČ, DANA VPLIV DIELS-ALDER ADUKTA NA ZAMREŽENE EPOKSIDNE SMOLE Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 5. 7. 2018 ZALAR, ANŽE SURFAKTANTI V ATMOSFERSKIH AEROSOLIH Mentor: doc. dr. Bojan Šarac Datum zagovora: 6. 7. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement HUDOVERNIK, ELA SINTEZA SILILIRANEGA KISLINSKEGA DERIVATA SIMVASTATINA ZA PRIPRAVO BIOLOŠKO AKTIVNIH KOMPLEKSOV S CISPLATINOM Mentor: prof. dr. Janez Košmrlj Datum zagovora: 6. 7. 2018 PENKO, ANA PRIMERNOST UPORABE METODE ELISA ZA UGOTAVLJANJE PRISOTNOSTI PROTEINOV V UMETNIŠKIH BARVAH Mentor: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 10. 7. 2018 LAVRIHA, PIA IZRAŽANJE IN IZOLACIJA NANOTELES PROTI MLKL N154 IN ANALIZA VEZAVE NANOTELESA 3MLK_84 NA MLKL N154 Mentor: doc. dr. Gregor Gunčar Datum zagovora: 10. 7. 2018 AGREŽ, BARBARA PEROVSKITNE SONČNE CELICE Mentor: izr. prof. dr. Matija Tomšič Datum zagovora: 10. 7. 2018 GAŠPARIČ, LEA VPLIV VODIKOVE VEZI NA LASTNOSTI KEMIJSKIH IN BIOLOŠKIH SISTEMOV Mentor: prof. dr. Andrej Jamnik Datum zagovora: 10. 7. 2018 ANTONČIČ, TEJA VREDNOTENJE UČINKOVITOSTI SISTEMA ZA PRIPRAVO PITNE VODE IZ POVRŠINSKEGA VIRA Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 11. 7. 2018 ŽIBERT, TAJA VINIL ALKOHOL V MEDZVEZDNEM PROSTORU Mentor: prof. dr. Tomaž Urbič Datum zagovora: 11. 7. 2018 OSTANEK JURINA, LEON Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 11. 7. 2018 RIJAVEC, MARŠA Mentor: prof. dr. Urška Šebenik Datum zagovora: 11. 7. 2018 PETROVIC, VITO NESREČA V NUKLEARNI ELEKTRARNI ČERNOBIL: VZROKI IN POSLEDICE Mentor: doc. dr. Mitja Robert Kožuh Datum zagovora: 12. 7. 2018 ANTOLIC, MATEJ INHALABILNOST KOVINSKIH DELCEV Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 12. 7. 2018 FERČEC, URBAN IZRAŽANJE, IZOLACIJA IN KARAKTERIZACIJA HUMANIH KASPAZ-4 IN -5 Mentor: prof. dr. Boris Turk Datum zagovora: 12. 7. 2018 ARH, NEJC OBLIKOVANJE IN PRIPRAVA NANOTELES ZA SIMETRIČNO DIMERIZACIJO NA OSNOVI KOORDINACIJE KOVINSKIH IONOV Mentor: doc. dr. Gregor Gunčar Datum zagovora: 13. 7. 2018 KRIŽAJ, NINA ENOSTAVNI ANALITIČNI MODEL VODE IN HIDROFOBNE HIDRATACIJE Mentor: izr. prof. dr. Miha Lukšič Datum zagovora: 13. 7. 2018 KLEMENČIČ, EVA VPLIV AKTIVACIJE MAKROFAGOV NA IZRAŽANJE IN IZLOČANJE KATEPSINA C Mentor: prof. ddr. Boris Turk Datum zagovora: 13. 7. 2018 LUKŠIČ, TJAŠA EVOLUCIJSKA DINAMIKA TRANSKRIPCIJSKIH FAKTORJEV PRI HOMINIDIH Mentor: izr. prof. dr. Dušan Kordiš Datum zagovora: 1. 8. 2018 MENCIGAR, MATEVŽ BIOKATALITSKI PROCESI S CELICAMI V MINIATURIZIRANIH REAKTORJIH Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 17. 8. 2018 KOVŠE, ALEN RAZVOJ KOMPOZITNEGA MATERIALA ZAVORNIH PLOŠČIC Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 22. 8. 2018 VEHAR, ANJA VPLIV KAVITACIJSKEGA PREDČIŠČENJA ODPADNE VODE NA RAZBREMENITEV ULTRAFILTRACIJSKIH MODULOV Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 23. 8. 2018 PRIMOŽIČ, LUCIJA RAZGRADNJA IN RAZBARVANJE ODPADNEGA PAPIRJA S POMOČJO GLIVE DICHOMITUS SQUALRNS Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 23. 8. 2018 HOČEVAR, JOŠT VLOGA HIDROFOBNEGA ŽEPA EPCAM IN TROP2 PRI SIGNALIZIRANJU PREKO REGULIRANE INTRAMEMBRANSKE CEPITVE Mentor: doc. dr. Miha Pavšič Datum zagovora: 24. 8. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S15 HEINDLER, ANŽE VARNO SKLADIŠČENJE IN UPORABA VODIKOVEGA KLORIDA Mentor: doc. dr. Barbara Novosel Datum zagovora: 29. 8. 2018 LIPOVŠEK, AGATA SINTEZA PRIPOJENIH DERIVATOV 3-BENZOILAMINO-2H-PIRAN-2-ONA, 5-BENZOILNEGA ANALOGA IN NADALJNJE [4+2] CIKLOADICIJE Z MALEINANHIDRIDOM IN N-FENILMALEIMIDOM Mentor: doc. dr. Krištof Kranjc Datum zagovora: 30. 8. 2018 KAMIN, REBEKA SINTEZA 6-SUBSTITUIRANIH 3-BENZOILAMINO-2H-PIRAN-2-ONSKIH DERIVATOV IN NJIHOVA UPORABA V DIELS-ALDERJEVIH REAKCIJAH Z MALEINANHIDRIDOM POD KLASIČNIMI IN MIKROVALOVNIMI POGOJI Mentor: doc. dr. Krištof Kranjc Datum zagovora: 30. 8. 2018 BERGLEZ, TILEN SIPANJE SVETLOBE NA OKROGLIH STRUKTURAH: TOGA KROGLA, VOTLA KROGLA IN VEZIKLI Mentor: prof. dr. Ksenija Kogej Datum zagovora: 30. 8. 2018 FRIŠKOVEC VONČINA, ŠPELA REAKCIJE BAKROVEGA(II) KLORIDA S PIRIDIN-2-ONOM IN PIRAZINOM Mentor: doc. dr. Saša Petriček Datum zagovora: 31. 8. 2018 MENART, SVIT PRETVORBA KETONOV V SILILNE DERIVATE GEM- DIHIDROPEROKSIDOV Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 3. 9. 2018 SLABAJNA, BLAŽ RENTGENSKA PRAŠKOVNA DIFRAKCIJA GEOLOŠKIH VZORCEV Mentor: prof. dr. Anton Meden Datum zagovora: 3. 9. 2018 ŠVIGELJ, NINA VPLIV UČINKOVINE NA OSNOVI NUKLEINSKIH KISLIN NA RAZPAD TARČNIH PROTEINOV Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 3. 9. 2018 KOTNIK, JAN MATEMATIČNI OPIS SPROŠČANJA UČINKOVIN IZ POLIMERNIH HIDROGELOV Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 3. 9. 2018 POGAČAR, ŽIGA MODELI PRENOSA SNOVI V HIDROGELIH Mentor: prof. dr. Matjaž Krajnc Datum zagovora: 3. 9. 2018 PONIKVAR, ŽIGA SINTEZA NEKATERIH 5,6-DISUBSTITUIRANIH 3-BENZOILAMINO-2H-PIRAN-2-ONOV IN NJIHOVA UPORABA V CIKLOADICIJAH Z MALEINANHIDRIDOM IN N-SUBSTITUIRANIM MALEIMIDOM Mentor: doc. dr. Krištof Kranjc Datum zagovora: 4. 9. 2018 DOLHAR, DAVID SINTEZA IN LASTNOSTI t-BUTIL AZIDOFORMATA Mentor: prof. dr. Janez Košmrlj Datum zagovora: 4. 9. 2018 STIBILJ, KRISTJAN IZOLACIJA IN IDENTIFIKACIJA GLIV IZ BAZENSKIH VODA TER VPLIV NATRIJEVEGA HIPOKLORITA NA RAST IZBRANIH GLIV Mentor: prof. dr. Iztok Turel Datum zagovora: 5. 9. 2018 BANFI, PRIMOŽ STRUKTURA IN KARAKTERISTIKE SIDEROFOROV Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 5. 9. 2018 BRCAR, IDA POSKUSI SONOGASHIROVEGA PRIPAJANJA Z ARIL KLORIDI Mentor: prof. dr. Janez Košmrlj Datum zagovora: 5. 9. 2018 CERNATIČ, FILIP UPORABA METOD KEMOINFORMATIKE PRI RAZVOJU ANTIMALARIKOV Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 5. 9. 2018 BORŠIC, ELVIRA SILILIRANJE KISLINSKEGA DERIVATA SIMVASTATINA Mentor: prof. dr. Janez Košmrlj Datum zagovora: 6. 9. 2018 ZUPANC, MAJA URAVNAVANJE SIGNALIZACIJE TLR4/NF-?B/IL-6 V POVEZAVI S PRESNOVNO IN ENDOKRINO VLOGO SKELETNE MIŠICE Mentor: prof. dr. Tomaž Marš Datum zagovora: 6. 9. 2018 MALNARIČ, IRIS EPOKSIDNI POLIMERI S SPOSOBNOSTJO SAMOCELJENJA Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 6. 9. 2018 PRIVŠEK, PETRA VPLIV RECIKLIRANJA NA REOLOŠKE IN TERMIČNE LASTNOSTI HDPE Mentor: doc. dr. Lidija Slemenik Perše Datum zagovora: 6. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement KAPŠ, ŠPELA SAMOCELJENJE POLIMERNIH MATERIALOV NA OSNOVI FOTOKEMIČNIH REAKCIJ Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 6. 9. 2018 HERMAN, JOŠT MORFOLOŠKE LASTNOSTI LIOFILIZIRANEGA GRAFEN OKSIDA IN GRAFENA Mentor: doc. dr. Boštjan Genorio Datum zagovora: 6. 9. 2018 ARNŠEK, MAŠA UPORABA POLIMEROV Z OBLIKOVNIM SPOMINOM V BIOMEDICINI Mentor: prof. dr. Urška Šebenik Datum zagovora: 6. 9. 2018 ČUJEŠ, ANA UPORABA NANOCELULOZE V BIOMEDICINI Mentor: prof. dr. Urška Šebenik Datum zagovora: 6. 9. 2018 BIZJAK, OŽBEJ POLIHIPE Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 6. 9. 2018 PELC, TIN ADSORPCIJA BIOLOŠKIH MAKROMOLEKUL NA POVRŠINE Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 6. 9. 2018 LEBAR, SERGEJA UPORABA MEMBRANSKIH SUPRESORJEV ZA ODSTRANJEVANJE MOTEČIH IONOV Mentor: prof. dr. Matevž Pompe Datum zagovora: 6. 9. 2018 MOŽINA, JERNEJ PAMETNE NAPRAVE NA OSNOVI POLIMEROV S SPOMINSKIM UČINKOM Mentor: prof. dr. Urška Šebenik Datum zagovora: 6. 9. 2018 KELHAR, NUŠA IZBOLJŠANJE PROTOKOLA BLOKADE NEŽELENE VEZAVE PROTITELES PRI IMUNOOZNAČEVANJU PROTEINOV V TKIVNIH PREPARATIH Mentor: prof. dr. Peter Veranič Datum zagovora: 7. 9. 2018 PILPAH, TILEN MIKROBIOREAKTORJI S TREMI FAZAMI Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 7. 9. 2018 ŠADL, ALEN SINTEZA IN DERIVATIZACIJA TIENIL KETONOV Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 7. 9. 2018 ERHATIČ, JERNEJA REAKCIJE BAKROVEGA(II) KLORIDA Z N-(2-PIRIDIL) PIRIDIN-2-TIOKARBOKSAMIDOM Mentor: doc. dr. Andrej Pevec Datum zagovora: 7. 9. 2018 ŽUNTAR, JAN MODIFICIRANI SEPARATORJI V LITIJ-KOVINSKIH AKUMULATORJIH Mentor: izr. prof. dr. Robert Dominko Datum zagovora: 7. 9. 2018 KOZAMERNIK, MARK FUNKCIONALIZACIJA GRAFENSKIH NANOTRAKOV ZA UPORABO V POLIMERNIH NANOKOMPOZITIH Mentor: doc. dr. Boštjan Genorio Datum zagovora: 7. 9. 2018 HRUP, MARTIN INFRARDEČI SPEKTRI ALKOHOLAMINOV Mentor: doc. dr. Barbara Modec Datum zagovora: 7. 9. 2018 BAVČAR, MOJCA ŠTUDIJ REAKCIJE ?-PINENA S HIDROKSILNIM RADIKALOM Mentor: prof. dr. Matevž Pompe Datum zagovora: 7. 9. 2018 RAJH, EVA HISTOKEMIJSKA KAREKTERIZACIJA ORGANSKEGA MATRIKSA PRI RAKU PORCELLIO SCABER Mentor: doc. dr. Nada Žnidaršič Datum zagovora: 10. 9. 2018 ROGAN, MATIC REAKTIVNOST ORGANSKIH SPOJIN PRI RAZLIČNIH POGOJIH Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 10. 9. 2018 HLEBŠ, TINA OSEBNA VAROVALNA OPREMA GASILCA IN PRAKTIČNI PROBLEMI PRI NJENI UPORABI Mentor: izr. prof. dr. Matija Tomšič Datum zagovora: 10. 9. 2018 HRUŠEVAR, PETRA IZOLACIJA IN KARAKTERIZACIJA BAKTERIJ ČLOVEŠKE RIBICE Mentor: doc. dr. Martina Turk Datum zagovora: 10. 9. 2018 GOSTENČNIK, ŽAN SINTEZA LIGANDOV ZA ORGANOKOVINSKE KOMPLEKSE Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 10. 9. 2018 MAROLT, ŠPELA OVREDNOTENJE VOLUMETRIČNIH OPERACIJ, IZVEDENIH S ČRPALKO NA OSNOVI BRIZGE Mentor: izr. prof. dr. Nataša Gros Datum zagovora: 10. 9. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S17 ŠMAJDEK, SVIT REDOKS TITRACIJE Z ELEKTROKEMIJSKO DETEKCIJO KONČNE TOČKE Mentor: izr. prof. dr. Nataša Gros Datum zagovora: 10. 9. 2018 KRUŠIČ, JANA ORGANOKATALIZIRANA SPIROCIKLIZACIJA METIL (5Z)- 5-BENZILIDEN-1,2-DIMETIL-4-OKSO-4,5-DIHIDRO-1H- PIROL-3-KARBOKSILATA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 11. 9. 2018 ČERNIČ, TINA VPLIV ANTIBIOTIKOV NA MALO VODNO LEČO Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 11. 9. 2018 ZUPANČIČ, KLEMEN ZMANJŠEVANJE EMISIJ PRAŠNIH DELCEV PM10 Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 12. 9. 2018 KREJAN, EVA PRIMERJAVA ORGANSKIH IN RUTENIJEVIH BARVIL V ELEKTROKEMIJSKIH SONČNIH CELICAH Mentor: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 12. 9. 2018 ŠIMENC, JAKA IZBIRA IN PRIPRAVA MATERIALA ZA TEHNOLOGIJO 3D TISKANJA Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 12. 9. 2018 LIPOVŠEK, JAN ANALIZA EVAKUACIJSKEGA ČASA V POSLOVNEM OBJEKTU S PROGRAMOM PATHFINDER Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 12. 9. 2018 SUŠNIK, MIHA UKREPI ZA VARNO UPORABO ČISTIL Mentor: doc. dr. Barbara Novosel Datum zagovora: 12. 9. 2018 KOZOLE, MOJCA ADSORPCIJA IZBRANIH KOVIN NA MIKROPLASTIKO Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 12. 9. 2018 PIRC, KARMEN ADSORPCIJA MIKROPLASTIKE NA AKTIVNO BLATO IZ BIOLOŠKE ČISTILNE NAPRAVE Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 12. 9. 2018 BELLINA, NIKOL ERGONOMSKO OBLIKOVANJE DELOVNEGA MESTA PRI IZDELAVI AVTOMOBILSKIH DELOV Mentor: doc. dr. Klementina Zupan Datum zagovora: 12. 9. 2018 ŽULA, MATEJ ČIŠČENJE ODPADNIH VOD S KAVITACIJO Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 12. 9. 