748 zyx zyxwvu Journal of Natural Prodwts Vol. 58, No. 5,pp. 748-750, May 1995 AN INSECT ANTIFEEDANT LIMONOID FROM TURRAEA NILOTICA MICHAEL D. BENTLEY,* GEOFFREY 0 .ADUL, Department of Chemistty, University of Maine, Orono, Maine 04469 A. RANDALL ALFORD, Department of Applied Ecology and Environmental Scimes, Uniwrsity of Maine, Orono, Maine 04469 FLI-YUNGHUANG, Department of Physics, Georgia Institute of Technology, Atlanta, Georgia 30332 &SUE GELBAUM, Research Centerfw Biotechnology, Georgia Institute of Technology, Atlanta, Gewgia 30332 zyxwvut and AHMEDHASANMI ICIPE, P.O. Box 30772, Nairobi, Kenya Assmm.-Nilotin 111,a new limonoid, has been isolated from the rootbark of Tuwaea nilotica and its structure established by spectroscopic methods. It displayed significant activity as an antifeedant against larvae of the Colorado potato beetle, Lptinotarsa deremliwta. The genus Tuwaea (Meliaceae)is comprised of a group of 60-70 species of shrubs and small trees occurring in the Indian Ocean region. Several speciesstudied have been shown to produce limonoids of the priurianin and havanensin classes (1-3). Tuwaea nilotica Kotschy et Peyr. is found in East'Africa and, in traditional medicine, adecoction of the roots is taken for upset stomachs (4).From an earlier study of the stem components of this plant, protolimonoids, but not limonoids, were reported (5). In continuing our investigations on the limonoid chemistry of this genus, we have examined a MeOH extract of the root bark of T. nilotica and here report the isolation of a new limonoid active as an insect antifeedant. Chromatography of the MeOH extract of T. nilotica root bark was performed on Si gel using a hexane/EtOAc gradient to obtain a fraction for which the presence of a limonoid was confirmed by 'H-nmr spectroscopy. This fraction was further purified by Si gel cc (toluene/ methyl ethyl ketone eluent) to obtain a new limonoid, nilotin {l], C4&&. The 'H-nmr spectrum of 1 (see Table 1) indicated the presence of four tertiary methyls, acarbomethoxy, four acetates, a I tiglate, a P-substituted furan, and an epoxide, and was consistent with the presence of a limonoid of the havanensin class. Substitution patterns and stereochemistry were determined with the aid of 'H COSY and 'H NOESY nmr experiments. Important proton connectivities observed in the COSY spectrum were H1 and H-3 with H-2; H-5 and H-7 with H-6; H-9 with H-11; and H-11 with H12. The small coupling constants of H- 1 and H-3 with H-2 were consistent with the commonly observed equatorial stereochemistry of the 1- and 3-protons. Fur- May 19951 zyxwv zyxwvutsrqp zyxwv zyxwvu Bentley et al. : Bioactive Limonoid from Tuwaea TABLE 1. 'H- and I3C-NmrData for 1. 'C 6 (ppm) Position 1 ........ 2 ........ 3 ........ 4 ........ 5 ........ 6 ........ 7 ........ 8 ........ 9 ........ 10 . . . . . . . 11 . . . . . . . 12 . . . . . . . 13 . . . . . . . 14 . . . . . . . 15 . . . . . . . 16 . . . . . . . 17 . . . . . . . 18 . . . . . . . 19 . . . . . . . 20 . . . . . . . 21 . . . . . . . 22 . . . . . . . 23 . . . . . . . 29 . . . . . . . 30 . . . . . . . 2' . . . . . . . . 3' . . . . . . . . 4' . . . . . . . . 5' . . . . . . . . Me(Ac). ... COfiMe. . . . c = o . .. . . 4.65 t (3) 2.20 m 4.97 t (3) 3.13 dd (12, 2) 1.70 m 4.70 t (2.5) 3.38 d (3.5) 5.16 t (3.5) 4.85 d (3.5) 3.61 s 2.34 m 2.80 m 1.06 s 1.25 s 7.10 m 6.40 m 7.27 m 1.22 s 1.34 s 6.83 qq ( 7 2 ) 1.7-1.76 m 1.7-1.76 m 1.94 s 2.05 s 2.10 s 2.14 s 3.49 s - 73.9 24.8' 72.4 40.1 33.4 24.2' 74.0 49.6b 40.3 40.1 74.4 79.6 48.7b 73.7 63.2 32.5 40.3 17.9 16.5 128.0' 140.6 112.3 142.3 749 the usual a-stereochemistry of the furan. Correlation of H-17 and H-12 further supported these stereochemical assignments. Correlation of H-7 with the 30methyl corroborated the assignment of equatorial stereochemistry for H-7 indicated by its coupling constant with H-6. 