Annals. Food Science and Technology 2009 ADULTICIDAL ACTIVITY AND TOXICITY OF EXTRACTIVES FROM TECLEA TRICHOCARPA AGAINST ADULT MAIZE WEEVIL (SITOPHILUS ZEAMAIS) ERASTUS S. KAMAU MWANGIa*, JOSEPH M. KERIKOa, ALEX K. MACHOCHOb, SUMESH.C. CHHABRAb, ALPHOSE W. WANYONYIb and PAUL K.TARUSc a Department of Chemistry, Jomo Kenyatta University of Agriculture and Technology, P.O Box 62000 Nairobi, Kenya b Department of Chemistry, Kenyatta University, P.O. Box 43844 00100, Nairobi, Kenya. c Department of Chemistry and Biochemistry, Chepkoilel University College (Moi University). P.O.Box 1125 30100 Eldoret, Kenya. Corresponding author:eskmwangi@yahoo.com Abstract With a growing world population and increased affluence leading to demand for more and higher quality foods, and given environmental problems such as soil degradation, water scarcity, and biodiversity loss, new and innovative solutions are required to minimize food losses caused by pests. Organic solvent extracts and thereof isolated compounds of Teclea trichocarpa Eng. were evaluated for adulticidal activity against maize weevil, Sitophilus zeamais Motchulsky, and for brine shrimp, Artemia salina, lethality. Hexane extract of the leaves of T. trichocarpa displayed mild brine shrimp toxicity (LD50 =153.2 g/ml), while the other extracts showed no significant toxicity (LD50 240 g/ml). Both hexane and dichloromethane extracts of leaves of T. trichocarpa showed dose dependent mean percentage adulticidal activity. At 600 and 800 ppm these extracts, respectively, were comparable to the positive control, actellic super, a synthetic pesticide which is in the market today. Considering the cost, increasing incidence of pesticide resistance and environmental concerns posed by synthetic pesticides, several pressures have accelerated the search for more environmentally and toxicologically safe, more selective and efficacious pesticides. Results discussed with regard to the use of the plant extractives as suitable and sustainable alternative to synthetic insecticide in maize grain storage and could be incorporated in integrated pest management. Keywords: brine shrimp; Teclea trichocarpa; adulticidal activity; maize weevil, Sitophilus zeamais post-harvest pests. However, the persistence, resistance, the cost and availability of these conventional insecticide and potential health hazard both to the consumers and to the environment have necessitated continued use of local plant products. Traditional methods involves admixture with local plant materials as repellents, sunning and use of wood ash (Mutambuki et al., 1989). Although these botanicals have been in use since time immemorial their efficacy, safety and their active principles deserve more attention (Balandrin et al., 1985). Plants have been screened for repellency, and antifeedant against maize weevil, and various classes of the natural products been identified to be responsible for the activity such as terpenoids, flavonoids, flavones, alkaloids and essential oils. (Hassanali and Lwande, 1989; Hassanali et al., 1990; Lwande et al., 1. INTRODUCTION Efficient production of good quality food grains is a big challenge to mankind. A variety of techniques have been applied to meet the challenge. One of the aspects is to improve efficiency in grain production and post harvest practices to ensure that food losses are minimized if not eliminated and that the grains produced is of good quality and safe for human consumption. Tropical countries suffer severe losses of stored food products due to pests. This is partly attributed to conducive climatic conditions. Apart from other causes of food losses like crop diseases and weeds, pre- and post-harvest pests are responsible for ~40% of Africa’s food losses (Mandava, 1985). Prophylactic methods have not constrained the pests to acceptable levels. Synthetic pesticides have been used against Available on-line at www.afst.valahia.ro 1 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 1983, Ndungu et al., 1999 and Bekele et al., 1996). light (254 and 366 nm), and by anisaldehyde and dragendorff’s visualization reagents. VLC column were packed with thin layer chromatography silica gel 60 (6-35 microns mesh, ASTM) and column