2018 BELINGAR, LUCIJA PRIPRAVA IN KARAKTERIZACIJA BIOOGLJA IZ LIGNOCELULOZNE BIOMASE Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 12. 9. 2018 VIDRIH, ŽIGA PRIPRAVA IN KARAKTERIZACIJA BIOOGLJA IZ LESNE BIOMASE Mentor: izr. prof. dr. Andreja Žgajnar Gotvajn Datum zagovora: 12. 9. 2018 BREZAR, TINA INŠPEKCIJA DELA NA PODROCJU VARNOSTI IN ZDRAVJA PRI DELU Mentor: Luka Tičar Datum zagovora: 12. 9. 2018 LUKANČIČ, KATJA OCENA ODSTRANJEVANJA CELOTNEGA FOSFORJA IZ ODPADNIH VOD NA CČN LJUBLJANA Mentor: doc. dr. Gabriela Kalčikova Datum zagovora: 12. 9. 2018 ZABRET, POLONA POSINTEZNA MODIFIKACIJA TITANOVIH IN CIRKONIJEVIH KOVINSKO-ORGANSKIH MREŽ Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 13. 9. 2018 TITOVŠEK, DAVID Mentor: prof. dr. Kristina Djinovic Carugo Datum zagovora: 13. 9. 2018 SEME, TANJA VPLIV KAVITACIJE NA RAZGRADJO BARVILA ZA TEKSTIL Mentor: prof. dr. Matevž Dular Datum zagovora: 13. 9. 2018 HALAS, JANEZ PADEC TLAKA NA POROZNIH NOSILCIH Mentor: izr. prof. dr. Aleš Podgornik Datum zagovora: 13. 9. 2018 PERIC, TANJA KARAKTERIZACIJA VPLIVA KOMPONENT PROTEINSKEGA KOMPLEKSA PERMES NA ACETILACIJO HISTONOV Mentor: izr. prof. dr. Uroš Petrovič Datum zagovora: 13. 9. 2018 ZORKO, DAVID SPEKTROFOTOMETRIČNO DOLOČEVANJE MONOSAHARIDOV Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 13. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement KORDEŽ, MAY DOLOČANJE HLAPNIH SPOJIN V PIVU Mentor: prof. dr. Helena Prosen Datum zagovora: 13. 9. 2018 ŠPEGEL, MATEJA DOLOČANJE KLORIRANIH TRIAZINSKIH PESTICIDOV V VODI Z GC-ECD Mentor: prof. dr. Helena Prosen Datum zagovora: 13. 9. 2018 VAN MIDDEN, KATARINA PETRA PRIPRAVA VZORCEV ZA IDENTIIKACIJO INTERAKCIJSKIH PARTNERJEV PROTEINA FUS Mentor: izr. prof. dr. Boris Rogelj Datum zagovora: 14. 9. 2018 KAVČIČ, SANDI STRUKTURA IN REAKTIVNOST PROSTIH RADIKALOV Mentor: prof. dr. Tomaž Urbič Datum zagovora: 14. 9. 2018 ŠTRANCAR, VIDA OSNOVNA BIOKEMIJSKA KARAKTERIZACIJA METAKASPAZE TIPA 2 MODELNEGA ALGNEGA ORGANIZMA CHLAMYDOMONAS REINHARDTII Mentor: izr. prof. dr. Marko Dolinar Datum zagovora: 14. 9. 2018 KLANČNIK, BOR SINTEZA IZOTIOCIANATNIH DERIVATOV ISATINA IN NJIHOVA UPORABA V ORGANOKATALIZIRANI KASKADNI REAKCIJI Mentor: doc. dr. Uroš Grošelj Datum zagovora: 14. 9. 2018 LOČNIŠKAR, JAN OKSIDACIJA CIMETOVE IN FERULNE KISLINE V BENZALDEHID IN VANILIN Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 14. 9. 2018 VALETIČ, VASJA Mentor: prof. dr. Iztok Turel Datum zagovora: 14. 9. 2018 POGORELC, EVA GOSTOTA IN VISKOZNOST VODNIH RAZTOPIN FUNKCIONALIZIRANIH KVARTERNIH OKTILAMONIJEVIH SURFAKTANTOV Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 14. 9. 2018 MLINAR, ŽIGA DOLOČANJE RAZGRADNIH PRODUKTOV KOFEINA S TEKOČINSKO KROMATOGRAFIJO Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 14. 9. 2018 GRIŽNIK, MOJCA SINTEZA IN KARAKTERIZACIJA CINK-ALUMINIJEVIH PLASTOVITIH DVOJNIH HIDROKSIDOV Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 14. 9. 2018 MASELJ, NIK SINTEZA DIHALOJODATOV IN NJIHOVIH AMONIJEVIH SOLI Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 14. 9. 2018 VESEL, MATEJ ERGONOMSKI PROBLEMI ZARADI PRETEŽNO SEDEČEGA DELA Mentor: doc. dr. Klementina Zupan Datum zagovora: 14. 9. 2018 BELAK VIVOD, MATIC PRIPRAVA IN POMENE NAČRTOVANIH BIOLOŠKIH MOLEKUL Mentor: prof. dr. Barbara Hribar Lee Datum zagovora: 14. 9. 2018 ROČNIK, TINA VPLIV VPELJAVE BIOTRANSFORMACIJ NA TRAJNOST KEMIJSKEGA PROCESA Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 14. 9. 2018 SINIČ, TADEJ MATERIALI ZA SOLARNE CELICE Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 17. 9. 2018 ŽIVIČ, ZALA VPLIV AMINOKISLINSKEGA ZAPOREDJA NA TERMODINAMIKO INTERAKCIJE INTRINZIČNO NEUREJENIH PROTEINOV S TARČO Mentor: prof. dr. Jurij Lah Datum zagovora: 17. 9. 2018 SEDEJ, NELI IDENTIFIKACIJA IN OPREDELITEV NARAVNIH VARIANT LANOSTEROL 14 -DEMETILAZE IN SILICO Mentor: prof. dr. Damjana Rozman Datum zagovora: 17. 9. 2018 HALUŽAN VASLE, ANA VPLIV AKTIVACIJE ADRENERGIČNIH RECEPTORJEV NA ŠTEVILO IN VELIKOST LIPIDNIH KAPLJIC V PODGANJIH ASTROCITIH V KULTURI Mentor: doc. dr. Nina Vardjan Datum zagovora: 17. 9. 2018 SMERDU, MARK GRAFEN OKSID KOT DODATEK PREMAZOM ZA SONČNE ABSORBERJE Mentor: doc. dr. Boštjan Genorio Datum zagovora:17. 9. 2018 MOTALN, KLEMEN UVOD V NOVO ENCIMSKO KINETIKO Mentor: prof. dr. Igor Plazl Datum zagovora: 17. 9. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S19 KOLARIČ, MITJA PRETVORBE ORGANSKIH SPOJIN S KISIKOVIMI FUNKCIONALNIMI SKUPINAMI Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 17. 9. 2018 MARINKO, ALJOŠA IZOLACIJA CRISP IZ STRUPA MODRASA (VIPERA AMMODYTES AMMODYTES) IN NJIHOV VPLIV NA CELIČNO LINIJO RAKA DOJKE Mentor: prof. dr. Igor Križaj Datum zagovora: 17. 9. 2018 DIZDAREVIC, ŠEJLA LITIJ-ŽVEPLOVE BATERIJE ZA KOMERCIALNO UPORABO Mentor: prof. dr. Miran Gaberšček Datum zagovora: 17. 9. 2018 RUPNIK, ALEN IZDELAVA PRETOČNIH REAKTORSKIH SISTEMOV S TEHNOLOGIJO 3D TISKANJA Mentor: doc. dr. Aleš Ručigaj Datum zagovora: 17. 9. 2018 GREBENC, ANDREJ PREGLED NAPREDNIH METOD ZA OPIS TRKOV V MREŽNI BOLTZMANNOVI METODI Mentor: prof. dr. Igor Plazl Datum zagovora: 17. 9. 2018 KAVČNIK, ANDREJA ELEKTROKEMIJA V MIKROREAKTORJIH Mentor: prof. dr. Igor Plazl Datum zagovora: 17. 9. 2018 ŽIVIČ, SUZANA REAKTIVNOST ORGANSKIH SPOJIN PRI KLASIČNIH IN ALTERNATIVNIH POGOJIH Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 17. 9. 2018 OCVIRK, MANCA DOLOČEVANJE HLAPNIH SPOJIN V MEDU Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 17. 9. 2018 REBERNIK, MIHAELA SINTEZA IN KARAKTERIZACIJA TERNARNIH ORGANORUTENIJEVIH KOMPLEKSOV S PIRITIONOM IN FOSFINI Mentor: doc. dr. Jakob Kljun Datum zagovora: 17. 9. 2018 KORENČIČ, EVA PRIMERJAVA ALKALNE ZALOGE V TESTNIH LISTIH IN KNJIGAH PO RAZKISLINJENJU Mentor: izr. prof. dr. Irena Kralj Cigic Datum zagovora:17. 9. 2018 KOLMAN, NEJC REOLOŠKE LASTNOSTI VODNIH RAZTOPIN POLIETILEN GLIKOLA OB PRISOTNOSTI RAZLIČNIH SOLI Mentor: izr. prof. dr. Janez Cerar Datum zagovora: 17. 9. 2018 MUŠIČ, PETER POŽARNA VARNOST PRI SKLADIŠČENJU GUM Mentor: doc. dr. Domen Kušar Datum zagovora:17. 9. 2018 KRAJNC, DOROTEJA ZAMENJAVA TIO2 Z ZRO2 V BELIH PIGMENTNIH PREMAZIH Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 17. 9. 2018 LESJAK, TINA ERGONOMSKA ANALIZA DELOVNEGA MESTA PRI PROIZVODNJI ULITKOV V PODJETJU TALUM D. D. KIDRIČEVO Mentor: doc. dr. Klementina Zupan Datum zagovora: 17. 9. 2018 KRAGELJ, MIHA VPLIV KAKOVOSTI SUROVINE NA LASTNOSTI ŽGANIH GLINENIH IZDELKOV Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 17. 9. 2018 KOTNIK, LOVRO OKSIDATIVNA ADICIJA 2-JODOETINILBENZENOVIH DERIVATOV NA PALADIJ Mentor: prof. dr. Janez Košmrlj Datum zagovora: 17. 9. 2018 ERHATIČ, KLEMEN IZDELAVA IN RAZVOJ MIKROFLUIDNE NAPRAVE ZA SUHI REFORMING BIOPLINA Mentor: prof. dr. Igor Plazl Datum zagovora: 17. 9. 2018 ADAMEK, MAKSIMILJAN VPLIVI GVAJAKOLA IN NEKATERIH NJEGOVIH DERIVATOV NA FIZIOLOGIJO RASTLIN Mentor: izr. prof. dr. Marko Novinec Datum zagovora: 17. 9. 2018 TOMC, ANŽE POVEČEVALNI EFEKTI PROCESA OZONACIJE BIORAZGRADLJIVIH SNOVI V MIKROSISTEMIH Mentor: prof. dr. Igor Plazl Datum zagovora: 18. 9. 2018 GREGO, MARTIN VPLIV SVETLOBE NA MELATONIN V ČLOVEKU Mentor: prof. dr. Grega Bizjak Datum zagovora: 18. 9. 2018 TRUDEN, ANJA SINTEZA IZBRANIH 1,4-DIARIL-1,2,3-TRIAZOLOV Mentor: prof. dr. Janez Košmrlj Datum zagovora: 18. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement LIPOVŠEK, MATEJ VARNOST PRI PRIPRAVI PREMAZOV ZA PROIZVODNJO KARTONA Mentor: doc. dr. Barbara Novosel Datum zagovora: 18. 9. 2018 JANKOVIČ, DOMINIK SINTEZA 1-ALKIL-4-ARIL-1,2,3-TRIAZOLOV Mentor: doc. dr. Martin Gazvoda Datum zagovora: 18. 9. 2018 SILJANOVSKA, ANA PRIPRAVA STABILNIH DIAZOIJEVIH SOLI Mentor: prof. dr. Janez Košmrlj Datum zagovora: 18. 9. 2018 ŽNIDARIČ, MATEJA 4-METOKSIBENZIL AZID KOT REAGENT ZA PRIPRAVO TRIAZOLOV Mentor: doc. dr. Martin Gazvoda Datum zagovora: 18. 9. 2018 KLANČIŠAR, DAVID PREPREČEVANJE ZASTRUPITEV Z OGLJIKOVIM OKSIDOM Mentor: doc. dr. Barbara Novosel Datum zagovora: 18. 9. 2018 NOVAK, VALENTINA PRIPRAVA ČLOVEŠKIH PROTEINOV ANEKSINA A11 IN TRANSPORTINA 1 TER KARAKTERIZACIJA NJUNE VEZAVE Mentor: doc. dr. Vera Zupunski Datum zagovora: 19. 9. 2018 VIDMAR, TIM ANALIZA HIPOTETIČNEGA POZARA NA KOLONSKO SKLADIŠČNEM PROSTORU V LESNO PREDELOVALNEM OBRATU Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 19. 9. 2018 ERZIN, ANJA VIRUSI V ODPADNIH VODAH TER NJIHOVO ODSTRANJEVANJE Mentor: izr. prof. dr. Andreja Zgajnar Gotvajn Datum zagovora: 19. 9. 2018 BEMBIČ, PRIMOŽ IDENTIFIKACIJA PROTEINSKIH TARČ MALOMOLEKULSKEGA LIGANDA Z AFINITETNO KROMATOGRAFIJO Mentor:izr. prof. dr. Marko Novinec Datum zagovora:19. 9. 2018 GERJOL, POLONA VPLIV NANODELCEV NA OKOLJE Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 19. 9. 2018 KOS, JAKA VPLIV NEKATERIH ARGININSKIH OSTANKOV NA VEZAVO HEPARINA NA KATEPSIN B Mentor: izr. prof. dr. Marko Novinec Datum zagovora: 19. 9. 2018 OJSTERŠEK, ADRIJANA VLOGA BIOTRANSFORMACIJ V BIOTEHNOLOGIJI Mentor: prof. dr. Polona Znidaršič Plazl Datum zagovora: 19. 9. 2018 HUDELJA, POLONA VPLIV CELULOZNIH NANOVLAKEN NA TRIBOLOŠKE LASTNOSTI IN FAZNE SPREMEMBE KERAMIKE 3Y-TZP, V NAMEN PREVERJANJA PLAGIATORSTVA Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 21. 9. 2018 KRANJC PEČENKO, JAN AVTOMATSKI PROTIPOZARNI SISTEM PROTENG FM200 Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 28. 9. 2018 MATEKOVIČ, JURE ALKALNO-SILIKATNA REAKCIJA V BETONU Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 1. 10. 2018 ŽIBERT, SARA VPLIV VISKOELASTIČNIH LASTNOSTI POLTRDNIH SNOVI (KRUHA, TESTA) NA PROCES GNETENJA Mentor: doc. dr. Lidija Slemenik Perše Datum zagovora: 22. 11. 2018 FINK, ANJA Mentor: prof. dr. Miran Gaberšček Datum zagovora: 7. 12. 2018 IZAK, SARA ANA SINTEZA IN DERIVATIZACIJA 3-PIRAZOLIDINONOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 12. 12. 2018 KODERMAC, META ŠPELA PALADIJ-KATALIZIRANO ARILIRANJE 4-HIDROKSIBENZIL AMINSKIH DERIVATOV Mentor: izr. prof. dr. Bogdan Štefane Datum zagovora: 12. 12. 2018 VRHUNEC, ENEJ VPLIV PH NA MEHANSKO TRDNOST MELAMIN-FORMALDEHIDNIH MIKROKAPSUL Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 14. 12. 2018 FIDEL, JAKA POSREDOVANJE GASILCEV PRI POŽARIH VOZIL NA ALTERNATIVNE POGONE Mentor: izr. prof. dr. Simon Schnabl Datum zagovora: 19. 12. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S21 DIPLOME - VISOKOŠOLSKI STROKOVNI ŠTUDIJ KEMIJSKA TEHNOLOGIJA - 1. STOPNJA _ BAHOR, JERNEJA BAKROVE SPOJINE Z ANIONOM AZELAINSKE KISLINE Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 12. 2. 2018 PROSEN, NIKA PROGRAMI ZA AVTOMATSKO POIMENOVANJE KEMIJSKIH STRUKTUR Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 14. 2. 2018 ROTER, ANJA INHALACIJSKA BIODOSTOPNOST POTENCIALNO STRUPENIH ELEMENTOV V OBCESTNIH SEDIMENTIH Z OBMOČJA IDRIJE Mentor: doc. dr. Marija Zupančič Datum zagovora: 16. 2. 2018 ZEC, SANDRA ŠTUDIJA INHALACIJSKE BIODOSTOPNOSTI POTENCIALNO STRUPENIH ELEMENTOV V CESTNIH PRAHOVIH INDUSTRIJSKEGA OBMOČJA EMALAHLENI V JUŽNI AFRIKI Mentor: doc. dr. Marija Zupančič Datum zagovora: 16. 