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Two tyrosinase inhibiting flavonol glycosides from Buddleia coriacea
Short Reports REFERENCES 1. Palmer, E. and Pitman, N. (1972) Trees ofSouthernAfrica Vol. 3, p. 1613. A. A. Balkema, Cape Town. 2. Wright, W. and Warren, F. L. (1967) J. Chem Sot. (C) 283. 3. Warren, F. L., Cooks, R. G. and Williams, D. H. (1967) J. Chem. Sot. (C) 286. 4. Morimoto, S., Nonaka, G. and Nishioka, I. (1986) Chem. Pharm. Bull. 34, 633. 5. Bonefeld, M., Friedrich, H. and Kolodziej, H. (1986) Phytochemistry 25, 1205. 6. Murakami, T., Wada, H., Tanaka, N., Kuraishi, T., Saiki, Y.
Phytochetisny, Vol. 31, No. 3, pp. 1075.1077, Printed m Great Britain.
TWO TYROSINASE
1075
and Chen, C. (1985) Yakugaku Zasshi 105, 649. 7. Ishimaru, K., Nonaka, G. and Nishioka, I. (1987) Phytcchemistry 26, 1167. Hsu, F., Nonaka, G. and Nishioko, I. (1985) Chem. Pharm. 8’ Bull. 33, 3142. 9 Hikino, H., Shimoyama, N., Kasahara, Y., Takahashi, M. and Konno, C. (1982) Heterocycles 19, 1381. 1O Rasoanaivo, P., Ratsimamanga-Urverg, S., Messana, I., De Vicente, Y. and Geleffi, C. (1990) Phytochemistry 29, 2040. 11. Bock, Pedersen, C. and Pedersen, H. (1984)Adu. Carbohydr. Chem. Biochem. 42, 193.
1992
003 I 9422/92 $5.00 + 0.00 Pergamon Press plc
INHIBITING
FLAVONOL
GLYCOSIDES
FROM
BUDDLEIA CORIACEA ISAO KUBO* and YOSHIHIROYOKOKAWA Division of Entomology and Parasitology, College of Natural Resources, University of California, Berkeley, CA 94720, U.S.A. (Received 23 August 1991) Key Word Index-Buddleia inhibitory activity.
coriacea; Loganiaceae;
flavonol
glucoside; buddlenoid
A, buddlenoid
B; tyrosinase
Abstract-Two new flavonol glycosides, buddlenoids A and B, have been isolated and identified as tyrosinase inhibitors from the aerial parts of Buddleia coriacea. Their structures were deduced from spectroscopic evidence to be kaempferol 7-(6”-p-coumaroylglucoside) and isorhamnetin 7-(6”-p-coumaroylglucoside). Both buddlenoids showed high inhibition of mushroom tyrosinase.
INTRODUCTION The aerial parts of Buddleia coriacea, known as ‘Quishuara’, are used as a remedy for venereal diseases in Bolivia Cl]. There has been no previous report on the chemical constituents. In our continuing search for alternative insect control agents, we have isolated tyrosinase inhibitors from several plants. Tyrosinase is one of the most important key enzymes in the insect moulting process [2]. Moreover, tyrosinase inhibitors have also become increasingly important for cosmetic products in relation to hyperpigmentation [S]. Thus, tyrosinase inhibitors may control over production of the dermal melanin pigment since tyrosinase plays an important role in the process of melanin biosynthesis [4, 53. In our preliminary screening of tyrosinase inhibitory activity, the methanol extract of Quishuara proved to exhibit potent inhibitory activity. Bioassay guided fractionation, after repeated various CC techniques, led to the isolation of two active principles. RESULTSANDDISCUSSION The methanol extract of the aerial parts of B. coriacea exhibited mushroom tyrosinase inhibitory activity in our
