Acta agriculturae Slovenica, 118/4, 1–6, Ljubljana 2022 doi:10.14720/aas.2022.118.4.2546 Original research article / izvirni znanstveni članek Chemical composition and insecticidal effect of methanol extract of Cap- paris spinosa L. fruits on Tribolium confusum Jacquelin du Val, 1863 and Sitophilus oryzae (L., 1763) adults Tayebe MAHMOUDI MANESH 1, Sultan RAVAN 1, 2, Abbas KHANI 1, Mansoor SARANI 3 Received February 28, 2022; accepted November 02, 2022. Delo je prispelo 28. februar 2022, sprejeto 2. novembra 2022 1 Department of Plant Protection, Agricultural Faculty, University of Zabol, Zabol, Iran 2 Corresponding author, e-mail: sultan_ravan@yahoo.com 3 Scientific Member of Agricultural and Natural Resources Research Center of Sistan, Zabol, Iran Chemical composition and insecticidal effect of methanol extract of Capparis spinosa L. fruits on Tribolium confusum Jacquelin du Val, 1863 and Sitophilus oryzae (L., 1763) adults Abstract: Tribolium confusum and Sitophilus oryzae are stored product pests found worldwide. Environmental dam- ages, human health issues and the emergence of resistance are driving scientists to seek alternatives to synthetic insecticides for its control. Under this scenario, plant secondary metabolites are being increasingly studied as bioinsecticides because they are renewable, natural, biodegradable, non-persistent in the environment and safe to non-target organism and humans. In this study, the chemical composition and lethal effects of meth- anol extract of Capparis spinosa fruits on Tribolium confusum and Sitophilus oryzae adults were studied. The LC50 of extract on T. confusum and S. oryzae in contact method were 14.7 and 10.5 mg cm-2, respectively, whereas in the dip method, the LC50 value determined 41.3 and 34.3 mg ml-1 for T. confusum and S. oryzae, respectively. The most important identified compounds were the thymol (22.5  %), methyl sulfonyl heptyl isothiocya- nate (13.3 %), butyl isothiocyanate (8.1 %), γ-terpinene (6.2%) and iso propyl isothiocyanate (5.8 %). The results confirmed the potential of the C. spinosa extract in controlling stored-product insects. Key words: Capparis spinosa; GC-Mass; insecticidal plant extract; stored-product pest; erpenoides Kemijska sestava in insekticidni učinki metanolnih izvlečkov plodov kaprovca Capparis spinose L. na odrasle osebke male- ga mokarja (Tribolium confusum Jacquelin du Val, 1863) in riževega žužka (Sitophilus oryzae (L., 1763)) Izvleček: Mali mokar (Tribolium confusum) in rižev žu- žek (Sitophilus oryzae) sta po vsem svetu razširjena skladiščna škodljivca. Okoljska škoda, škodljivi vplivi na zdravje ljudi in pojav odpornosti na insecticide vodijo raziskovalce k iskanju alternativ sintetičnim insekticidom za uravnavanje teh ško- dljivcev. V tem pogledu se povečuje preučevanje sekundarnih metabolitov rastlin, ker so obnovljivi, naravni, razgradljivi, neobstojni, okoljsko varni za neciljne organizme in človeka. V raziskavi so bili preučevani letalni učinki metanolnih izvlečkov plodov kaprovca (Capparis spinose) na odrasle osebke malega mokarja (Tribolium confusum) in riževega žužka (Sitophilus oryzae). LC50 vrednosti izvlečkov za malega mokarja in riževe- ga žužka sta bili pri kontaktni metodi 14,7 in 10,5 mg cm-2, pri metodi potapljanja pa 41,3 in 34,3 mg ml-1. Najpomembnejše sestavine, določene v izvlečkih plodov kaprovca so bile timol (22,5 %), metil sulfonil heptil izotiocianat (13,3 %), butil izo- tiocianat (8,1 %), γ-terpinen (6,2 %) in izopropil izotiocianat (5,8  %). Rezultati so potrdili potencialno uporabo izvlečkov plodov kaprovca za uravnavanje škodljivih skladiščnih žuželk. Ključne besede: Capparis spinosa; masni