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Qualitative Determination, Quantitative Evaluation and Comparative Sustainable Development Research; Vol. 1, No. 1; 2019 ISSN 2690-9898 E-ISSN 2690-9901 https://doi.org/10.30560/sdr.v1n1p55 Qualitative Determination, Quantitative Evaluation and Comparative Insecticidal Potential of Ruta Graveolens Essential Oil and Its Major Constituents in the Management of Two Stored Pests Sitophilus Zeamais (Coleoptera: Curculionidae) And Corcyra Cephalonica (Lepidoptera: Pyralidae) A.G.W.U. Perera1, M.M.S.C. Karunaratne1 & S.D.M. Chinthaka2 1 Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka 2 Department of Chemistry, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka Correspondence: A.G.W.U. Perera, Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Nugegoda, Sri Lanka. E-mail: [email protected] Received: December 1, 2019 Accepted: December 15, 2019 Online Published: December 30, 2019 Abstract Eco-chemical control based on essential oil mediated plant-insect interactions is an alternative method to the unsystematic use of insecticides, due to advanced structural diversity and allelopathic potential of essential oils. In this sense, present work was aimed at qualitative and quantitative investigation of chemical composition and the evaluation of insecticidal activities of Ruta graveolens essential oil and its major constituents against Sitophilus zeamais and Corcyra cephalonica in stored maize. Fresh leaves were subjected to hydrodistillaton and the chemical composition of oil was studied. Essential oil and its major constituents were then assessed for their allelopathic activity on test insects. Fifty components were identified, where long chain aliphatic 2-methyl ketones predominated the oil as major constituents. Results revealed strong concentration-, insect species- and time- dependent toxicities, in which oil caused 100 % mortalities at concentrations of 1.52 and 0.46 µL/cm2 against S. zeamais and C. cephalonica respectively after 24 hours of exposure. In aliphatic 2-methyl ketone series, respective congeners having odd and even number of carbon atoms were more effective on S. zeamais and C. cephalonica. These findings provide a scientific basis for the eco-potential of using essential oil of R. graveolens and its major constituents in integrated insect pest management programs. Keywords: Ruta graveolens, Sitophilus zeamais, Corcyra cephalonica, contact insecticidal activity, long chain aliphatic methyl ketones 1. Introduction Synthetic insecticides are being extensively used with the view of protecting stored grains in increasing yields which also have triggered many undesirable impacts on entire ecosystems, society and every aspect of life on earth. Thus, the development of green insecticides has been focused as a viable pest management strategy in IPM programs in recent years (Khani and Rahdari, 2012). Particularly, plant essential oils and their constituents have been extensively studied and proposed as being a high potential option (Pumnuan et al., 2015; Guo et al., 2015) due to their specificity in exerting wide array of known biological influences on insects including repellent, deterrent or antifeedant, inhibition of digestion, increasing oviposition or contrarily decreasing reproduction by ovicidal and larvicidal effects (Regnault-Roger, 1997). The high volatility of plant essential oils and their constituents reduce the concern for their residues on stored grains (Kim et al., 2016). Different content ratios among the authentic compounds of essential oils will strongly influence its quality which varies with geographical and seasonal variation (Porel et al., 2014). Even the minor compounds should not be disregarded as they contribute significantly to the quality of oil. Hence the accurate quantification of individual compounds of essential oils is a key element in research or industrial analysis which may be used to understand the relationship not only between the constituent structure and aroma of essential oils (Zhu et al., 2005), but also between the constituent structures and their broad spectrum of biological properties against insect pests. During our screening process oriented in search of bioactive constituents to replace the use of synthetic insecticides, the essential oil of Ruta graveolens was found to possess contact insecticidal activity against the maize weevil, 55 Published by IDEAS SPREAD sdr.ideasspread.org Sustainable Development Research Vol. 1, No. 1; 2019 Sitophilus zeamais Motschulsky, and the rice moth, Corcyra cephalonica Stainton. which are two key pests of cereal grains in tropical and subtropical regions of the world (Krishna-Ayyar, 1930). Even though Jayaweera (1982) reported the major constituents of R. graveolens essential oil as 2-nonanone and 