preliminary assay. Fractionation guided by the tyrosinase inhibitory activity led to the isolation of two new active principles which were designated as buddlenoids A (1) and B (2). Buddlenoid A (1) had a molecular formula of C3,,H260,1, which was established by observation of m/z 593 [M-H]by negative ion FAB mass spectrometry, in conjunction with the ‘H and 13CNMR data. Extensive analysis of the NMR data (Tables 1 and 2) indicated that 1 consists of kaempferol, glucose and pcoumaroyl moieties. Also the signals of meta coupling between H-6 and H-8 (66.70 and 6.71, each d, J= 1 Hz) and of AA’BB’ type of H-2’, H-6’ and H-3’, H-S’ (67.48 and 7.15, each d, .I=5 Hz) in the ‘HNMR spectrum indicated the characteristic A- and B-ring moieties of kaempferol. The UV spectrum showed absorption maxima at 267 and 317 nm and bathochromic shifts on addition of NaOAc, indicated that the C-5 and C-4 hydroxyls were unsubstituted [6]. This was also partially supported by the observation of a downfield signal at 6 12.45 in the ‘H NMR spectrum indicating the presence of an intramolecular hydrogen bond between the OH-5 and C=O groups of flavonols. In addition, the signals of C-2 and C-3 observed at 6149.5 and 135.1 in the ‘“C NMR spectrum indicated that the hydroxyl group at
1076
Short Reports Table 1. ‘HNMR spectral data of compounds 1 and 2 (500 MHz, pyridine-d,, J values in parentheses) H
1
6 8 2 3’ 5’ 6
6.70 d 6.71 d 7.48 d 7.15 d 7.15 d 7.48 d
2”‘, 6”’ 3”‘, 5”’ 7”’ 8”’ OH-5
1”
6.06 8.40 7.20 7.80 6.46 12.45
OMe-3’
_
Table 2. ‘%JNMR
spectral data of compounds (125 MHz, pyridine-d,)
C
1
2
2 3 4 5 6 7 8 9 10 1’ 2 3’ 4 5’ 6
149.5 135.1 178.4 158.0 100.7 162.8 95.1 157.7 104.6 122.0 130.7 116.9 161.5 116.9 130.7
149.5 135.1 178.7 157.9 100.0 162.8 94.7 157.7 105.2 122.0 116.8 149.5 151.1 111.5 115.9
C
1 and 2
1
2
104.6 76.0 78.5 71.3 76.0 64.3 126.1 131.9 116.1 161.8 145.2 114.9 167.3
104.3 76.0 78.5 71.3 76.0 64.3 126.6 131.9 116.1 161.8 145.6 115.1 167.3 167.3
2
d d d d d br
(1) (1) (5) (5)
6.68 br s 6.68 br s 7.20 d (2)
(5) (5)
7.18 d (5) 7.12 dd (2, 5)
(5) (5) (5) (20) (20)
6.16 8.40 7.22 7.81 6.49
d d d d d 12.45 br 3.82 s
(5) (5) (5) (20) (20)
C-3 was free, as the signal of C-2 was not observed around 6156 171. Therefore, only the C-7 hydroxyl group was substituted. Regarding the glucose moiety, the ‘H NMR spectrum indicated the signal of an anomeric proton at 66.06 (d, J = 5 Hz). This suggested that the glucose moiety of 1 was an esteritied p-glucopyranose. The acylation shifts of C-5 (676.0) and C-6 (664.3) in the glucose residue clearly indicated the attachment of an acyl group at the 6position of the inner glucose. Furthermore, the ‘H NMR spectrum contained the characteristic signals of an AB type of a p-hydroxylphenyl group (6 7.15 and 7.48, each d, J = 5 Hz) and trans-olefine protons (66.46 and 7.80, each d, J = 20 Hz). In addition, the ’ 3C NMR signal of d 167.3 and IR absorption of 1690 cm-’ demonstrated the presence of a conjugated ester. These data indicated that the acyl group in 1 is p-coumaroyl and that the 6-p-coumaroylglucosyl group was attached to the 7-position of kaempferol. A fragment ion peak at m/z 459 corresponding to ion 3 in the negative ion FAB mass spectrometry
