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Canthin-6-one and β-carboline alkaloids from Eurycoma harmandiana
Phytochemistry 56 (2001) 383±386
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Canthin-6-one and b-carboline alkaloids from Eurycoma harmandiana Tripetch Kanchanapoom a, Ryoji Kasai a, Phannipha Chumsri b, Yoshikazu Hiraga c, Kazuo Yamasaki a,* a
Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan b Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand c Department of Chemistry, Graduate School of Science, Hiroshima University 1-3-1, Kagamiyama, Higashi-Hiroshima 739-8526, Japan Received 31 May 2000; received in revised form 11 August 2000 Dedicated to Professor Vichiara Jirawongse on the occasion of his 82nd birthday
Abstract Two alkaloids, canthin-6-one 9-O-b-glucopyranoside and 7-hydroxy-b-carboline 1-propionic acid, were isolated from the roots of Eurycoma harmandiana, together with the ®ve known canthin-6-one alkaloids, 9-hydroxycanthin-6-one, 9-methoxycanthin-6-one, 9,10-dimethoxycanthin-6-one, canthin-6-one and canthin-6-one N-oxide, and the two known b-carboline alkaloids, b-carboline 1propionic acid and 7-methoxy-b-carboline 1-propionic acid. Their structures were based on analyses of spectroscopic data. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Eurycoma harmandiana; Simaroubaceae; Alkaloid; Canthin-6-one 9-O-b-glucopyranoside; 7-hydroxy-b-carboline 1-propionic acid; Canthin-6-one; b-Carboline
1. Introduction Eurycoma harmandiana Pierre (Simaroubaceae, Thai name: Ian-don) is a small shrub distributed on the border regions between Thailand and Laos. In North-eastern Thai traditional medicine, the root is used as a bitter tonic, as well as an aphrodisiac, anticancer and antimalarial agent. No phytochemical investigation has been carried out on this species. The present study deals with the isolation and structural elucidation of nine alkaloids (1±9), consisting of the two new alkaloids (1, 8) together with the known canthin-6-one alkaloids (2± 6) and the known b-carboline alkaloids (7,9) from the roots of this plant. 2. Results and discussion The ethanolic extract of the roots of E. harmandiana was suspended in H2O and extracted with CH2Cl2, * Corresponding author. Tel.: +81-82-257-5285; fax: 81-82-2575289. E-mail address: [email protected] u.ac.jp (K. Yamasaki).
EtOAc and n-BuOH, successively. The n-BuOH extract was subjected to a column of a highly porous copolymer of styrene and divinylbenzene, and eluted successively with H2O, MeOH and Me2CO. The fraction eluted with MeOH, and the CH2Cl2 extract were then repeatedly subjected to silica gel column chromatography, and reversed phase siliga gel chromatography to aord nine compounds (1±9). Five were identi®ed as the known canthin-6-one alkaloids; 9-hydroxycanthin-6-one (2), 9methoxycanthin-6-one (3) (Kardono et al., 1991), 9,10dimethoxycanthin-6-one (4) (Mitsunaga et al., 1994), canthin-6-one (5) (Koike and Ohmoto, 1985) and canthin-6-one N-oxide (6) (Ohmoto et al., 1981) together with the two known b-carboline alkaloids; b-carboline 1-propionic acid (7) and 7-methoxy-b-carboline 1-propionic acid (9) (Kardono et al., 1991) by analysis of both physical and spectroscopic data. Compound 1 was obtained as a yellow amorphous powder and determined as C20H18O7N2 by HR-FAB mass spectrometry. The 13C NMR spectrum revealed the presence of one b-glucopyranosyl unit in addition to 14 carbon signals for the aglycone. Enzymatic hydrolysis of 1 with crude hesperidinase (Kohda and Tanaka, 1975) gave 9-hydroxycanthin-6-one (2). The 1H NMR