2-undecanone, the entire profile on the chemical composition of the oil of R. graveolens, growing in Sri Lanka, has never been established. While, the number of reports the insecticidal properties of essential oil of R. graveolens (Jeon et al., 2015) is very limited, literature survey has shown that there is neither a report on quantitative evaluation of the major essential oil constituents nor on their comparative insecticidal activities. In this phytocentric study, we performed qualitative and quantitative determination of Ruta graveolens essential oil and its constituents. Using the results thus obtained, we comparatively evaluated the contact insecticidal effect of the essential oil together with its constituents with the view of identifying and characterizing bio-active constituents of the essential oil to be utilized in the management of S. zeamais and C. cephalonica under laboratory conditions. 2. Materials and Methods 2.1 Test Insects Maize weevils (Sitophilus zeamais) and rice moths (Corcyra cephalonica) were obtained from the cultures reared on respective media of whole, un-infested maize grains and coarsely ground maize in the Agricultural Insect Pest Management Laboratory of Department of Zoology, Faculty of Applied Sciences, University of Sri Jayewardenepura, Sri Lanka. The relative humidity, light regime and ambient temperature during the experimental period were 84 ± 2% RH, 12h:12h light:dark, and 29 ± 20C respectively. One week old, unsexed adult weevils and ˂24h old rice moths were used in all bioassays. 2.2 Plant Material Fresh, mature and healthy leaves of Ruta graveolens were collected from a local market at Colombo Fort, Sri Lanka. These were coarsely powdered by using a domestic electric grinder (Multinational®, 2101, India). 2.3 Essential Oil Extraction The essential oil was extracted by hydro-distillation of the ground plant leaves (250 g of sample in 2.5 L of distilled water) using a modified Clevenger-type apparatus for 5 h with 3 replications. The superior phase was collected from the condenser, dried over anhydrous sodium sulphate and stored in sealed glass vials at 40C prior to analysis. The essential oil extraction was carried out in the laboratory at the Ayurvedic Research Institute, Navinna, Maharagama, Sri Lanka. 2.4 Reference Compounds and Reagents Pure commercial standard compounds of 2-octanone (C8), 2-nonanone (C9), 2-decanone (C10), 2-undecanone (C11), 2-dodecanone (C12), 2-tridecanone (C13) representing the 2-ketone series were purchased from Sigma- Aldrich (USA). These reference compounds were selected due to their top-tier presence in the essential oil composition of R. graveolens. Acetone (analytical grade quality) for Gas Chromatography-Mass Spectrometry (GC-MS) Analysis was also purchased from Sigma-Aldrich, USA. The other standard compounds were all in GC purities. 2.5 Sample Preparation For the characterization of essential oil composition of R. graveolens through GC-MS analysis, 12 µL of accurately measured essential oil sample was dissolved in 1 mL of Acetone. Then 2 µL of the solution was injected into the GC-MS system. For GC-MS analysis, a standard cocktail comprising long chain aliphatic methyl ketone constituents was prepared separately, in order to detect and quantify the compounds of interest. The cocktail which is a mixed stock solution containing reference standards (C8, C9, C10, C11, C12, and C13) was prepared by dissolving 12 μL of accurately measured standard of each compound in acetone targeting a stock concentration of 100 μgmL-1 for the cocktail. Dilutions of the standard cocktail were made in acetone to produce concentrations of the compounds ranged at 10- 50 μgmL-1. 2.6 Gas Chromatography-Mass Spectrometry (GC-MS) Analysis Chemical standards and EO of R. graveolens were analyzed using an Agilent Technologies 7890A GC system (Palo Alto, CA) equipped with an Agilent Technologies 5975C inert XL EI/CI mass spectrometer. A DB-5MS fused silica capillary column of 30m×0.25mm (J & W Scientific, Folsom, CA) was used. The temperature was programmed as follows: initial oven temperature was 700C, held for 2 minutes, and then ramped at 100C/min up 56 Published by IDEAS SPREAD sdr.ideasspread.org Sustainable Development Research Vol. 1, No. 1; 2019 to 2800C, where it was held for 7 minutes. The injector and detector temperatures were set at 2750C using helium as the carrier gas, at a flow rate of 1mL/min. The injection volume of the sample was 2 μL using the split flow mode (split ratio, 5:1). The identification of EO components was based on the NIST reference mass spectral library provided with the GC-MS equipment. The composition was reported as a relative percentage of the total peak area. EO constituents were ranged into 2 molecular
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