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Insecticidal bioactivity of crude extracts of Aglaia species (meliaceae)
( ~ Pergamon
BiochemicalSystematicsand Ecology,Vol. 22, No. 2, pp. 121-127, 1994 Copyright© 1994ElsevierScienceLtd Printed in GreatBritain.All rights reserved 0305-1978/94 $6.00+0.00
Insecticidal Bioactivity of Crude Extracts of Aglaia Species (Meliaceae) C. SATASOOK,*t M. B. ISMAN,*$ F. ISHIBASHI,*§ S. MEDBURY,IIP. WlRIYACHITRA¶ and G. H. N. TOWERS* *Departments of Plant Science and Botany, University of British Columbia, Vancouver, B.C., Canada, V6T 1Z4; IIHonolulu Botanical Gardens, 50 North Vineyard Boulevard, Honolulu, HI 96817, U.S.A.; ¶Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50002, Thailand
Key Word Index--Aglaia; Peridroma saucia; screening; natural insecticides. Abstract--Crude foliar extracts of 19 species of Aglaia (Meliaceae), mostly of Indo-Malaysian origin, were screened for larval growth inhibiting and insecticidal effects on the polyphagous lepidopteran Peridroma saucia (Noctuidae). Extracts of at least seven of these species significantly reduce larval growth of P. saucia. Aglaia odorata yielded the most inhibitory extracts, but there is significant (35-fold) geographical variation in the bioactivity of extracts within this widespread species. In addition, extracts of bark are significantly more active than foliar extracts. Foliar extracts significantly deter neonate larvae, but nutritional analyses of fourth instar larvae fed artificial diets laced with A. odorata extracts indicate that both diet consumption and dietary utilization are impaired. These results are similar to that obtained with pure (-)-rocaglamide, an insecticidal principle isolated from A. odorata. This species should provide a useful starting point for the development of a botanical insecticide.
Introduction In the last two decades considerable effort has been devoted to the discovery of new sources of botanical insecticide and antifeedants. Among plant families studied, the Meliaceae and Rutaceae are perhaps the most promising (Schoonhoven, 1982), at least partly owing to the presence of limonoids, triterpenes characteristic of the order Rutales. The biological activity of limonoids from the Rutales has recently been reviewed (Champagne et al., 1992). Azadirachtin, a limonoid from the neem tree (Azadirachta indica) is well known for its antifeeding and growth disruption activities against various insect pests (Champagne et al., 1992). Some neem-based botanical insecticides have already been developed and marketed (Schmutterer, 1990). Screening for feeding deterrency and growth inhibitory effects of extracts from other members of the Meliaceae against a number of insect pests revealed that extracts from some species of Aglaia possessed promising bioactivity (Chiu, 1985; Mikolajczak and Reed, 1987; Champagne et aL, 1989). These reports suggest the potential of Aglaia as a source for natural pesticides and provided the impetus for a systematic investigation of the genus for bioactive taxa. About 130 species of Aglaia are found in Indo-Malaysia, South China and the Pacific Islands. They exist as dioecious trees, bushes or shrubs with minute or small fragrant flowers (Ridley, 1922; Li, 1977). Aglaia odorata is the type species and probably a native of Southeast Asia (Backer and van den Brink, 1965; Pennington and Styles, 1975). The present paper reports studies on screening the bioactivities of crude extracts from 19 Aglaia species against a polyphagous lepidopteran, the variegated cutworm, Peridroma saucia (HL~bner). tPresent address: Faculty of Science, Prince of Songkla University, Hat Yai 90112, Thailand. $Author to whom reprint requests and correspondence should be addressed. §Present address: Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan. (Received 23 June 1993) 121
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Materials and Methods Plant extracts. Forty-two samples of 19 species of Aglaia collected from various parts of Asia and Hawaii (Table 1) were studied. Samples collected at the Kebun Raya Indonesia (Bogor Botanical Garden, Bogor, Indonesia) were from living specimens in their collection. Samples collected in Thailand are represented by voucher specimens in the Herbarium of Chiang Mai University, Chiang Mai, Thailand. Samples of A. chittagonga and A. luzoniensis are represented by voucher specimens held by Professor N. R. Farnsworth, College of Pharmacy, University of Illinois at Chicago. For the majority of species examined, only a singte specimen was sampled and tested, and therefore there is no replication within these species. Ground dried leaves were extracted with methanol, and the methanolic extracts were used for screening experiments. In the case of A. odorata, acetone extracts were used for further detailed study as acetone was found to yield greater bioactivity (unpublished data). Insects. The variegated cutworms, R saucia, used in the present study were taken from a laboratory culture maintained for several years on artificial diet (F9795, Bioserv, Inc., Frenchtown, N J) at 27+1°C and 16L:8D. Screening o f plant extracts. Foliar methanolic extracts of Aglaia species were mixed with the dry portion of the artificial diet at a concentration of 10 mg g 1 dry wt. diet resulting in a final screening concentration of 0.2% fresh wt. (2000 ppm). The carrier solvent was evaporated overnight in a fume hood; control diet was treated with the carrier solvent (methanol) alone. The artificial diet used in this study was prepared by the method of Isman and Rodriguez (1983). Upon hatching, two neonate R saucia were placed on 1 g fresh wt. diet in an individual cell of an injection molded plastic tray. Twenty replicates were set up for each plant extract. These plastic trays were kept in a plastic box lined with moistened paper to maintain humidity. The experiment was carried out in a growth chamber at 25±1°C and 16L:8D. The number of insects in each cell was thinned to one neonate on day 3 of the experiment. Larval growth was assessed as a percentage of the controls after 7 days, based on larval weights. Analysis of variance (ANOVA) was performed on the basis of the actual numbers observed if the variances of the sample means were tested to be homogeneous, and a multiple range test for differences between means was done using Tukey's test (Zar, 1984). Aglaia odorata, the most potent species based on the initial screening, was selected for further study aimed at identifying the plant tissue with the greatest bioactivity. Crude acetone extracts from leaves, barks, wood and flowers of A. odorata were prepared. Plant extracts were mixed into artificial diet at concentrations of 5.0 and 10.0 mg g ~ dry wt. as described in the previous experiment. Experimental procedures and assessment were otherwise identical to those of the screening experiment.