plinski kroma- tograf; insekticidni rastlinski izvlečki; skladiščni škodljivci; ter- penoidi Acta agriculturae Slovenica, 118/4 – 20222 T. MAHMOUDI MANESH et al. 1 INTRODUCTION Food production and preservation has always been a major concern of human beings. Likewise, the increas- ing trend of human population has made people experi- ence numerous problems such as hunger and environ- mental pollution. In fact, over 600 species of beetles, 70 species of butterflies, and about 355 species of mites, which infest stored agricultural products, reduce the quantity and quality of these warehoused crops. As much as 50 %–60 % cereal yields can be lost during the storage stage due to the lack of technical possibilities for their proper harvesting and storage (Kumar and Kalita, 2017). In Iran, in rural areas, mainly due to the traditional and unsuitable conditions of warehousing, this rate arrives to 80 % (Forghani and Marouf, 2015). Confused flour beetle (Tribolium confusum Jaqc- quelin du Val, 1863) and rice weevil Sithophilus oryzae (L., 1763) are regarded two of the most important pests of stored cereals around the world, which not only causes significant losses due to feeding, but also, they infect the stored crop with their fecal pellets and larval shells, and reduces the crop’s quality greatly (Forghani and Marouf, 2015). In recent years, a great number of researchers have been attracted to the ways of pest management by using herbal essential oils and plant extracts. The plant extracts have useful insecticide properties such as less effect on natural enemies, no plant-burning, minimal lethality on the vertebrates and rapid degradation in the environ- ment and can be partly replaced by artificial insecticides (Wink, 2018). Capparis spinosa L. is a plant of the family Cappridaceae, a flowering bush with two bases branching from the base and broadly exfoliated on the ground. The leaves are plain and light green, and flowers are reddish- white with a large number of long flags and the fruit is oval and meaty and bright green, gradually reddish. It grows in different regions of Iran with different soils and has a high tolerance to heavy soil texture (Ahmadi and Saeidi, 2018). Based on our knowledge, few studies have been reported previously relating to the activity of C. spinosa extract against insect pests. However, insecticidal activi- ties of other species of Capparis genus were reported in various studies (Hussein et al., 2006; El-Shershaby, 2010; Upadhyay, 2012; Ladhari et al., 2013). In this research, chemical composition and insec- ticidal effect of methanol extract of Capparis spinosa on two species of storage product pests (confused flour bee- tle and rice weevils) was investigated. 2 MATERIALS AND METHODS 2.1 INSECTS CULTURES The two species of insects were obtained from the infected storages located in Zabol, Iran and maintained in the dark in incubators (27 ±1 °C; relative humidity = 65 ± 5%) for propagation. The larvae of T. confusum were picked up and reared in glass containers (0.5 l) contain- ing wheat flour mixed with yeast (10:1, w/w). S. oryzae were reared on cracked rice seeds. Insects used in all of the experiments were one week old adults. 