2. 2018 PEČJAK, SARA ANALIZA NEKATERIH PREHRANSKIH DOPOLNIL Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Mentor: prof. dr. Anton Meden Datum zagovora: 16. 2. 2018 LULIC, ELVISA FIZIKALNO-KEMIJSKE LASTNOSTI VZORCEV BIOOGLJA RAZLIČNIH IZVOROV Mentor: doc. dr. Marija Zupančič Datum zagovora: 26. 2. 2018 OBLAK, PETRA NIKLJEVI(II) KOMPLEKSI S PIRIDIN-2-ONOM IN 3-HIDROKSIPIRIDIN-2-ONOM Mentor: doc. dr. Saša Petriček Datum zagovora: 28. 2. 2018 KRAGOLNIK, INES KOORDINACIJSKE SPOJINE BAKRA(II) S KINALDINATOM IN 3-AMINO-1-PROPANOLOM Mentor: doc. dr. Barbara Modec Datum zagovora: 28. 2. 2018 ŽIVEC, NINA PRIPRAVA PCP KLEŠČASTIH KOMPLEKSOV S KOVINA-OH VEZJO Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 1. 3. 2018 JANČAR, ANA DOLOČITEV FLUORIDA V RAZLIČNIH VRSTAH VZORCEV S FLUORIDNO IONOSELEKTIVNO ELEKTRODO Mentor: viš. pred. dr. Tatjana Zupančič Datum zagovora: 8. 3. 2018 INTIHAR, KLARA UPORABA GALVANSKIH ČLENOV V VSAKDANJEM ŽIVLJENJU Mentor: prof. dr. Andrej Jamnik Datum zagovora: 8. 3. 2018 MUHIČ, TEJA SINTEZA (2E,5E)-BIS(DIMETILAMINOMETILIDEN) CIKLOHEKSAN-1,4-DIONA IN NJEGOVE PRETVORBE Z DIAMINI Mentor: prof. dr. Jurij Svete Datum zagovora: 13. 3. 2018 DOJČINOVIČ, ANA SINTEZA IN PRETVORBA 5-(HIDROKSIMETIL) FURFURALA Z BIOTEHNOLOŠKIMI PROCESI Mentor: prof. dr. Polona Žnidaršič Plazl Datum zagovora: 14. 3. 2018 BORAK, GAŠPER NAPOVEDNI MODELI ZA KLASIFIKACIJO LIGANDOV KINAZE B-RAF Mentor: doc. dr. Črtomir Podlipnik Datum zagovora: 19. 3. 2018 GRUBAR, KATJA PRIPRAVA KLEŠČASTIH KOMPLEKSOV Z NEKATERIMI KOVINAMI PREHODA Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 26. 3. 2018 JAZBEC, BENJAMIN SINTEZA IZBRANE 3-OKSOBUTANOJSKE KISLINE Mentor: prof. dr. Janez Košmrlj Datum zagovora: 27. 3. 2018 TOMAŽIN, MATEJA ISKANJE ORGANOKATALIZATORJA ZA REAKCIJO 5-(3-(BENZILOKSI)-3-OKSOPROPIL)PIROLONA Z DERIVATOM ISATINIMINA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 29. 3. 2018 KOVAČ, BLAŽ OPTIMIZACIJA SINTEZ BAKROVIH IN KOBALTOVIH KOORDINACIJSKIH SPOJIN Z DIATRIZOATNIM LIGANDOM Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 5. 4. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement GLIHA, ALEKSANDRA SINTEZA RASTLINSKEGA HORMONA 2-(3,4-DIKLOROFENOL)TRIETILAMINA Mentor: izr. prof. dr. Jernej Iskra Datum zagovora: 12. 4. 2018 BALKOVEC, BARBARA REGENERACIJA IN TESTIRANJE HPLC KOLON Mentor: izr. prof. dr. Irena Kralj Cigic Datum zagovora: 3. 5. 2018 CIGUT, ANA TITRACIJSKE KRIVULJE KISLIN IN BAZ Mentor: izr. prof. dr. Nataša Gros Datum zagovora: 25. 5. 2018 ARNEŽ, KLEMEN VPLIV UV SVETLOBE NA ABSORPCIJSK SPEKTER BARVILA 1,1-DIETIL-4,4 KARBOCIANIN JODID Mentor: prof. dr. Barbara Hribar Lee Datum zagovora: 4. 6. 2018 PFLAUM, MARGARETA SREBROVE KOORDINACIJSKE SPOJINE Z B-DIKETONATO LIGANDI Mentor: izr. prof. dr. Franc Perdih Datum zagovora: 7. 6. 2018 VODA, EVA TRDNE FAZE VODE Mentor: izr. prof. dr. Miha Lukšič Datum zagovora: 14. 6. 2018 GRABNAR, GAŠPER REOLOŠKA KARAKTERIZACIJA NANOCELULOZNEGA HIDROGELA Mentor: prof. dr. Urška Šebenik Datum zagovora: 15. 6. 2018 PAVKOVIČ, VERONIKA GOSTOTE ALKOHOLOV IN NJIHOVIH MEŠANIC Mentor: prof. dr. Tomaž Urbič Datum zagovora: 20. 6. 2018 ARHAR, SONJA VPLIV HALS-OV NA UV STABILNOST ALKIDNEGA PREMAZA Mentor: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 21. 6. 2018 KOLENC, MATEJ VPLIV DIALIL FTALATA NA ZAMREŽEVANJE IN LASTNOSTI NENASIČENE POLIESTRSKE SMOLE Mentor: viš. pred. dr. Branko Alič Datum zagovora: 21. 6. 2018 GORŠEK, SIMONA PRETVORBE KARBONILNIH SPOJIN Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 29. 6. 2018 KRŽIŠNIK, ZALA GRADBENI MATERIALI ZA VARČEVANJE Z ENERGIJO Mentor: doc. dr. Klementina Zupan Datum zagovora: 5. 7. 2018 ČOSIČ, JANA DOLOČEVANJE TEŽKIH KOVIN NA OBMOČJU RUDARSTVA Z METODAMA AAS IN MP-AES Mentor: viš. pred. dr. Tatjana Zupančič Datum zagovora: 10. 7. 2018 KORITNIK, IZTOK PRIMERJAVA KVALITETE MIKOFENOLAT MOFETILA, SINTETIZIRANEGA IZ MIKOFENOLNE KISLINE RAZLIČNIH KVALITET Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 11. 7. 2018 STANKIČ, ALEKSANDRA SINTEZA NOVIH IMIDAZOLIDINONSKIH ORGANOKATALIZATORJEV NA OSNOVI FENILALANINA Mentor: doc. dr. Uroš Grošelj Datum zagovora: 12. 7. 2018 ZAFRAN, ANJA VPLIV ŠTEVILA NANOSOV FOTOKATALITSKO AKTIVNIH TANKIH PLASTI TIO2 NA HITROST RAZGRADNJE BARVILA PLASMOCORINTH B Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 13. 7. 2018 ROSIČ, NIVES VPLIV PRETOKA KISIKA NA HITROST RAZGRADNJE BARVILA PLASMOCORINTHA B S FOTOKATALITSKO AKTIVNIMI TANKIMI PLASTMI TIO2 Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 13. 7. 2018 PIŠKUR, KRISTINA DOLOČANJE ANORGANSKIH PIGMENTOV Z RENTGENSKO PRAŠKOVNO DIFRAKCIJO Mentor: prof. dr. Anton Meden Datum zagovora: 13. 7. 2018 RESNIK, SIMONA HIDROFOBNI EFEKT Mentor: izr. prof. dr. Miha Lukšič Datum zagovora: 13. 7. 2018 MARKIČ, DANIJEL POLIURETANSKI BARVNI PREMAZI NA VODNI OSNOVI Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 22. 8. 2018 PORENTA, EMA PRIMERJAVA PRIPRAVE IN FOTOKATALITSKE UČINKOVITOSTI PLASTI TIO2 PO SOL-GEL POSTOPKU IN S TEHNIKO NANAŠANJA PASTE Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 22. 8. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S23 OBLAK, JURIJ SINTEZA 2,3-DIARIL-SUBSTITUIRANIH PIRAZINSKIH DERIVATOV Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 24. 8. 2018 PERŠOLJA, PETER SINTEZA IN KARAKTERIZACIJA 1,4-DIHETEROARIL SUBSTITUIRANIH BENZENOV Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 24. 8. 2018 SURINA, ROK UPORABA PAMETNEGA TELEFONA KOT DETEKTORJA SVETLOBE V KEMIJI Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 27. 8. 2018 TOLAR, KATARINA ELEKTROKEMIČNA PRETVORBA IN HRANJENJE ELEKTRIČNE ENERGIJE V GALVANSKEM ČLENU Mentor: izr. prof. dr. Matija Tomšič Datum zagovora: 28. 8. 2018 KAVALIČ, TADEJ KATALITSKO ARILIRANJE C-H VEZI 2-FENILPIRIMIDINA V VODI - PRIMERJAVA REAKTIVNOSTI ARIL BROMIDOV IN ARIL KLORIDOV Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 29. 8. 2018 PRAH, NINA VPLIV TEMPERATURE, ČASA IN PH MEDIJA NA TVORBO TANIN-PROTEIN KOMPLEKSA Mentor: izr. prof. dr. Franc Požgan Datum zagovora: 29. 8. 2018 ZAPLATAR, VALERIJA ODVISNOST TERMIČNE STABILNOSTI DODEKAMERNE DVOVERIŽNE DNA OD ZAPOREDJA BAZNIH PAROV IN KONCENTRACIJE DNA Mentor: prof. dr. Jurij Lah Datum zagovora: 29. 8. 2018 GREGORC, LUCIJA TERMIČNA ANALIZA MANGANOVEGA(II) IN NIKLJEVIH(II) KOMPLEKSOV S PIRIDIN-2-ONOM Mentor: doc. dr. Saša Petriček Datum zagovora: 29. 8. 2018 KOBE, TADEJ ACETILPIRIDINI KOT KATIONI V HEKSAFLUORIDOSILIKATNIH SOLEH Mentor: doc. dr. Andrej Pevec Datum zagovora: 30. 8. 2018 BARTOLJ, IRENA KARAKTERIZACIJA IN SINTEZA SPOJINE BAKROVEGA BENZOATA TRIHIDRATA Mentor: doc. dr. Nives Kitanovski Datum zagovora: 3. 9. 2018 PERKO, MARK KARAKTERIZACIJA IN SINTEZA SPOJINE [MoBr3(C5H5N)3] Mentor: doc. dr. Nives Kitanovski Datum zagovora: 3. 9. 2018 BAVDEK, MAJA KARAKTERIZACIJA IN SINTEZA SPOJINE trans-((C6H7N)2H)[Mo(NCS)4(C6H7N)2] Mentor: doc. dr. Nives Kitanovski Datum zagovora: 3. 9. 2018 GRACAR, KLAVDIJA KARAKTERIZACIJA IN SINTEZA K[Cr(NCS)4py2]x4py Mentor: doc. dr. Nives Kitanovski Datum zagovora: 3. 9. 2018 LISJAK, ANA VPLIV NEKATERIH KOVIN NA BARVO PAPIRJA PRI POSPEŠENEM TERMIČNEM STARANJU Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 3. 9. 2018 MRGOLE, KRISTJAN IZOLACIJA ELAGITANINOV IZ EKSTRAKTA KOSTANJEVEGA LESA IN ANALIZA NA HPLC-DAD Mentor: viš. pred. dr. Tatjana Zupančič Datum zagovora: 4. 9. 2018 RAJK, LUKA PRIPRAVA FENIL SUBSTITUIRANIH KLEŠČASTIH KOMPLEKSOV Z NIKLJEM IN PALADIJEM Mentor: izr. prof. dr. Janez Cerkovnik Datum zagovora: 4. 9. 2018 PEČOVNIK, NINA RAZVOJ METODE ZA DOLOČEVANJE TRIAZINSKIH PESTICIDOV V SADNIH SOKOVIH Mentor: prof. dr. Helena Prosen Datum zagovora: 5. 9. 2018 ZAFRAN, ANA VPLIV PROCESNIH POGOJEV NA UV INICIRANO POLIMERIZACIJO AKRILAMIDA V RAZTOPINI Mentor: viš. pred. dr. Branko Alič Datum zagovora: 7. 9. 2018 LAZIC, VALERIJA PRIMERJAVA ICP-OES IN ICP-MS ZA DOLOČANJE NIZKIH KONCENTRACIJ MANGANA Mentor: izr. prof. dr. Drago Kočar Datum zagovora: 7. 9. 2018 STRNAD, LIZA MARIJA FAZNA ANALIZA RAZLIČNIH VZORCEV TAL S PODROČJA LJUBLJANE S PRAŠKOVNO DIFRAKCIJO Mentor: izr. prof. dr. Amalija Golobič Datum zagovora: 10. 9. 2018 TURK, MARKO VPLIV RAZLIČNIH PARAMETROV NA UČINKOVITOST SONČNIH CELIC Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 10. 9. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement POVŠIČ, LIDIJA IZRAČUN FAZNEGA RAVNOTEŽJA TEKOČINA - PLIN V NEIDEALNIH MEŠANICAH VODE IN ETANOLA Mentor: prof. dr. Jurij Reščič Datum zagovora: 10. 9. 2018 BRULC, ANJA VPLIV MAKROMOLEKUL NA STABILNOST PROTEINOV Mentor: prof. dr. Jurij Reščič Datum zagovora: 11. 9. 2018 ŽUPEVEC, NATALIJA KEMIJSKA KINETIKA OZONA V STRATOSFERI Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 11. 9. 2018 KEJŽAR, KATJA OPTIMIZACIJA SINTEZE ANALOGOV LIGANDA SALEN NA OSNOVI O-VANILINA IN NJIHOVIH KOMPLEKSOV PREHODNIH KOVIN Mentor: doc. dr. Jakob Kljun Datum zagovora: 11. 9. 2018 BRATKOVIČ, MAŠA BAKROVE KOORDINACIJSKE SPOJINE Z ANIONOM SUKCINSKE KISLINE Mentor: doc. dr. Bojan Kozlevčar Datum zagovora: 12. 9. 2018 MISLAJ, MATEJ DOLOČITEV ŽELEZA V ZELENJAVI IN VEGETARIJANSKI PREHRANI Mentor: viš. pred. dr. Tatjana Zupančič Datum zagovora: 14. 9. 2018 LUŠTEK, KARMEN DOLOČANJE PARACETAMOLA V TABLETAH LEKADOLA Mentor: prof. dr. Barbara Hribar Lee Datum zagovora: 14. 9. 2018 PLESNIK, KRISTINA PRIPRAVA POLIENAMINONOV NA OSNOVI CIKLOHEKSAN-1,2-DIONA IN DIAMINOV Mentor: prof. dr. Jurij Svete Datum zagovora: 14. 9. 2018 MLAKAR, TINA VPLIV AMINSKIH STABILIZATORJEV NA REOLOŠKE LASTNOSTI ALKIDNEGA PREMAZA Mentor: prof. dr. Urška Lavrenčič Štangar Datum zagovora: 17. 9. 2018 FILIPIČ, NIKA KOROZIJSKA KARAKTERIZACIJA MEDENIN V STIKU S PITNO VODO Mentor: izr. prof. dr. Marjan Marinšek Datum zagovora: 17. 9. 2018 LEVC, ŽIVA VPLIV ZAČETNE KONCENTRACIJE BARVILA PLASMOCORINTH B NA HITROST NJEGOVE RAZGRADNJE S FOTOKATALITSKO AKTIVNIMI TANKIMI PLASTMI TIO2 Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 17. 9. 2018 ŠTEPEC, MIHA SINTEZA 1-ALKIL-N-BOC-ISATIN IMINOV Mentor: prof. dr. Jurij Svete Datum zagovora: 19. 9. 2018 TRELC, MAJA FIZIKALNO-KEMIJSKI PROCESI OB VNOSU ZDRAVIL V TELO Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 20. 9. 2018 KOZMUS, JASMINA HALOGENIRANJE TERMINALNIH ACETILENOV Mentor: doc. dr. Martin Gazvoda Datum zagovora: 24. 9. 2018 SIMČIC, PATRIK PRIPRAVA NEKATERIH HALOGENIH DERIVATOV Mentor: izr. prof. dr. Marjan Jereb Datum zagovora: 26. 9. 2018 PEZDIREC, MAJA VPLIV TLAKA NA TRDNE FAZE VODE V LEDENIKIH Mentor: viš. pred. dr. Andrej Godec Datum zagovora: 27. 9. 2018 HROVAT, BLAŽ PEROVSKITE SOLAR CELLS WITH MIXED HTMS Mentor: prof. dr. Ksenija Kogej Datum zagovora: 27. 9. 2018 MAL, SUZANA IONOTERMALNA SINTEZA MIKROPOROZNEGA ALUMINOFOSFATA S KABAZITNIM STRUKTURNIM TIPOM Mentor: izr. prof. dr. Amalija Golobič Datum zagovora: 28. 9. 2018 URBIHA, ERIKA ANALIZNE METODE ZA KARAKTERIZACIJO KROMOVEGA(III) OKSIDA Mentor: izr. prof. dr. Mitja Kolar Datum zagovora: 4. 