TABLE 1. AGLAIA SPECIESTESTED AND COUNTRY OF COLLECTION Aglaia species*
Country of collection
A. argentea BI
Kebun Raya Indonesia (-- Bogon Philippines; nora. excL iloNo (Blanco) Merr. Thailand Philippines; syn. diffusa Merr Bogor; syn. latifolia Miq. Bogor Bogor; syn. formosana Hayata Bogor Bogor; syn. harmsiana Perkins Thailand Thailand Bogor Bogor Bogor; syn. eusideroxylon Koord. & Valet. Philippines Bogor, China, Thailand, Hawaii Bogor, Thailand Thailand Thailand; syn. barbatula Koord. & Valet. India; syn. maiae Bourdillon Philippines; syn. Ilanosiana C.DC. Bogor Bogor Bogor; syn. tufa Miq.
A. chittagonga Miq. ,4. edufis (Roxb.) Wall. A. elaeagnoidea (A. Juss.) Bentham A. e/hptica BI. A. eximia Miq. A. forbes//King A. glabrata Teijsm. & Binn. A. grandis Korth. ex Miq. A. lawii(Wight) Saldenha ex. Ramamoorthy A. luzoniensls (Vidal) Merril et Rolfe A. odorata Lour. A. odoratissima BI. A. oligophy/la Miq. A. pachyphylla Miq. .4. perviridis Hiern A. rimosa (Blanco) Merrill A. smithfi Koord. A. tomentosa Teijsm. & Binn.
*Specific names according to Dr C. M. Pannell, Oxford University (personal communication). Dr Pannell did not examine actual voucher specimens of the material investigated.
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Feeding deterrence. The most potent foliar extracts selected from the screening experiment were bioassayed to establish whether they specifically deterred larval feeding. Artificial diets containing extract at 5 mg g-1 dry wt. were prepared as in the previous experiment. A choice test was performed in a small Petri dish (5 cm dia.) lined with moistened filter paper. The arena was divided into equal quadrats, each quadrat containing approximately 1 g of fresh control and treated diet placed alternately. Ten newly hatched neonate R saucia were put in the centre of the arena, with five replicates for each extract. The experimental dishes were kept in a plastic box maintained in complete darkness for 24 h. The number of neonates on control or treated diet and/or in each quadrat were then compared using a Chi-square test (Zar, 1984). Nutritional analysis experiment. This experiment was carried out using fourth-instar P. saucia larvae (40+5 mg). For each concentration, 20 larvae were provided individually with crude acetone extract of A. odorata twigs at dietary concentrations of 0, 300 and 600 ppm prepared as previously described. All nutritional indices were calculated on the basis of dry wts, and therefore, at the outset of the experiment, 10 diet samples (ca 0.5 g each) and 20 individual larvae were taken and dried to constant weight at 60°C to determine fresh wt/dry wt. ratios. Insect larvae were allowed to feed for 3 days before dry wts of larvae, remaining diet, and frass were recorded. Nutritional indices were calculated as previously described (Farrar et al., 1989): relative growth rate (RGRi)= mg of biomass gained per mg of initial larval biomass per day; relative consumption rate (RCRi) =mg of biomass ingested per mg of initial larval biomass per day; approximate digestibility (AD) = (food ingested--feces)/(food ingested)×100; efficiency of conversion of ingested food (ECI)= (biomass gained)/(food ingested)×100; efficiency of conversion of digested food (ECD)=(biomass gained)/(food ingested--feces)×100. Data were analyzed by ANOVA with means separation determined using Tukey's test (Zar, 1984).
Results
Screening bioassays Of the 42 extracts screened in the initial experiment, 16 were highly inhibitory to larval growth of neonate R saucia (reducing growth by at least two-thirds compared to controls, Table 2). These extracts represent seven of the 19 species examined. Foliar extracts from six of the species reduced larval growth by more than 90% relative to controls at the screening concentration (2000 ppm). The most bioactive extracts are those from foliage of A. odorata and A. oligophylla. However, A. odorata appears to be highly variable with respect to its potency against R saucia. Material collected in southern Thailand was almost 10-fold more inhibitory than that collected in northern Thailand, and potency of extracts from foliage of two A. odorata trees growing just meters apart in Hawaii differed by more than 35-fold (Table 3). Similarly, there are significant differences in the potency of extracts obtained from different tissues of A. odorata (Table 4). When acetone extracts were bioassayed at equivalent concentrations, those from bark were the most potent, followed in order of descending potency by those of flowers, foliage and wood.
