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Mutangin, a dihydroagarofuranoid sesquiterpene insect antifeedant from Elaeodendron buchananii
C031-9422/93 f 6.00+0.00 01993 Pergamon Press Ltd
Phytochemistry, Vol. 34, No. 3, pp. 665667, 1993 Printedin Great Britain
MUTANGIN, A DIHYDROAGAROFURANOID SESQUITERPENE ANTIFEEDANT FROM ELAEODENDRON BUCHANANII MUNIRU K. TSANUO, AHMED HASSANALI,* ISAAC J. 0. JoNDIKot and The
International
INSECT
BALDWYN TORTO
Centre of Insect Physiology and Ecology, Box 30772, Nairobi, Kenya; TJDepartment of Chemistry, Kenyatta University, P.O. Box 43844, Nairobi, Kenya; (Receioed
in revised form 5 April 1993)
Key Word Index-Elaeodendron buchananii; Celastraceae; dihydroagarofuran; gin; antifeedant; Chilo partellus.
sesquiterpene; mutan-
Abstract-A
novel sesquiterpene (mutangin) of the eudesmane type with five hydroxyl functions has been isolated from unripe fruits of Elaeodendron buchananii. Its structure was elucidated by MS, 1D and 2D NMR spectroscopy. Mutangin has demonstrated moderate antifeeding activity against the lepidopteran, Chile partellus (Swinhoe).
INTRODUCTION
The tree Elaeodendron buchananii Loes (Eukanda, Mhakumo, Murundu, Mutanga, Sawanet, Sunwa, in local dialects) is one of the better known poisonous plants in East Africa [l]. Ingestion of its leaves has resulted in many deaths to domestic stock although, interestingly, wild animals (e.g. giraffes) are apparently unaffected [l]. The root bark of the plant is widely used in local medicine as a remedy for wounds, syphilis and diarrhoea [Z]. Previous phytochemical investigations of some Elaeodendron species have revealed the presence of inter alia, cardiac steroids (from the bark of E. glaucum Pers.) [3], tetranortriterpenoid cardiac glycosides (from seeds of E. glaucum) [4,5,6], triterpenoid quinone methides (from the root bark of E. bake) [7,8] and flavonoids (from the root bark of E. bake) [9]. One recent study on the root bark of E. buchananii led to the isolation of a steroidal compound with moderate cytotoxicity against L-1210 leukaemic cells [lo]. As part of our search for anti-insect phytochemicals, we have examined the unripe fruits of E. buchananii and herein we describe the isolation and structure of a novel sesquiterpene, mutangin, with moderate insect antifeedant activity.
RESULTS AND DISCUSSION
The sesquiterpenoid nature of mutangin (1) and its close relationship to eudesmane esters, e.g. celangulin and eumaitenin, previously isolated from Celastrus angulatus and Muytenus boaria, respectively [ll, 121, was apparent from spectroscopic evidence. The mass (CI, EI) spectrum gave a 1w, of 636. DEPT and broad band “CNMR spectra showed the presence of six methyl, three methyl*Author to whom correspondence should be addressed.
