Caroline Karimi Karemu*, Mary Ndungu, Mercy Githua

Transcription

1 ONLINE ISSN : Volume 4, Issue 2, Research Article Evaluation of repellent effects of the oils from Eucalyptus Camaldulensis (Schlecht) and Eucalyptus globulus (Labill.) against Sitophilus zeamais (Motschulsky) Caroline Karimi Karemu*, Mary Ndungu, Mercy Githua Received: 5, September 2012 Accepted: 31, October 2012 Published: 3, September 2013 ABSTRACT The essential oils of Eucalyptus camaldulensis and Eucalyptus globulus were extracted by steam distillation using a Clevenger-type apparatus. Repellency bioassays of the oilswere carried out using a Y-shape olfactometer. The oils were found to be significantly repellent against S. zeamais. Percentage repellency increased with increasing concentration of the oils. Gas chromatography- mass spectrometry (GCMS) and GC co-injections with authentic samples indicated the presence of the following constituents: 1, 8-cineole, α-pinene, β-phellandrene, β-myrcene, γ-terpinene, caryophyllene and α-cardinol. Although synthetic chemicals are still widely used as repellents, natural products have the potential to provide efficient and safer repellents for humans and the environment. KEYWORDS: Eucalyptus camaldulensis, Eucalyptus globulus, essential oil, Sitophilus zeamais, maize, repellency. INTRODUCTION Maize (Zea mays) grain losses contribute to food insecurity and low farm incomes not only in Kenya, but also in Sub-Saharan African countries. The losses are directly measurable in economic, quantitative and qualitative (nutritional) terms. Such losses lead to lower levels of food security, hunger and low farm incomes (Komen et al, 2010). Food security in Sub Saharan Africa largely depends upon improved food productivity through the use of sustainable good agricultural practices (GAPs) and the reduction of postharvest losses caused by pests and diseases. For decades, the pest control policy in developing countries has been dependent upon the use of synthetic pesticides. Although synthetic pesticides are known to have undoubted benefits, their adoption rate and use for insect control in grain storage has remained remarkably low in resource-poor farming environments. The subsistence nature of agriculture in developing countries coupled with the high cost, poor information and erratic supply of synthetic pesticides have emerged as reasons for farmers reluctance to adopt pesticides. Further, there have been revelations that synthetic insecticides penetrate into stored grain and may be toxic and that pests have become more resistant to synthetic compounds. Therefore cheap and effective methods for reducing pest damage are required in these countries (Ogendo, et al 2004). Different types of aromatic plant preparations such as powders, solvent extracts, essential oils and whole plants have been investigated for their toxicity, repellence, anti feedant activity, insect growth regulatory activities against pests of agricultural importance (Prakash, 2008). * Corresponding author karimi.karemu@gmail.com

2 627 The losses due to the maize weevils can be more than one type: actual weight loss, which is due to the eating as well as opening of the grain to more drying; quality loss, which is a function of the percent grain bored and the amount of insect parts and excrement in the grain; nutritional loss due to insects heading preferentially to the high protein, vitamin containing germ; loss in seed viability in grain stored for planting. Much of this sums up as commercial loss and can be greater than all other losses when grain becomes no longer marketable. Thus lead to lowering levels of food security, hunger and low on farm incomes (Komen, et al. 2010). Previous research done using the various species of Eucalyptus has shown the repellency of the extracted oils. It has been determined that the essential oil of Eucalyptus presented insecticidal repellency against S. zeamais using the oils of E. dunii, E. saligna, E. benthamii, E. globulus and E.viminalis. About 100% of mortality was achieved with doses of 65, 100 and 400 µl for of E. dunii, E. saligna and E. benthamii respectively (Mossi et al, 2010).According to Renault-Rogers and her co-workers, the effects of essential oils depend on several things, including, most importantly their chemical composition. EXPERIMENTS AND METHODS Extraction of essential oils The leaves of Eucalyptus camaldulensis and Eucalyptus globulus were harvested and taken to the laboratory immediately to avoid loss of essential oils through evaporation. The fresh plant material was cut into small pieces and the essential oil extracted by steam distillation using a Clevenger-type apparatus. The steam distillate was collected every hour and extracted with hexane. This prevented possible decomposition of the oil due to heat and exposure to light. The combined oil and solvent extract were dried with anhydrous sodium sulphate. The solvent was then extracted using a microdistillation apparatus to give the essential oil. The oils were stored in sealed amber coloured vials at about 4 C. The relative yield (% w/w) of the essential oil from the Eucalyptus camaldulensis was 0.86% while that of Eucalyptus globulus was 0.61%. The oils optical rotation and refractive index were determined to ascertain the purity of the oil (Author, 1995). The results were as follows: Source of oil Refractive index at 20 C Optical rotation at 20 C E. globulus E. camaldulensis TABLE 1: Optical rotation and refractive index of the essential oils Repellency bioassay tests The maize weevils (Sitophilus zeamais) were obtained from a laboratory colony reared under ambient conditions (25±3 C, 70±5% humidity) on insecticide free maize grain. Four concentrations (0.1, 0.01, and μl/μl) were used for the bioassay. Twenty microliters of the diluted test material was taken for the S. zeamais bioassay The repellent activity of the essential oils against the maize weevils was assessed using a Y-shape olfactometer. It consists of a glass tube with a Y shape bifurcation where an insect walks and decides between two choices at the Y ends, the volatile oil (treated arm) and the standard (control arm). The olfactometer hence comprised of three compartments A, B and C connected by means of glass tubing