Diet choice test Extracts of three of the most potent species (A. odorata, A. oligophylla and A. pervl'ridis) significantly deterred feeding of neonate R saucia larvae when added to artificial diets at 1000 ppm in a binary choice test (Table 5). Nutritional experiment When incorporated into artificial diet at 300 ppm, the crude acetone extract of A. odorata twigs reduced the relative growth rate by more than 75%, and the relative consumption rate by 65%, compared to controls (Table 6). Indices of dietary utilization (ECI, ECD) were reduced by approximately one-third compared to controls, but the differences were not statistically significant. At a dietary concentration of 600 ppm, the twig extract reduced consumption rate by over 90%, and larvae lost biomass over the 3-day course of the experiment (Table 6). Discussion We have examined less than one-sixth of the approximately 130 currently recognized species in the genus Aglaia, yet over one-third of the taxa we screened are highly
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TABLE 2. GROWTH INHIBITORY EFFECT OF AGLAIA EXTRACTS ON NEONATE PERIDROMA SAUCIA (10 mg g ~ dry wt. diet, n = 20)
Aglaia species* A. glabrata A. grandis A. Ilanosiana A. diffusa A. tufa A. latifolia A. harmsiana A. elliptica A. chittagonga A. diffusa A. Ilanosiana A. forbesii A. tomentosa A. eximia A. luzoniensis A. glabrata A, barbitu#~ A, odoratlssJrna A. odoratlssima A, odoratlssm~a A. ellipticc~ A. argentea A. smithfi A. harrnslana A. odorata A. elaegno/dea A, elaegnoidea A. odorata A. elaegnoidea ,4. formosana A. lat/folia A. eusidoxylorl A. odorata A. 17oilo A. odorata A. maiae A. iloilo A. odorata A. ohgophy/la A. odorata
Larval growth (% relative to control) mean+_S.E. 149.3-+10.3 at 139.9+_7.8 ab 123.5_+8.1 abc 118.6_+10.4 abcd 116.9+7.8 abcde 114.6_+6.8 bcdef 112.5+_5.2 bcdefg 105.6-+ 11.3 bcdefgh 103,3+6,8 bcdefgh 100.1+4.5 cdefghi 97.8_+8.5 cdefghi] 97.0_+5.5 cdefghij 94.7-+11.4 cdefghlj 87.6_+4,2 defghij 86.1 ±3.6 defghl) 85.4;7.8 defgh# 83,6+4.2 efghil 82.0_+4.4 fghl/k 78.2:,'-6.9 ghJ/k 76.3-+6.3 hljk 69.9_+4.6 ~k 66.8+6.5 #~ 52.6_+2.6 jkl 45.4_+5.1 kl 35.6+3.7 /m 31.2_+1.9 /m 28.9_+.2.7/m 27.9+1.1 /m 25.9_+ 1.8 m 21.14:1.6 tn 10.4+1 0 m 8.8+0.7 m 8,0:;0.8 rn 5.7+0.4 ,'n 5.6_+0.3 m 5.5±0.4 m 4.5+05 m 3.2+0.3 m 2.3+0.2 nl 1.0+0.1 m
*Original specific n a m e s at the time/site of coFlection. Synonymy and correct nomenclature indicated in Table 1. Where a specific name is listed more than once, extracts were collected from specimens in different countries or locations within a country. tMeans followed by the same letters are not significantly different at the 5% level
bioactive against R saucia larvae. These results suggest that the probability of uncovering additional species in the genus with insecticidal properties is high, and additional investigation along this line is clearly warranted. In fact, our study may even underestimate the number of species with potent bioactivity from among the taxa examined. In many cases we were only able to obtain plant material from single specimens, whereas where several individuals were examined, as in the case of A. odorata, a wide range in bioactivity was observed within a species. In an investigation of the insecticidal action of extracts from 80 trees representing three species of Zanthozylum (Rutaceae), Marr and Tang (1992) found
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TABLE 3. COMPARATIVE POTENCY OF FOLIAR A. ODORATA METHANOLIC EXTRACTS COLLECTED FROM DIFFERENT LOCATIONS ON GROWTH OF NEONATE R SAUCIA (10 mg g-1 dry wt. diet, n=20)
Location
Larval growth (% relative to control) mean+S.E.
Hawaii Northern Thailand China Maui Southern Thailand Hawaii
35.6±3.7 27.9±1.1 8.0±0.8 5.6±0.3 3.2±0.3 1.0±0.1
TABLE 4. EFFECT OF CRUDE ACETONE EXTRACTS FROM DIFFERENT PARTS OF A. ODORATA INCORPORATED INTO ARTIFICIAL DIET ON GROWTH OF NEONATE P. SAUCIA AFTER 7 DAYS OF FEEDING (n = 20)
Crude extract
Larval growth (% relative to control) mean+S.E. 5.0 mg g 1 dry wt. 10.0 mg g-1 dry wt.
Bark Flower Leaf Wood
3.6±0.5 a* 5.9±0.5 b 11.9_+0.7 c 30.8±2.7 d
0.9±0.1 2.9±0.3 3.8±0.3 129±0.9
a b c d
*Means within a column followed by the same letter are not significantly different (P>0.05) based on Tukey's test.
TABLE 5. EFFECT OF SOME CRUDE AGLAIA EXTRACTS (5 mg g 1 dry wt.) ON DIET CHOICE BY NEONATE P. SAUCIA (n= 50)
Species
Percentage of neonates on control (C) or treated (T) diet C T
A. odorata A. maiae A. oligophylla
72 70 76
38* 30** 24**
*Significantly different (7.2, P<0.01) **Significantly different (X2, P
TABLE 6. FEEDING, GROWTH AND DIETARY UTILIZATION BY FOURTH-INSTAR P. SAUCIA FED ARTIFICIAL DIET CONTAINING CRUDE ACETONE EXTRACT OF A. ODORATA(n= 20) Concentration in diet (ppm) 0.0 300 600
Nutritional index (mean_+S.E.) RGRi RCR~ (rag mg -1 day -1) (rag m9 -1 day 1)
AD (%)
ECI (%)
ECD (%)
1.41 a* +0.09 0.33 b ±0.04 --0.03 c ±0.01
46.24 a ±2.55 59.63 a ±3.38 58.68 a ±5.51
29.08 a ±1.40 18.74 a ±1.82 --7.40 b ±2.71
69,79 a +7.79 44,01 a ±12.46 --26.36 b ±17.38
4.95 a +0.32 1.69 b +0.14 0.43 c ±0.02
*Means within a column followed by the same letter are not significantly different (P>0.05), based on Tokey's test.