10. 2018 EGART, MARJETA POVRŠINSKO AKTIVNE SNOVI: UPORABA NA PODROČJU DOSTAVE ZDRAVILNIH UČINKOVIN Mentor: prof. dr. Ksenija Kogej Datum zagovora: 4. 10. 2018 PETEH, TOMAŽ POLŠARŽNA POLIMERIZACIJA AKRILAMIDA IN ITAKONSKE KISLINE Mentor: viš. pred. dr. Branko Alič Datum zagovora: 22. 10. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S25 ŠIREC, JASNA VPLIV INERTNIH SNOVI NA MINIMALNO VŽIGNO ENERGIJO GORLJIVIH PRAHOV Mentor: doc. dr. Barbara Novosel Datum zagovora: 30. 10. 2018 JONTEZ, URŠKA REAKCIJE BAKROVEGA(II) KINALDINATA Z 1-AMINO-2- PROPANOLOM Mentor: doc. dr. Barbara Modec Datum zagovora: 8. 11. 2018 MIKLAVČIČ, CIRIL SKLOPITEV DVODIMENZIONALNE KROMATOGRAFIJE Z MASNIM SPEKTROMETROM (2D-LCMS) - PRIKAZ PRINCIPA DELOVANJA SISTEMA NA UČINKOVINI KLOPIDOGREL HIDROGENSULFAT Mentor: prof. dr. Matevž Pompe Datum zagovora: 15. 11. 2018 BOLTES, TANJA VPLIV TEMPERATURE NA MICELIZACIJO POVRŠINSKO AKTIVNIH SNOVI Mentor: prof. dr. Marija Bešter Rogač Datum zagovora: 29. 11. 2018 JANEŽIČ, BLAŽ VLOGA IN UPORABA PROGRAMSKE OPREME ZA OBDELAVO KROMATOGRAFSKIH PODATKOV PRI HPLC ANALIZAH Mentor: doc. dr. Bojan Šarac Datum zagovora: 30. 11. 2018 POGORELC, PETRA FOTOKATALITSKO UČINKOVITE PLASTI TIO2 ZA ČIŠČENJE STREŠNIH KRITIN Mentor: izr. prof. dr. Romana Cerc Korošec Datum zagovora: 14. 12. 2018 OKIC, EMA KEMIJSKO LUŽENJE DUPLEKSNEGA NERJAVNEGA JEKA Mentor: doc. dr. Bojan Šarac Datum zagovora: 18. 12. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement UNIVERZA V MARIBORU FAKULTETA ZA KEMIJO IN KEMIJSKO TEHNOLOGIJO 1. januar - 31. december 2018 DOKTORATI DOKTORSKI ŠTUDIJ - 3. STOPNJA HORVAT GABRIJELA PRIPRAVA, KARAKTERIZACIJA IN APLIKACIJA POLISAHARIDNIH AEROGELOV Mentor: red. prof. dr. ŽELJKO KNEZ, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZORAN NOVAK, univ. dipl. inž. kem. tehnol. Datum zagovora: 23. 03. 2018 IVANOVIC MILENA RAZVOJ ANALIZNIH METOD ZA SOČASNO IDENTIFIKACIJO IN DOLOČEVANJE FENOLNIH SPOJIN Mentor: izr. prof. dr. MITJA KOLAR, univ. dipl. inž. kem. Somentor: doc. dr. MAŠA ISLAMČEVIC RAZBORŠEK, prof. biol. in kem. Datum zagovora: 30. 03. 2018 KORENAK JASMINA BIOMIMETIČNE MEMBRANE ZA PROCES OSMOZE PRI OBDELAVI INDUSTRIJSKE ODPADNE VODE Mentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Somentor: izr . prof. dr. CLAUS HELIX NIELSEN Datum zagovora: 06. 07. 2018 KRAVANJA GREGOR NAČRTOVANJE VISOKOTLAČNIH PROCESOV ZA PREDELAVO POLIMEROV IN IZBOLJŠAVE PRENOSA TOPLOTE Mentor: red. prof. dr. ŽELJKO KNEZ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. , red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Datum zagovora: 15. 05. 2018 STAVBAR SEVERINA PROCESI ODSTRANJEVANJA ANTIBIOTIKOV IZ BOLNIŠNIČNIH ODPADNIH VOD Mentor: red. prof. dr. SONJA ŠOSTAR TURK, univ. dipl. inž. tekst. Somentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. , izr. prof. dr. MITJA KOLAR, univ. dipl. inž. kem. Datum zagovora: 18. 04. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S27 MAGISTERIJI MAGISTRSKI ŠTUDIJ - 2. STOPNJA CMAGER NUŠA KEMIJSKA MODIFIKACIJA POVRŠINE VINILBENZIL KLORIDNIH POLIHIPE MATERIALOV Z METODO RAFT GRAFTIRANJA Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. Somentor: asist. dr. MUZAFERA PALJEVAC, prof. kem. in biol. Datum zagovora: 21. 11. 2018 DEŽELAK BLAŽ HIDROTERMIČNA Degradacija polietilena nizke gostote Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZELJKO KNEZ, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 10. 2018 DRETAR ROK SINTEZA VELIKIH OMREZIJ TOPLOTNIH PRENOSNIKOV Mentor: red. prof. dr. ZDRAVKO KRAVANJA, univ. dipl. inž. kem. tehnol. Somentor: doc. ddr. ANDREJA NEMET, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 GOJZNIKAR MATEJA POROZNI KOPOLIMERI IZ VINILESTROV IN TIOLOV KOT NOSILCI BIOLOŠKIH CELIC Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. Somentor: asist. dr. MUZAFERA PALJEVAC, prof. kem. in biol. Datum zagovora: 24. 09. 2018 GOVEJŠEK TAMARA DOLOČANJE ARZENA V TITANOVEM DIOKSIDU Mentor: doc. dr. MATJAZ FINŠGAR, univ. dipl. kem. ZUNANJI SOMENTOR Datum zagovora: 29. 08. 2018 GRAČNAR MAJA IZOLACIJA BIOLOŠKO AKTIVNIH KOMPONENT IZ PLODOV GOZDNIH BOROVNIC (Vaccinium Myrtillus L. ) Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. PETRA KOTNIK,univ. dipl. inž. kem. tehnol. asis. DARIJA COR, , univ. dipl. inž. kem. tehnol. dr. URŠKA ROZMAN Datum zagovora: 11. 07. 2018 HERIC ANDREJA PRIPRAVA EKSTRAKTOV ARONIJE ARONIA MELANOCARPA IN TESTIRANJE NJIHOVE BIOLOŠKE AKTIVNOSTI Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. UROŠ MAVER, mag. farm. doc. dr. PETRA KOTNIK,univ. dipl. inž. kem. tehnol Datum zagovora: 24. 10. 2018 HREN MAŠA VPLIV AGRESIVNEGA MEDIJA IN VNETNIH PROCESOV NA KOROZIJO BIOKOMPATIBILNIH KOVINSKIH IMPLANTATOV V UMETNI SLINI Mentor: doc. dr. MOJCA SLEMNIK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 IVANOVSKI MAJA FUNKCIONALIZIRANI KOPOLIMERI IZ MAKROLAKONOV Z ENCIMSKO POLIMERIZACIJO ODPIRANJA OBROČA Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. ZUNANJI SOMENTOR Datum zagovora: 24. 09. 2018 JEROMEL KAJA DOLOČEVANJE ANTIMIKROBNEGA DELOVANJA ALOE VERE Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MATEJA PRIMOZIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 10. 2018 KEGL TINA NOVI NANOMATERIALI ZA ADSORPCIJO REDKOZEMELJSKIH ELEMENTOV IZ VODNIH RAZTOPIN Mentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. ZUNANJI SOMENTOR Datum zagovora: 04. 09. 2018 KERMC DOMEN KARAKTERIZACIJA VISKIJEV Z GC-MS IN ICP-MS TER NJIHOVA KEMOMETRIJSKA KLASIFIKACIJA Mentor: doc. dr. MAŠA ISLAMČEVIC RAZBORŠEK, prof. biol. in kem. ZUNANJI SOMENTOR Datum zagovora: 24. 09. 2018 KORES KATARINA NAČRTOVANJE IN RAZVOJ NOVIH PROTIMIKROBNIH SPOJIN Z UPORABO ProBiS RAČUNALNIŠKIH PRISTOPOV Mentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. ZUNANJI SOMENTOR Datum zagovora: 25. 10. 2018 KUHAR DOROTEJA ČIŠČENJE POVRŠINSKE VODE Z ULTRAFILTRACIJO Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 03. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement LEDINEK NINA OPTIMIZACIJA HPLC METODE ZA DOLOČANJE SORODNIH SUBSTANC PREDNISOLONA V UČINKOVINI Mentor: red. prof. dr. ZORAN NOVAK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Datum zagovora: 17. 01. 2018 LJUBEC BARBARA FORMULIRANJE FARMACEVTSKIH UČINKOVIN V POLIMERE S PLINI VISOKE GOSTOTE Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZORAN NOVAK, univ. dipl. inž. kem. teh asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 LUKAČ DAMJANA RAZKROJ NEKATERIH ACETILACETONATOV PREHODNIH KOVIN S TERMIČNO IN ULTRAZVOČNO METODO Mentor: izr. prof. dr. MATJAŽ KRISTL, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 MATIS STAŠA SINTEZA IN IZOLACIJA NANOCELULOZE IZ BAKTERIJE Gluconacetobacter xylinusd Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MATEJA PRIMOŽIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 OHMAN MIHA PROIZVODNJA TEKOČEGA OGLJIKOVEGA DIOKSIDA Mentor: izr. prof. dr. DARKO GORIČANEC, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. DANIJELA URBANCL, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 03. 2018 OSMIC AZRA PRIPRAVA HIERARHIČNO POROZNIH MATERIALOV IZ MULTIPLIH ŠABLON Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. Somentor: asist. dr. MUZAFERA PALJEVAC, prof. kem. in biol. Datum zagovora: 24. 10. 2018 PETEK REGORŠEK VITA Karakterizacija zeta potenciala votlo-vlaknastih membrand Mentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Zunanji somentor Datum zagovora: 23. 05. 2018 POTRČ SANJA PROIZVODNJA TRDNIH BIOGORIV S TOREFIKACIJO BIOMASE Mentor: izr. prof. dr. DARKO GORIČANEC, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. DANIJELA URBANCL, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 ROMANIC LUKA FOTOKATALITSKI FILTER NA OSNOVI TiO2 ZA ČIŠČENJE ZRAKA Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Somentor: izr. prof. dr. REGINA FUCHS GODEC, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 SEITL MAŠA UČINKOVITOST RAZGRADNJE BISFENOLA A PO HIDROTERMIČNEM PROCESIRANJU ODPADNE VODE Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. PETRA KOTNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 SIMONIČ TAJDA IZBOLJŠANJE UČINKOVITOSTI VAKUUMSKEGA UPARJANJA TEKOČE FRAKCIJE DIGESTATA Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZORKA NOVAK PINTARIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 02. 2018 ŠTIH VESNA VODOTOPNE EMULZIJE KAROTENOIDOV Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. ZUNANJI SOMENTOR Datum zagovora: 11. 07. 2018 ŠTUMPF SARA PROTIMIKROBNE LASTNOSTI TANINSKIH EKSTRAKTOV Mentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. Somentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol asist. dr. GREGOR HOSTNIK Datum zagovora: 26. 09. 2018 TEVŽ NEJA RAZVOJ KINETIČNEGA MODELA IN SPREMLJANJE RAZGRADNJE SPECIFIČNEGA ANTIBIOTIKA MED PROCESOM ODSTRANJEVANJA IZ ODPADNE VODE Mentor: red. prof. dr. ANDREJA GORŠEK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. DARJA PEČAR, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 03. 2018 TOMPA SAŠA MIKROBIOLOŠKA KINETIKA RASTI NARAVNE STARTER KULTURE V SIROTKI Mentor: doc. dr. DARJA PEČAR, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ANDREJA GORŠEK, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 VODOPIVEC KATJA RAZVOJ IN VALIDACIJA MODIFICIRANE Bi-Sn ELEKTRODE Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Somentor: asist. BARBARA PETOVAR, mag. kem. Datum zagovora: 04. 09. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S29 ŽALIG VALENTINA SINTEZE IN KARAKTERIZACIJA NANODELCEV ZA UPORABO V OSMOTSKIH PROCESIH ČIŠČENJA ODPADNE VODE Mentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. doc. dr. MOJCA SLEMNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 ŽITEK TAJA VPLIV EKSTRAKTOV IZ NARAVNIH MATERIALOV NA METABOLNO AKTIVNOST ČLOVEŠKIH MELANOMSKIH CELIC (WM-266-4) Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. teh. Datum zagovora: 04. 09. 2018 DIPLOME - UNIVERZITETNI ŠTUDIJ UNIVERZITETNI ŠTUDIJ - 1. STOPNJA __ AMBROŽ ANA PRIPRAVA OKOLJU PRIJAZNIH MIKROKAPSUL ZA KONTROLIRANO SPROŠČANJE HLAPNIH ORGANSKIH SPOJIN Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Somentor: izr. prof. dr. JULIJA VOLMAJER VALH, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 ARBEITER DAMJAN PRIPRAVA IN ANALIZA NAPREDNIH PREVLEK NA OSNOVI HIDROKSICELULOZE NA KOVINSKIH SUBSTRATIH ZA BIOMEDICINSKE APLIKACIJE Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Somentor: doc. dr. UROŠ MAVER, mag. farm. Datum zagovora: 27. 07. 