1
ene, 16 methine and 10 quaternary carbon atoms. This was consistent with ‘HNMR signal integration which suggested the presence of 40 hydrogen atoms in the molecule (Table 1). The IR (YE: cm- I: 1755, 1735, 1720) and UV (n&tH nm: 231 and 279) spectra and ester carbony1 signals (6 165.3, 165.6,169.3,169.9 and 170.6) in the 13CNMR spectrum, together with other benzoyl and acetyl signals in the ‘H and 13CNMR spectra, showed the presence of three acetate and two benzoate ester groups. A summation of these data gave the formula C35H40010 with the total mass weight of 620. This left a balance of 16 mass units assignable to an ethereal oxygen atom giving a formula C3sH,o0,i to mutangin. The r3CNMR spectrum (see Experimental) was suggestive of a penta-substituted dihydroagarofuran skeleton. Sesquiterpenes based on this skeleton appear to be quite common in Celastraceae [ 1l-131. The COSY spectrum gave ‘H-‘H interaction pattern (Table 1) indicative of ester substitution at C-l, C-2, C-6, C-9 and C-15. The location of one of the benzoate groups at C-9 was based on the presence of a prominent ion at m/z 202 (62%) in EI spectrum which was attributable to the radical cation [BzOCH = CHC (Me),]+. arising from a fragmentation pathway analogous to that previously proposed by Wakabayashi et al. [ll]. The assignment of the axial position to the benzoate was based on the
665
666
M. K. TSANUO
absence of an axial-axial coupling between the hydrogens of C-9 and C-8 (Table 1). The observed coupling of 7.2 Hz associated with H-9 must, therefore, be due to spin interaction with the axial H-S. Equatorial H-8 and H-9 are known to demonstrate very weak coupling (J - 0 Hz) in this class ofcompounds [13-161. The axial assignment of the C-9 benzoate was corroborated by the NOESY spectrum which showed an interaction between the C-12 methyl and the benzoate signal complex. The location of the remaining benzoate at C-15 was inferred from the occurrence of a high field signal (6 1.49) attributable to the methyl of the equatorial C-6 acetate. Inspection of a model and a NOESY interaction between the methyl of this acetate and the benzoate signal complex confirmed the close proximity of the two groups. The remaining two acetates, therefore, belonged to C-l and C-2. Stereochemical assignments which were based on coupling constants of H-l, H-2 and H-6 (Table 1) are in conformity with the stereochemistry observed at these positions in this class of natural products [ll, 131. A summation of these assignments gave the structure la,2a,6/3-triacetoxy-9/?,15-dibenzoyioxy-/?-dihydroagarofuran (1) for mutangin. In a leaf disc bioassay [19], mutangin reduced the feeding by third instar larvae of the spotted stem-borer Chilo partellus by 64.9 f 5.0 and 54.8 + 2.8 at 100 and 50 pg disc-‘, respectively. It thus joins a number of other agarofuran polyol esters with anti-insect properties [17, 181. EXPERIMENTAL
‘H, 13CNMR and 2DNMR: CDCI,, Varian XL 200 and GLT-360 spectrometers; EIMS and CIMS: VG12250 and VGTRIO-3, respectively; UV: EtOH; IR: KBr. Extraction and isolation. The unripe fruits of E. buchananii (3 kg), collected from trees growing near Nairobi National Park, were successively soaked (each time for 24 hr) in methanol (5 l), n-hexane (6 1)and CHCI, (5 1)at room temp. and the extracts coned. The resulting aq. methanolic concentrate was successively shaken with hexane and CHCI,, and the organic layers were combined with the corresponding extracts obtained earlier. Antifeedant bioassay (see below) showed that only the combined CHCI, extract was significantly active. The extract (10.8 g) was, therefore, chromatographed on silica gel (50 x 3.5 cm; 230-400 mesh) using petrol-Me&O (4:1)/Me,CO gradient to elute the column. The more active set of frs containing common components (TLC) were combined (0.38 g) and rechromatographed on silica gel (25 x 1.2 cm) with 15% EtOAc in petrol. Compound 1 eluted relatively early in an active set of frs, the crude product crystallizing from petrol as needles (161 mg) mp 234” (uncorr.). CIMS m/z: 654 [M+NHJ+; EIMS m/z (rel.int.); 636[M]+ (36.3), 621 (33.8), 410 (41.0), 350 (9.8), 335 (9.9), 202 (62.0), 123 (18.0), 105 (99.0), 77 (100); IR vi:: cm-‘: 1755,1735,1720,1610; UVIEzH nm: 231,272, 279; ‘HNMR (200 MHz, CDCl,): 61.20 (3H, d, J =7.5 Hz,H-14), 1.49(6H,s,6-OAcandH-13), 1.56(3H,s, H-12), 1.82 (lH, m, H,,-3), 2.08 (3H, s, AC), 2.24 (lH, m,
et al.