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wide intraspecific variation in both secondary chemistry and toxicity in their bioassay. Their study underscores the need to examine several individuals of a taxon to properly characterize its biological activity. Unfortunately, as in the case of the present study, it is not always possible to collect material from different individuals of the same species, particularly in tropical forests where species diversity is high and many suitable habitats of a species are not readily accessible. In the case of A. odorata, from which a botanical insecticide is under development in Thailand, it was essential to first select an elite tree from which clonal propagation could yield a large number of individuals suitable for harvest. As many of the taxa reported in our study are represented only by single specimens, caution should be exercised in extrapolating from our results. Other individual trees from species which we report as having significant bioactivity could easily be far less active. However, we feel at the least that our preliminary results point to certain species of Aglaia which may be better leads than others for more detailed explorations for insecticidal principles. When screening for bioactivity, it is desirable to obtain as many different parts of the plant as possible for extraction. Our examination of A. odorata indicates that the stembark and twigs are the most potent parts of the plant. In our parallel investigation of Trichilia (Meliaceae) species, we found that extracts of wood and bark were consistently more bioactive than those of foliage (Xie et al., 1994). Therefore, in collecting additional species of Aglaia, it might be advisable to focus on collecting bark and stems, rather than foliage. Bioassay-driven fractionation of A. odorata twigs led to the isolation of (-)-rocaglamide, a highly-substituted benzofuran, as the insecticidal principle (Janprasert et al., 1993). Further phytochemical investigation of A. odorata foliage led to the isolation of three additional analogues of rocaglamide, all of which are insecticidal (Ishibashi et al., 1993). Rocaglamide is a potent, but slow-acting toxin against R saucia, requiring at least 4 days for full toxicity to be expressed (Satasook et aL, 1992). Although rocaglamide has a direct antifeedant effect on P. saucia larvae, this action is insufficient to account for the complete bioactivity of the compound. In the present study, extracts of A. odorata, A. perviridis and A. oligophylla were found to deter feeding by neonate larvae (Table 5). Such a behavioural effect probably contributes substantially to the inhibition of larval growth observed when larvae are fed continuously on artificial diets containing extracts of these, and other Aglaia species. However, we believe that the extreme inhibition of larval growth observed is likely more a consequence of physiological toxicity. When added to artificial diet at a concentration of 300 ppm, a crude acetone extract of A. odorata twigs significantly reduced the relative consumption rate of fourth instar R saucia larvae, concomitant with a reduction in the relative growth rate (Table 6). A similar result was obtained using pure rocaglamide at a dietary concentration of 1.5 ppm (Satasook et al., 1992). Although these results would seem to implicate an antifeedant effect as the major cause of growth suppression, topical application of pure rocaglamide produced the same result, precluding the mouthpart chemosensilla as the primary site-of-action and therefore peripherally-mediated feeding deterrence as the primary mode-of-action. Instead, the rapid reduction in feeding following topical administration of rocaglamide points to a centrally-mediated anorexic effect (Satasook et al., 1992). This rapid cessation of feeding of larvae exposed either orally or by contact, should prove a valuable asset for a botanical insecticide based on A. odorata. In this regard, A. odorata is comparable to botanical insecticides derived from neem, Azadirachta indica, which have both antifeedant and insect growth-regulating effects on pests. Isolation and chemical identification of active compounds and further biological study in other promising Aglaia species are also being investigated.
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Acknowledgements--Wethank
Nancy Brard and Yanfen Zheng for technical assistance, Dr Y. S. Xie for reading the manuscript and Dr C. M. Pannell for correcting our taxonomy. Supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada to M. B. Isman and G. H. N. Towers, and a grant from the International Development Research Centre of Canada to P. Wiriyachitra.
References Backer, C. A. and van den Brink, R. C. B. (1965) Flora of Java. pp. 126-129. N. V. P. Noordhoff-Groningen, The Netherlands. Champagne, D. E., Isman, M. B. and Towers, G. H. N. (1989) Insecticidal activity of phytochemicals and extracts of the Meliaceae. In Insecticides of Plant Origin (Arnason, J. T., Philogene, B. J. R. and Morand, P., eds), pp. 95-109, ACS Symposium Series 387, 95-109. Champagne, D. E., Koul, O., Isman, M. B., Scudder, G. G. E. and Towers, G. H. N. (1992) Biological activity of limonoids from the Rutales. Phytochemistry 31, 377-394. Chiu, S.-E (1985). Recent research findings on Meliaceae and other promising botanical insecticides in China. J. Plant Disease Protect. 92, 310-319. Farrar, R. R., Jr., Barbour, J. D. and Kennedy, G. G. (1989) Quantifying food consumption and growth in insects. Ann. Entomol. Soc. Am. 82, 593-598. Ishibashi, F., Satasook, C., Isman, M. B. and Towers, G. H. N. (1993) Insecticidal 1H-Cyclopentatetrahydro [b] benzofurans from Aglaia odorata (Lour.)(Meliaceae). Phytochemistry 32, 307-310. Isman, M. B. and Rodriguez, E. (1983) Larval growth inhibitors from species of Parthenium (Asteraceae). Phytochemistry 22, 2709-2713. Janprasert, J., Satasook, C., Sukumalanand, P., Champagne, D. E., Isman, M B., Wiriyachitra, P. and Towers, G. H. N. (1993) Rocaglamide, a new natural insecticide from Aglaia odorata (Lour.)