chromatography on silica gel 60 (0.040-0.063 mm 230-400 mesh, Merck). Solvents were laboratory grade and were obtained from BDH, Nairobi and were double distilled before use. Teclea trichocarpa is reported to be used by traditional healers belonging to the Akamba tribe for malaria treatment and as anthelmintic, while the Giriama tribe of Kenya steam the leaves and inhale the vapour as a cure for fever (Watt and BreyerBrandwijk, 1962). The plant bark has been shown to have antifeedant activity against the African armyworm, Spodoptera exempta (Lwande et al., 1983). The leaves were reported to possess antiprotozoa activities against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi and Leishmania donovani. (Muriithi et al., 2002; Mwangi et al., 2010). The leaves and stem bark of T. trichocarpa is also traditionally used to control maize weevil by Keiyo community living in the Rift Valley, Kenya. This study aimed at evaluating the pesticidal activity and toxicity of extractives from T. trichocarpa against adult maize weevil (Sitophilus zeamais) and a strategy of improving food security in the communities. Plant Materials The leaves of T. trichocarpa (Rutaceae) (2.0 kg) were collected from Siroch also in Keiyo District in rift Valley, Kenya. The samples of T. trichocarpa were authenticated by a taxonomist at the National Museums of Kenya in Nairobi, Kenya and given voucher specimen number SKM/JKUAT/002/006. The leaves were dried in the shade, and ground into powdered material using a grinding mill (Christy and Norris Ltd, England). The powdered plant material were hermetically sealed in polythene bags and stored in a refrigerator at 4oC in the dark until the time of extraction. 1.2.2 Extraction, Isolation 1.2 MATERIALS AND METHODS Melting points were determined on an electro thermal melting point apparatus and expressed in degree centigrade (oC) and were uncorrected. IR spectra were taken in KBr pellets and recorded on a Shimazdu (model FT-IR-8400 CE) with absorption given in wave numbers (cm-1). NMR spectra were recorded on a Bruker DPX- 400 NMR. The spectra were recorded in CDCl3 as the solvent and TMS as the internal standard. The chemical shifts reported in  (ppm) units relative to TMS signal. TLC was performed on aluminium sheets pre-coated with silica gel 60 F254 (Merck) with a 0.2 mm layer thickness, Preparative TLC was done using normal phase silica gel (F254 Merck) precoated on aluminium plate (20 x 20 cm) and a layer thickness of 0.25 mm. Spots on chromatograms were examined under UV Available on-line at www.afst.valahia.ro Fractionation and The air-dried, powdered leaves of T. trichocarpa (2.0 kg), were extracted sequentially with 7.5 litres each of hexane, dichloromethane (CH2Cl2) ethyl acetate and methanol exhaustively at room temperature. Each extract was concentrated under reduced pressure at 45oC. The yields and percentage yields of the extracts are presented in Table 1. The extracts were screened for toxicity using brine shrimp. Leaves of T. trichocarpa yielded a yellow paste of hexane extract (25.0 g) and a green paste of dichloromethane extracts (48.5 g). These were subjected to vacuum liquid chromatography (VLC) separation on silica gel 60 each at a time, eluted with n-hexane with increasing amount of CH2Cl2 and later increasing amount of methanol in CH2Cl2 up 2 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 to 1:5.Fifty five and 65 fractions were collected, respectively, from TLC analysis similar fractions pooled together. UV active spots on TLC were considered for further separation. From the hexane extract of T. trichocarpa, fraction (31-37) 2128.9 mg that eluted with n-hexane: CH2Cl2 (1:4) was further chromatographed on sephadex and eluted with a mixture of CH2Cl2 and Methanol (1:1) to give 32 sub-fractions, sub fraction 31-32 on crystallization in methanol afforded -amyrin [6] (32.7 mg). Fractions 34-38 (8034.1 mg) that eluted with 2:3 (nHexane: CH2Cl2) was loaded onto VLC and eluted with hexane and increasing amount of CH2Cl2 and then increasing amount of methanol. Twenty-eight sub fractions were obtained from which fraction 13-21 was further chromatographed on silica gel and eluted with 2:3 (n-Hexane: CH2Cl2) this yielded -sitosterol [5]. 