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Journal of Stored Products Research 43 (2007) 9296 www.elsevier.com/locate/jspr

Antifeedant activities of terpenoids isolated from tropical Rutales


S. Omara, M. Marcottea, P. Fieldsb, P.E. Sanchezc, L. Povedac, R. Matad, A. Jimenezd, ` nea, T. Dursta, J. Zhanga, S. MacKinnona, D. Leamana, J.T. Arnasona, B.J.R. Philoge
a

Departments of Biology and Chemistry, University of Ottawa, Ottawa, Ont., Canada ON K1N 6N5 b Agriculture and Agri-Food Canada, Winnipeg, Canada MB R3T 2M9 c Herbario Juvenal, Universidad Nacional, Heredia 3000, Costa Rica d Departamento de Farmacia, Facultad de Quimica, UNAM, Coyoacan, Mexico, D.F., Mexico Accepted 30 November 2005

Abstract Terpenes isolated from tropical species of the Rutales were tested for insect antifeedant activity against rice weevil, Sitophilus oryzae (L.) using a our disk bioassay that requires only small amounts of compounds (0, 0.05, 0.25 and 0.50% w/w). At 0.50% (w/w) ve compounds isolated from Lansium domesticum (iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, onoceradienedione, lansiolic acid and lansiolic acid A) were shown to exhibit signicant antifeedant activity. Humilinolide C and D isolated from Swietenia humilis, and gedunin from Cedrela odorata, were also active at 0.50% (w/w). The most interesting results were obtained from the spirocaracolitones from Ruptiliocarpon caracolito which produced total feeding inhibition at 0.50% and potent antifeedant activity at concentrations as low as 0.05%. In conclusion, the antifeedant bioassay provides a rapid and inexpensive method for screening novel compounds available in small quantities to assess their activity as insect antifeedants. r 2006 Elsevier Ltd. All rights reserved.
Keywords: Insect control agents; Antifeedants; Sitophilus oryzae

1. Introduction In the New World tropics as well as in Southeast Asia, native trees of the order Rutales are well known for the production of bioactive terpenoids (Isman et al., 1997; Arnason et al., 1993, 1987). In particular, the plant family Meliaceae is noted for the production of useful bitter principles which are insect antifeedant and growth-reducing substances with low mammalian toxicity (Butterworth and Morgan, 1968; Arnason et al., 1985; Schmutterer, 1990; Isman et al., 1997). In recent years, bioactive terpenoids have been isolated from different neotropical species including Cedrela odorata L., Ruptilocarpon caracolito L. (MacKinnon, 1995), and Swietenia humilis Zucc. (Jimenez et al., 1997).

Corresponding author. Tel.: +1 613 562 5800, 4166; fax: +1 613 562 5765. ` ne). E-mail address: bphilog@science.uottawa.ca (B.J.R. Philoge

Lansium domesticum Corr. Serr. (Meliaceae) is a tree native to Southeast Asia producing a sweet and aromatic fruit, which is a popular dessert (Wong et al., 1994). The leaves have been used by indigenous people in the Philippines for the control of mosquitoes (Monzon et al., 1994). The peel of this fruit is traditionally known to be toxic to domestic animals. Phytochemical investigations of the peel revealed the presence of triterpene glycosides, and seco-onoceranoids such as lansic acid (Nishizawa et al., 1983). The volatile constituents of the fruit are sesquiterpene hydrocarbons including germacrene-D (Wong et al., 1994). The seed and leaf contain tetranortriterpenoids named dukunolides (Nishizawa et al., 1985, 1989). The major triterpenoid compound of the leaf is lansiolic acid and the minor triterpene is characterized as cycloartanoid type carboxylic acid. The bark of L. domesticum is used traditionally as an antimalarial remedy by the native people of Borneo (Leaman et al., 1995). Despite the importance of such use, there was no published phytochemical investigation on

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the bark until our group successfully isolated six novel triterpenes namely iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, onoceradienedione, lansiolic acid, lansiolic acid A and 3-keto lansiolic acid (unpublished). In this paper, we report the bioactivity of these compounds and related terpenes isolated from neotropical species using a Sitophilus oryzae L. antifeedant bioassay well suited to study of small amounts of pure compounds. 2. Material and methods 2.1. Insect rearing Rice weevils, S. oryzae (Coleoptera: Curculionidae), were obtained from Agriculture and Agri-Food Canada, Winnipeg, MB. Insects were reared in a growth chamber (30 1C, 85% r.h. and 18 h light:6 h dark photoperiod) on Western Hard Red Spring wheat at 14% moisture content.