R’
Rz
1
H
OH
2
OMe
OH
a
I,
1 2”
3” 4’ 5” 6” 1”’ 2”, 6”’ 3”‘, 5”’ 4 11, 711, I,, 8
I<, 9 OMe
caused by usual retro-Diels-Alder cleavage [8] supported the placement of the 6-p-coumaroylglucosyl residue at this position. However, the position of the p-coumaroylglucosyl moiety was finally established by direct comparison with a similar flavonol glycoside, tiliroside (3) which has been isolated from several plants [9, lo]. The structure of 3 was previously reported to be kaempferol3fl-D-(6”-p-coumaroylglucoside) [l l--13]. The only difference between buddlenoid A (1) and tiliroside (3) is the position of the 6-p-coumaroylglucosyl group. Although the two compounds showed similar properties, melting points and some spectroscopic data (e.g. the i3C NMR C2 signal of 3 at 6 156.3 and of 1 at 149.5) were distinct. Therefore, 1 was characterized as kaempferol 7-/3-D-(6”-pcoumaroylglucoside). The spectroscopic data of buddlenoid B (2) were similar to those of 1. However, in the ‘H NMR spectrum, the characteristic AA’BB’ signals of the B-ring of the kaempferol moiety in 1 were replaced by an ABX system at 67.12
Short Reports (dd, 5=2, 5 Hz), 7.18 (d, J=5 Hz) and 7.20 (d, 5=2 Hz) indicating the presence of only three aromatic protons in the B-ring of 2. In the negative ion FAB mass spectrum, the molecular ion was detected at m/z 623 [M -H] -. In conjunction with a methoxyl group observed at 6 3.82 (s) in the ‘H NMR spectrum, these results suggested that 2 has an additional methoxyl group in the B-ring. Moreover, the NOESY spectrum of 2 showed that an NOE was present btween H-2’ and OMe-3’. This led us to characterize buddlenoid B as isorhamnetin 7-/?-D-(6”-p-coumaroylglucoside) (2). Bioassay with the purified compounds indicated that both buddlenoids (1 and 2) exhibited potent mushroom tyrosinase inhibitory activity. Thus, the ID,,s were 0.39 mM for buddlenoid A and 0.44 mM for buddlenoid B. EXPERIMENTAL
1077
[a-H]-; ‘H and 13CNMR are listed in Tables 1 and 2, respectively. Enzyme assay. Mushroom tyrosinase was purchased from Sigma (St. Louis, MO). The assay was performed as previously described in ref. [3]. Thus, an aqueous soln (0.1 ml) of mushroom tyrosinase (138 unit) was mixed with 0.1 ml of inhibitory soln and 1.8 ml of 0.1 M Pi buffer (pH 6.8), and incubated at 25” for 10 min. Then, 1 ml of 2 mM DOPA was added to the mixture to measure, immediately, the initial rate of linear increase in optical density at 475 run, based on the formation of dopachrome. The extent of inhibition by the addition of samples is expressed as a percentage necessary for 50% inhibition (ID,,).
Acknowledgements-The authors are indebted to Promenat (Proyecto de Medicina Native), La Paz, Bolivia for providing the plant material, to Professor F. C. Chen for providing an authentic sample of tiliroside, and Dr M. Adamczeski for measuring the NMR data.