1.2.3 Toxicity Testing Against the Brine Shrimp The hatching brine shrimp eggs, Artemia salina leach were hatched in artificial seawater prepared by dissolving 38 g of sea salt (Sigma chemicals Co., UK) in 1 litre of distilled water. After 48 hrs incubation at room temperature (25oC), the larvae (nauplii) were attracted to one side of the vessel with a light source and collected with pipette. Nauplii were separated from eggs by aliquoting them three times in small beakers containing seawater. The bioactivity of the extracts was monitored by the brine shrimp lethality test (Meyer et al., 1982). Samples were dissolved in dimethylsulphoxide (DMSO) and diluted with artificial sea salt water so that final concentration of DMSO did not exceed 0.05%. Fifty microlitres of sea salt water was placed in all the 96-well microtitre plates. Fifty microlitres of 4000 ppm of the plant extract was placed in the row one and a twofold dilution carried out down the column. The last row left with sea salt water and DMSO only served as the drug free control. Hundred microlitres of suspension of nauplii containing 10 larvae was added into each well and incubated for 24 h. the plates were then examined under a microscope (12.5X and the number of dead napulii in each well counted and recorded. Lethality concentrations fifties (LC50 values) for each assay were calculated by taking average of three experiments using a Finney Probit analysis program on an IBM computer (McLaughlin et al., 1991). From VLC of the CH2Cl2 extract, fraction 3440 (1014 mg) was loaded onto sephadex column and eluted with CH2Cl2: methanol (1:1) to give 16 sub fractions. Sub fraction 34 was subjected to column chromatography and eluted with ethyl acetate: CH2Cl2 (1:3). This afforded 38.1 mg of melicopicine [1]. Sub Fraction 5-18 showed UV active spots, column chromatography of this fraction eluted with CH2Cl2: ethyl acetate (2:1) mixture gave 25 fractions from which sub fractions 9-11, 12-15 and 21-25 were further subjected to chromatographic separation. Sub fractions 9-11 were subjected to preparative thin layer chromatography, this afforded skimmianine [4] (22.2 mg), sub fractions 2125 was subjected to preparative thin layer chromatography. This afforded two compounds Melicopicine [1] (64.8 mg), and normelicopicine [2] (46.9 mg). Fraction 41-42 from VLC was subjected to column chromatography and eluted with ethyl acetate: CH2Cl2 (1:9). The sub fraction (5-8) that eluted with CH2Cl2: ethyl acetate (1:1) afforded yellow needle like compound, arborinine [3] (99.0 mg) on partitioning between methanol and CH2Cl2 (2:1). Available on-line at www.afst.valahia.ro 1.2.4 Sitophilus zeamais Culture Adult Sitophilus zeamais was obtained from a laboratory colony reared under ambient conditions with natural photoperiods on untreated (insecticide-free) whole maize grains obtained and maintained at National Agricultural Research Laboratories (NARL), Nairobi, Kenya. One hundred S. zeamais of mixed sexes were introduced into 2 litre glass 3 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 separated by using Tukey’s studentised range (HSD) test. jars containing 400 g weevil susceptible maize grains following the methods of Bekele and Hassanali (2001). The mouths of the jars were then covered with nylon mesh held in place with rubber bands. Freshly emerged adults of S. zeamais were then used for the experiments (Asawalam and Emosairue, 2006). 1.3 RESULTS AND DISCUSSION The potential of using the T. trichocarpa extracts and the constituent components of extracts as protectant for stored maize grains against maize weevil, and toxicity against brine shrimp, were the main objectives for this study. On extraction with various organic solvents the yields were as shown in Table 1. The percentage yields of hexane extract was lower than the other extracts, with thrice and twelve fold percentage yield of DCM and methanol extracts, respectively. These extracts were subjected to brine shrimp lethality test and adulticidal test against maize weevil before embarking on fractionation of the crude extract. 