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Quassinoids from Eurycoma harmandiana
Phytochemistry 57 (2001) 1205–1208 www.elsevier.com/locate/phytochem
Quassinoids from Eurycoma harmandiana Tripetch Kanchanapooma,b, Ryoji Kasaia, Phannipha Chumsric, Kazuo Yamasakia,* a Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
b
Received 21 December 2000; received in revised form 12 April 2001 Dedicated to Professor Vichiara Jirawongse on the occasion of his 83rd birthday
Abstract Three quassinoids, iandonosides A and B and iandonone, were isolated from the root of Eurycoma harmandiana, along with five known quassinoids, casteloside B, 13 b, 21-dihydroeurycomanone, chaparrinone, glaucarubolone and ailanquassin B as well as the coumarin, scopoletin. The structural elucidations were based on analyses of spectroscopic data. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Eurycoma harmandiana; Simaroubaceae; Quassinoids, Iandonoside A; Iandonoside B; Iandonone; Iandonol
1. Introduction Eurycoma harmandiana Pierre is a small Simaroubaceous plant (Thai name: Ian-don) distributed in the border regions between Thailand and Laos. In a previous paper (Kanchanapoom et al., 2001), we reported the isolation and structural elucidation of canthin-6-one and b-carboline alkaloids. Further investigation of the root of the same plant afforded nine compounds (1–9), which included three new unusual 15a-OH quassinoids (2, 3, 5) together with five known quassinoids (1, 4, 6–8) and one known coumarin (9). The present paper deals with the structural determination of these compounds.
2. Results and discussion From the ethanolic extract of the roots of E. harmandiana, nine compounds (1–9) were isolated. Five were identified as known quassinoids; casteloside B (1) (Chaudhuri and Kubo, 1992), l3b,21-dihydroeurycomanone (4) (Morita et al., 1990), chaparrinone (6), glaucarubolone (7) (Chaudhuri and Kubo, 1992) and ailanquassin B (8) (Aono et al., 1994) together with one
* Corresponding author. Tel.: +81-82-257-5285; fax: +81-82-2575289. E-mail address: [email protected] (K. Yamasaki).
known coumarin, scopoletin (9) by physical data and spectroscopic evidence. Iandonoside A was obtained as an amorphous powder and determined as C26H38O13 by HR–FAB mass spectrometry. The 13C NMR spectrum revealed the presence of one b-glucopyranosyl unit together with 20 carbon signals for the aglycone, which suggested a C20 quassinoid skeleton. DEPT experiments indicated the presence of three methyls, two methylenes, 10 methines, as well as five quaternary carbons in the aglycone moiety. The signals at 72.3 (C-20) and 110.9 (C-11) in the 13 C NMR spectrum were characteristic of an 11b,20hemiketal moiety. Comparison of the 1H and 13C NMR spectral data of 2 with those of 1 revealed the same relative arrangement of carbons and protons in rings A and B. The chemical shifts of rings C and D suggested differences in the relative configuration of hydroxyl group at C-15. The complete assignments were established by using HSQC, NOESY, difference NOE spectral experiments (Fig. 1) and the coupling constants in the 1H NMR spectrum (Table 1). Irradiation of the H20 signal ( 3.70) gave rise to NOE enhancements at H13 ( 2.81), H-14 ( 2.10) indicating the presence of aconfiguration of Me-21 ( 1.24) and b-configuration of H-14. The coupling constant of H-12 ( 3.97, d, J=5.0 Hz) confirmed the a-configuration for the hydroxyl group at C-12. The configuration of the hydroxyl group at C-15 was assigned as a by the coupling constant of H-15 ( 4.50, d, J=4.6 Hz), and further support was
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00235-7
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Fig. 1. NOESY and NOE enhancements of 2, 3 and 5.