General. Mps: uncorr., ‘H and 13CNMR were taken in
pyridined, at 500 MHz for ‘H and 125 MHz for 13C. FAB-MS were obtained using glycerol matrix. PIant material. Dried aerial parts of B. coriacea (Quishuara) were provided by Promenat (Proyecto de Medicina Native), La Paz, Bolivia. Extraction and isolation. Dried ground aerial parts (330 g) were extracted with MeOH ( x 3) at ambient temp. for 10 days. After removal of the solvent, H,O (1 1)was added and the H,O based soln was partitioned with n-hexane, EtOAc and n-BuOH. The EtOAc extract (3.5 g) was chromatographed on silica gel using a CHCl, and MeOH gradient to obtain 5 frs. Subsequent bioassay indicated fr. 3 to be active. Hence, this bioactive fr. (46Omg) was further chromatographed on ODS eluted with M&H to afford buddlenoid A (2O.Omg) and buddlenoid B (31.2 mg). Buddlenoid A (1). C,,H,,O,,; amorphous powder (MeOH); mp 173-174”, UV (MeOH) 317, 267 nm (~28000, 22000), (+NaOMe) 375, 313, 275 nm (~20000, 26000, 30000); IR vKBr: 3300, 1690, 165Ocm-‘; FAB-MS m/z 593 [M-H]-, 459 [a-H]-; ‘H and ‘sCNMR are listed in Tables 1 and 2, respectively. Buddlenoid B (2). C31Has014; amorphous powder (MeOH); mp 165-166”, UV lMcOH 238, 267mn (~20000, ISOOO), (+NaOMe) 378,318,276nm (~14000,18000,22000); IR vKBr: 3400, 1646, 1605cm-‘; FAB-MS m/z 623 [M-H]-, 459
REFERENCES
1. Girault, L. (1978) in Kallawaya, p. 350. Unicef, Bolivia. 2. Neville, A. C. (1975) in Zoophysiology and Ecology 4/S. Springer, New York. 3. Fitzpatrick, T. B., Seiji, M. and McGugan, A. D. (1961) New Engl. J. Med. MS, 328, 374,430. 4. Pomerantz, S. H. (1963) J. Biol. Chem. 238, 2351. 5. Mason, H. S. and Peterson, E. W. (1965) B&hem. Biophys. Acta. 111, 134. 6. Markham, K. R. and Mabry, T. J. (1975) in The FZauonoids (Harborne, J. B., ed.), p. 45. Academic Press, New York. 7. Markham, K. R., Ternal, B., Stanley, R., Geiger, H. and Mabry, T. J. (1978) Tetrahedron 34, 1389. 8. Mabry, T. J. and Markham, K. R. (1975) in 7’he Flauonoids, (Harborne, J. B., ed.), p. 78. Academic Press, New York. 9. &Seth, von D. and Nordal, A. (1957) Pharm. Acta Helo. 32, 109. 10. H&hammer, L., Stich, L. and Wagner, H. (1961) Arch. Pharm. 294,685.
11. Harborne, J. B. (1964) Phytochemistry 3, 151. 12. Lin, J.-H., Lin, Y.-M. and Chen, F.-C. (1976) J. Chin. Chem. sot. 23, 57. 13. Kuroyanagi, M., Fukuoka, M., Yoshihira, K., Natori, S. and Yamasaki, K. (1978) Chem. Pharm. Bull. 26, 3594.
Phytochetisny, Vol. 31, No. 3, pp. 1075.1077, Printed m Great Britain.
TWO TYROSINASE
1075
and Chen, C. (1985) Yakugaku Zasshi 105, 649. 7. Ishimaru, K., Nonaka, G. and Nishioka, I. (1987) Phytcchemistry 26, 1167. Hsu, F., Nonaka, G. and Nishioko, I. (1985) Chem. Pharm. 8’ Bull. 33, 3142. 9 Hikino, H., Shimoyama, N., Kasahara, Y., Takahashi, M. and Konno, C. (1982) Heterocycles 19, 1381. 1O Rasoanaivo, P., Ratsimamanga-Urverg, S., Messana, I., De Vicente, Y. and Geleffi, C. (1990) Phytochemistry 29, 2040. 11. Bock, Pedersen, C. and Pedersen, H. (1984)Adu. Carbohydr. Chem. Biochem. 42, 193.
1992
003 I 9422/92 $5.00 + 0.00 Pergamon Press plc
INHIBITING
FLAVONOL
GLYCOSIDES
FROM
BUDDLEIA CORIACEA ISAO KUBO* and YOSHIHIROYOKOKAWA Division of Entomology and Parasitology, College of Natural Resources, University of California, Berkeley, CA 94720, U.S.A. (Received 23 August 1991) Key Word Index-Buddleia inhibitory activity.
coriacea; Loganiaceae;
flavonol
glucoside; buddlenoid
A, buddlenoid
B; tyrosinase
Abstract-Two new flavonol glycosides, buddlenoids A and B, have been isolated and identified as tyrosinase inhibitors from the aerial parts of Buddleia coriacea. Their structures were deduced from spectroscopic evidence to be kaempferol 7-(6”-p-coumaroylglucoside) and isorhamnetin 7-(6”-p-coumaroylglucoside). Both buddlenoids showed high inhibition of mushroom tyrosinase.