(Family Meliaceae). Phytochemistry 32, 67-69. Li, H. L. (1977) Flora of Taiwan, pp. 544-545. Epoch Publishing Co, Ltd. Taipei, Taiwan. Marr, K. L. and Tang, C. S. (1992) Volatile insecticidal compounds and chemical variability of Hawaiian Zanthoxylum (Rutaceae) species. Biochem. Syst. Ecol. 20, 209-217. Mikolajczak, K. L. and Reed, D. K. (1987). Extractives of seeds of the Meliaceae: Effects on Spodoptera frugipera (J. F. Smith), Acalymma vittatum (F.), and Artemia salina Leach, J. Chem. Ecol. 13, 99-111. Pennington, T. D. and Styles, B. 1". (1975) A generic monograph of the Meliaceae. Blumea 22, 480-482. Ridiey, H. N. (1922) The Flora of the Malay Peninsula, pp. 401-410. L. Reeve and Co. Ltd., London. Satasook, C., Isman, M. B. and Wiriyachitra, P. (1992) Toxicity of rocaglamide, an insecticidal natural product, to the variegated cutworm, Peridroma saucia (Lepidoptera: Noctuidae). Pestic. ScL 36, 53-58. Schmutterer, H. (1990) Properties and potential of natural pesticides from the neem tree, Azadirachta indica. A. Rev. Entomol. 35, 271-297. Schoonhoven, L. M. (1982) Biological aspects of antifeedants. Entomol. Exp. Appl. 31, 57-69. Xie, Y. S., Gunning, P., MacKinnon, S., Isman, M. B., Hasbun, C., Sanchez, P., Arnason, J. T., Towers, G. H. N. and Taylor, D. R. (1994) Biological activity of crude extracts of Trich#ia species and the limonoid hirtin against lepidopteran larvae. Biochem. Syst. Ecol. 22, 129-136. Zar, J. H. (1984) BiostatisEcalAnalysis, pp. 168-190. Prentice-Hall Inc., Englewood Cliffs, NJ.
BiochemicalSystematicsand Ecology,Vol. 22, No. 2, pp. 121-127, 1994 Copyright© 1994ElsevierScienceLtd Printed in GreatBritain.All rights reserved 0305-1978/94 $6.00+0.00
Insecticidal Bioactivity of Crude Extracts of Aglaia Species (Meliaceae) C. SATASOOK,*t M. B. ISMAN,*$ F. ISHIBASHI,*§ S. MEDBURY,IIP. WlRIYACHITRA¶ and G. H. N. TOWERS* *Departments of Plant Science and Botany, University of British Columbia, Vancouver, B.C., Canada, V6T 1Z4; IIHonolulu Botanical Gardens, 50 North Vineyard Boulevard, Honolulu, HI 96817, U.S.A.; ¶Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50002, Thailand
Key Word Index--Aglaia; Peridroma saucia; screening; natural insecticides. Abstract--Crude foliar extracts of 19 species of Aglaia (Meliaceae), mostly of Indo-Malaysian origin, were screened for larval growth inhibiting and insecticidal effects on the polyphagous lepidopteran Peridroma saucia (Noctuidae). Extracts of at least seven of these species significantly reduce larval growth of P. saucia. Aglaia odorata yielded the most inhibitory extracts, but there is significant (35-fold) geographical variation in the bioactivity of extracts within this widespread species. In addition, extracts of bark are significantly more active than foliar extracts. Foliar extracts significantly deter neonate larvae, but nutritional analyses of fourth instar larvae fed artificial diets laced with A. odorata extracts indicate that both diet consumption and dietary utilization are impaired. These results are similar to that obtained with pure (-)-rocaglamide, an insecticidal principle isolated from A. odorata. This species should provide a useful starting point for the development of a botanical insecticide.
Introduction In the last two decades considerable effort has been devoted to the discovery of new sources of botanical insecticide and antifeedants. Among plant families studied, the Meliaceae and Rutaceae are perhaps the most promising (Schoonhoven, 1982), at least partly owing to the presence of limonoids, triterpenes characteristic of the order Rutales. The biological activity of limonoids from the Rutales has recently been reviewed (Champagne et al., 1992). Azadirachtin, a limonoid from the neem tree (Azadirachta indica) is well known for its antifeeding and growth disruption activities against various insect pests (Champagne et al., 1992). Some neem-based botanical insecticides have already been developed and marketed (Schmutterer, 1990). Screening for feeding deterrency and growth inhibitory effects of extracts from other members of the Meliaceae against a number of insect pests revealed that extracts from some species of Aglaia possessed promising bioactivity (Chiu, 1985; Mikolajczak and Reed, 1987; Champagne et aL, 1989). These reports suggest the potential of Aglaia as a source for natural pesticides and provided the impetus for a systematic investigation of the genus for bioactive taxa. About 130 species of Aglaia are found in Indo-Malaysia, South China and the Pacific Islands. They exist as dioecious trees, bushes or shrubs with minute or small fragrant flowers (Ridley, 1922; Li, 1977). Aglaia odorata is the type species and probably a native of Southeast Asia (Backer and van den Brink, 1965; Pennington and Styles, 1975). The present paper reports studies on screening the bioactivities of crude extracts from 19 Aglaia species against a polyphagous lepidopteran, the variegated cutworm, Peridroma saucia (HL~bner). tPresent address: Faculty of Science, Prince of Songkla University, Hat Yai 90112, Thailand. $Author to whom reprint requests and correspondence should be addressed. §Present address: Faculty of Fisheries, Nagasaki University, Nagasaki 852, Japan. (Received 23 June 1993) 121