13 C-Nmr assignments were made with the aid ofAPT and HETCOR techniques and are presented in Table 1. The structure of nilotin 111 has the same skeleton and substitution pattern as several limonoids previously isolated from Tuwaeafizbud (3),but differs in groups attached at C-3, C-11, and C-12. Nilotin 111 showed significant antifeedant activity in no-choice feeding assays using 4th instar Colorado potato beetles. The dose required for 50% feeding reduction (ED,,) was 7kg/ml, and was thus comparable to that determined earlier for the citrus limonoid, limonin (ED,,=8 p.g/ml) (6).This is structurally consistent with our previous findings in structure-activity studies on citrus limonoids as Colorado potato beetle antifeedants that demonstrated that the furan and epoxide, both present in 1,are primarily responsible for the observed activity (6). zyxwvutsr 16.5 23.6 128.5' 137.8 11.6 14.3 21.4 21.9 20.9 20.9 zyxwvuts EXPERIMENTAL 174.0 170.7 169.7 169.5 169.2 165.7 a< Values with the same superscripts are interchangeable. ther correlations were H- 11 with H- 12, H-17 with H-16, H-5' with H-3', and H-22 with H-21 and H-23. Using the NOESY technique, correlations between H-9 and H-11 confirmed the p stereochemistry of the ester at C-11. Correlation of the 18-methyl protonsand a tiglate methyl placed the tiglate at the 12a position, while correlation of the 18methyl with the furan H-23 confirmed GENERALEXPERIMENTALPROCEDURES.-M~S were determined on a Fisher hot-stage apparatus and are uncorrected. Ir spectra were recorded on a Bio-Rad FTS-60 spectrometer. 'H- and "C-nmr spectra were obtained on Varian XL-200 and XL400 instruments, respectively, using CDCI, as solvent and TMS as internal standard. PLANT mnRlhL.-The roots of T. ndorica were collected in May 1989, from Shimba Hills, Kwale District, near Mombasa, Kenya. The plant was authenticated by S.G. Mathenge of the Herbarium of the Department of Botany, University of Nairobi, and a reference specimen is on file in that department. zyxwv EXTRACTIONANDISOLATION.-The root bark of T. nilotzcu was airdried for a week and ground into a powder. T h e powder (103 g) was soaked in MeOH (1 liter) for 4 weeks at room temperature, filtered, and the extract concentrated in vawo. Water was added and the aqueous MeOH extract partitioned with petroleum ether. The aqueous 750 Journal of Natural Products MeOH phase was evaporated to yield an oil (20 g), of which 18 g were chromatographed on 600 g of Si gel (Merck Kieselgel, 230-400 mesh) using a hexaneiEtOAc gradient. A limonoid-containing fraction was collected. Cc of this fraction (680 rng) on Si gel eluted with toluene-methyl ethyl ketone (8:2) led to the isolation of 1 (180 rng) as a white solid: rnp 138-140'; hrfabrns mlz CMfLi]' 763.3530, C4,H5,0,,Li, calcd 763.3493; ir (KBr) v rnax 2951, 1737 (br), 1365, 1232, 1126, 1032 -1 1 crn ; H- and "C-nmr spectral data, see Table 1. {Vol. 58, No. 5 %FR=61 (SEM=15) (30 insects); and for 3 pgi disk, %FR=20 (SEM=11) (8 insects). zyxwvutsrq zyxw zyxwvutsrqp zyxw ANTIFEEDANT BIOASSAY.-compound 1 was subjected to no-choice antifeedant assays at three dose levels (30, 10, and 3 pgiml) on potato leaf disks using 4th instar Colorado potato beetles according to our previously published procedure (6). Percent feeding reduction (%FR) was calculated by the equation %FR=[l-treatment consumption/control consurnption)X 100 and standard errors (SEM) were calculated (6). For an applied concentration of 30 pgiml leaf disk, %FRZ78(SEM=19)(38 insects);for 10 pgidisk, ACKNOWLEDGMENTS We are grateful to Mr. Edward Nyandat for the plant collection. LITERATURE CITED 1. 2. 3. 4. 5. 6. M.D. Bentley, F. Gaul, M.S. Rajab, and A. Hassanali,]. "a.Prod., 55, 84 (1992). M.S. Rajab, M.D. Bentley, A. Hassanali, and A. Chapya, PhpchemistryJ 2 7 , 2353 (1988). J . A . Akinniyi, J . D . Connolly, D . A . Mulholland, D.S. Rycroft, and D.A.H. Taylor, Phytochemistry, 2 5 , 2187 (1986). J.O. Kokwaro, "Medicinal Plants of East Africa," East Africa Literature Bureau, Nairobi, Kenya, 1976, p. 168. D.A. Mulholland and D.A.H. Taylor, Phytochemistry, 2 7 , 1220 (1988). M.D. Bentley,M.S. Rajab,A.R. Alford, M.J. Mendel, and A. Hassanali, Entomol. Exp. Appl,, 49, 189 (1988). Received 26 August 1994