Table 1. ‘H NMR (200 Hz) and COSY spectral gin 1 in CDCI,
data of mutan-
H on C
6
COSY linkage with
1
2
5.77 d (3.4)* 5.61 dd (3.4, 3.1)
H-2 H-l, H,,-3,
3 (ax)
1.82 m
3 (es)
2.54 m
4
2.56 m
6 7
6.24 s 2.41 m
8 (ax) 8 (es)
2.24 m 2.60 m
9 12 13 14 15 (i, j) Acetates Benzoates Betuoates
(ortho) (meta, para)
5.41 d (7.2) 1.56 s 1.49 s 1.20 d (7.5) 4.40 d (12.6) 5.08 d (12.6) 1.48 s 2.08 s, 2.32 s 8.08 m 7.40-7.14 m
Ha,-3
H-2, H,,-3, H-4 H-2, H&,-3, H-4 H,,-3, Ha,-4 H-14 H-7, ~-AC Me H-6, H,,-8, Ha,-8 H-7, H,-8 H-7, H,,-8, H-9 H.?,-8
H-4 Hi-15 Hj-15 H-6
* J (Hz) in parenthesis.
H,,-8), 2.32(3H,s, AC),2.41 (lH,m, H-7), 2.54(1H, WI,H,,3), 2.56 (lH, m, H-4), 2.60 (lH, m, H,-8), 4.40 and 5.08 (2H,ABq,J=12.6 Hz,H-15),5.47(1H,d,J=7.2 Hz,H-9), 5.60 (lH, dd, J=3.4 and 3.1 Hz, H-2), 5.74 (lH, d, J = 3.4 Hz, H-l), 6.24 (lH, s, H-6) 7.40-7.74 (6H, m, benzene-meta, para), 8.08 (4H, m, benzene-ortho); 13C NMR (90 MHz): 6 170.5, 169.9 and 169.2 (acetate C = 0), 165.5 and 165.3 (benzoate C=O), 129.1 and 129.9 (benzoate, 1’ and l”), 129.5 and 130.1 (benzoate 2’,2”,6’ and 6”), 128.3 and 128.7 (benzoate 3’,3”,5’ and 5”), 133.3 and 133.4 (benzoate 4’ and 4”), 89.5 (C-5), 82.7(C-ll), 78.8(C-9), 71.5(C-2), 69S(C-6), 69.4(C-l), 65.5(C-15), 53.5(C-lo), 48.9 (C-7), 35.O(C-3 or C-8), 33.6(C-4), 30.9 (C-3 or C-8), 30.6(C-13), 26.O(C-12), 21.4, 21.4, 20.4(acetyl methyls). The 13C NMR assignments are based upon a comparison with the values for celanguilin [l l] and eumaitenin [12]. Bioassay. Zea mays leaf discs of known size (1.8 cm diameter) were treated with either known amounts (100, 50,25,12.5 pg) of test samples dissolved in Me,CO (20 ~1) or only Me&O (20 d). A choice of the 2 discs was offered to 2 unsexed third instar C. partellus larvae starved for 24 hr in a 7-cm diameter Petri-dish as previously described [19]. The larvae were allowed to feed in the dark at 29f 2” and 78 _+2% relative humidity for 24 hr. The experiment, conducted in 20 replicates, was then terminated and the disc fragments dried for 1 hr at 110” and weighed. The amounts of consumed control and treated leaf discs were computed with reference to the average weight of a similarly dried unconsumed set of leaf discs
Mutangin from E. buchananii and deterrence calcd using the expression: % deterrence=
l[
wt test disc eaten wt cont. disc eaten
1x
100.
667
(1985) Phytochemistry 24, 1345. 7. Fernando, H. C., Gunatilaka, A. A. L., Kumar, V., Weeratunga, G., Tezuka, Y. and Kikuchi, T. (1988) Tetrahedron Letters 28, 287.
The following % deterrence were obtained: 64.9kS.O (100 pg disc- ‘), 54.8 + 2.8 (50 pg disc- I), 9.2 + 7.0 (25 pg disc- ‘) and 3.1+ 1.8 (12.5 pg disc- ‘). Differences between consumed control and treated discs for the lower two doses were not significant (Students’ t-test).