2018 BANDUR PATRICIJA ŠTUDIJA AKTIVNOSTI NEKATERIH ENCIMOV V GELU ALOE VERE Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: asist. KATJA VASIC, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 DAMIJAN TJAŠA ANTIOKSIDATIVNI POTENCIAL IN VSEBNOST TOTALNIH FENOLOV V EKSTRAKTU STEVIE REBAUDIANA BERT Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 DEGEN ALJAŽ OPTIMIZACIJA SILIKATNE PREVLEKE NiCu NANODELCEV ZA APLIKACIJE V MEDICINI Mentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MOJCA SLEMNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 DOKL MONIKA IZOLACIJA MIKRO IN NANOCELULOZE IZ RASTLINSKIH MATERIALOV Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: asist. KATJA VASIC, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 DROBNIČ TEA ANALIZA IN OPTIMIZACIJA PIGMENTNIH PAST ZA KOVINSKE PREMAZE NA VODNI OSNOVI Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. ZUNANJI SOMENTOR Datum zagovora: 04. 09. 2018 ERMENC TEJA OPTIMIZACIJA PROCESIRANJA VZORCEV SLINE PRI RAKU GLAVE IN VRATU ZA ŠTUDIJE BIOLOŠKIH OZNAČEVALCEV Mentor: prof. dr. UROŠ POTOČNIK, univ. dipl. kem. Somentor: doc. dr. HELENA SABINA ČELEŠNIK, dipl. mikrobiolog Datum zagovora: 12. 09. 2018 FLUCHER VIKTORIJA VPLIV TEMPERATURE NA VOLUMETRIČNE LASTNOSTI BINARNIH MEŠANIC ALKOHOLOV Mentor: izr. prof. dr. REGINA FUCHS GODEC, univ. dipl. inž. kem. tehnol. Somentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. Datum zagovora: 11. 09. 2018 GIDER EVA UVAJANJE DIGITALIZACIJE V LABORATORIJ Z UPORABO PROGRAMA sciNote® Mentor: doc. dr. DUŠAN KLINAR, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MILOŠ BOGATAJ, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement GLAVAČ KAJA SEPARACIJA AKTIVNIH KOMPONENT IZ LUPIN KAKAVOVCA Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Somentor: asist. MAJA ČOLNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 GYURKAČ MARCELL SINTEZA IN KARAKTERIZACIJA MANGANOVIH KOORDINACIJSKIH SPOJIN Z AMINOPIRIDINI Mentor: izr. prof. dr. MATJAŽ KRISTL, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 23. 05. 2018 ITERNIČKA BERNARD PRIPRAVA POLI(GLICIDIL METAKRILATA) Z VEČ NIVOJSKO POROZNOSTJO Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. Somentor: asist. dr. MUZAFERA PALJEVAC, prof. kem. in biol. Datum zagovora: 12. 09. 2018 JANČIČ NATALIJA BIOSORPCIJA Cr6+ IONOV NA IMOBILIZIRANI MEŠANICI ALG NA ALGINATNIH NOSILCIH Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ANDREJA GORŠEK, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 KELC IRIS EKSPRESIJSKI IN EPIGENETSKI VZORCI PRI BOLNIKIH S KRONIČNO VNETNO ČREVESNO BOLEZNIJO Mentor: red. prof. dr. UROŠ POTOČNIK, univ. dipl. kem. Somentor: doc. dr. HELENA SABINA ČELEŠNIK, dipl. mikrobiolog Datum zagovora: 18. 04. 2018 KERŠIČ PIA GENETSKA ANALIZA BOLNIKOV Z REVMATOIDNIM ARTRITISOM ZA POLIMORFIZME SNP V GENIH ZA VNETNE CITOKINE Mentor: red. prof. dr. UROŠ POTOČNIK, univ. dipl. kem. ZUNANJI SOMENTOR Datum zagovora: 18. 04. 2018 KOCBEK SIMON VPLIV MIKROVALOV NA ZVIJANJE a-PEPTIDA: RAČUNALNIŠKI PRISTOP Mentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. Somentor: Martin Gladovic, mag. kem. Datum zagovora: 12. 09. 2018 KOGELNIK REBEKA DOLOČEVANJE PRISOTNOSTI PROTEINOV IN AKTIVNOSTI NEKATERIH ENCIMOV V FIGAH Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: asist. KATJA VASIC, univ. dipl. inž. kem. tehnol. doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 KORPIČ ŠPELA EKSTRAKCIJE NARAVNIH PIGMENTOV IZ ALG Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. ZUNANJI SOMENTOR Datum zagovora: 11. 07. 2018 KOTNIK NUŠA UGOTAVLJANJE UČINKOVITOSTI KLOROVEGA DIOKSIDA NA ESCHERICHIA COLI V PITNI VODI Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. ZUNANJI SOMENTOR Datum zagovora: 24. 09. 2018 KOVŠE TJAŠA HIDROTERMIČNA DEGRADACIJA PET EMBALAŽE Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Somentor: asist. MAJA ČOLNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 KRAVANJA KATJA ANDRINA BIOLOŠKA AKTIVNOST KONVENCIONALNIH EKSTRAKTOV CEYLONSKEGA CIMETA (CINNAMOMUM ZEYLANICUM) Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: dr. URŠKA ROZMAN Datum zagovora: 04. 09. 2018 MAJER DAVID VALIDACIJA METODE ZA ANALIZO Pb(II) Z UPORABO SPE SENZORJA Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Somentor: asist. BARBARA PETOVAR, mag. kem. Datum zagovora: 29. 08. 2018 MALIC MARINA ANTIKARCINOGENI POTENCIALI POLIFENOLNIH SPOJIN IZ SMILJA - RAČUNALNIŠKI PRISTOP Mentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. Somentor: asist. VERONIKA FURLAN, mag. kem. Datum zagovora: 12. 09. 2018 MARINČEK EVA SINTEZA POROZNIH KOPOLIMERNIH POLIELEKTROLITOV NA OSNOVI 2-AKRILAMIDO-2-METILPROPANSULFONSKE KISLINE Mentor: dr. SEBASTIJAN KOVAČIČ, prof. kem. in biol. Datum zagovora: 04. 09. 2018 MERSLAVIČ MANCA ZASNOVA IN ANALIZA NOVIH POLIMERNIH OBLOG Z VGRAJENIMI ZDRAVILNIMI UČINKOVINAMI NA ZLITINI TITAN/ALUMINIJ/VANADIJ Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem Somentor: doc. dr. UROŠ MAVER, mag. farm. Datum zagovora: 12. 09. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S31 NAVODNIK ALEN UPORABA PLAZEMSKIH REAKTORJEV ZA PRETVORBO METANA V VIŠJE OGLJIKOVODIKE IN SINTEZNI PLIN Mentor: dr. BLAŽ LIKOZAR Somentor: doc. dr. DARJA PEČAR, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 OREL MATIC UPORABA MIKROEMULZIJ ZA SINTEZO MAKROPOROZNIH POLISTIRENSKIH DERIVATOV Mentor: red. prof. dr. PETER KRAJNC, univ. dipl. inž. kem. Somentor: asist. dr. MUZAFERA PALJEVAC, prof. kem. in biol. Datum zagovora: 12. 09. 2018 OSTROŠKO URŠKA PREDOBDELAVA STUDENČNE VODE ZA PROCES REVERZNE OSMOZE Mentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Somentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 PFEILER SEBASTIAN DOLOČITEV OBRATOVALNIH KARAKTERISTIK ENO IN DVOSTOPENJSKE TOPLOTNE ČRPALKE Mentor: izr. prof. dr. DARKO GORIČANEC, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. DANIJELA URBANCL, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 PLAJNŠEK ALEN DEAKTIVACIJA KATALIZATORJEV PRI POSTOPKU PROIZVODNJE METANOLA IZ OGLJIKOVEGA DIOKSIDA IN VODIKA Mentor: dr. BLAŽ LIKOZAR Somentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 PODRIČNIK MANCA HITRA PRESOJA IN RAZVRŠČANJE VEČJIH NESREČ Z NEVARNIMI SNOVMI Mentor: red. prof. dr. ZORKA NOVAK PINTARIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. ddr. ANDREJA NEMET, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 PRELOG MIHA PROIZVODNJA TRANSPORTNEGA GORIVA IZ BIOPLINA Mentor: izr. prof. dr. DARKO GORIČANEC, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. PETER TROP, univ. dipl. inž. kem. tehnol. Datum zagovora: 21. 03. 2018 PUHAR JAN SIMULACIJA RAZPENJALNIKA S PROGRAMSKIMA ORODJEMA SCILAB IN GAMS Mentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. ZUNANJI SOMENTOR Datum zagovora: 11. 09. 2018 ROŠKARIČ MATEVŽ ZAŠČITNO DELOVANJE IZBRANIH POLIFENOLNIH SNOVI PROTI GENOTOKSIČNIM UČINKOM, KI JIH POVZROČA MIKOTOKSIN AFLATOKSIN B1 Mentor: izr. prof. dr. URBAN BREN, univ. dipl. kem. ZUNANJI SOMENTOR Datum zagovora: 12. 09. 2018 VERDNIK ALEKSANDRA FAZNA RAVNOTEŽJA PRI POVIŠANIH TLAKIH ZA SISTEMA RESVERATROL/OGLJIKOV DIOKSID IN RESVERATROL/ETANOL/OGLJIKOV DIOKSID Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 VOGRINC MARKO REGULACIJA PRETOČNEGA SISTEMA Z UPORABO PRETOČNEGA SENZORJA, AVTOMATSKEGA VENTILA IN POVRATNO-ZANČNEGA REGULACIJSKEGA SISTEMA Mentor: doc. dr. MILOŠ BOGATAJ, univ. dipl. inž. kem. tehnol. Somentor: doc. ddr. ANDREJA NEMET, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 VOZLIČ ANA KINETIKA PROIZVODNJE BIOPLINA MED ANAEROBNO FERMENTACIJO BIOLOŠKIH ODPADKOV PRECEPLJENIH Z GLIVAMA PLEUROTUS OSTREATUS IN TRAMETES VERSICOLOR Mentor: doc. dr. DARJA PEČAR, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ANDREJA GORŠEK, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement DIPLOME - VISOKOŠOLSKI STROKOVNI ŠTUDIJ KEMIJSKA TEHNOLOGIJA - 1. STOPNJA _ BABIČ JERNEJA INHIBITORNE LASTNOSTI BOROVNIC NA RAST MIKROBNIH CELIC Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MATEJA PRIMOŽIČ, univ. dipl. inž. kem. tehnol. doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 CIZL JASNA BIOLOŠKA PREDOBDELAVA LIGNOCELULOZNIH MATERIALOV Z GLIVAMI BELE TROHNOBE Mentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MAŠA ISLAMČEVIC RAZBORŠEK, prof. biol in kem. Datum zagovora: 26. 09. 2018 GABERC JERNEJ KOROZIJSKA HITROST BIOKOMPATIBILNIH KOVINSKIH MATERIALOV Z DODATKOM AGRESIVNIH IONOV V UMETNI SLINI Mentor: doc. dr. MOJCA SLEMNIK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 GORENC PETRA DOLOČANJE MAKROHRANIL V RAZLIČNIH DIGESTATIH IN UMETNIH GNOJILIH Mentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. MAŠA ISLAMČEVIC RAZBORŠEK, prof. biol in kem. izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 20. 08. 2018 KNECHTL IVAN VPELJAVA TRAJNOSTNIH POSTOPKOV ZA LOČITEV BIO-OSNOVANIH GRADNIKOV IZ ODPADNE MORSKE BIOMASE ZA NAPREDNO UPORABO Mentor: dr. BLAŽ LIKOZAR Somentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 KOBALE TAMARA DOLOČEVANJE AKTIVNOSTI NEKATERIH ENCIMOV V GRANATNEM JABOLKU Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: KATJA VASIC, univ. dipl. inž. kem. tehnol doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 KRIVEC ROLANDO PROIZVODNJA BIOPLINA Z ANAEROBNO DIGESTIJO BIOLOŠKIH ODPADKOV PRECEPLJENIH Z GLIVAMI NA SPECIFIČNEM SUBSTRATU Mentor: red. prof. dr. ANDREJA GORŠEK, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. Darja Pečar, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 KUZMAN NEJKA RECIKLIRANJE ODPADNIH VODA ZDRAVILNE TERMALNE VODE, Z UPORABO RAZLIČNIH KOLON STEKLENIH SMOL Mentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Somentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Datum zagovora: 20. 06. 2018 MAKOVEC MAŠA VPLIV PREDOBDELAVE PIŠČANČJE STELJE Z GLIVAMI NA PROIZVODNJO BIOPLINA Mentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZDRAVKO KRAVANJA, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 09. 2018 MIKŠ NEŽA MOŽNOST VRAČANJA DESTILATA PO ČIŠČENJU ODPADNE VODE V TEHNOLOŠKE PROCESE IZDELAVE Al ULITKOV Mentor: izr. prof. dr. MARJANA SIMONIČ, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA PETRINIC, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 MILOŠIČ LILIJANA AKTIVNOST ENCIMOV V TEKOČIH IN TRDNIH PRALNIH SREDSTVIH Mentor: red. prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: asist. MAJA ČOLNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 10. 2018 PALČNIK POLONA Izolacija AKTIVNIH komponent iz semen chia z uporabo različnih tehnik ekstrakcije Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 24. 10. 2018 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S33 SIHER ANJA PRIPRAVA NANODELCEV Zn, Ni in Co TELURIDOV Z VISOKOTEMPERATURNO IN Z MEHANOKEMIJSKO METODO Mentor: izr. prof. dr. MATJAŽ KRISTL, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 20. 08. 2018 STERNAD LEA PROIZVODNJA BIOPLINA S PREDOBDELAVO LIGNOCELULOZE Z ORGANSKIM TOPILOM IN VROČO VODO Mentor: doc. dr. LIDIJA ČUČEK, univ. dipl. inž. kem. tehnol. Somentor: red. prof. dr. ZDRAVKO KRAVANJA, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 ŠAŠEK ŽAN OPTIMIZACIJA IN VALIDACIJA Cu MODIFICIRANE ELEKTRODE IZ STEKLASTEGA OGLJIKA ZA ANALIZO Pb(II) Mentor: doc. dr. MATJAŽ FINŠGAR, univ. dipl. kem. Somentor: asist. BARBARA PETOVAR, mag. kem. Datum zagovora: 27. 