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Materials and Methods Plant extracts. Forty-two samples of 19 species of Aglaia collected from various parts of Asia and Hawaii (Table 1) were studied. Samples collected at the Kebun Raya Indonesia (Bogor Botanical Garden, Bogor, Indonesia) were from living specimens in their collection. Samples collected in Thailand are represented by voucher specimens in the Herbarium of Chiang Mai University, Chiang Mai, Thailand. Samples of A. chittagonga and A. luzoniensis are represented by voucher specimens held by Professor N. R. Farnsworth, College of Pharmacy, University of Illinois at Chicago. For the majority of species examined, only a singte specimen was sampled and tested, and therefore there is no replication within these species. Ground dried leaves were extracted with methanol, and the methanolic extracts were used for screening experiments. In the case of A. odorata, acetone extracts were used for further detailed study as acetone was found to yield greater bioactivity (unpublished data). Insects. The variegated cutworms, R saucia, used in the present study were taken from a laboratory culture maintained for several years on artificial diet (F9795, Bioserv, Inc., Frenchtown, N J) at 27+1°C and 16L:8D. Screening o f plant extracts. Foliar methanolic extracts of Aglaia species were mixed with the dry portion of the artificial diet at a concentration of 10 mg g 1 dry wt. diet resulting in a final screening concentration of 0.2% fresh wt. (2000 ppm). The carrier solvent was evaporated overnight in a fume hood; control diet was treated with the carrier solvent (methanol) alone. The artificial diet used in this study was prepared by the method of Isman and Rodriguez (1983). Upon hatching, two neonate R saucia were placed on 1 g fresh wt. diet in an individual cell of an injection molded plastic tray. Twenty replicates were set up for each plant extract. These plastic trays were kept in a plastic box lined with moistened paper to maintain humidity. The experiment was carried out in a growth chamber at 25±1°C and 16L:8D. The number of insects in each cell was thinned to one neonate on day 3 of the experiment. Larval growth was assessed as a percentage of the controls after 7 days, based on larval weights. Analysis of variance (ANOVA) was performed on the basis of the actual numbers observed if the variances of the sample means were tested to be homogeneous, and a multiple range test for differences between means was done using Tukey's test (Zar, 1984). Aglaia odorata, the most potent species based on the initial screening, was selected for further study aimed at identifying the plant tissue with the greatest bioactivity. Crude acetone extracts from leaves, barks, wood and flowers of A. odorata were prepared. Plant extracts were mixed into artificial diet at concentrations of 5.0 and 10.0 mg g ~ dry wt. as described in the previous experiment. Experimental procedures and assessment were otherwise identical to those of the screening experiment.
TABLE 1. AGLAIA SPECIESTESTED AND COUNTRY OF COLLECTION Aglaia species*
Country of collection
A. argentea BI
Kebun Raya Indonesia (-- Bogon Philippines; nora. excL iloNo (Blanco) Merr. Thailand Philippines; syn. diffusa Merr Bogor; syn. latifolia Miq. Bogor Bogor; syn. formosana Hayata Bogor Bogor; syn. harmsiana Perkins Thailand Thailand Bogor Bogor Bogor; syn. eusideroxylon Koord. & Valet. Philippines Bogor, China, Thailand, Hawaii Bogor, Thailand Thailand Thailand; syn. barbatula Koord. & Valet. India; syn. maiae Bourdillon Philippines; syn. Ilanosiana C.DC. Bogor Bogor Bogor; syn. tufa Miq.
A. chittagonga Miq. ,4. edufis (Roxb.) Wall. A. elaeagnoidea (A. Juss.) Bentham A. e/hptica BI. A. eximia Miq. A. forbes//King A. glabrata Teijsm. & Binn. A. grandis Korth. ex Miq. A. lawii(Wight) Saldenha ex. Ramamoorthy A. luzoniensls (Vidal) Merril et Rolfe A. odorata Lour. A. odoratissima BI. A. oligophy/la Miq. A. pachyphylla Miq. .4. perviridis Hiern A. rimosa (Blanco) Merrill A. smithfi Koord. A. tomentosa Teijsm. & Binn.
*Specific names according to Dr C. M. Pannell, Oxford University (personal communication). Dr Pannell did not examine actual voucher specimens of the material investigated.
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Feeding deterrence. The most potent foliar extracts selected from the screening experiment were bioassayed to establish whether they specifically deterred larval feeding. Artificial diets containing extract at 5 mg g-1 dry wt. were prepared as in the previous experiment. A choice test was performed in a small Petri dish (5 cm dia.) lined with moistened filter paper. The arena was divided into equal quadrats, each quadrat containing approximately 1 g of fresh control and treated diet placed alternately. Ten newly hatched neonate R saucia were put in the centre of the arena, with five replicates for each extract. The experimental dishes were kept in a plastic box maintained in complete darkness for 24 h. The number of neonates on control or treated diet and/or in each quadrat were then compared using a Chi-square test (Zar, 1984). Nutritional analysis experiment. This experiment was carried out using fourth-instar P. saucia larvae (40+5 mg). For each concentration, 20 larvae were provided individually with crude acetone extract of A. odorata twigs at dietary concentrations of 0, 300 and 600 ppm prepared as previously described. All nutritional indices were calculated on the basis of dry wts, and therefore, at the outset of the experiment, 10 diet samples (ca 0.5 g each) and 20 individual larvae were taken and dried to constant weight at 60°C to determine fresh wt/dry wt. ratios. Insect larvae were allowed to feed for 3 days before dry wts of larvae, remaining diet, and frass were recorded. Nutritional indices were calculated as previously described (Farrar et al., 1989): relative growth rate (RGRi)= mg of biomass gained per mg of initial larval biomass per day; relative consumption rate (RCRi) =mg of biomass ingested per mg of initial larval biomass per day; approximate digestibility (AD) = (food ingested--feces)/(food ingested)×100; efficiency of conversion of ingested food (ECI)= (biomass gained)/(food ingested)×100; efficiency of conversion of digested food (ECD)=(biomass gained)/(food ingested--feces)×100. Data were analyzed by ANOVA with means separation determined using Tukey's test (Zar, 1984).