*. Fernando, H. C., Gunatilaka, A. A. L.,Tezuka,Y. and Kikuchi, T. (1989) Tetrahedron 45, 5867. Weeratunga, G., Kumar, V., Sultanbawa, M. and 9. Urais, S. (1982) Tetrahedron Letters 23, 3031. 10. Kubo, I. and Fukuhara, K. (1990) J. Nat. Prod. 53,
Acknowledgements-The authors wish to thank Professor Thomas R. Odhiambo, Director, ICIPE for granting facilities to M.K.T. to undertake MSc work at ICIPE, and to Mr Lawrence Lester of the University of Maine for running NMR spectra.
ll. Wakabayashi, N., Wu, W. J., Waters, R. M., Redfern, R. C., Mills, G. D. Jr, De Millo, A. B., Lusby, W. R. and Andrzejewski, D. (1988) J. Nat. Prod. 51, 537. 12. Becerra, J., Gaete, L., Silva, M., Behlman, F., Jekuporic, J. (1987) Phytochemistry 26, 3073. 13. Bruning, R. and Wagner, H. (1978) Phytochemistry
968.
REFERENCES
B. and Trump, E. C. (1969) Common Poisonous Plants of East Africa, p.100. Collins, St.
1. Verdcourt,
Jame’s Place, London. 2. Kokwaro, J. 0. (1976) Medicinal PIants of East Africa, p. 51. East African Literature Bureau, Nairobi. Anjaneyulu, A. and Narayana, R. M. (1980) Phytochemistry 19, 1163. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I. (1981) HeterocycZes 15, 355. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I. (1982) Chem. Pharm. Bull. 30,4075. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I.
17, 1821.
14. Gonzalez, A. G., Gonzalez, C. M., Buzzoacchi, I. I., Ravelo, A. G., Luis, J. G. and Damingnez, X. A. (1967) Phytochemistry 7, 2133.
15. Tu, Y. Q., Wu, T. X., Li, Z. Z., Zhen, T. and Chen, Y. Z. (1991) J. Nat. Prod. 54, 1383. 16. Tu, Y. Q. and Chen, Y. Z. (1991) Phytochemistry 30, 4169.
17. Wu, W. J., Tu, Y. Q., Liu, H. X. and Zhu, J. B. (1992) J. Nat. Prod. 55, 1294.
18. Tu, Y. Q., Huang, E. S., Ma, Y. X., Wu, X. L. and Song, Q. B. (1992) J. Nat. Prod. 55, 1320. 19. Hassanali, A., Bentley, M. D., Ole-Sitayo, E. N., Njoroge, P. E. W. and Yatagai, M. (1986) Insect Sci. Applic. 7, 495.
Phytochemistry, Vol. 34, No. 3, pp. 665667, 1993 Printedin Great Britain
MUTANGIN, A DIHYDROAGAROFURANOID SESQUITERPENE ANTIFEEDANT FROM ELAEODENDRON BUCHANANII MUNIRU K. TSANUO, AHMED HASSANALI,* ISAAC J. 0. JoNDIKot and The
International
INSECT
BALDWYN TORTO
Centre of Insect Physiology and Ecology, Box 30772, Nairobi, Kenya; TJDepartment of Chemistry, Kenyatta University, P.O. Box 43844, Nairobi, Kenya; (Receioed
in revised form 5 April 1993)
Key Word Index-Elaeodendron buchananii; Celastraceae; dihydroagarofuran; gin; antifeedant; Chilo partellus.
sesquiterpene; mutan-
Abstract-A
novel sesquiterpene (mutangin) of the eudesmane type with five hydroxyl functions has been isolated from unripe fruits of Elaeodendron buchananii. Its structure was elucidated by MS, 1D and 2D NMR spectroscopy. Mutangin has demonstrated moderate antifeeding activity against the lepidopteran, Chile partellus (Swinhoe).