2.2. Bioassay The bioassay was performed according to a method developed by Xie et al. (1996). Hard red spring wheat our (200 mg) was added to 1 mL of an aqueous solution containing the test substance at a concentration of 0.5% w/w and mixed using a magnetic stirrer. Ten aliquots (100 mL each) of the stirred suspension were placed in a 100 15 mm plastic Petri dish and allowed to dry overnight in air at room temperature. The next day, ve of the our disks were weighed and placed in a new Petri dish with 25 insects. The Petri dish was then sealed with paralm paper and kept at 30 1C and 85% r.h. for 3 days. Finally, the uneaten parts of the our disks were weighed. The insect consumption for the different test substances was compared to the control group. As only very small quantities of isolated compounds were available, experiments were done in duplicates.

CH3 H3C CH3 CH3 H3C H3C CH2 O H3C CH3 H3C CH3 H3C CH2 H3C

O CH3 CH3

ONOCERATRIENE

ONOCERADIENEDIONE COOH OH

H3C

CH3 CH3 CH3 H3C

H3C

CH2 CH3

H3C

H3C

CH2 O H3C

CH2

O H3C

CH3

CH3

3-KETO-22-HYDROXYONOCERADIENE COOH H3C CH3 H3C CH2 CH2 CH3

3-KETOLANSIOLIC ACID

COOH H3C CH3 H2C CH2 HO CH2 CH3

HO H3C

CH3

O H3C

CH3 LANSIOLID ACID A

LANSIOLIC ACID

Fig. 1. Structures of triterpenes isolated from Lansium domesticum from Borneo.

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94 S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296

2.3. Compound isolation The isolation of all compounds used in this study is described in detail in Jimenez et al. (1997), MacKinnon (1995), and Omar (2001). Their chemical structures are given in Figs. 1 and 2. 3. Results and discussion To date, numerous triterpenoids are known to possess strong feeding deterrent activity (Arnason et al., 1987, 1985). The results of feeding deterrence studies against S. oryzae by the crude extracts and six pure compounds isolated from L. domesticum further support the phagorO

epressive effect of triterpenoids (Table 1). Flour disks prepared using the crude extracts (ethyl acetate and hexane fractions) exhibited total inhibition of diet consumption at 0.50% (w/w), but the water extract was found to be phagostimulatory. Five of the pure compounds namely, iso-onoceratriene, 3-keto-22-hydroxyonoceradiene, onoceradienedione, lansiolic acid and lansiolic acid A exhibited signicant antifeedant activities at 0.5% (w/w); however, 3keto lansiolic acid was not active at this concentration. The crude extracts were more active than the isolated compounds. This may be the result of synergistic effects of the compounds in a mixture (Xie et al., 1996) and/or the presence of other unidentied active compounds. However, no clear structureactivity relationships were evident in this
HO OAc

CH3 CH3 CH3 O O H3C CH3 GEDUNIN HO OH OAc O

O O O MeO OAC

OAc

OAc O

SPIROCARACOLITONE B AcO AcOH2C

O O MeO OAc

OCOPh

OAc H3CO O O OAc O H3CO R=CO C(CH3)=CHCH3 OR O

OAc O

SPIROCARACOLITONE D O

SPIROCARACOLITONE E O

Me OAc Me H MeO2C O Me OH Me OCOCH(CH3)2 HUMILINOLIDE B O H O O H H MeO2C O Me Me H Me H

Me O H Me O H OAc OCOC(CH3)=CHCH3 HUMILINOLIDE C


Fig. 2. Structures of triterpenes isolated from neotropical plants.

Me O H O O Me OH Me OCOCH3 HUMILINOLIDE D H

H MeO2C

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S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296 Table 1 Antifeedant activity of crude extracts and pure compounds isolated from Lansium domesticum against Sitophilus oryzae (0.5% w/w) Treatment Consumption of diet (% control7SEM) 100.0710.2a 68.9717.4a,b 64.777.5b 63.273.8b 56.174.5b 53.873.4b 40.176.2b 0.0c 0.0c 184.6729d P value Table 2 Antifeedant activity of spirocaracolitones against Sitophilus oryzae Compound Concentration (% w/w) Consumption of diet (% control7SEM) 100.078.5a 67.176.1b 8.873.9c 0.0 74.676.3b 2.971.3c 0.0 78.3710.1a,b 19.079.9d 0.0 P value 95

Control 3-ketolansiolic acid Iso-onoceratriene Lansiolic acid Lansiolic acid A 3-keto-22-hydroxyonoceradiene Onoceradienedione Ethyl acetate fraction Hexane fraction Water fraction

40.05 o0.05 o0.01 o0.01 o0.01 o0.001 o0.001 o0.001 o0.001

Control Spirocaracolitone B

0 0.05 0.25 0.50 0.05 0.25 0.50 0.05 0.25 0.50

o 0.05 o 0.001 o 0.001 0.05 o0.001 o0.001 40.08 o0.001 o0.001

Spirocaracolitone D

Spirocaracolitone E

Multiple range test using Tukeys test (Po0.05). The same letters denote treatments not signicantly different from each other.