3 628 (10mm in diameter). The compartments were fitted with ground glass female joints D, E and F each fitted with a glass stopper of appropriate size. The stoppers have two narrow grooves which allowed airflow into the olfactometer during the bioassays. Maize weevils were introduced into compartment A and were induced to migrate to a choice of B or C. The test sample was applied onto the treated filter paper discs (Whatman No.1, 1.8cm in diameter) while hexane alone was applied onto the control filter paper discs. The solvent was allowed to evaporate and the resulting treated and control discs were then placed in one or other of the compartments B and C. Prior to introduction of the test material, air-suction was applied at the Y junction (G) by means of an aspirator pump, Barnant Air Cadet (model No ). This ensured that the olfactometer did not become saturated with the test material (volatile oil), which was confined to the treated arm of the olfactometer. All odorous air from the olfactometer to the aspirator pump was carried out away in a Tygon tube (10mm diameter) from the pump outlet. For each assay thirty randomly selected adult maize weevils of mixed sex and age were introduced into the olfactometer. Advantage was taken of the fact that weevils are negatively photo tactic by illuminating the introduction compartment A of the weevils into the olfactometer with light from a 60 watt bulb (I) placed 15cm away and screening the rest of the olfactometer in a paper carton (H). An average of 80-85% of the weevils was thus induced to migrate from compartment A towards B or C during the period of the bioassay (Author, 1995).All bioassays were conducted at 25±5 C and 70±5% humidity. The assay was left to run for thirty minutes and then the number of weevils in the control arm (NC) and the treated arm (NT) were counted. After each test the olfactometer was thoroughly cleaned and dried. The assay for each dose of the test material was replicated five times and the same for the standard N, N- diethyl-m-toluamide (DEET). Percentage repellency (PR) values were computed using the formula

4 629 PR = {(N C N T ) / (N C + N T )} 100 Analysis of the essential oils Gas Chromatography Mass spectrometry (GC-MS) GC MS was performed on an Agilent Technologies instrument Model 7890 A (GC System) and Model 5975 C inert XL Electron Impact/ Chemical Ionization MSD with triple axis detector (MS compartment). The instrument was equipped with a 30metre methyl silicone (HP-5ms capillary column) with an internal diameter of 250µm and a film thickness of 0.25µm. The helium carrier gas had a delivery rate of 4ml/min. The automatic sampling injector (Model 7683 B) operated in the splitless mode and the sample concentration was 250mg/ µl. The column temperature program was 40 C then raised at 10 C/min to 140 C and maintained for 15 minutes, then raised at 10 C/min to 280 C then maintained for 1 minute. For Mass Spectrometry the electron impact was 70eV. The mass analyzer used was Quadrupole Analyzer. The compounds were identified using the program Agilent 5975C MSD Data Analysis by their GC retention time and by comparison of the test compound mass spectra with the National Institute for Standard Technology (NIST), CHEMECOL and Adams2 mass spectral data bases. Gas Chromatography (GC) Analysis The identity of the constituents was established by comparison with retention times of the authentic samples and peak enhancement of the components by co-injection of the essential oil with the standards. GC analysis of the oils was performed using a Shimadzu Gas Chromatograph (Model GC-14B) fitted with a Flame Ionization Detector and a Shimadzu C-R8A integrator. The instrument was equipped with a 60metre carbowaxcolumn with an internal diameter of 0.25mm and a film thickness. The Nitrogen carrier gas had a flow rate of 2ml/min. the temperature was programmed from 70 C (25 minutes) then raised at 10 C/min every three minutes to 230 C and held for two minutes. Repellency Bioassay of the pure identified compounds. The compounds identified from the essential oils were then assayed for their repellent activity against S. zeamais. The data obtained was then analyzed as in repellency bioassay section above. RESULTS AND DISCUSSION Repellency effects of the essential oils on S.zeamais The mean repellency values of the essential oils at the four dose levels against S. zeamais are provided in Table 4. All the dosages were repellent to S. zeamais in a dose-response manner. Analysis of variance indicated significant differences (P < 0.05) between weevil reponses to the four dosages tested. The repellency of the essential oils was also compared to that of the standard DEET as indicated in Table 4. The oils had higher percentage repellency values than DEET. Essential oil composition The chemical composition of the oils were studied usinggas Chromatography (GC )and Gas Chromatography Mass Spectrometry (GC-MS). Constituents of the essential oils were identified by analysis of their mass spectra, by direct comparison of their mass spectra to the NIST, Adams2 and Chemecol libraries of mass spectra using computer routing and by co-injection with authentic samples in the GC. This procedure could be stated in three parts; a - Mass spectra analysis b - Retention time comparison c - Peak enhancement by co-injection The main constituent of the oil of Eucalyptus camaldulensis was 1, 8-cineole followed by α-pinene, β- pinene and 4-terpineol. Other constituents were α- sabinene, γ-terpinene and β-myrcene. TABLE 2: Chemical composition of the essential oil of Eucalyptus camaldulensis