1.2.4.1 Adulticidal Assessments Bioassay tests were carried in the laboratory to determine the efficacy of the botanicals under different dosage levels against S. zeamais. Three doses of each plant extracts, were used as treatment to assess adulticidal activity against maize weevil. For pure compounds and blend mixtures the concentrations were double, equal and half that of positive control (Actellic super). A synthetic insecticide Actellic super 2% dust at 0.05 % w/w and untreated maize grains were included as positive and negative controls, respectively. Table 1. Percentage yield of T. trichocarpa organic extracts Extract Yields (g) Percentage yields (%) The test samples were mixed with talc thoroughly and the dust were admixed with 50 g of maize held in jam jars covered with ventilated lids. To ensure a thorough admixture, the grain was put in plastic jam jar, dust applied and top lid replaced. The grain was then swirled within the jar until a proper admixture was realized. Twenty, 5-day old S. zeamais adults were introduced into treated and untreated maize grains and confined by perforated lids placed over muslin cloth that was held in place by a rubber band. The design of the experiment was Completely Randomized Design (CRD) with three replications. The treatments were kept on at room temperature for seven days before mortality was assessed. Percentage mean mortality for S. zeamais was recorded after seven days exposure period as described by Bekele et al., (1996). CH2Cl2 52.4 2.6 EtOAc 41.6 2.1 MeOH 199.2 10 1.3.1 The Toxicity Assay The hexane, dichloromethane, ethyl acetate and methanol crude extracts of T. trichocarpa were tested for their toxicity against brine shrimp lethality assay. The results are shown in Table 2. The hexane extracts of T. trichocarpa leaves with LD50 values of 153.2 g/ml was considered active, while CH2Cl2, EtOAc and MeOH extracts showed mild toxicity against brine shrimp (Table 2). Since a crude sample is considered active up to a concentration of 240 g/ml (Meyer et al., 1982); and brine shrimp test is an indicator of toxicity, various pharmacological actions, and pesticidal effects (Meyer et al., 1982), it was deduced that both hexane and CH2Cl2 extracts of T. trichocarpa had greater potential as insecticide. Data were subjected to analysis of variance (ANOVA) procedure (SAS, 2000) and significantly different (P>0.05) means were Available on-line at www.afst.valahia.ro hexane 26.0 1.3 4 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 Table 2. The mean LD50 values  s.d. for the T. trichocarpa leaves organic crude extracts screened against brine shrimp (Artemia salina, leach). Plants extract Hexane extract DCM ( CH2Cl2) extract EtOAc extract MeOH extract activity at almost all doses being hexane extracts showing 75% adulticidal at a concentration of 200 ppm and 100% adulticidal from 600 ppm, at which was comparable to the positive control at 95% confidence level; at 200 ppm, actellic super, a synthetic pesticide at the recommended rate of 0.05%, which is in the market today. LD50 153.2 ± 1.0 279.9 ± 0.7 416.1± 0.9 567.8 ±1.8 1.3.2 Adulticidal Screening The fact that, the crude extracts at high concentration had significant mean percentage adulticidal against maize weevil is interesting and led support to the traditional use of this plant material as grain protectant against destructive pests. Both extracts represents an attractive candidate for field evaluation as a protectant of stored maize. It is also expected that, the crude plant extract could offer suitable and sustainable alternative to synthetic pesticide. However, conclusive recommendation of their use can only be made after exhaustive analysis of the effect of the crude on the quality of grain and safety. From the adulticidal assay against maize weevil (S. zeamais) the methanol and ethyl acetate extracts showed no activity. The crude extracts (hexane and dichloromethane) of T. trichocarpa were therefore subjected to further adulticidal test against maize weevil (S. zeamais). The effects of different doses of hexane and CH2Cl2 extracts on maize weevil after seven days were determined and LD50 values computed and the results are summarized in Table 3. Table 3. Percent mortality of adult S. zeamais on maize grains treated with different concentrations of hexane and CH2Cl2 