Multiple range test using Tukeys test (Po0.05). The same letters denote treatments not signicantly different from each other.

study. The concentration used to observe antifeedant effects was also much higher than for the commercially available products such as Margosan-O, active at a 3.75 ppm azadirachtin level (Xie et al., 1996) or toosendanin at 20 ppm (Champagne et al., 1992). Thus, the commercial application of L. domesticum compounds as a stored-product antifeedant may not be practical. Nevertheless, the bioassay employed in this study clearly allowed a fast screening and isolation of bitter compounds present at low concentrations in the active crude extracts. For example, other secondary compounds were also isolated from tropical sources (Liche xanthone, Formylorcinolcarboxyl, methyl orsellinate, gyrophoric acid) but were found inactive when tested with this bioassay (data not shown). The chemical structures of other triterpenes isolated from the Rutales are shown (Fig. 2). The CD spirocaracolitones B, D and E isolated from R. carocolito caused total feeding inhibition at 0.50% (w/w) and were signicantly active at 0.25 % (w/w) (Table 2). Spirocaracolitone B and D also showed signicantly different activity at 0.05%. Other studies also revealed that these compounds were very active when tested against the European corn borer, Ostrinia nubilalis Hu bner (MacKinnon, 1995) and showed modest antifungal and antimalarial activities (MacKinnon et al., 1997). The main active compound in the C. odorata wood, gedunin, had previously shown moderate antifeedant activity against many insect species (Arnason et al., 1987; Kubo and Klocke, 1986; Champagne et al., 1992). Humilinolides isolated from Swietenia humilis showed signicant growth-reducing activity including delay in time of pupation and adult emergence against O. nubilalis (Jimenez et al., 1997). In this study, gedunin and humilinolide C and D were active but humilinolide B was inactive at 0.50% (w/w) (Table 3). The most active compound was humilinolide C. A similar spectrum of activity was noted with O. nubilalis, humilinolide C

Table 3 Antifeedant activity of humilinolides and gedunin against Sitophilus oryzae Compound Concentration (% w/w) Consumption of diet (% control7SEM) 10077.4a 79.7716.7a 24.871.0b 65.2711.1c 31.276.3b P value

Control Humilinolide B Humilinolide C Humilinolide D Gedunin

0.50 0.50 0.50 0.50

40.05 o0.05 o0.05 o0.05

Multiple range test using Tukeys test (Po0.05). The same letters denote treatments not signicantly different from each other.

exhibiting the highest growth reducing activity (Jimenez et al., 1997). These studies suggested the mode of action of these compounds to be a combination of antifeedant action and post-digestive toxicity as seen in other limonoids (Isman et al., 1997; Xie et al., 1995). Kubo and Klocke (1986) indicated that most antifeedant compounds they had isolated exhibited pharmacological activities as well and suggested that the antifeedant bioassay has unexpectedly provided them with a unique system for screening bioactive compounds. Studies conducted in our laboratory have demonstrated that the extracts from L. domesticum and C. odorata exhibited potent in vitro antiplasmodial activities (Leaman et al., 1995; MacKinnon et al., 1997). Furthermore, the antifeedant bioassay provides a rapid and inexpensive method for screening such compounds.

Acknowledgements Funding was provided by the Natural Science and Engineering Research Council of Canada and Ontario Ministry of Science and Technology.