INTRODUCTION The aerial parts of Buddleia coriacea, known as ‘Quishuara’, are used as a remedy for venereal diseases in Bolivia Cl]. There has been no previous report on the chemical constituents. In our continuing search for alternative insect control agents, we have isolated tyrosinase inhibitors from several plants. Tyrosinase is one of the most important key enzymes in the insect moulting process [2]. Moreover, tyrosinase inhibitors have also become increasingly important for cosmetic products in relation to hyperpigmentation [S]. Thus, tyrosinase inhibitors may control over production of the dermal melanin pigment since tyrosinase plays an important role in the process of melanin biosynthesis [4, 53. In our preliminary screening of tyrosinase inhibitory activity, the methanol extract of Quishuara proved to exhibit potent inhibitory activity. Bioassay guided fractionation, after repeated various CC techniques, led to the isolation of two active principles. RESULTSANDDISCUSSION The methanol extract of the aerial parts of B. coriacea exhibited mushroom tyrosinase inhibitory activity in our
preliminary assay. Fractionation guided by the tyrosinase inhibitory activity led to the isolation of two new active principles which were designated as buddlenoids A (1) and B (2). Buddlenoid A (1) had a molecular formula of C3,,H260,1, which was established by observation of m/z 593 [M-H]by negative ion FAB mass spectrometry, in conjunction with the ‘H and 13CNMR data. Extensive analysis of the NMR data (Tables 1 and 2) indicated that 1 consists of kaempferol, glucose and pcoumaroyl moieties. Also the signals of meta coupling between H-6 and H-8 (66.70 and 6.71, each d, J= 1 Hz) and of AA’BB’ type of H-2’, H-6’ and H-3’, H-S’ (67.48 and 7.15, each d, .I=5 Hz) in the ‘HNMR spectrum indicated the characteristic A- and B-ring moieties of kaempferol. The UV spectrum showed absorption maxima at 267 and 317 nm and bathochromic shifts on addition of NaOAc, indicated that the C-5 and C-4 hydroxyls were unsubstituted [6]. This was also partially supported by the observation of a downfield signal at 6 12.45 in the ‘H NMR spectrum indicating the presence of an intramolecular hydrogen bond between the OH-5 and C=O groups of flavonols. In addition, the signals of C-2 and C-3 observed at 6149.5 and 135.1 in the ‘“C NMR spectrum indicated that the hydroxyl group at
1076
Short Reports Table 1. ‘HNMR spectral data of compounds 1 and 2 (500 MHz, pyridine-d,, J values in parentheses) H
1
6 8 2 3’ 5’ 6
6.70 d 6.71 d 7.48 d 7.15 d 7.15 d 7.48 d
2”‘, 6”’ 3”‘, 5”’ 7”’ 8”’ OH-5
1”
6.06 8.40 7.20 7.80 6.46 12.45
OMe-3’
_
Table 2. ‘%JNMR
spectral data of compounds (125 MHz, pyridine-d,)
C
1
2
2 3 4 5 6 7 8 9 10 1’ 2 3’ 4 5’ 6
149.5 135.1 178.4 158.0 100.7 162.8 95.1 157.7 104.6 122.0 130.7 116.9 161.5 116.9 130.7
149.5 135.1 178.7 157.9 100.0 162.8 94.7 157.7 105.2 122.0 116.8 149.5 151.1 111.5 115.9
C
1 and 2
1
2
104.6 76.0 78.5 71.3 76.0 64.3 126.1 131.9 116.1 161.8 145.2 114.9 167.3
104.3 76.0 78.5 71.3 76.0 64.3 126.6 131.9 116.1 161.8 145.6 115.1 167.3 167.3
2
d d d d d br
(1) (1) (5) (5)
6.68 br s 6.68 br s 7.20 d (2)
(5) (5)
7.18 d (5) 7.12 dd (2, 5)
(5) (5) (5) (20) (20)
6.16 8.40 7.22 7.81 6.49
d d d d d 12.45 br 3.82 s
(5) (5) (5) (20) (20)