crude extracts from T. trichocarpa leaves against maize weevil (S. zeamais). Plants extract Hexane DCM, (CH2Cl2) EtOAc Actellic super Negative control 100 ppm 25.0 ± 5.0 b 25.0 ± 5.0 b 25.0 ± 5.0 b 75.0 ± 5.0 a 200 ppm 75.0 ± 5.0 b 400 ppm 75.0 ± 5.0 b 45.0 ± 0.0 d 600 ppm 100.0 ± 0.0 a 70.0± 10.0 b 800 ppm 100.0 ± 0.0 a 95.0 ± 5.0 a 40.0 ± 0.0 d 50.0 ± 0.0 d 60.0 ± 5.0 c 70.0± 10.0 b 87.0 ± 5.0 a 100.0 100.0 ± 0.0 ± 0.0 a a 5.0 ± 5.0 c 100.0 ± 0.0 a 100.0 ± 0.0 a Adoption of these natural plant products could improve efficiency in post-harvest practices as a strategy of providing people with sufficient and healthy food in an ecologically sustainable manner. Being natural, protectants from plant materials would be easily degraded by biological factors, and cases of pollution and poisoning would be reduced. Improving grain storage would mean less hunger, improved nutrition for mankind, a higher standard of living and a sounder economy for the nation. Examining Tables 2 and 3, the brine shrimp lethality and adulticidal activity results for the crude extracts, respectively, the hexane extracts of T. trichocarpa showed higher toxicity as well as adulticidal activity against maize. It was evident that toxicity against brine shrimp may be a basis of deducing an active adulticidal extract, similarly blending hexane and CH2Cl2 crude extract lowered activity showing antagonistic effect. For this Key: Mean values with the same letters within the same column are not significantly different at 95% confidence level (Tukey’s studentized test). From the results in Table 3, it is evident that adulticidal activities are dose dependent for both organic extracts. The most active extracts, with the highest mean adulticidal Available on-line at www.afst.valahia.ro 5 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 reason, hexane and CH2Cl2 crude extract was fractionated and pure compounds isolated. included as the negative control. Results are summarized in Table 4. From the results in Table 4, the mean percentage adulticidal was dose dependent. However, all the compounds showed low activities at both 0.1 and 0.05 w/w, being between 10% to 22% at 0.1% w/w when compared to actellic super. The adulticidal activity of the three-acridone alkaloids melicopicine [1], normelicopicine [2] and arborinine [3] were noted to be low with the mortality being between 10% to 22% at 0.1% w/w of the compound. Comparing the two terpenoids, 3–sitosterol [5] showed higher activity (12.5±2.5) than -amyrin [6] (5.0±0.0) at 0.05 w/w and were significantly different (p < 0.05). Although the two compounds share a common biosynthetic pathway, the difference in activity may be attributed to their structural difference. – sitosterol has also been reported to show weak feeding inhibitory activities against the larvae of Chilo partellus 26 (Tsanuo, 1992). This compound could be a better protectant against destructive pests due to its feeding inhibitory and adulticidal activities. The TLC profile of T. trichocarpa revealed the presence of several UV active and fluorescing compounds in the crude extracts. Chromatographic separation of the hexane and dichloromethane extracts afforded two terpenoids (-amyrin and –sitosterol) and four alkaloids; melicopicine, arborinine, normelicopicine (acridone alkaloids) and skimmianine (furoquinoline alkaloid). The structures of the compounds were characterized and identified by their IR., 1H NMR and 13C NMR, and comparing with data of authentic samples -amyrin (Mahato and Kundu, 1994), arborinine (Bergenthal et al., 1979), melicopicine (Rasoanaivo et al., 1999), normelicopicine (Muriithi et al., 2002), skimmianine and –sitosterol (Knight, 1974) . O R1 OCH3 R2 N R3 H3CO N CH3 R4 O OCH3 1 R1, R2,R3, R4 = OCH3 Melicopicine 4 Skimmianine Table 4: Mean percentage adulticidal  s.d. of isolated compounds from T. trichocarpa against maize weevil. 2 R1 = OH, R2, R3,R4 = OCH3 Normelicopicine 3 R1 = OH, R2, R3 = OCH3, R4 = H Arborinine Compounds HO Melicopicine [1] Normelicopicine [2] Arborinine [3] Skimmianine [4] β–Sitosterol [5] α–Amyrin [6] Actellic super Negative control HO 5. 