07. 2018 ŠTUKOVNIK ZALA EKSTRAKCIJA KOMPONENT IZ SEMEN NAVADNE AJDE (FAGOPYRUM ESCULENTUM) Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 TALJAN MIHAELA AKTIVNOST LAKAZE IN CELULAZE V GLIVI PLEUROTUS OSTREATUS Mentor: prof. dr. MAJA LEITGEB, univ. dipl. inž. kem. tehnol. Somentor: asist. KATJA VASIC, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 TRŽAN PRIMOŽ VPLIV EKSTRAKCIJSKEGA POSTOPKA NA KVALITETO EKSTRAKTOV IZ LIOFILIZIRANIH HRUŠK (Pyrus communis) Mentor: doc. dr. MAŠA KNEZ HRNČIČ, univ. dipl. inž. kem. tehnol. Somentor: asist. dr. DARIJA COR , univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 TURK ANDREJA EKSTRAKCIJA LUBJA KOSTANJA S SUBKRITIČNO VODO Mentor: red. prof. dr. MOJCA ŠKERGET, univ. dipl. inž. kem. tehnol. Somentor: asist. MAJA ČOLNIK, univ. dipl. inž. kem. tehnol. Datum zagovora: 04. 09. 2018 VOLOVLEK ROK ČISTILNO SREDSTVO ZA SOUPORABO S PRODUKTOM CAFELIER ZA ČIŠČENJE KAVNIH APARATOV Mentor: doc. dr. ANITA KOVAČ KRALJ, univ. dipl. inž. kem. Somentor: doc. ddr. ANDREJA NEMET, univ. dipl. inž. kem. tehnol. Datum zagovora: 11. 07. 2018 ZUPANC TOMAŽ MENJAVA METODE DOLOČANJA OPTIČNIH LASTNOSTI PIGMENTNEGA TITANOVEGA DIOKSIDA Mentor: izr. prof. dr. MATJAŽ KRISTL, univ. dipl. inž. kem. tehnol. Somentor: doc. dr. IRENA BAN, univ. dipl. inž. kem. tehnol. Datum zagovora: 12. 09. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement FAKULTETA ZA ZNANOSTI O OKOLJU 1. januar - 31. december 2018 DOKTORATI MOJCA 2or2 furlan THE DETECTION AND STUDY OF BIOLOGICALLY ACTIVE COMPOUNDS IN ENVIRONMENTAL PROCESSES AND SAMPLES Mentor: prof. dr. Mladen Franko Datum zagovora: 31. 5. 2018 JOSÉ MANUEL CARITA GONÇALVES DISTRIBUTION OF ENTERIC VIRUSES IN THE GULF OF TRIESTE AND THEIR INTERACTIONS WITH ENVIRONMENTALAND BIOLOGICAL PARAMETERS Mentor: prof. dr. Valentina Turk Somentor: dr. Jon Gutierrez Aguirre Datum zagovora: 29. 6. 2018 BARBARA DEBELJAK STRUCTURE AND FUNCTIONING OF THE HYPORHEIC ZONES IN THE GRAVEL-BEDS OF FIVE RIVERS IN RELATION TO CATCHMENT LAND USE Mentor: prof. dr. Anton Brancelj Datum zagovora: 29. 6. 2018 MAGISTERIJI ŠTUDIJSKI PROGRAM OKOLJE - 2. STOPNJA NIJAT RAHIMLI EFFECTS OF POTENTIAL CLIMATE CHANGES ON THE BEHAVIOUR, FEEDING RATE AND REPRODUCTION OF SELECTED SOIL INVERTEBRATES Mentor: doc. dr. Suzana Žižek Datum zagovora: 9. feb. 18 ANA KARAT OKOLJSKA OCENA TVEGANJA FUNGICIDA FOLPETA ZA TALNE EKOSISTEME Mentor: doc. dr. Suzana Žižek Datum zagovora: 15. feb. 18 MOJCA VRČON MIHELJ OPTOELEKTRONSKE IN FOTOKATALITSKE LASTNOSTI Z VANADIJEM DOPIRANEGA FE2TEO6 Mentor: prof. dr. Matjaž Valant Datum zagovora: 18. maj. 18 LARA VALENTIC ANALIZA MIKROPLASTIKE V IZBRANIH POVRŠINSKIH IN PODZEMELJSKIH KRAŠKIH VODAH Mentor: izr. prof. dr. Tanja Pipan Datum zagovora: 27. avg. 18 ŠPELA MAČEK KOLIČINSKO IN KAKOVOSTNO OVREDNOTENJE VODNIH VIROV V RAZPOKLINSKEM IN KRAŠKEM VODONOSNIKU NA OBMOČJU OBČINE POSTOJNA Mentor: izr. prof. dr. Metka Petrič Datum zagovora: 10. sep. 18 DOROTEJA GOŠAR ARSENIC IN NATURAL WATERS: HYDROGEOCHEMISTRY CHARACTERIZATION AND TOXICITY EFFECTS Mentor: doc. dr. Martina Bergant Marušič prof. dr. Maria do Rosario Melo da Costa Datum zagovora: 25. sep. 18 TJAŠA BIRSA DOLOČANJE ACETILHOLINESTERAZE V HUMANIH KRVNIH VZORCIH Mentor: doc. dr. Martina Bergant Marušič izr. prof. dr. Dorota Korte Datum zagovora: 19. okt. 18 TJAŠA LOJPUR VPLIV PROTEINOV APOBEC NA INFEKCIJO Z VIRUSI HPV Mentor: doc. dr. Martina Bergant Marušič Datum zagovora: 29. okt. 18 ANDREJ JERKIČ DOLOČEVANJE AKTIVNOSTI ACHE ENCIMA V ČLOVEŠKEM SERUMU Z UPORABO FIA-TLS METODE Mentor: prof. dr. Dorota Korte Datum zagovora: 28. nov. 18 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S35 DIPLOME ŠTUDIJSKI PROGRAM OKOLJE - 1. STOPNJA _ TANJA BATKOVIČ OS - EKOLOŠKO SPREJEMLJIVI PRETOK Mentor: univ. dipl. inž. in kom. inž. Anja Potokar Datum zagovora: 31. 5. 2018 ANŽE KURAJ PRAKTIČNO USPOSABLJANJE NA KMETIJSKO GOZDARSKEM ZAVODU NOVA GORICA Mentor: dr. Tjaša Jug Datum zagovora: 14. 9. 2018 JAN DAVID TRETIRANJE BALASTNIH VOD IN PROCESI CERTIFICIRANJA SISTEMOV Mentor: Dr. Matej David Datum zagovora: 9. 10. 2018 META KRIŽAJ OBDELAVA PODATKOV S PODROČJA KAKOVOSTI ZRAKA V RAZLIČNIH TEKOČIH PROJEKTIH Mentor: dr. David Kocman Datum zagovora: 9. 10. 2018 MOJCA ZOTLER KOMPOZIT IZ DIGESTATA IZ ANAEROBNE RAZGRADNJE MEŠANIH KOMUNALNIH ODPADKOV, GLINE IN PEPELA KOT ALTERNATIVEN GRADBENI MATERIAL Mentor: doc. Dr. Ana Mladenovič Somentor: dr. Primož Oprčkal dr. Vesna Zalar Serjun Datum zagovora: 9. 10. 2018 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S37 KOLEDAR VAŽNEJŠIH ZNANSTVENIH SREČANJ S PODROČJA KEMIJE IN KEMIJSKE TEHNOLOGIJE SCIENTIFIC MEETINGS -CHEMISTRY AND CHEMICAL ENGINEERING June 2019 _ 2 - 6 Information: 16 - 19 Information: 16 - 19 Information: 19 - 21 Information: 16 - 20 Information: 16 - 20 Information: 26 - 28 Information: 26 - 30 Information: 14th INTERNATIONAL SYMPOSIUM ON MACROCYCLIC AND SUPRAMOLECULAR CHEMISTRY Lecce, Italy https://ismsc2019.eu/ LOSS PREVENTION 2019 Delft, The Netherlands http://lossprevention2019.org/ 29th EUROPEAN SYMPOSIUM ON COMPUTER-AIDED PROCESS ENGINEERING Eindhoven, The Netherlands https://escape29.nl/ IBERO-AMERICAN CONGRESS OF CHEMICAL ENGINEERING (CIBIQ) Santander, Spain https://anque-icce2019.com/es/ 17th INTERNATIONAL CONFERENCE ON CHEMISTRY AND THE ENVIRONMENT -ICCE2019 Thessaloniki, Greece http://www.euchems.eu/events/17th-international-conference-chemistry-environment-icce2019/ 12th IWA INTERNATIONAL CONFERENCE ON WATER RECLAMATION AND REUSE Berlin, Germany http://efce.info/IWA+Conference+2019.html THERMODYNAMICS 2019 Huelva, Spain http://efce.info/Thermodynamics+2019.html 6th EUROPEAN CONFERENCE ON ENVIRONMENTAL APPLICATIONS OF ADVANCED OXIDATION PROCESSES - EAAOP-6 Ljubljana, Slovenia http://eaaop6.ki.si/ July 2019 1 - 3 Information: 3 - ó Information: CONGRESS ON NUMERICAL METHODS IN ENGINEERING Guimaraes, Portugal www.cmn2019.pt 8th INTERNATIONAL CONFERENCE ON EXPERIMENTS / PROCESS / SYSTEM MODELING / SIMULATION / OPTIMIZATION Athens, Greece http://www.epsmso.gr/ Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement 5 - 12 IUPAC 2019 Paris, France Information: https://www.iupac2019.org/ 10 - 11 10th WORLD CONGRESS ON GREEN CHEMISTRY AND TECHNOLOGY Paris, France Information: https://greenchemistry.chemistryconferences.org/ 17 - 19 8th EUROPEAN VARIETY IN UNIVERSITY CHEMISTRY EDUCATION Tuscany, Italy Information: https://www.monash.edu/eurovariety-2019/_nocache 17 - 19 INTERNATIONAL CONFERENCE ON MATERIALS AND NANOMATERIALS Paris, France Information: https://mns-19.com/ 21 - 26 THE 18th INTERNATIONAL SYMPOSIUM ON NOVEL AROMATIC COMPOUNDS (ISNA- 18) Sapporo City, Japan Information: https://iupac.org/event/18th-international-symposium-novel-aromatic-compounds-isna-18/ 22 - 24 5th INTERNATIONAL CONGRESS ON WATER, WASTE AND ENERGY MANAGEMENT Paris, France Information: https://waterwaste-19.com/ 30 - Aug. 1 8th INTERNATIONAL CONFERENCE FOR NETWORK FOR INTER-ASIAN CHEMISTRY EDUCATORS (NICE) Taipei, Taiwan Information: https://iupac.org/event/8th-international-conference-network-inter-asian-chemistry-educators/ August 2019 4 - 8 Information: 36th INTERNATIONAL CONFERENCE ON SOLUTION CHEMISTRY (ICSC) Xining, China http://icsc2019.csp.escience.cn/dct/page/1 September 2019 8 - 13 Information: 9 - 13 Information: 11 - 14 Information: 15 - 19 Information: 25 - 27 Information: 33ND CONFERENCE OF EUROPEAN COLLOID AND INTERFACE SOCIETY (ECIS) Leuven, Belgium https://kuleuvencongres.be/ecis2019/ EUROPEAN CORROSION CONGRESS Seville, Spain https://eurocorr.org/EUROCORRt2019.html 7th INTERNATIONAL CONFERENCE ON SEMICONDUCTOR PHOTOCHEMISTRY - SP7 Milano, Italy https://www.sp7.unimi.it/ 11th EUROPEAN CONGRESS OF CHEMICAL ENGINEERING - ECCE11 & 4TH EUROPEAN CONGRESS OF APPLIED BIOTECHNOLOGY - ECAB5 Florence, Italy http://efce.info/ECCE12_ECAB5-p-112545.html SLOVENIAN CHEMICAL SOCIETY ANNUAL MEETING 2019 Portorož, Slovenia http://chem-soc.si/slovenian-chemical-society-annual-meeting-2019 Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S39 October 2019 1 - 6 Information: 17 - 18 Information: 21 - 24 Information: 14th CONFERENCE ON SUSTAINABLE DEVELOPMENT OF ENERGY, WATER AND ENVIRONMENT SYSTEMS (SDEWES) Dubrovnik, Croatia http://www. dubrovnik2019. sdewes. org/ 2019 WATER DAYS, INTERNATIONAL SYMPOSIUM - CONNECTED TO WATER FOR 25 YEARS Bernardin Congress Centre, Portorož, Slovenia https://sdzv-drustvo.si/en/water-days-2019/ 24RD INTERNATIONAL CONGRESS OF CHEMICAL AND PROCESS ENGINEERING CHISA 2019 Seč, Czech Republic http://2019.chisa.cz/ November 2019 27 - 29 EAPRIL Tartu, Estonia Information: https://eapril.org/eapril-2019 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement Acta Chimica Slovenica Author Guidelines Submissions Submission to ACSi is made with the implicit understanding that neither the manuscript nor the essence of its content has been published in whole or in part and that it is not being considered for publication elsewhere. All the listed authors should have agreed on the content and the corresponding (submitting) author is responsible for having ensured that this agreement has been reached. The acceptance of an article is based entirely on its scientific merit, as judged by peer review. There are no page charges for publishing articles in ACSi. The authors are asked to read the Author Guidelines carefully to gain an overview and assess if their manuscript is suitable for ACSi. Additional information • Citing spectral and analytical data • Depositing X-ray data Submission material Typical submission consists of: • full manuscript (PDF file, with title, authors, abstract, keywords, figures and tables embedded, and references) • supplementary files - Full manuscript (original Word file) - Statement of novelty (Word file) - List of suggested reviewers (Word file) - ZIP file containing graphics (figures, illustrations, images, photographs) - Graphical abstract (single graphics file) - Proposed cover picture (optional, single graphics file) - Appendices (optional, Word files, graphics files) Incomplete or not properly prepared submissions will be rejected. Submission process Before submission, authors should go through the checklist at the bottom of the page and prepare for submission. Submission process consists of 5 steps. 