C-3 was free, as the signal of C-2 was not observed around 6156 171. Therefore, only the C-7 hydroxyl group was substituted. Regarding the glucose moiety, the ‘H NMR spectrum indicated the signal of an anomeric proton at 66.06 (d, J = 5 Hz). This suggested that the glucose moiety of 1 was an esteritied p-glucopyranose. The acylation shifts of C-5 (676.0) and C-6 (664.3) in the glucose residue clearly indicated the attachment of an acyl group at the 6position of the inner glucose. Furthermore, the ‘H NMR spectrum contained the characteristic signals of an AB type of a p-hydroxylphenyl group (6 7.15 and 7.48, each d, J = 5 Hz) and trans-olefine protons (66.46 and 7.80, each d, J = 20 Hz). In addition, the ’ 3C NMR signal of d 167.3 and IR absorption of 1690 cm-’ demonstrated the presence of a conjugated ester. These data indicated that the acyl group in 1 is p-coumaroyl and that the 6-p-coumaroylglucosyl group was attached to the 7-position of kaempferol. A fragment ion peak at m/z 459 corresponding to ion 3 in the negative ion FAB mass spectrometry
R’
Rz
1
H
OH
2
OMe
OH
a
I,
1 2”
3” 4’ 5” 6” 1”’ 2”, 6”’ 3”‘, 5”’ 4 11, 711, I,, 8
I<, 9 OMe
caused by usual retro-Diels-Alder cleavage [8] supported the placement of the 6-p-coumaroylglucosyl residue at this position. However, the position of the p-coumaroylglucosyl moiety was finally established by direct comparison with a similar flavonol glycoside, tiliroside (3) which has been isolated from several plants [9, lo]. The structure of 3 was previously reported to be kaempferol3fl-D-(6”-p-coumaroylglucoside) [l l--13]. The only difference between buddlenoid A (1) and tiliroside (3) is the position of the 6-p-coumaroylglucosyl group. Although the two compounds showed similar properties, melting points and some spectroscopic data (e.g. the i3C NMR C2 signal of 3 at 6 156.3 and of 1 at 149.5) were distinct. Therefore, 1 was characterized as kaempferol 7-/3-D-(6”-pcoumaroylglucoside). The spectroscopic data of buddlenoid B (2) were similar to those of 1. However, in the ‘H NMR spectrum, the characteristic AA’BB’ signals of the B-ring of the kaempferol moiety in 1 were replaced by an ABX system at 67.12
Short Reports (dd, 5=2, 5 Hz), 7.18 (d, J=5 Hz) and 7.20 (d, 5=2 Hz) indicating the presence of only three aromatic protons in the B-ring of 2. In the negative ion FAB mass spectrum, the molecular ion was detected at m/z 623 [M -H] -. In conjunction with a methoxyl group observed at 6 3.82 (s) in the ‘H NMR spectrum, these results suggested that 2 has an additional methoxyl group in the B-ring. Moreover, the NOESY spectrum of 2 showed that an NOE was present btween H-2’ and OMe-3’. This led us to characterize buddlenoid B as isorhamnetin 7-/?-D-(6”-p-coumaroylglucoside) (2). Bioassay with the purified compounds indicated that both buddlenoids (1 and 2) exhibited potent mushroom tyrosinase inhibitory activity. Thus, the ID,,s were 0.39 mM for buddlenoid A and 0.44 mM for buddlenoid B. EXPERIMENTAL
1077
[a-H]-; ‘H and 13CNMR are listed in Tables 1 and 2, respectively. Enzyme assay. Mushroom tyrosinase was purchased from Sigma (St. Louis, MO). The assay was performed as previously described in ref. [3]. Thus, an aqueous soln (0.1 ml) of mushroom tyrosinase (138 unit) was mixed with 0.1 ml of inhibitory soln and 1.8 ml of 0.1 M Pi buffer (pH 6.8), and incubated at 25” for 10 min. Then, 1 ml of 2 mM DOPA was added to the mixture to measure, immediately, the initial rate of linear increase in optical density at 475 run, based on the formation of dopachrome. The extent of inhibition by the addition of samples is expressed as a percentage necessary for 50% inhibition (ID,,).