2.2 PLANT MATERIAL AND EXTRACTIONS For the extraction of secondary metabolites, fresh fruits of C. spinosa were collected from the central part of Hirmand (61° 32’ E and 31° 0’ N), Sistan and Balu- chistan province, Iran, during July and August, 2013. The fruits were dried naturally and grounded into powder. The powder was weighed and extraction was also done by soaking the powdered fruits in methanol solvent (ra- tio 1:10 w/v) and shaking the mixture on a shaker (350 rpm) for 24 hours at 25 °C. After the completion of extraction, the liquid extract was filtered through Whatman No.1 filter paper fitted in a Buchner funnel using suction. The extracted plant sub- stances were stored in airtight containers covered with aluminum foils in refrigerator at 4 °C. To determine the dry mass of pure extract at one milliliter of solution, the small parts of solution (5 ml) were collected in three rep- licate, separately and allowed to dry completely at 100 °C in oven (Ghaemi et al., 2006). 2.3 INVESTIGATION OF LETHAL EFFECT BY CONTACT METHOD (RESIDUAL TOXICITY) For determination contact toxicity, each experimen- tal unit consisted of a 6 cm Petri dish (with an area of 28.26 cm2). In order to evaluate the lethal effect of the plant extract on both insect pests, different volumes ranging between 1.5 to 4.8 ml of the plant extract with the concentration of 117 mg ml-1 (equivalent to 6.21, 8.69, 11.8, 13.66, 14.91, 16.15, and 19.87 mg cm-2) were uniformly poured by a 1 ml sampler into a Petri dish. In the control samples, various volumes of 1.5 to 4.8 ml of methanol were also poured through a Petri dish. The Petri dish cap was left open for 30 minutes to let the sol- Acta agriculturae Slovenica, 118/4 – 2022 3 Chemical composition and insecticidal effect of methanol extract of Capparis spinosa L. fruits ... vent evaporate. Then, ten 1-7- day- old adult insects were put in each dish and the Petri dish was covered by sele- phon nylon. The Petri’s patch was placed in the incuba- tor at 27 ± 1 °C and relative humidity of 65 ± 5 °C in darkness for 24 h (Akbar et al., 2022; Tavares et al., 2021; Seremet et al., 2018). The number of the dead insects was calculated after 24 hours based on the movement of the legs and tenta- cles. In case of observation of mortality in the control, the percentage of the lethality in different concentrations of the extract was corrected by Abbott’s formula (Abbott, 1925). 2.4 INVESTIGATION OF LETHAL EFFECT BY INSECT-DIP METHOD (TOPICAL TOXICITY) To evaluate the toxicity of metanol extract in dip method, different concentrations of extracts, including 26.5, 29.5, 35.4, 41.3, 44.2, 47.2 and 59 mg ml-1 were pre- pared, and the insects were immersed in each concentra- tion for 10 seconds. Then, the insects were placed on the filter paper for 5 minutes to dry the bodies. The insects of each replicate were transferred into a 6 cm Petri dish on a filter paper and the Petri dish was closed. In the control, immersion was performed in the methanol solvent for 10 seconds. The number of dead insects was determined after 24 hours based on the movement of the legs and tentacles (Akbar et al., 2022; Tavares et al., 2021; Liu et al., 2016). In both bioassay method, experiment was conduct- ed on ten 1-7- day- old adult insects in a completely ran- dom design with four replicates for each concentration. To determine the 50 % lethal concentration (LC50), five concentrations causing between 20 % and 80 % mortality were used mainly based on the preliminary test results of the contact toxicity of the extract. The experiment was carried out in the same way as the above test, and the number of live and dead insects was counted. The LC50 values were calculated using Probit analysis (IBM SPSS V21.0). 2.5 GAS CHROMATOGRAPHY–MASS SPECTROS- COPY OF EXTRACT Gas chromatography–mass spectroscopy (GC–MS) analyses of metanol extract were carried out on a Hewl- ett-Packard 5890 gas chromatograph coupled to a HP 5970 mass-selective detector (MSD) using a fused silica ultra performance cross linked methyl silicone column (50 m × 0.2 mm i.d.; 0.25 µm film thickness) at 4 °C/ min ramp rate. Helium was the carrier gas at 1 ml/min flow rate. Mass spectra were recorded over 40–500 amu range at 1 scan/s with ionization energy 70 eV and ion source temperature 250 °C. Constituent’s identification was made by comparison of their mass spectra with those stored in NIST and Wiley libraries or with mass spectra from the literature (Ravan et al., 2019; Rojht et al., 2012). 