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00363-0
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T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
spectrum of 1 showed two pairs of doublets ascribed to ortho-coupled signals at 8.15 (1H, d, J=5.0 Hz), 8.75 (1H, d, J=5.0 Hz) and at 8.08 (1H, d, J=9.8 Hz), 6.93 (1H, d, J=9.8 Hz), and the AMX type signals at 8.12 (1H, d, J=2.0 Hz), 7.27 (1H, dd, J=8.6, 2.0 Hz) and 8.23 (1H, d, J=8.6 Hz). These spectral data were similar to those of 2, except that the 1H NMR signals for H-8 and H-10 were down®eld shifted by about 0.16 and 0.30 ppm, respectively, which established the attachment of the glucosyl unit to C-9. Furthermore, the 13C NMR spectrum also revealed the attachment of a glucosyl unit to C-9 due to the down®eld shift of C-9 (0.9 ppm) together with up®eld shifts of C-8 (1.6 ppm) and C-10 (2.5 ppm). Thus, compound 1 was concluded to be canthin-6-one 9-O-b-glucopyranoside. Compound 8 was obtained as an amorphous powder and determined as C14H12O3N2 by HR-FAB mass spectrometry. The 1H NMR spectrum revealed a pair of vicinal protons at 8.13 (1H, d, J=5.3 Hz) and 7.74 (1H, d, J=5.3 Hz), and the AMX type signals at 7.93 (1H, d, J=8.6 Hz), 6.69 (1H, dd, J=8.6, 2.0 Hz) and 6.90 (1H, d, J=2.0 Hz) together with two sets of methylene protons [ 2.82 (2H, t, J=7.3 Hz) and 3.27 (2H, t, J=7.3 Hz)]. The 1H and 13C NMR spectra were very similar to those of 7-methoxyl-b-carboline 1-propionic acid (9) but no methoxy signal was observed, indicating the presence of a hydroxyl group. The hydroxyl group was assigned to C-7 by the irradiation of N±H at 11.28 which induced a 5% NOE at H-8 [ 6.90 (1H, d, J=2.0 Hz)]. Consequently, 7-hydroxy-b-carboline 1propionic acid was proposed for compound 2. The biological activities of the isolated compounds have not been investigated. However, there are own biological activities for the known compounds 2, 3, 5, and 6 which are cytotoxic (Anderson et al., 1983; Kardono et al., 1991), and compound 7 (Kardono et al.,
1991) has antimalarial activity. These activities are in agreement with their traditional uses in North-east Thailand. 3. Experimental 3.1. General NMR spectra were recorded in DMSO-d6 or CDCl3 using a JEOL JNM A-400 spectrometer (400 MHz for 1 H NMR and 100 MHz for 13C NMR) and JNM-ECP 500 (500 MHz for 1H NMR and 125 MHz for 13C NMR) with tetramethylsilane (TMS) as internal standard. MS were recorded on a JEOL JMS-SX 102 spectrometer. For CC, silica gel G 60 (Merck), RP-18 (50 mm, YMC) and highly porous copolymer of styrene and divinylbenzene (Mitsubishi Chem. Ind. Co. Ltd) were used. The solvent systems were: (I) EtOAc±MeOH (9:1), (II) EtOAc±MeOH±H2O (4:1:0.1), (III) EtOAc± MeOH±H2O (7:3:0.3), (IV) EtOAc±MeOH±H2O (6:4:1), (V) 30±50% MeOH, (VI) CH2Cl2, (VII) CH2Cl2±MeOH (19:1), (VIII) CH2Cl2±MeOH (9:1), (IX) CH2Cl2±MeOH (4:1) and (X) 40±100% MeOH. 3.2. Plant material The roots of E. harmandiana were collected in May, 1998 from the Nong Khai Province, North-east Thailand. The identity of the plant was con®rmed by Professor Vichiara Jirawongse, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher sample is kept in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.