3–sitosterol 6. -amyrin The six compounds thus isolated from hexane and CH2Cl2 extracts were tested against maize weevil (adulticidal) at different doses. Actellic super, a synthetic insecticide, was used as positive control and no treatment was Available on-line at www.afst.valahia.ro Mean percentage adulticidal at different concentration in w/w 0.1 w/w 0.05 w/w 12.5 ± 2.5 c 2.5 ± 2.5 c 15.0 ± 0.0 a 7.5 ± 2.5 ac 22.5 ± 2.5 a 10.0 ± 0.0 a 17.5 ± 2.5 a 7.5 ± 2.5 ac 20.0 ± 0.0 a 12.5 ± 2.5 a 10.0 ± 5.0 ac 5.0 ± 0.0 c 95.0 ± 0.0 b 87.5 ± 2.5 b 5.0 ± 0.0 c Key: Mean values with the same letters within the same column are not significantly different at 95% confidence The isolated compounds were less active than the crude extracts, from which they were 6 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 of -amyrin and arborinine did not show significant change in activity at high concentration but at 0.05% w/w there was increased activity, implying synergism is in play. isolated, an indication of possible loss of synergism in the isolation process. In order to ascertain these observations, pure isolated compounds were blended in the same ratio and subjected to adulticidal test. The adulticidal assay results at different dosage of thereof blended mixture of isolated compounds; actellic super (positive control) and drug free (negative control) are summarized in Table 5. Although all the test mixtures used were in the ratio of 1: 1, their occurrence in the crude extracts of the plant is not in these ratios hence their effects could differ. Similarly, the isolated compounds were not the only compounds present in the crude extracts as evidenced from TLC analysis and therefore, it is evident adulticidal activity is caused by additive effect of most constituent components with different levels of activity. Table 5: Mean percentage adulticidal  s.d. of the blended compounds from Teclea trichocarpa against maize weevil. Compounds Skimmianine/ Arborinine -Amyrin/ Normelicopicine Arborinine /Melicopicine -Amyrin/ Arborinine 3–sitosterol/ Arborinine Actellic super Negative control Mean percentage adulticidal at different concentration in w/w 0.1 w/w 0.05 w/w 20.0 ± 0.0 c 12.5 ± 2.5 a 17.5 ± 2.5 a 20.0 ± 0.0 a 22.5 ± 2.5 c 17.5 ± 2.5 a 1.4 CONCLUSION The study has shown that hexane and CH2Cl2 extracts of T. trichocarpa displayed higher toxicity against brine shrimp as well as adulticidal activity against maize weevil. The results provide a scientific rationale for the use of T. trichocarpa in post-harvest protection. There is, therefore, a good promise to use of this botanical pesticide as alternative to the synthetic pesticide, Actellic super 2% dust. 10.0 ± 5.0 a 15.0 ± 5.0 a 17.5 ± 2.5 a 12.5 ± 2.5 a 95.0 ± 0.0 b 87.5 ± 2.5 b 5.0 ± 0.0 c 2. ACKNOWLEDGEMENTS Key: Mean values with the same letters within the same column are not significantly different at 95% confidence level (Tukey’s studentized test). The authors are grateful to AICAD for financially sponsoring this research (in part) under the contract number AICAD/RD06/FPP/03-017 AICAD research fund. The Staff at National Agricultural Research Laboratories (NARL) and Botany and Chemistry Department of JKUAT for their guidance during adulticidal activity and lethality tests, respectively. Thanks to Mr. Malebo, for running the NMR spectra. From the results in Table 5, it is evident that the adulticidal activities are concentration dependent. However, comparing these results with those presented in Table 4, mixture of amyrin/ normelicopicine, and skimmianine/ arborinine, at higher concentration showed higher activity than corresponding pure compounds, implying some synergism. Whether this implies, a mixture of terpenoids and alkaloids or different types of alkaloids are more effective remains to be investigated. Arborinine/normelicopicine and –sitosterol/ arborinine mixtures showed lower activity than corresponding pure compounds, implying there was loss of activity (antagonist). Mixture Available on-line at www.afst.valahia.ro 3. REFERENCES [1] Asawalam, E.F. and Emosairue, S.O. (2006). Comparative Efficacy of Piper guineense (Schum And Thonn) and Pirimiphos Methyl on Sitophilus Zeamais (Motsch.). Tropical and Subtropical Agroecosystems, 6: 143-148. 