2.6 STATISTICAL ANALYSIS OF DATA Data were analyzed by SPSS software (IBM SPSS V21.0). Normality of raw data was surveyed using Non- Parametric One-Sample Kolmogorov-Smirnov test. Sta- tistical analysis of the mortality data was performed by one-way analysis of variance (ANOVA) with a post-hoc Tukey test at p < 0.05. 3 RESULTS AND DISCUSSION 3.1 INSECTICIDIAL ACTIVITIES The results showed that by increasing the concen- tration of metanol extract of plant, the mortality rate of the tested insects increased significantly in both bioassay methods (Figure 1, F5, 23 = 27, p < 0.001 for T. confusum and F4, 19 = 39.6, p < 0.001 for S. oryzae in contact method and F5, 23 = 32.6, p < 0.001 for T. confusum and F5, 23 = 108, p < 0.001 for S. oryzae in insect-dip method). The 50  % lethal concentration (LC50) in contact method was determined as 10.5 and 14.7 mg cm-2 for S. oryzae and T. confusum, respectively. LC50 was calculated as 34.3 and 41.3 mg ml-1, 24 hours after dipping in the metanol extract of C. spinosa, for S. oryzae and T. con- fusum, respectively (Table 1). Due to the non-overlap- ping of the 95 % confidence level of LC50, the toxicity of metanol extract of C. spinosa on S. oryzae was signifi- cantly more than that of T. confusum (Table 1). No study has been reported concerning the contact toxicity of Capparis spinosa extract on confused flour beetle and rice weevil. However, insecticidal activities of other species of Capparis genus were reported in vari- ous studies. The essential oil of another species Capparis genus, Capparis decidua (Forssk.) Edgew., had an insec- ticide and repellency effect on Sitophilus oryzae and Rhy- zopertha dominica (Fabricius, 1792) (Upadhyay 2012). Besides, the lethality and repellency of Capparis aegyptia Lam. on Tetranychus urticae Koch, 1836 (Acari, Tetra- nychidae) was reported (Hussein et al. 2006). Moreover, the biological and toxic effects of the leaf extract of Capparis aegyptia on Agrotis segetum (Denis and Schiffermüller, 1775) (Lepidoptera: Noctuidae) and A. ipsilon (Hufnagel, 1766) was studied and according to Acta agriculturae Slovenica, 118/4 – 20224 T. MAHMOUDI MANESH et al. 3.2 CHEMICAL COMPOSITION OF PLANT EX- TRACT Gas chromatography-mass spectrometry analysis indicated that there were 33 compounds in the metanol extract of C. spinosa fruits, comprising 98.6 % of the to- tal mass, among which the most notably were thymol (22.5 %), methyl sulfonyl heptyl isothiocyanate (13.3 %), butyl isothiocyanate (8.1  %), γ-terpinene (6.22  %) and isopropyl isothiocyanate (5.8 %) (Table 2). The present result was similar to the other literature reported previously. The main components of C. spinosa leaf oil were thymol (26.4  %), isopropyl isothiocyanate (11  %), 2- hexenol (10.2  %) and butyl isothiocyanate (6.3 %). The volatile oils of the fruits composed mainly of isopropyl isothiocyanate (52.2 %) and methyl isothiocy- anate (41.6 %) (Afsharypuor et al., 1998). Existence of the glucosinolates degradation prod- ucts as isothiocyanate derivatives in metanol extract of C. spinosa fruits, are of pharmacological interest because they have insecticidal effect and so contribute to the plant’s overall defense mechanism (Bohinc et al., 2014; Gupta et al., 1999). In addition, investigation of the plant extract showed the presence of terpenoid compounds such as thymol (22.5 %), γ-terpinene (6.2 %), carvacrol (3.1 %), caryophyllene (2.3 %), linalool (1.5 %) α-thujene (1.3 %), β-pinene and 1,8-cineole (1.1 %) in extract of C. spinosa fruits that have been well documented as active fumi- gants, repellents, and insecticides toward stored-product insects (Rojht et al., 2012; Papachristos et al., 2004). 