T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
385
Table 1 1 H NMR spectral data of compounds 1 and 8 (400 MHz, DMSO-d6) H
1
1 2 4 5 8 10 11 Glc-1
8.15 (1H, 8.75 (1H, 8.08 (1H, 6.93 (1H, 8.12 (1H, 7.27 (1H, 8.23 (1H, 5.02 (1H,
d, J=5.0 Hz) d, J=5.0 Hz) d, J=9.8 Hz) d, J=9.8 Hz) d, J=2.0 Hz) dd, J=8.6, 2.0 Hz) d, J=8.6 Hz) d, J=8.0 Hz)
Table 2 13 C NMR spectral data of compounds 1 and 8 (100 MHz, DMSO-d6) C
1
C
8
1 2 4 5 6 8 9 10 11 12 13 14 15 16 G-1 G-2 G-3 G-4 G-5 G-6
114.3 145.9 139.9 128.2 158.9 104.5 159.7 116.5 124.4 118.2 139.9 129.3 131.8 135.3 101.0 73.2 77.2 69.5 76.5 60.5
1 3 4 5 6 7 8 9 10 11 12 10 20 30
142.9 137.4 111.8 122.5 109.7 158.3 96.6 142.3 113.8 127.8 133.9 31.6 27.9 174.2
H
8
3 4 5 6 8 10 20 N±H
8.13 (1H, d, J=5.3 Hz) 7.74 (1H, d, J=5.3 Hz) 7.93 (1H, d, J=8.6 Hz) 6.69 (1H, dd, J=8.6, 2.0 Hz) 6.90 (1H, d, J=2.0 Hz) 2.82 (2H, t, J=7.3 Hz) 3.27 (2H, t, J=7.3 Hz) 11.28 (1H, bs)
compounds 2 (367.0 mg), 3 (900.0 mg), 4 (8.0 mg), 5 (250.4 mg) and 6 (27.5 mg). 3.4. Compound 1 Yellow amorphous powder. UV lMeOH+HCl (log ") max nm: 285 (3.16), 321 (3.14), 367 (3.22); 1H NMR: Table 1; 13C NMR: Table 2; Positive HR-FAB-MS, m/z: 399.1205 [M+H]+ (C20H19O7N2 requires 399.1192). 3.5. Enzymatic hydrolysis of compound 1 Compound 1 (25.0 mg) was dissolved in 0.5 ml of DMSO. A solution of crude hesperidinase (Kohda and Tanaka, 1975) (100 mg in 20 ml of H2O) was added. After stirring at 37 C for 12 h, the mixture was extracted with CH2Cl2. The CH2Cl2 extract was evaporated to provide 9-hydroxycanthin-6-one (2) (14.0 mg) whose structure was identi®ed by 1H and 13C NMR spectra analysis. 3.6. Compound 2
3.3. Extraction and isolation The dried root (7.0 kg) of E. harmandiana was extracted with 95% EtOH. After removal of the solvent by evaporation, the residue (200 g) was extracted with CH2Cl2, EtOAc and n-BuOH, successively. The n-BuOH extract (45.0 g) was subjected to a column of highly porous copolymer of styrene and divinylbenzene, and eluted with H2O, MeOH and Me2CO, successively. The fraction eluted with MeOH was chromatographed on a column of silica gel (systems I, II, III and IV, successively) aording nine fractions. Fraction 5 and 6 were further puri®ed by RP-18 using system V to provide compounds 1 (75.0 mg), 7 (35.0 mg), 8 (30.7 mg) and 9 (20.7 mg). The CH2Cl2 extract (90.0 g) was subjected to a column of silica gel (systems VI, VII, VIII and IX) aording six fractions. Fraction 2 was subjected to repeated chromatography on silica gel column using a gradient system (CH2Cl2 to 5% MeOH in CH2Cl2) followed by RP-18 reversed phase column chromatography to aord
nm (log "): 300 Amorphous powder. UV lMeOH+HCl max (2.74), 318 (2.76), 346 (2.80); 1H NMR: Table 1; 13C NMR: Table 2; Positive HR-FAB-MS, m/z: 257.0891 [M+H]+ (C14H13O3N2 requires 257.0926). Acknowledgements We would like to thank the Research Center for Molecular Medicine, Hiroshima University for the use of its NMR facilities. References Anderson, L.A., Harris, A., Phillipson, J.D., 1983. Production of cytotoxic canthin-6-one alkaloids by Ailanthus altissima plant cell cultures. Journal of Natural Products 46 (3), 374±378. Kardono, L.B.S., Angerhofer, C.K., Tsauri, S., Padmawinata, K., Pezzuto, J.M., Kinghorn, A.D., 1991. Cytotoxic and antimalarial constituents of the roots of Eurycoma longifolia. Journal of Natural Products 54 (5), 1360±1367.
386
T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
Kohda, H., Tanaka, O., 1975. Enzymatic hydrolysis of ginseng saponins and their related glycosides. Yakugaku Zasshi 95 (2), 246±249. Koike, K., Ohmoto, T., 1985. Carbon-13 nuclear magnetic resonance study of canthin-6-one alkaloids. Chemical & Pharmaceutical Bulletin 33 (12), 5239±5244.
Mitsunaga, K. et al., 1994. Canthin-6-one alkaloids from Eurycoma longifolia. Phytochemistry 35 (3), 799±802. Ohmoto, T., Koike, K., Sakamoto, Y., 1981. Studies on the constituents of Ailanthus altissima Swingle. II. Akaloidal constituents. Chemical & Pharmaceutical Bulletin 29 (2), 390±395.