7 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 research laboratories annual report, Nairobi. pp.53-56. [2] Balandrin, M.F., Klocke, A.J., Wurtele, S.E. and Bollinger, H.W. (1985). Natural plant source of industrial and medicinal materials. Science 228: 1154-1160. [12] Mahato, S. B. and Kundu, A. P. (1994). Review articles number 98, 13C NMR spectra of pentacyclic triterpenoids a compilation and some salient features. Phytochemistry 37: 1517 - 1575. [3] Bergernthal, D., Mester, J. and Rozsa, Z. (1979). Spektren einiger Acridon-Alkaloide. Phytochemistry 18: 161 - 163. [13] McLaughlin, J.L., Chang, C.J., Smith, D.L., 1991. “Bench-top” bioassays for the discovery of bioactive natural products: an update. Study of Natural Product Chemistry 9: 383-409. [4] Bekele, A.J. and Hassanali, A. (2001). Blend effects in the toxicity of the essential oil constituents of Ocimum kilmandscharicum and Ocimum kenyense (Labiateae) on two Post-harvest insect pests. Phytochemistry 57: 385-391. [14] Meyer, B.N., Ferrgn, N.R., Jacobsen, L.B., Nicholas, D.E. and Mc Laughlin; J.L. (1982). A convenient general bioassay for active plant constituents. Planta Medica 45: 31. [5] Bekele, A.J., Obeng-Ofori, D. and Hassanali, A. (1996). Evaluation of Ocimum suave (wild) as a source of repellents, toxicant and protectants in storage against three stored product insect pests. International journal of pest management, 42: 139-142. [15] Muriithi, M.W., Abraham, W.R., AddaeKyeme, J., Scowen, I., Croft, S.L., Gitu, P.M., Kendrick, H., Njagi, E.M. and Wright C.W. (2002). Isolation and in-vitro antiplasmodial activities of alkaloid from Teclea trichocarpa, in vivo antimalarial activity and x-ray crystal structure of normelicopicine. Journal of Natural Products 65: 7: 956-959. [6] Hassanali, A. and Lwande, W. (1989). Antipest secondary metabolites from African plants. In: Arnason, J.T, Philogene, B.J.R. and Morands, P. (eds). Insecticides of plant origin; ACs symposium series No. 387, American chemical society, Washington D. C; pp. 78-94. [16] Mwangi, E.S.K., Keriko, J.M., Machocho, A.K., Wanyonyi, A.W., Malebo, H.M., Chabbra, S.C. and Tarus, P.K., (2010). Antiprotozoal activity and cytotoxicity of metabolites from leaves of Teclea trichocarpa. Journal of Medicinal Plants 4(9), pp. 726-731 [7] Hassanali, A., Lwande, W., Ole-Sitayo, N., Moreka, L., Nokoe, S. and Chapya, A. (1990). Weevil repellent constituents of Ocimum suave leaves and Eugenia caryophylata cloves used as grain protectants in parts of East Africa, Discovery and Innovation 2: 91 – 95. [17] Ndungu, W.M., Chhabra, S.C. and Lwande, W., (1999). Cleome hirta essential oils livestock and maize weevil repellent, Elsevier science B.V.. Fitoterapia 70: 514 – 516. [8] Knight, S.A. (1974). Carbon-13 NMR spectra of some tetra- and pentacyclic triterpenoids. Org. Magn. Res 6: 603 - 611. [18] Rasoanaivo, P., Federici., Palazzino, G. and Galeffi, C. (1999). Acridone of Vepris sclerophylla: their 13C NMR data. Fitoterapia 70: 625 - 627. [9] Lwande, W., Gebregesus, T., Chapya, A., Macfoy, C., Hassanali, A. and Okech, M., (1983). 9-Acridone insect antifeedant alkaloids from T. trichocarpa bark. Insects Science and its application 4: 393 – 395. [19] SAS Institute 2000. Statistical Analytical Systems SAS/STAT User's guide version 8 (2) Cary NC: SAS Institute Inc. [10] Mandava N.B. (1985). Handbook of natural pesticides; methods, theory, practice and detection. CRD Boca Raton, F lorida. pp. 534. [20] Tsanuo, M.K. (1992). Bioassay guided isolation and structural elucidation of anti-feeding compounds against Chilo partellus (Swinhoe) in fruits of Elaeodendron buchananii L. MSc. Thesis, Kenyatta University. [11] Mutabuki, K., Wekesa, P.W., Koech, S., Mbugua, J.N. an Kibata, G.N. (1989) An assessment of effectiveness of various insecticidal dusts on the control of the large grain borer Prostephanus truncates (Horn) and other major stored product pests. In: National agricultural Available on-line at www.afst.valahia.ro [21] Watt, J.M., Breyer-Brandwijk, M.G. (1962) The Medicinal and Poisonous Plants of Southern 8 Volume 10, Issue 1, 2009 Annals. Food Science and Technology 2009 and Eastern Africa; 2nd ed. E & S. Livingstone Ltd., Edinburg; pp.923 Available on-line at www.afst.valahia.ro 9 Volume 10, Issue 1, 2009