4 CONCLUSIONS The metanol extracts of C. spinosa fruits showed insecticidal activities against S. oryzae and T. confusum adults. The main components of plant extract have insec- ticidal properties such as isothiocyanate derivatives and terpenoid compounds. However, further tests are needed the results, its extract reduced the fertility of adult female insects by 50 % (El-Shershaby 2010). The metanol extracts of C. spinosa leaf in the con- centration of 10000 ppm caused 46  % mortality of the third instar larvae of Spodoptera littoralis (Boisduval, 1833) (Lepidoptera, Noctuidae) after three days of treat- ment. This rate increased by 100 % after a 7- day treat- ment. The anti-nutritional index of the metanol extract of this plant in the same concentration on larvae was 49.7 % (Ladhari et al. 2013). Figure 1: Mortality percentage (Mean ± SE) of T. confusum and S. oryzae exposed to metanol extract of C. spinosa in con- tact (above) and dip method (below) bioassay Means with the different letters above the columns for each pest species are significantly different (p < 0.05) (Tukey post- hoc test after analysis of variance) Bioassay method Insect LC50 95 % CL P-value Slope ± SE x 2(df) Contact T. confusum 14.7* (13.4-16.6) 0.67 4.24 ± 0.61 2.33 (4) S. oryzae 10.5 (9.5-11.6) 0.49 4.56 ± 0.73 2.73 (3) Dipping T. confusum 41.3 (38.3-45.8) 0.34 5.47 ± 1.29 3.34 (3) S. oryzae 34.3 (32.3-36.4) 0.92 7.68 ± 1.18 0.48 (3) Table 1: LC50 values of methanol extract of C. spinosa against T. confusum and S. oryzae after 24 h * Ten individuals per replicate, four replicates per concentration, 5 or 6 concentrations per assay, total number of tested insects were 200 or 240 adults for every test; LC: lethal concentration (mg cm-2 in Contact method and mg ml-1 in insect-dip method), CL: confidence limits Acta agriculturae Slovenica, 118/4 – 2022 5 Chemical composition and insecticidal effect of methanol extract of Capparis spinosa L. fruits ... to develop a formulation and to improve the potency and stability of these potential insecticides for practical use. 5 ACKNOWLEDGMENTS The research was supported by the university of Zabol (Grant No 9618-63), which is greatly appreciated. 6 REFERENCES Abbott, W. 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Reducing postharvest losses Kovats index Rate (%)Compounds Peak No 7412.7Methyl isothiocyanate 1 8225.8Isopropyl isothiocyanate 2 8581.52-Hexeneal3 8691.1Benzyl aldehyde4 90213.3Methyl sulfonyl heptyl isothiocyanate 5 9192Methyl furan6 9368.1Butyl isothiocyanate7 9571.3α-Thujene8 9710.9Comphene9 9881.1β-pinene10 10113.43-octanone11 10201.5α-Terpinene12 10511Ocymene13 10801.11,8-cineole14 11066.2γ-Terpinene15 11281.5Linalool16 11751.8Cis-sabinene hydrate17 12202.6n-Dodecane18 12481Comphor19 12701.3Para-menta-6,8,dien- 2-ol-acetate 20 13111.4Carvone21 134522.5Thymol22 13822.3Caryophyllene23 14601n-tetradecane24 14810.9α -farnesene25 14953.1Carvacrol26 15201Cadinol27 15880.8Geranyl acetone28 17072.5Hexadecanoic acid29 17310.8Frulic acid30 18151.2Octa decanoic acid31 18781.1n-Eicosane32 18970.8Beta-sesquiphellandrene33 98.6Total Table 2: Chemical constituents of methanol extract of Cap- paris spinosa fruits Acta agriculturae Slovenica, 118/4 – 20226 T. 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