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Canthin-6-one and b-carboline alkaloids from Eurycoma harmandiana Tripetch Kanchanapoom a, Ryoji Kasai a, Phannipha Chumsri b, Yoshikazu Hiraga c, Kazuo Yamasaki a,* a
Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan b Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok, Thailand c Department of Chemistry, Graduate School of Science, Hiroshima University 1-3-1, Kagamiyama, Higashi-Hiroshima 739-8526, Japan Received 31 May 2000; received in revised form 11 August 2000 Dedicated to Professor Vichiara Jirawongse on the occasion of his 82nd birthday
Abstract Two alkaloids, canthin-6-one 9-O-b-glucopyranoside and 7-hydroxy-b-carboline 1-propionic acid, were isolated from the roots of Eurycoma harmandiana, together with the ®ve known canthin-6-one alkaloids, 9-hydroxycanthin-6-one, 9-methoxycanthin-6-one, 9,10-dimethoxycanthin-6-one, canthin-6-one and canthin-6-one N-oxide, and the two known b-carboline alkaloids, b-carboline 1propionic acid and 7-methoxy-b-carboline 1-propionic acid. Their structures were based on analyses of spectroscopic data. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Eurycoma harmandiana; Simaroubaceae; Alkaloid; Canthin-6-one 9-O-b-glucopyranoside; 7-hydroxy-b-carboline 1-propionic acid; Canthin-6-one; b-Carboline
1. Introduction Eurycoma harmandiana Pierre (Simaroubaceae, Thai name: Ian-don) is a small shrub distributed on the border regions between Thailand and Laos. In North-eastern Thai traditional medicine, the root is used as a bitter tonic, as well as an aphrodisiac, anticancer and antimalarial agent. No phytochemical investigation has been carried out on this species. The present study deals with the isolation and structural elucidation of nine alkaloids (1±9), consisting of the two new alkaloids (1, 8) together with the known canthin-6-one alkaloids (2± 6) and the known b-carboline alkaloids (7,9) from the roots of this plant. 2. Results and discussion The ethanolic extract of the roots of E. harmandiana was suspended in H2O and extracted with CH2Cl2, * Corresponding author. Tel.: +81-82-257-5285; fax: 81-82-2575289. E-mail address: [email protected] u.ac.jp (K. Yamasaki).
EtOAc and n-BuOH, successively. The n-BuOH extract was subjected to a column of a highly porous copolymer of styrene and divinylbenzene, and eluted successively with H2O, MeOH and Me2CO. The fraction eluted with MeOH, and the CH2Cl2 extract were then repeatedly subjected to silica gel column chromatography, and reversed phase siliga gel chromatography to aord nine compounds (1±9). Five were identi®ed as the known canthin-6-one alkaloids; 9-hydroxycanthin-6-one (2), 9methoxycanthin-6-one (3) (Kardono et al., 1991), 9,10dimethoxycanthin-6-one (4) (Mitsunaga et al., 1994), canthin-6-one (5) (Koike and Ohmoto, 1985) and canthin-6-one N-oxide (6) (Ohmoto et al., 1981) together with the two known b-carboline alkaloids; b-carboline 1-propionic acid (7) and 7-methoxy-b-carboline 1-propionic acid (9) (Kardono et al., 1991) by analysis of both physical and spectroscopic data. Compound 1 was obtained as a yellow amorphous powder and determined as C20H18O7N2 by HR-FAB mass spectrometry. The 13C NMR spectrum revealed the presence of one b-glucopyranosyl unit in addition to 14 carbon signals for the aglycone. Enzymatic hydrolysis of 1 with crude hesperidinase (Kohda and Tanaka, 1975) gave 9-hydroxycanthin-6-one (2). The 1H NMR