Results
Screening bioassays Of the 42 extracts screened in the initial experiment, 16 were highly inhibitory to larval growth of neonate R saucia (reducing growth by at least two-thirds compared to controls, Table 2). These extracts represent seven of the 19 species examined. Foliar extracts from six of the species reduced larval growth by more than 90% relative to controls at the screening concentration (2000 ppm). The most bioactive extracts are those from foliage of A. odorata and A. oligophylla. However, A. odorata appears to be highly variable with respect to its potency against R saucia. Material collected in southern Thailand was almost 10-fold more inhibitory than that collected in northern Thailand, and potency of extracts from foliage of two A. odorata trees growing just meters apart in Hawaii differed by more than 35-fold (Table 3). Similarly, there are significant differences in the potency of extracts obtained from different tissues of A. odorata (Table 4). When acetone extracts were bioassayed at equivalent concentrations, those from bark were the most potent, followed in order of descending potency by those of flowers, foliage and wood.
Diet choice test Extracts of three of the most potent species (A. odorata, A. oligophylla and A. pervl'ridis) significantly deterred feeding of neonate R saucia larvae when added to artificial diets at 1000 ppm in a binary choice test (Table 5). Nutritional experiment When incorporated into artificial diet at 300 ppm, the crude acetone extract of A. odorata twigs reduced the relative growth rate by more than 75%, and the relative consumption rate by 65%, compared to controls (Table 6). Indices of dietary utilization (ECI, ECD) were reduced by approximately one-third compared to controls, but the differences were not statistically significant. At a dietary concentration of 600 ppm, the twig extract reduced consumption rate by over 90%, and larvae lost biomass over the 3-day course of the experiment (Table 6). Discussion We have examined less than one-sixth of the approximately 130 currently recognized species in the genus Aglaia, yet over one-third of the taxa we screened are highly
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TABLE 2. GROWTH INHIBITORY EFFECT OF AGLAIA EXTRACTS ON NEONATE PERIDROMA SAUCIA (10 mg g ~ dry wt. diet, n = 20)
Aglaia species* A. glabrata A. grandis A. Ilanosiana A. diffusa A. tufa A. latifolia A. harmsiana A. elliptica A. chittagonga A. diffusa A. Ilanosiana A. forbesii A. tomentosa A. eximia A. luzoniensis A. glabrata A, barbitu#~ A, odoratlssJrna A. odoratlssima A, odoratlssm~a A. ellipticc~ A. argentea A. smithfi A. harrnslana A. odorata A. elaegno/dea A, elaegnoidea A. odorata A. elaegnoidea ,4. formosana A. lat/folia A. eusidoxylorl A. odorata A. 17oilo A. odorata A. maiae A. iloilo A. odorata A. ohgophy/la A. odorata
Larval growth (% relative to control) mean+_S.E. 149.3-+10.3 at 139.9+_7.8 ab 123.5_+8.1 abc 118.6_+10.4 abcd 116.9+7.8 abcde 114.6_+6.8 bcdef 112.5+_5.2 bcdefg 105.6-+ 11.3 bcdefgh 103,3+6,8 bcdefgh 100.1+4.5 cdefghi 97.8_+8.5 cdefghi] 97.0_+5.5 cdefghij 94.7-+11.4 cdefghlj 87.6_+4,2 defghij 86.1 ±3.6 defghl) 85.4;7.8 defgh# 83,6+4.2 efghil 82.0_+4.4 fghl/k 78.2:,'-6.9 ghJ/k 76.3-+6.3 hljk 69.9_+4.6 ~k 66.8+6.5 #~ 52.6_+2.6 jkl 45.4_+5.1 kl 35.6+3.7 /m 31.2_+1.9 /m 28.9_+.2.7/m 27.9+1.1 /m 25.9_+ 1.8 m 21.14:1.6 tn 10.4+1 0 m 8.8+0.7 m 8,0:;0.8 rn 5.7+0.4 ,'n 5.6_+0.3 m 5.5±0.4 m 4.5+05 m 3.2+0.3 m 2.3+0.2 nl 1.0+0.1 m
*Original specific n a m e s at the time/site of coFlection. Synonymy and correct nomenclature indicated in Table 1. Where a specific name is listed more than once, extracts were collected from specimens in different countries or locations within a country. tMeans followed by the same letters are not significantly different at the 5% level
bioactive against R saucia larvae. These results suggest that the probability of uncovering additional species in the genus with insecticidal properties is high, and additional investigation along this line is clearly warranted. In fact, our study may even underestimate the number of species with potent bioactivity from among the taxa examined. In many cases we were only able to obtain plant material from single specimens, whereas where several individuals were examined, as in the case of A. odorata, a wide range in bioactivity was observed within a species. In an investigation of the insecticidal action of extracts from 80 trees representing three species of Zanthozylum (Rutaceae), Marr and Tang (1992) found
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TABLE 3. COMPARATIVE POTENCY OF FOLIAR A. ODORATA METHANOLIC EXTRACTS COLLECTED FROM DIFFERENT LOCATIONS ON GROWTH OF NEONATE R SAUCIA (10 mg g-1 dry wt. diet, n=20)
Location
Larval growth (% relative to control) mean+S.E.
Hawaii Northern Thailand China Maui Southern Thailand Hawaii
35.6±3.7 27.9±1.1 8.0±0.8 5.6±0.3 3.2±0.3 1.0±0.1
TABLE 4. EFFECT OF CRUDE ACETONE EXTRACTS FROM DIFFERENT PARTS OF A. ODORATA INCORPORATED INTO ARTIFICIAL DIET ON GROWTH OF NEONATE P. SAUCIA AFTER 7 DAYS OF FEEDING (n = 20)
Crude extract
Larval growth (% relative to control) mean+S.E. 5.0 mg g 1 dry wt. 10.0 mg g-1 dry wt.