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Step 4: Upload supplementary files • Original Word file (original of the PDF uploaded in the step 2) • Statement of novelty in a Word file must be uploaded • All graphics have to be up I oaded in a single ZIP file. Graphics should be named Figure 1.jpg, Figure 2.eps, etc. • Graphical abstract image must be uploaded separately • Proposed cover picture (optional) should be up-l oaded separatel y. • Any additional appendices (optional) to the paper may be uploaded. Appendices may be published as a supplementary material to the paper, if accepted. • For each uploaded file the author is asked for additional metadata which may be provided. Depending of the type of the file please provide the relevant title (Statement of novelty, List of suggested reviewers, Figures, Graphical abstract, Proposed cover picture, Appendix). Step 5: Confirmation • Final confirmation is required. Article Types Feature Articles are contributions that are written on editor's invitation. They should be clear and concise summaries of the most recent activity of the author and his/her research group written with the broad scope of ACSi in mind. They are intended to be general overviews of the authors' subfield of research but should be written in a way that engages and informs scientists in other areas. They should contain the following (see also general directions for article structure in ACSi below): (1) an introduction that acquaints readers with the authors' research field and outlines the important questions to which answers are being sought; (2) interesting, new, and recent contributions of the author(s) to the field; and (3) a summary that presents possible future directions. Manuscripts normally should not exceed 40 pages of one column format (letter size 12, 33 lines per page). Generally, experts in a field who have made important contribution to a specific topic in recent years will be invited by an editor to contribute such an Invited Feature Article. Individuals may, however, send a proposal (one-page maximum) for an Invited Feature Article to the Editorin-Chief for consideration. Scientific articles should report significant and innovative achievements in chemistry and related sciences and should exhibit a high level of originality. They Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S41 should have the following structure: 1. Tit I e (max. 150 characters), 2. Authors and af I iations, 3. Abstract (max. 1000 characters), 4. Keywords (max. 6), 5. Introduction, 6. Experimental, 7. Results and Discussion, 8. ConcI usions, 9. Acknowledgements, 10.References. The sections should be arranged in the sequence generally accepted for publications in the respective fields and should be successively numbered. Short communications generally follow the same order of sections as Scientific articles, but should be short (max. 2500 words) and report a significant aspect of research work meriting separate publication. Editors may decide that a Scientific paper is categorized as a Short Communication if its length is short. Technical articles report applications of an already described innovation. Typically, technical articles are not based on new experiments. Preparation of Submissions Text of the submitted articles must be prepared with Microsoft Word. Normal style set to single column, 1.5 line spacing, and 12 pt Times New Roman font is recommended. Line numbering (continuous, for the whole document) must be enabled to simplify the reviewing process. For any other format, please consult the editor. Articles should be written in English. Correct spelling and grammar are the sole responsibility of the author(s). Papers should be written in a concise and succinct manner. The authors shall respect the ISO 80000 standard [1], and IUPAC Green Book [2] rules on the names and symbols of quantities and units. The Système International d'Unités (SI) must be used for all dimensional quantities. Graphics (figures, graphs, illustrations, digital images, photographs) should be inserted in the text where appropriate. The captions should be self-explanatory. Lettering should be readable (suggested 8 point Arial font) with equal size in all figures. Use common programs such as MS Excel or similar to prepare figures (graphs) and ChemDraw to prepare structures in their final size. Width of graphs in the manuscript should be 8 cm. Only in special cases (in case of numerous data, visibility issues) graphs can be 17 cm wide. All graphs in the manuscript should be inserted in relevant places and aligned left. The same graphs should be provided separately as images of appropriate resolution (see below) and submitted together in a ZIP file (Graphics ZIP). Please do not submit figures as a Word file. In graphs, only the graph area determined by both axes should be in the frame, while a frame around the whole graph should be omitted. The graph area should be white. The legend should be inside the graph area. The style of all graphs should be the same. Figures and illustrations should be of sufficient quality for the printed version, i.e. 300 dpi minimum. Digital images and photographs should be of high quality (minimum 250 dpi resolution). On submission, figures should be of good enough resolution to be assessed by the referees, ideally as JPEGs. High-resolution figures (in JPEG, TIFF, or EPS format) might be required if the paper is accepted for publication. Tables should be prepared in the Word file of the paper as usual Word tables. The captions should appear above the table and should be self-explanatory. References should be numbered and ordered sequentially as they appear in the text, likewise methods, tables, figure captions. When cited in the text, reference numbers should be superscripted, following punctuation marks. It is the sole responsibility of authors to cite articles that have been submitted to a journal or were in print at the time of submission to ACSi. Formatting of references to published work should follow the journal style; please also consult a recent issue: 1. J. W. Smith, A. G. White, Acta Chim. Slov. 2008, 55, 1055-1059. 2. M. F. Kemmere, T. F. Keurentjes, in: S. P. Nunes, K. V. Peinemann (Ed.): Membrane Technology in the Chemical Industry, Wiley-VCH, Weinheim, Germany, 2008, pp. 229-255. 3. J. Levec, Arrangement and process for oxidizing an aqueous medium, US Patent Number 5,928,521, date of patent July 27, 1999. 4. L. A. Bursill, J. M. Thomas, in: R. Sersale, C. Coll e I a, R. Aiell o (Eds.), Recent Progress Report and Discussions: 5th International Zeo I ite Conference, Naples, Italy, 1980, Gianini, Naples, 1981, pp. 25-30. 5. J. Szegezdi, F. Csizmadia, Prediction of dissociation constant using microconstants, http://www. che-maxon.com/conf/Prediction_of_dissociation _con-stant_using_microco nstants.pdf, (assessed: March 31, 2008) Titles of journals should be abbreviated according to Chemical Abstracts Service Source Index (CASSI). Special Notes • Complete characterization, including crystal structure, should be given when the synthesis of new compounds in crystal form is reported. • Numerical data should be reported with the number of significant digits corresponding to the magnitude of experimental uncertainty. • The SI system of units and IUPAC recommendations for nomenclature, symbols and abbreviations should be followed closely. Additionally, the authors should follow the general guidelines when citing spectral and analytical data, and depositing crystallographic data. • Characters should be correctly represented throughout the manuscript: for example, 1 (one) and l (ell), 0 (zero) and O (oh), x (ex), D7 (times sign), B0 (degree sign). Use Symbol font for all Greek letters and mathematical symbols. • The rules and recommendations of the IUBMB and the International Union of Pure and Applied Chemistry (IUPAC) should be used for abbreviation of chemical names, nomenclature of chemical compounds, enzyme nomenclature, isotopic compounds, optically active isomers, and spectroscopic data. • A conflict of interest occurs when an individual (author, reviewer, editor) or its organization is involved in multiple interests, one of which could possibly corrupt the motivation for an act in the Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement other. Financial relationships are the most easily identifiable conflicts of interest, while conflicts can occur also as personal relationships, academic competition, etc. The Editors will make effort to ensure that conflicts of interest will not compromise the evaluation process; potential editors and reviewers will be asked to exempt themselves from review process when such conflict of interest exists. When the manuscript is submitted for publication, the authors are expected to disclose any relationships that might pose potential conflict of interest with respect to results reported in that manuscript. In the Acknowledgement section the source of funding support should be mentioned. The statement of disclosure must be provided as Comments to Editor during the submission process. • Published statement of Informed Consent. Research described in papers submitted to ACSi must adhere to the principles of the Declaration of Helsinki (http://www.wma.net/e/policy/ b3.htm). These studies must be approved by an appropriate institutional review board or committee, and informed consent must be obtained from subjects. The Methods section of the paper must include: 1) a statement of protocol approval from an institutional review board or committee and 2), a statement that informed consent was obtained from the human subjects or their representatives. • Published Statement of Human and Animal Rights.When reporting experiments on human subjects, authors should indicate whether the procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008. If doubt exists whether the research was conducted in accordance with the Helsinki Declaration, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly approved the doubtful aspects of the study. When reporting experiments on animals, authors should indicate whether the institutional and national guide for the care and use of laboratory animals was followed. • To avoid conflict of interest between authors and referees we expect that not more than one referee is from the same country as the corresponding au-thor(s), however, not from the same institution. • Contributions authored by Slovenian scientists are evaluated by non-Slovenian referees. • Papers describing microwave-assisted reactions performed in domestic microwave ovens are not considered for publication in Acta Chimica Slovenica. • Manuscripts that are not prepared and submitted in accord with the instructions for authors are not considered for publication. Appendices Authors are encouraged to make use of supporting information for publication, which is supplementary material (appendices) that is submitted at the same time as the manuscript. It is made available on the Journal's web site and is linked to the article in the Journal's Web edition. The use of supporting information is particularly appropriate for presenting additional graphs, spectra, tables and discussion and is more likely to be of interest to specialists than to general readers. When preparing supporting information, authors should keep in mind that the supporting information files will not be edited by the editorial staff. In addition, the files should be not too large (upper limit 10 MB) and should be provided in common