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(00)00363-0
384
T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
spectrum of 1 showed two pairs of doublets ascribed to ortho-coupled signals at 8.15 (1H, d, J=5.0 Hz), 8.75 (1H, d, J=5.0 Hz) and at 8.08 (1H, d, J=9.8 Hz), 6.93 (1H, d, J=9.8 Hz), and the AMX type signals at 8.12 (1H, d, J=2.0 Hz), 7.27 (1H, dd, J=8.6, 2.0 Hz) and 8.23 (1H, d, J=8.6 Hz). These spectral data were similar to those of 2, except that the 1H NMR signals for H-8 and H-10 were down®eld shifted by about 0.16 and 0.30 ppm, respectively, which established the attachment of the glucosyl unit to C-9. Furthermore, the 13C NMR spectrum also revealed the attachment of a glucosyl unit to C-9 due to the down®eld shift of C-9 (0.9 ppm) together with up®eld shifts of C-8 (1.6 ppm) and C-10 (2.5 ppm). Thus, compound 1 was concluded to be canthin-6-one 9-O-b-glucopyranoside. Compound 8 was obtained as an amorphous powder and determined as C14H12O3N2 by HR-FAB mass spectrometry. The 1H NMR spectrum revealed a pair of vicinal protons at 8.13 (1H, d, J=5.3 Hz) and 7.74 (1H, d, J=5.3 Hz), and the AMX type signals at 7.93 (1H, d, J=8.6 Hz), 6.69 (1H, dd, J=8.6, 2.0 Hz) and 6.90 (1H, d, J=2.0 Hz) together with two sets of methylene protons [ 2.82 (2H, t, J=7.3 Hz) and 3.27 (2H, t, J=7.3 Hz)]. The 1H and 13C NMR spectra were very similar to those of 7-methoxyl-b-carboline 1-propionic acid (9) but no methoxy signal was observed, indicating the presence of a hydroxyl group. The hydroxyl group was assigned to C-7 by the irradiation of N±H at 11.28 which induced a 5% NOE at H-8 [ 6.90 (1H, d, J=2.0 Hz)]. Consequently, 7-hydroxy-b-carboline 1propionic acid was proposed for compound 2. The biological activities of the isolated compounds have not been investigated. However, there are own biological activities for the known compounds 2, 3, 5, and 6 which are cytotoxic (Anderson et al., 1983; Kardono et al., 1991), and compound 7 (Kardono et al.,
1991) has antimalarial activity. These activities are in agreement with their traditional uses in North-east Thailand. 3. Experimental 3.1. General NMR spectra were recorded in DMSO-d6 or CDCl3 using a JEOL JNM A-400 spectrometer (400 MHz for 1 H NMR and 100 MHz for 13C NMR) and JNM-ECP 500 (500 MHz for 1H NMR and 125 MHz for 13C NMR) with tetramethylsilane (TMS) as internal standard. MS were recorded on a JEOL JMS-SX 102 spectrometer. For CC, silica gel G 60 (Merck), RP-18 (50 mm, YMC) and highly porous copolymer of styrene and divinylbenzene (Mitsubishi Chem. Ind. Co. Ltd) were used. The solvent systems were: (I) EtOAc±MeOH (9:1), (II) EtOAc±MeOH±H2O (4:1:0.1), (III) EtOAc± MeOH±H2O (7:3:0.3), (IV) EtOAc±MeOH±H2O (6:4:1), (V) 30±50% MeOH, (VI) CH2Cl2, (VII) CH2Cl2±MeOH (19:1), (VIII) CH2Cl2±MeOH (9:1), (IX) CH2Cl2±MeOH (4:1) and (X) 40±100% MeOH. 3.2. Plant material The roots of E. harmandiana were collected in May, 1998 from the Nong Khai Province, North-east Thailand. The identity of the plant was con®rmed by Professor Vichiara Jirawongse, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher sample is kept in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.