5 630 E. camaldulensis oil Compound Retention time Relative % Method of identification α-pinene a, b, c β-phellandrene a β-myrcene a, b, c 2-carene a 1,8-cineole a, b, c γ-terpinene a terpinolene a α-terpineol a 4-terpineol a copaene a caryophyllene a isoledene a α-caryophyllene a epi-bicyclosesquiphellandrene a α-cardinol a Due to unavailability of standards, only a few constituents of the essential oils were identified in detail. Repellency effects of the pure identified compounds against S. zeamais The mean repellency values of the essential oils main constituents at the four dose levels against S. zeamais are provided in Table 4. All the doses were repellent to S. zeamais in a dose-response manner. The values are the mean repellency (%) ± Standard Error Margin of five replicates of 30 maize weevils each. The means within the same column followed by the same lower case letters are not significantly different (p<0.05, ANOVA and Tukey s tests). At the 0.002µl dose the oil of E. globulus and citral behaved as attractants, that is, there were more weevils in the test arm than the control arm of the olfactometer. During the data analysis this was treated as negative repellency (or attractance). The formula: PR = {(N C N T ) / (N C + N T )} 100 was reversed to become PR = {(N T N C ) / (N C + N T )} 100 The transformed mean thus calculated for the dose was called attractance hence the negative sign indicating negative repellence. The essential oils had higher repellent activity than the standard DEET at all the doses except for the oil of E. globulus at the 0.02μl and at the 0.002µl dose where it behaved as an attractant. From the data obtained it was possible to conclude that the different dosages (DOSE) have a definite significant effect (P < 0.05) on the percent repellency of the different compounds (REPEL) based on the test statistic computed by the Tukey s tests.

6 631 TABLE 4:Repellent activity of the essential oil of Eucalyptus camaldulensis, Eucalyptus globulus, their constituents and the standard DEET against Sitophilus zeamais Compound / oil α pinene Dose μl/disc ±8.75 a,b,c,d 22.45±5.10 b 11.03±3.27 a 17.54±7.04a β pinene 25.17±10.66 c,d 32.87±5.87 a,b 28.50±8.25 a 28.65±7.48 a 1,8 cineole 70.87±2.64 a,b 58.29±9.92 a 29.00±4.0 a 24.08±10.58 a linalool 63.63±5.95 a,b 26.08±8.79 b 14.27±5.40 a 6.60±5.40 a citral 25.63± 7.14 c,d ±7.76 b 18.58± 8.85 a DEET 25.00± 0 c,d 20.00± 0 b 6.7± 0 a 4.00± 0 a E. camaldulensis 74.35±6.29a 41.97±11.40a,b 12.12±3.42 a 10.51±3.80 a E. globulus 53.68± a,b,c 32.09± 4.35 a,b 5.55 ±1.84 a citral ± 0.0 E. globulus ± 0.0 DISCUSSION α pinene has been shown to possess repellent activity against many insects, such as American cockroaches, Periplaneta Americana, mosquitoes and the common tick (Liu et al, 2011). Alpha pinene is emitted in substantial amounts by vegetation, and these emissions are affected by temperature and light intensity. It has been shown to show moderate repellency against S. zeamais in this study. 1, 8 cineole has also been known to show strong repellent activity against several stored product insects such as the grain weevil Sitophilus granarius, T. castaneumand Protephanus truncates (Liu et al,2011). In this study it has been shown to have high repellent activity, with the highest value among the identified constituents of the eucalyptus oils. The strong repellent activity of 1, 8 cineole explains the high repellent activity of the oil of E. camaldulensis and considerable repellency of E. globulus oil against S. zeamaisin which it was the major constituent. The oils also contained α- pinene and linalool which could have also contributed to their high repellent activity.linalool also showed significant repellent activity against S. zeamais as revealed by this study. Linalool has been used in some mosquito repellent products and has been revealed to demonstrate repellency against mosquito larvae when used alone (Tiwaryet al, 2007). CONCLUSION In this study, the repellent activity of the oil of Eucalyptus camaldulensis was tested and shown to exhibit the higher repellency effect against the maize weevil, Sitophilus zeamais. The main chemical constituents were also tested against S. zeamais and showed appreciable repellent activity. Hence this oil constitutes a viable alternative for the control of the maize weevil, the major storage pest of the maize grain.

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