Bark Flower Leaf Wood
3.6±0.5 a* 5.9±0.5 b 11.9_+0.7 c 30.8±2.7 d
0.9±0.1 2.9±0.3 3.8±0.3 129±0.9
a b c d
*Means within a column followed by the same letter are not significantly different (P>0.05) based on Tukey's test.
TABLE 5. EFFECT OF SOME CRUDE AGLAIA EXTRACTS (5 mg g 1 dry wt.) ON DIET CHOICE BY NEONATE P. SAUCIA (n= 50)
Species
Percentage of neonates on control (C) or treated (T) diet C T
A. odorata A. maiae A. oligophylla
72 70 76
38* 30** 24**
*Significantly different (7.2, P<0.01) **Significantly different (X2, P
TABLE 6. FEEDING, GROWTH AND DIETARY UTILIZATION BY FOURTH-INSTAR P. SAUCIA FED ARTIFICIAL DIET CONTAINING CRUDE ACETONE EXTRACT OF A. ODORATA(n= 20) Concentration in diet (ppm) 0.0 300 600
Nutritional index (mean_+S.E.) RGRi RCR~ (rag mg -1 day -1) (rag m9 -1 day 1)
AD (%)
ECI (%)
ECD (%)
1.41 a* +0.09 0.33 b ±0.04 --0.03 c ±0.01
46.24 a ±2.55 59.63 a ±3.38 58.68 a ±5.51
29.08 a ±1.40 18.74 a ±1.82 --7.40 b ±2.71
69,79 a +7.79 44,01 a ±12.46 --26.36 b ±17.38
4.95 a +0.32 1.69 b +0.14 0.43 c ±0.02
*Means within a column followed by the same letter are not significantly different (P>0.05), based on Tokey's test.
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wide intraspecific variation in both secondary chemistry and toxicity in their bioassay. Their study underscores the need to examine several individuals of a taxon to properly characterize its biological activity. Unfortunately, as in the case of the present study, it is not always possible to collect material from different individuals of the same species, particularly in tropical forests where species diversity is high and many suitable habitats of a species are not readily accessible. In the case of A. odorata, from which a botanical insecticide is under development in Thailand, it was essential to first select an elite tree from which clonal propagation could yield a large number of individuals suitable for harvest. As many of the taxa reported in our study are represented only by single specimens, caution should be exercised in extrapolating from our results. Other individual trees from species which we report as having significant bioactivity could easily be far less active. However, we feel at the least that our preliminary results point to certain species of Aglaia which may be better leads than others for more detailed explorations for insecticidal principles. When screening for bioactivity, it is desirable to obtain as many different parts of the plant as possible for extraction. Our examination of A. odorata indicates that the stembark and twigs are the most potent parts of the plant. In our parallel investigation of Trichilia (Meliaceae) species, we found that extracts of wood and bark were consistently more bioactive than those of foliage (Xie et al., 1994). Therefore, in collecting additional species of Aglaia, it might be advisable to focus on collecting bark and stems, rather than foliage. Bioassay-driven fractionation of A. odorata twigs led to the isolation of (-)-rocaglamide, a highly-substituted benzofuran, as the insecticidal principle (Janprasert et al., 1993). Further phytochemical investigation of A. odorata foliage led to the isolation of three additional analogues of rocaglamide, all of which are insecticidal (Ishibashi et al., 1993). Rocaglamide is a potent, but slow-acting toxin against R saucia, requiring at least 4 days for full toxicity to be expressed (Satasook et aL, 1992). Although rocaglamide has a direct antifeedant effect on P. saucia larvae, this action is insufficient to account for the complete bioactivity of the compound. In the present study, extracts of A. odorata, A. perviridis and A. oligophylla were found to deter feeding by neonate larvae (Table 5). Such a behavioural effect probably contributes substantially to the inhibition of larval growth observed when larvae are fed continuously on artificial diets containing extracts of these, and other Aglaia species. However, we believe that the extreme inhibition of larval growth observed is likely more a consequence of physiological toxicity. When added to artificial diet at a concentration of 300 ppm, a crude acetone extract of A. odorata twigs significantly reduced the relative consumption rate of fourth instar R saucia larvae, concomitant with a reduction in the relative growth rate (Table 6). A similar result was obtained using pure rocaglamide at a dietary concentration of 1.5 ppm (Satasook et al., 1992). Although these results would seem to implicate an antifeedant effect as the major cause of growth suppression, topical application of pure rocaglamide produced the same result, precluding the mouthpart chemosensilla as the primary site-of-action and therefore peripherally-mediated feeding deterrence as the primary mode-of-action. Instead, the rapid reduction in feeding following topical administration of rocaglamide points to a centrally-mediated anorexic effect (Satasook et al., 1992). This rapid cessation of feeding of larvae exposed either orally or by contact, should prove a valuable asset for a botanical insecticide based on A. odorata. In this regard, A. odorata is comparable to botanical insecticides derived from neem, Azadirachta indica, which have both antifeedant and insect growth-regulating effects on pests. Isolation and chemical identification of active compounds and further biological study in other promising Aglaia species are also being investigated.
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Acknowledgements--Wethank
Nancy Brard and Yanfen Zheng for technical assistance, Dr Y. S. Xie for reading the manuscript and Dr C. M. Pannell for correcting our taxonomy. Supported by a Strategic Grant from the Natural Sciences and Engineering Research Council of Canada to M. B. Isman and G. H. N. Towers, and a grant from the International Development Research Centre of Canada to P. Wiriyachitra.
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