INTRODUCTION
The tree Elaeodendron buchananii Loes (Eukanda, Mhakumo, Murundu, Mutanga, Sawanet, Sunwa, in local dialects) is one of the better known poisonous plants in East Africa [l]. Ingestion of its leaves has resulted in many deaths to domestic stock although, interestingly, wild animals (e.g. giraffes) are apparently unaffected [l]. The root bark of the plant is widely used in local medicine as a remedy for wounds, syphilis and diarrhoea [Z]. Previous phytochemical investigations of some Elaeodendron species have revealed the presence of inter alia, cardiac steroids (from the bark of E. glaucum Pers.) [3], tetranortriterpenoid cardiac glycosides (from seeds of E. glaucum) [4,5,6], triterpenoid quinone methides (from the root bark of E. bake) [7,8] and flavonoids (from the root bark of E. bake) [9]. One recent study on the root bark of E. buchananii led to the isolation of a steroidal compound with moderate cytotoxicity against L-1210 leukaemic cells [lo]. As part of our search for anti-insect phytochemicals, we have examined the unripe fruits of E. buchananii and herein we describe the isolation and structure of a novel sesquiterpene, mutangin, with moderate insect antifeedant activity.
RESULTS AND DISCUSSION
The sesquiterpenoid nature of mutangin (1) and its close relationship to eudesmane esters, e.g. celangulin and eumaitenin, previously isolated from Celastrus angulatus and Muytenus boaria, respectively [ll, 121, was apparent from spectroscopic evidence. The mass (CI, EI) spectrum gave a 1w, of 636. DEPT and broad band “CNMR spectra showed the presence of six methyl, three methyl*Author to whom correspondence should be addressed.
1
ene, 16 methine and 10 quaternary carbon atoms. This was consistent with ‘HNMR signal integration which suggested the presence of 40 hydrogen atoms in the molecule (Table 1). The IR (YE: cm- I: 1755, 1735, 1720) and UV (n&tH nm: 231 and 279) spectra and ester carbony1 signals (6 165.3, 165.6,169.3,169.9 and 170.6) in the 13CNMR spectrum, together with other benzoyl and acetyl signals in the ‘H and 13CNMR spectra, showed the presence of three acetate and two benzoate ester groups. A summation of these data gave the formula C35H40010 with the total mass weight of 620. This left a balance of 16 mass units assignable to an ethereal oxygen atom giving a formula C3sH,o0,i to mutangin. The r3CNMR spectrum (see Experimental) was suggestive of a penta-substituted dihydroagarofuran skeleton. Sesquiterpenes based on this skeleton appear to be quite common in Celastraceae [ 1l-131. The COSY spectrum gave ‘H-‘H interaction pattern (Table 1) indicative of ester substitution at C-l, C-2, C-6, C-9 and C-15. The location of one of the benzoate groups at C-9 was based on the presence of a prominent ion at m/z 202 (62%) in EI spectrum which was attributable to the radical cation [BzOCH = CHC (Me),]+. arising from a fragmentation pathway analogous to that previously proposed by Wakabayashi et al. [ll]. The assignment of the axial position to the benzoate was based on the
665
666
M. K. TSANUO