T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
385
Table 1 1 H NMR spectral data of compounds 1 and 8 (400 MHz, DMSO-d6) H
1
1 2 4 5 8 10 11 Glc-1
8.15 (1H, 8.75 (1H, 8.08 (1H, 6.93 (1H, 8.12 (1H, 7.27 (1H, 8.23 (1H, 5.02 (1H,
d, J=5.0 Hz) d, J=5.0 Hz) d, J=9.8 Hz) d, J=9.8 Hz) d, J=2.0 Hz) dd, J=8.6, 2.0 Hz) d, J=8.6 Hz) d, J=8.0 Hz)
Table 2 13 C NMR spectral data of compounds 1 and 8 (100 MHz, DMSO-d6) C
1
C
8
1 2 4 5 6 8 9 10 11 12 13 14 15 16 G-1 G-2 G-3 G-4 G-5 G-6
114.3 145.9 139.9 128.2 158.9 104.5 159.7 116.5 124.4 118.2 139.9 129.3 131.8 135.3 101.0 73.2 77.2 69.5 76.5 60.5
1 3 4 5 6 7 8 9 10 11 12 10 20 30
142.9 137.4 111.8 122.5 109.7 158.3 96.6 142.3 113.8 127.8 133.9 31.6 27.9 174.2
H
8
3 4 5 6 8 10 20 N±H
8.13 (1H, d, J=5.3 Hz) 7.74 (1H, d, J=5.3 Hz) 7.93 (1H, d, J=8.6 Hz) 6.69 (1H, dd, J=8.6, 2.0 Hz) 6.90 (1H, d, J=2.0 Hz) 2.82 (2H, t, J=7.3 Hz) 3.27 (2H, t, J=7.3 Hz) 11.28 (1H, bs)
compounds 2 (367.0 mg), 3 (900.0 mg), 4 (8.0 mg), 5 (250.4 mg) and 6 (27.5 mg). 3.4. Compound 1 Yellow amorphous powder. UV lMeOH+HCl (log ") max nm: 285 (3.16), 321 (3.14), 367 (3.22); 1H NMR: Table 1; 13C NMR: Table 2; Positive HR-FAB-MS, m/z: 399.1205 [M+H]+ (C20H19O7N2 requires 399.1192). 3.5. Enzymatic hydrolysis of compound 1 Compound 1 (25.0 mg) was dissolved in 0.5 ml of DMSO. A solution of crude hesperidinase (Kohda and Tanaka, 1975) (100 mg in 20 ml of H2O) was added. After stirring at 37 C for 12 h, the mixture was extracted with CH2Cl2. The CH2Cl2 extract was evaporated to provide 9-hydroxycanthin-6-one (2) (14.0 mg) whose structure was identi®ed by 1H and 13C NMR spectra analysis. 3.6. Compound 2
3.3. Extraction and isolation The dried root (7.0 kg) of E. harmandiana was extracted with 95% EtOH. After removal of the solvent by evaporation, the residue (200 g) was extracted with CH2Cl2, EtOAc and n-BuOH, successively. The n-BuOH extract (45.0 g) was subjected to a column of highly porous copolymer of styrene and divinylbenzene, and eluted with H2O, MeOH and Me2CO, successively. The fraction eluted with MeOH was chromatographed on a column of silica gel (systems I, II, III and IV, successively) aording nine fractions. Fraction 5 and 6 were further puri®ed by RP-18 using system V to provide compounds 1 (75.0 mg), 7 (35.0 mg), 8 (30.7 mg) and 9 (20.7 mg). The CH2Cl2 extract (90.0 g) was subjected to a column of silica gel (systems VI, VII, VIII and IX) aording six fractions. Fraction 2 was subjected to repeated chromatography on silica gel column using a gradient system (CH2Cl2 to 5% MeOH in CH2Cl2) followed by RP-18 reversed phase column chromatography to aord
nm (log "): 300 Amorphous powder. UV lMeOH+HCl max (2.74), 318 (2.76), 346 (2.80); 1H NMR: Table 1; 13C NMR: Table 2; Positive HR-FAB-MS, m/z: 257.0891 [M+H]+ (C14H13O3N2 requires 257.0926). Acknowledgements We would like to thank the Research Center for Molecular Medicine, Hiroshima University for the use of its NMR facilities. References Anderson, L.A., Harris, A., Phillipson, J.D., 1983. Production of cytotoxic canthin-6-one alkaloids by Ailanthus altissima plant cell cultures. Journal of Natural Products 46 (3), 374±378. Kardono, L.B.S., Angerhofer, C.K., Tsauri, S., Padmawinata, K., Pezzuto, J.M., Kinghorn, A.D., 1991. Cytotoxic and antimalarial constituents of the roots of Eurycoma longifolia. Journal of Natural Products 54 (5), 1360±1367.
386
T. Kanchanapoom et al. / Phytochemistry 56 (2001) 383±386
Kohda, H., Tanaka, O., 1975. Enzymatic hydrolysis of ginseng saponins and their related glycosides. Yakugaku Zasshi 95 (2), 246±249. Koike, K., Ohmoto, T., 1985. Carbon-13 nuclear magnetic resonance study of canthin-6-one alkaloids. Chemical & Pharmaceutical Bulletin 33 (12), 5239±5244.
Mitsunaga, K. et al., 1994. Canthin-6-one alkaloids from Eurycoma longifolia. Phytochemistry 35 (3), 799±802. Ohmoto, T., Koike, K., Sakamoto, Y., 1981. Studies on the constituents of Ailanthus altissima Swingle. II. Akaloidal constituents. Chemical & Pharmaceutical Bulletin 29 (2), 390±395.