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96 S. Omar et al. / Journal of Stored Products Research 43 (2007) 9296 MacKinnon, S., 1995. Bioactive triterpenoids of the Rutales. Ph.D. Thesis, University of Ottawa. MacKinnon, S., Durst, T., Arnason, J.T., Angerhofer, C., Pezzuto, J.M., Sachez-Vidas, P.E., Poveda, L.J., Gbeassor, F.M., 1997. Antimalarial activity of tropical Meliaceae extracts and gedunin derivatives. Journal of Natural Products 60, 336341. Monzon, R.B., Alvior, J.P., Luczon, L.L.C., Morales, A.S., Mutuc, R.E.S., 1994. Larvicidal potential of ve Philippines plants against Aedes aegypti (Linnaeus) and Culex quinquefasciatus (Say). Southeast Asian Journal of Tropical Medicine and Public Health 25, 755759. Nishizawa, M., Nishide, H., Kosela, S., Hayashi, Y., 1983. Structure of lansiosides: biologically active new triterpene glycosides from Lansium domesticum. Journal of Organic Chemistry 48, 44624466. Nishizawa, M., Nademoto, Y., Sastrapradja, S., Shiro, M., Hayashi, Y., 1985. Structure of dukunolides, bitter principles of Lansium domesticum. Journal of Organic Chemistry 50, 54875490. Nishizawa, M., Emura, M., Yamada, H., Shiro, M., Hayashi, Y., Tokuda, H., 1989. Isolation of a new cycloartanoid triterpene from leaves of Lansium domesticum novel skin-tumor promotion inhibitor. Tetrahedron Letters 30 41, 56155618. Omar, S. 2001. Phytochemical discovery of antifeedant, antimicrobial and antimalarial principles. Ph.D. Thesis. University of Ottawa. Schmutterer, H., 1990. Properties and potential of natural pesticides from the neem tree, Azadirachta indica. Annual Review of Entomology 35, 271297. Wong, K.C., Wong, S.W., Siew, S.S., Tie, D.Y., 1994. Volatile constituents of the fruits of Lansium domesticum and Baccaurea motleyana. Flavour and Fragrance 9, 319324. Xie, Y.S., Fields, P.G., Isman, M.B., Chen, W.K., Zhang, X., 1995. Insecticidal activity of Melia toosendan extracts and toosendanin against three stored-product insects. Journal of Stored Products Research 31, 259265. Xie, Y.S., Bodnaryk, R.P., Fields, P.G., 1996. A rapid and simple ourdisk bioassay for testing substances active against stored-product insects. The Canadian Entomologist 128, 865875.

References
` ne, B.J.R., Donskov, N., Hudon, M., McDougall, Arnason, J.T., Philoge C., Fortier, G., Morand, P., Gardner, D., Lambert, J., Morris, C., Nozzolillo, C., 1985. Antifeedant and insecticidal properties of azadirachtin to the European corn borer, Ostrinia nubilalis Hubner. Entomologia Experimentalis et Applicata 38, 2934. ` ne, B.J.R., Donskov, N., Kubo, I., 1987. Limonoids Arnason, J.T., Philoge from the Meliaceae and Rutaceae reduce feeding, growth and development of Ostrinia nubilalis. Entomologia Experimentalis et Applicata 43, 221226. ` ne, B.J.R., Arnason, J.T., MacKinnon, S., Isman, M.B., Durst, T., Philoge Hasburn, C., Sanchez, P., Poveda, L., Roman, L.S., Isman, M.B., Satasook, C., Towers, G.H.N., Wiriyachitra, P., MacLaughlin, J.L., 1993. Insecticides in tropical plants with non-neurotoxic modes of action. Recent Advances in Phytochemistry 27, 107131. Butterworth, J.H., Morgan, E.D., 1968. Isolation of a substance that suppresses feeding in locusts. Entomologia Experimentalis et Applicata 38, 2934. Champagne, D., Koul, O., Isman, M., Scudder, G.G.E., Towers, G.H.N., 1992. Biological activity of limonoids from Rutales. Phytochemistry 31, 377394. Isman, M.B., Matsuura, H., MacKinnon, S., Durst, T., Towers, G.H.N., Arnason, J.T., 1997. Phytochemistry of the Meliaceae. Recent Advances in Phytochemistry 30, 155178. Jimenez, A., Mata, R., Pereda-Miranda, R., Calderon, J., Isman, M.B., Nicol, R., Arnason, J.T., 1997. Insecticidal limonoids from Swietenia humilis and Cedrela salvadorensis. Journal of Chemical Ecology 23, 12251234. Kubo, I., Klocke, J.A., 1986. Some terpenoid insect antifeedants from tropical sources. Advances in Pesticide Science 2, 284291. Leaman, D.J., Arnason, J.T., Yusuf, R., Sangat-Roemantyo, H., Soedjito, H., Angerhofer, C.K., Pezzuto, J.M., 1995. Malarial remedies of the Kenyah of the Apo Kayan, East Kalimantan, Indonesian Borneo: a quantitative assessment of local consensus as an indicator of biological efcacy. Journal of Ethnopharmacology 49, 116.