Acknowledgements-The authors are indebted to Promenat (Proyecto de Medicina Native), La Paz, Bolivia for providing the plant material, to Professor F. C. Chen for providing an authentic sample of tiliroside, and Dr M. Adamczeski for measuring the NMR data.
General. Mps: uncorr., ‘H and 13CNMR were taken in
pyridined, at 500 MHz for ‘H and 125 MHz for 13C. FAB-MS were obtained using glycerol matrix. PIant material. Dried aerial parts of B. coriacea (Quishuara) were provided by Promenat (Proyecto de Medicina Native), La Paz, Bolivia. Extraction and isolation. Dried ground aerial parts (330 g) were extracted with MeOH ( x 3) at ambient temp. for 10 days. After removal of the solvent, H,O (1 1)was added and the H,O based soln was partitioned with n-hexane, EtOAc and n-BuOH. The EtOAc extract (3.5 g) was chromatographed on silica gel using a CHCl, and MeOH gradient to obtain 5 frs. Subsequent bioassay indicated fr. 3 to be active. Hence, this bioactive fr. (46Omg) was further chromatographed on ODS eluted with M&H to afford buddlenoid A (2O.Omg) and buddlenoid B (31.2 mg). Buddlenoid A (1). C,,H,,O,,; amorphous powder (MeOH); mp 173-174”, UV (MeOH) 317, 267 nm (~28000, 22000), (+NaOMe) 375, 313, 275 nm (~20000, 26000, 30000); IR vKBr: 3300, 1690, 165Ocm-‘; FAB-MS m/z 593 [M-H]-, 459 [a-H]-; ‘H and ‘sCNMR are listed in Tables 1 and 2, respectively. Buddlenoid B (2). C31Has014; amorphous powder (MeOH); mp 165-166”, UV lMcOH 238, 267mn (~20000, ISOOO), (+NaOMe) 378,318,276nm (~14000,18000,22000); IR vKBr: 3400, 1646, 1605cm-‘; FAB-MS m/z 623 [M-H]-, 459
REFERENCES
1. Girault, L. (1978) in Kallawaya, p. 350. Unicef, Bolivia. 2. Neville, A. C. (1975) in Zoophysiology and Ecology 4/S. Springer, New York. 3. Fitzpatrick, T. B., Seiji, M. and McGugan, A. D. (1961) New Engl. J. Med. MS, 328, 374,430. 4. Pomerantz, S. H. (1963) J. Biol. Chem. 238, 2351. 5. Mason, H. S. and Peterson, E. W. (1965) B&hem. Biophys. Acta. 111, 134. 6. Markham, K. R. and Mabry, T. J. (1975) in The FZauonoids (Harborne, J. B., ed.), p. 45. Academic Press, New York. 7. Markham, K. R., Ternal, B., Stanley, R., Geiger, H. and Mabry, T. J. (1978) Tetrahedron 34, 1389. 8. Mabry, T. J. and Markham, K. R. (1975) in 7’he Flauonoids, (Harborne, J. B., ed.), p. 78. Academic Press, New York. 9. &Seth, von D. and Nordal, A. (1957) Pharm. Acta Helo. 32, 109. 10. H&hammer, L., Stich, L. and Wagner, H. (1961) Arch. Pharm. 294,685.
11. Harborne, J. B. (1964) Phytochemistry 3, 151. 12. Lin, J.-H., Lin, Y.-M. and Chen, F.-C. (1976) J. Chin. Chem. sot. 23, 57. 13. Kuroyanagi, M., Fukuoka, M., Yoshihira, K., Natori, S. and Yamasaki, K. (1978) Chem. Pharm. Bull. 26, 3594.