absence of an axial-axial coupling between the hydrogens of C-9 and C-8 (Table 1). The observed coupling of 7.2 Hz associated with H-9 must, therefore, be due to spin interaction with the axial H-S. Equatorial H-8 and H-9 are known to demonstrate very weak coupling (J - 0 Hz) in this class ofcompounds [13-161. The axial assignment of the C-9 benzoate was corroborated by the NOESY spectrum which showed an interaction between the C-12 methyl and the benzoate signal complex. The location of the remaining benzoate at C-15 was inferred from the occurrence of a high field signal (6 1.49) attributable to the methyl of the equatorial C-6 acetate. Inspection of a model and a NOESY interaction between the methyl of this acetate and the benzoate signal complex confirmed the close proximity of the two groups. The remaining two acetates, therefore, belonged to C-l and C-2. Stereochemical assignments which were based on coupling constants of H-l, H-2 and H-6 (Table 1) are in conformity with the stereochemistry observed at these positions in this class of natural products [ll, 131. A summation of these assignments gave the structure la,2a,6/3-triacetoxy-9/?,15-dibenzoyioxy-/?-dihydroagarofuran (1) for mutangin. In a leaf disc bioassay [19], mutangin reduced the feeding by third instar larvae of the spotted stem-borer Chilo partellus by 64.9 f 5.0 and 54.8 + 2.8 at 100 and 50 pg disc-‘, respectively. It thus joins a number of other agarofuran polyol esters with anti-insect properties [17, 181. EXPERIMENTAL
‘H, 13CNMR and 2DNMR: CDCI,, Varian XL 200 and GLT-360 spectrometers; EIMS and CIMS: VG12250 and VGTRIO-3, respectively; UV: EtOH; IR: KBr. Extraction and isolation. The unripe fruits of E. buchananii (3 kg), collected from trees growing near Nairobi National Park, were successively soaked (each time for 24 hr) in methanol (5 l), n-hexane (6 1)and CHCI, (5 1)at room temp. and the extracts coned. The resulting aq. methanolic concentrate was successively shaken with hexane and CHCI,, and the organic layers were combined with the corresponding extracts obtained earlier. Antifeedant bioassay (see below) showed that only the combined CHCI, extract was significantly active. The extract (10.8 g) was, therefore, chromatographed on silica gel (50 x 3.5 cm; 230-400 mesh) using petrol-Me&O (4:1)/Me,CO gradient to elute the column. The more active set of frs containing common components (TLC) were combined (0.38 g) and rechromatographed on silica gel (25 x 1.2 cm) with 15% EtOAc in petrol. Compound 1 eluted relatively early in an active set of frs, the crude product crystallizing from petrol as needles (161 mg) mp 234” (uncorr.). CIMS m/z: 654 [M+NHJ+; EIMS m/z (rel.int.); 636[M]+ (36.3), 621 (33.8), 410 (41.0), 350 (9.8), 335 (9.9), 202 (62.0), 123 (18.0), 105 (99.0), 77 (100); IR vi:: cm-‘: 1755,1735,1720,1610; UVIEzH nm: 231,272, 279; ‘HNMR (200 MHz, CDCl,): 61.20 (3H, d, J =7.5 Hz,H-14), 1.49(6H,s,6-OAcandH-13), 1.56(3H,s, H-12), 1.82 (lH, m, H,,-3), 2.08 (3H, s, AC), 2.24 (lH, m,
et al.
Table 1. ‘H NMR (200 Hz) and COSY spectral gin 1 in CDCI,
data of mutan-
H on C
6
COSY linkage with
1
2
5.77 d (3.4)* 5.61 dd (3.4, 3.1)
H-2 H-l, H,,-3,
3 (ax)
1.82 m
3 (es)
2.54 m
4
2.56 m
6 7
6.24 s 2.41 m
8 (ax) 8 (es)
2.24 m 2.60 m
9 12 13 14 15 (i, j) Acetates Benzoates Betuoates
(ortho) (meta, para)
5.41 d (7.2) 1.56 s 1.49 s 1.20 d (7.5) 4.40 d (12.6) 5.08 d (12.6) 1.48 s 2.08 s, 2.32 s 8.08 m 7.40-7.14 m
Ha,-3
H-2, H,,-3, H-4 H-2, H&,-3, H-4 H,,-3, Ha,-4 H-14 H-7, ~-AC Me H-6, H,,-8, Ha,-8 H-7, H,-8 H-7, H,,-8, H-9 H.?,-8
H-4 Hi-15 Hj-15 H-6
* J (Hz) in parenthesis.