widely known file formats to be accessible to readers without difficulty. All files of supplementary materials are loaded separately during the submission process as supplementary files. Proposed Cover Picture and Graphical Abstract Image Graphical content: an ideally full-colour illustration of resolution 300 dpi from the manuscript must be proposed with the submission. Graphical abstract pictures are printed in size 6.5 x 4 cm (hence minimal resolution of 770 x 470 pixels). Cover picture is printed in size 11 x 9.5 cm (hence minimal resolution of 1300 x 1130 pixels) Authors are encouraged to submit illustrations as candidates for the journal Cover Picture*. The illustration must be related to the subject matter of the paper. Usually both proposed cover picture and graphical abstract are the same, but authors may provide different pictures as well. * The authors will be asked to contribute to the costs of the cover picture production. Statement of novelty Statement of novelty is provided in a Word file and submitted as a supplementary file in step 4 of submission process. Authors should in no more than 100 words emphasize the scientific novelty of the presented research. Do not repeat for this purpose the content of your abstract. List of suggested reviewers List of suggested reviewers is a Word file submitted as a supplementary file in step 4 of submission process. Authors should propose the names, full affiliation (department, institution, city and country) and e-mail addresses of three potential referees. Field of expertise and at least two references relevant to the scientific field of the submitted manuscript must be provided for each of the suggested reviewers. The referees should be knowledgeable about the subject but have no close connection with any of the authors. In addition, referees should be from institutions other than (and preferably countries other than) those of any of the authors. How to Submit Users registered in the role of author can start submission by choosing USER HOME link on the top of the page, then choosing the role of the Author and follow the relevant link for starting the submission process. Prior to submission we strongly recommend that you familiarize yourself with the ACSi style by browsing the journal, particularly if you have not submitted to the ACSi before or recently. Društvene vesti in druge aktivnosti Acta Chim. Slov. 2019, 66, (2), Supplement S43 Correspondence All correspondence with the ACSi editor regarding the paper goes through this web site and emails. Emails are sent and recorded in the web site database. In the correspondence with the editorial office please provide ID number of your manuscript. All emails you receive from the system contain relevant links. Please do not answer the emails directly but use the embedded links in the emails for carrying out relevant actions. Alternatively, you can carry out all the actions and correspondence through the online system by logging in and selecting relevant options. Proofs Proofs will be dispatched via e-mail and corrections should be returned to the editor by e-mail as quickly as possible, normally within 48 hours of receipt. Typing errors should be corrected; other changes of contents will be treated as new submissions. Submission Preparation Checklist As part of the submission process, authors are required to check off their submission's compliance with all of the following items, and submissions may be returned to authors that do not adhere to these guidelines. 1. The submission has not been previously published, nor is it under consideration for publication in any other journal (or an explanation has been provided in Comments to the Editor). 2. All the listed authors have agreed on the content and the corresponding (submitting) author is responsible for having ensured that this agreement has been reached. 3. The submission files are in the correct format: manuscript is created in MS Word but will be submitted in PDF (for reviewers) as well as in original MS Word format (as a supplementary file for technical editing); diagrams and graphs are created in Excel and saved in one of the file formats: TIFF, EPS or JPG; illustrations are also saved in one of these formats. The preferred position of graphic files in a document is to embed them close to the place where they are mentioned in the text (See Author guidelines for details). 4. The manuscript has been examined for spelling and grammar (spell checked). 5. The title (maximum 150 characters) briefly explains the contents of the manuscript. 6. Full names (first and last) of all authors together with the affiliation address are provided. Name of author(s) denoted as the corresponding author(s), together with their e-mail address, full postal address and telephone/fax numbers are given. 7. The abstract states the objective and conc I usions of the research concisely in no mo re than 150 words. 8. Keywords (minimum three, maximum six) are provided. 9. Statement of novelty (maximum 100 words) clearly explaining new findings reported in the manuscript should be prepared as a separate Word file. 10. The text adheres to the stylistic and bibliographic requirements outlined in the Author guidelines. 11. Text in normal style is set to single column, 1.5 line spacing, and 12 pt. Times New Roman font is recommended. All tables, figures and illustrations have appropriate captions and are placed within the text at the appropriate points. 12. Mathematical and chemical equations are provided in separate lines and numbered (Arabic numbers) consecutively in parenthesis at the end of the line. All equation numbers are (if necessary) appropriately included in the text. Corresponding numbers are checked. 13. Tables, Figures, illustrations, are prepared in correct format and resolution (see Author guidelines). 14. The lettering used in the figures and graphs do not vary greatly in size. The recommended lettering size is 8 point Arial. 15. Separate files for each figure and illustration are prepared. The names (numbers) of the separate files are the same as they appear in the text. All the figure files are packed for uploading in a single ZIP file. 16. Authors have read special notes and have accordingly prepared their manuscript (if necessary). 17. References in the text and in the References are correctly cited. (see Author guidelines). All references mentioned in the Reference list are cited in the text, and vice versa. 18. Permission has been obtained for use of copyrighted material from other sources (including the Web). 19. The names, full affiliation (department, institution, city and country), e-mail addresses and references of three potential referees from institutions other than (and preferably countries other than) those of any of the authors are prepared in the word file. At least two relevant references (important papers with high impact factor, head positions of departments, labs, research groups, etc.) for each suggested reviewer must be provided. 20. Full-colour illustration or graph from the manuscript is proposed for graphical abstract. 21. Appendices (if appropriate) as supplementary material are prepared and will be submitted at the same time as the manuscript. Privacy Statement The names and email addresses entered in this journal site will be used exclusively for the stated purposes of this journal and will not be made availab I e for any other purpose or to any other party. ISSN: 1580-3155 Društvene vesti in druge aktivnosti S10 Acta Chim. Slov. 2019, 66, (2), Supplement Koristni naslovi chemical Society Slovensko kemijsko društvo www.chem-soc.si e-mail: chem.soc@ki.si Wessex Institute of Technology www.wessex .ac.uk SETAC www.setac.org European Water Association http://www.ewa-online.eu/ European Science Foundation www.esf.org & EFCE European Federation of Chemical Engineering https://efce.info/ International Union of Pure and Applied Chemistry https://iupac.org/ Novice europske zveze kemijskih društev (EuCheMS) najdete na: EuCheMS: Brussels News Updates http://www.euchems.eu/newsletters/ Društvene vesti in druge aktivnosti ZASTOPAMO: 0DD3RAUI1 VSE NA ENEM MESTU: PRODAJA SERVIS REZERVNI DELI POTROŠNI MATERIAL Donau Lab d.o.o., Ljubljana Tbilisijska 85 SI-1000 Ljubljana www.donaulab.si office-si@donaulab.com Razvoj in inovacije za globalno uspešnost Znanje, kreativnost zaposlenih in inovacije so ključnega pomena v okolju, kjer nastajajo Heliosovi pametni premazi. Z rešitvami, ki zadostijo široki paleti potreb, kontinuiranim razvojem ter s kakovostnimi produkti Helios predstavlja evropski center za inovacije, know-how in poslovni razvoj skupine Kansai Paint. HELIOS www.helios-group.eu Designing Excellence BODITE NEUSTAVLJIVI MAGNEZIJ Krka 300 ^ Granulat za pripravo napitka vsebuje magnezijev citrat in vitamin B2 Magnezij in vitamin B2 prispevata k zmanjševanju utrujenosti in izčrpanosti ter normalnemu delovanju živčnega sistema. t Magnezij prispeva tudi k delovanju mišic. MAGNEZIJ Krka 300 granulat za napitek MAGNEZIJEV CITRAT 300 mg magnezija ^ rog vitamina Ba j"krwn«dsn "SSimSB" Mg ""»«I«,, '^KRKK www.magnezijkrka.si G* I ^ KRKk @ Okus po pomaranči in limeti. 0 Brez konzervansov. ® Brez umetnih barvil, arom in sladil. 0 Ena vrečka na dan. Prehransko dopolnilo ni nadomestilo za uravnoteženo in raznovrstno - prehrano. Skrbite tudi za zdrav življenjski slog. \ ActaChimica Slovenica Acta ChimicaSlovenica It appears that, in our current state of knowledge, fluoride illustrates surprisingly well the classical medical concept that the effect of a substance depends on the dosage regimen. As Paracelsus (1493-1541) said: "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy" (page 255). Acta ChimicaSlc ActaChimicaSlc Slovenica Year 2019, Vol. 66, No. 2 m* Acta ChimicaSlc ActaChimicaSlc Slovenica Acta ChimicaSlc ActaChimicdiSlc Slovenica Study of the Influence, of Key Process Parameters on Furfural Product™ :nt of Hantupcptin-A Experimental am i of Trivaleni Cobalt Schiff Ba« Compile Acta ChimicaSlc ActaChimicaSlc Slovenica Acta ChimicaSlc ActaChimicdiSlc Slovenica Selected Titles: DNA Minor Grow Binders-Inspired by Nature The I Acta ChimicaSlc ActaChimicdiSlc Slovenica t-* »> .T® SpS Acta ChimicaSlc ActaChimicdiSlc Slovenica Acta ChimicaSlc ActaChimicdiSlc Slovenica ,-v -Co Dedicated to the Cutting Edge 2017 Conference of Young Scientists. Selected titles: Disintegrins from the Venom of llpem mimxhies mmodjta Efficiently Inhibit Migration of Breast Cancer Cells Chemometric Character,ition of Slovenian Red Wines Modelling the Correlation between Molecular Electrostatic Potential and pKa on Sets of Carboxylic Acids, Phenols, and Mines Cold Plasma and Acid Treatment Modification Effects on Phonolite Acta ChimicaSlc ActaChimicdiSlc Slovenica ActdChimicaSlc ActaChimicdiSlc Slovenica AcidiChimicaSlc ActaChimicdiSlc Slovenica Acta ChimicaSlc ActaChimicdiSlc Slovenica j_ 65/2018