H,,-8), 2.32(3H,s, AC),2.41 (lH,m, H-7), 2.54(1H, WI,H,,3), 2.56 (lH, m, H-4), 2.60 (lH, m, H,-8), 4.40 and 5.08 (2H,ABq,J=12.6 Hz,H-15),5.47(1H,d,J=7.2 Hz,H-9), 5.60 (lH, dd, J=3.4 and 3.1 Hz, H-2), 5.74 (lH, d, J = 3.4 Hz, H-l), 6.24 (lH, s, H-6) 7.40-7.74 (6H, m, benzene-meta, para), 8.08 (4H, m, benzene-ortho); 13C NMR (90 MHz): 6 170.5, 169.9 and 169.2 (acetate C = 0), 165.5 and 165.3 (benzoate C=O), 129.1 and 129.9 (benzoate, 1’ and l”), 129.5 and 130.1 (benzoate 2’,2”,6’ and 6”), 128.3 and 128.7 (benzoate 3’,3”,5’ and 5”), 133.3 and 133.4 (benzoate 4’ and 4”), 89.5 (C-5), 82.7(C-ll), 78.8(C-9), 71.5(C-2), 69S(C-6), 69.4(C-l), 65.5(C-15), 53.5(C-lo), 48.9 (C-7), 35.O(C-3 or C-8), 33.6(C-4), 30.9 (C-3 or C-8), 30.6(C-13), 26.O(C-12), 21.4, 21.4, 20.4(acetyl methyls). The 13C NMR assignments are based upon a comparison with the values for celanguilin [l l] and eumaitenin [12]. Bioassay. Zea mays leaf discs of known size (1.8 cm diameter) were treated with either known amounts (100, 50,25,12.5 pg) of test samples dissolved in Me,CO (20 ~1) or only Me&O (20 d). A choice of the 2 discs was offered to 2 unsexed third instar C. partellus larvae starved for 24 hr in a 7-cm diameter Petri-dish as previously described [19]. The larvae were allowed to feed in the dark at 29f 2” and 78 _+2% relative humidity for 24 hr. The experiment, conducted in 20 replicates, was then terminated and the disc fragments dried for 1 hr at 110” and weighed. The amounts of consumed control and treated leaf discs were computed with reference to the average weight of a similarly dried unconsumed set of leaf discs
Mutangin from E. buchananii and deterrence calcd using the expression: % deterrence=
l[
wt test disc eaten wt cont. disc eaten
1x
100.
667
(1985) Phytochemistry 24, 1345. 7. Fernando, H. C., Gunatilaka, A. A. L., Kumar, V., Weeratunga, G., Tezuka, Y. and Kikuchi, T. (1988) Tetrahedron Letters 28, 287.
The following % deterrence were obtained: 64.9kS.O (100 pg disc- ‘), 54.8 + 2.8 (50 pg disc- I), 9.2 + 7.0 (25 pg disc- ‘) and 3.1+ 1.8 (12.5 pg disc- ‘). Differences between consumed control and treated discs for the lower two doses were not significant (Students’ t-test).
*. Fernando, H. C., Gunatilaka, A. A. L.,Tezuka,Y. and Kikuchi, T. (1989) Tetrahedron 45, 5867. Weeratunga, G., Kumar, V., Sultanbawa, M. and 9. Urais, S. (1982) Tetrahedron Letters 23, 3031. 10. Kubo, I. and Fukuhara, K. (1990) J. Nat. Prod. 53,
Acknowledgements-The authors wish to thank Professor Thomas R. Odhiambo, Director, ICIPE for granting facilities to M.K.T. to undertake MSc work at ICIPE, and to Mr Lawrence Lester of the University of Maine for running NMR spectra.
ll. Wakabayashi, N., Wu, W. J., Waters, R. M., Redfern, R. C., Mills, G. D. Jr, De Millo, A. B., Lusby, W. R. and Andrzejewski, D. (1988) J. Nat. Prod. 51, 537. 12. Becerra, J., Gaete, L., Silva, M., Behlman, F., Jekuporic, J. (1987) Phytochemistry 26, 3073. 13. Bruning, R. and Wagner, H. (1978) Phytochemistry
968.
REFERENCES
B. and Trump, E. C. (1969) Common Poisonous Plants of East Africa, p.100. Collins, St.
1. Verdcourt,
Jame’s Place, London. 2. Kokwaro, J. 0. (1976) Medicinal PIants of East Africa, p. 51. East African Literature Bureau, Nairobi. Anjaneyulu, A. and Narayana, R. M. (1980) Phytochemistry 19, 1163. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I. (1981) HeterocycZes 15, 355. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I. (1982) Chem. Pharm. Bull. 30,4075. Shimada, K., Kyuno, T., Nambara, T. and Uchida, I.
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14. Gonzalez, A. G., Gonzalez, C. M., Buzzoacchi, I. I., Ravelo, A. G., Luis, J. G. and Damingnez, X. A. (1967) Phytochemistry 7, 2133.
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