obtained by irradiation of the signal at H-15 which exhibited the NOE enhancements at H-14 and Me-21 but no enhancement at H-9 ( 3.53). Consequently, the structure of 2 was elucidated as shown. Iandonoside B (3) was obtained as an amorphous powder and determined as C26H38O14 by HR–FAB
mass spectrometry. Inspection of the 13C NMR spectrum showed the presence of one b-glucopyranosyl unit and 20 carbon signals for the aglycone, which indicated it to be a C20 quassinoid glucoside. On enzymatic hydrolysis with crude hesperidinase (Kohda and Tanaka, 1975), 3 gave the new aglycone, named iandonol (3a). On going from 3 to 3a, the chemical shifts were changed for C-2, C-1 and C-3 by +11.2, 1.3 and 2.3 ppm, respectively. This confirmed the location of the glucopyranosyl moiety on C-2. The 1H and 13C NMR spectral data were similar with those of 2. However, the signal for H-14 was absent as well as the signal for H-15 ( 4.73) appeared as singlet. In addition, the carbon signals for C-l4, C-13 and C-15 showed different chemical shifts at 76.6, 41.4 and 76.1 (Table 2), respectively, indicating the replacement of a proton (H-14) by a hydroxyl group. The relative configurations were confirmed by using the coupling constants in the 1H NMR spectrum (Table 1), NOESY and difference NOE spectral experiments (Fig. 1). On irradiation of the signal at 2.95 (H-13), the intensity of H-20 ( 4.53) was enhanced, and irradiation of the H-15 signal ( 4.73) caused as determined increase in the NOE enhancement at Me-21 only ( 1.45). Upon irridiation of the H-9 signal ( 3.88), NOE enhancement was observed for the H-5 a signal ( 2.76) but not for either the H-12 ( 4.17) or H-15 ( 4.73) signals. Thus, the stereochemistry of the hydroxyl groups at C-12 and C-15 were assigned as a, a-configurations, and the also Me-21 has an a-configuration. On the basis of these spectral data, the structure of 3 was determined as the 14b-hydroxy analogue of iandonoside A. Iandonone (5) was obtained as an amorphous powder, with a molecular formula of C20H24O9 by HR– FAB mass spectrometry. The 13C NMR spectrum revealed the presence of 20 carbons, including a -lactone carbonyl ( 171.9), an a,b-unsaturated ketone carbonyl carbon ( 197.4) and two olefinic carbons ( 126.2 and 162.2) indicative of a C20 quassinoid skeleton. Iandonone showed the same chemical shifts for rings B, C and D with those of 3, and the chemical shifts of ring A were in full agreement with those of 4. The stereochemistry of three hydroxyl groups at C-1, C-12 and C-15 were also confirmed by using NOESY difference NOE experiments (Fig. 1) and the coupling constants in the 1H NMR spectrum (Table 1), led to deduce as b,a,aconfigurations, respectively. Therefore, the structure of 5 was elucidated as shown.
3. Experimental 3.1. General NMR spectra were recorded in C5D5N using a JEOL JNM A-400 spectrometer (400 MHz for 1H NMR and
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T. Kanchanapoom et al. / Phytochemistry 57 (2001) 1205–1208 Table 1 1 H NMR spectral data for compounds 2, 3, 3a and 5 (C5D5N, 400 MHz) Proton
2
3
3a
5
H-1 H-2 H-3 H-5 H-6e H-6a H-7 H-9 H-12 H-13 H-14 H-15 H-18 H-19 H-20
4.02, d, J=8.0 Hz 4.58, m 5.77, bs 2.67, bd, J=14.0 Hz 2.02, dt, J=14.0, 2.7 Hz 1.86, td, J=14.0, 2.7 Hz 4.46 t, J=2.7 Hz 3.53, s 3.97, d, J=5.0 Hz 2.81, m 2.10, dd, J=7.8, 5.1 Hz 4.50, d, J=4.6 Hz 1.43, bs 1.54, s 3.70, d, J=8.6 Hz 4.10, d, J=8.6 Hz 1.24, d, J=7.1 Hz 5.19, d, J=7.6 Hz 4.06, dd, J=8.1, 7.6 Hz 4.19, dd, J=8.5, 8.1 Hz 4.24, dd, J=9.1, 8.5 Hz 3.95, m 4.54, bd, J=11.5 Hz 4.38, dd, J=11.5, 4.7 Hz
4.14, d, J=8.0 Hz 4.57, m 5.80, bs 2.76, bd, J=14.0 Hz 2.13, dt, J=140, 2.7 Hz 1.89, td, J=14.0, 2.7 Hz 5.06, t, J=2.7 Hz 3.88, s 4.17, d, J=4.9 Hz 2.95, m – 4.73, s 1.44, bs 1.61, s 4.03, d, J=9.5 Hz 4.53, d, J=9.5 Hz 1.45, d, J=7.1 Hz 5.18, d, J=7.8 Hz 4.06, dd, J=8.5, 7.8 Hz 4.17, dd, J=9.0, 8.5 Hz 4.20, dd, J=9.0, 8.5 Hz 3.94, m 4.51, dd, J=12.0, 2.6 Hz 4.35, dd, J=12.0, 5.1 Hz
4.11, d, J=7.8 Hz 4.62, m 5.78, bs 2.82, bd, J=13.4 Hz 2.18, dt, J=14.0, 2.5 Hz 1.96, td, J=14.0, 2.5 Hz
4.36, s – 6.11, bs 3.15, bd, J=14.0 Hz 2.31, dt, J=14.0, 2.2 Hz 2.04, td, J=14.0, 2.2 Hz 5.13, bs 4.13, s 4.08, d, J=5.1 Hz 2.97, m – 4.80, s 1.76, bs 1.57, s 4.04, d, J=9.0 Hz 4.53, d, J=9.0 Hz 1.48, d, J=7.1 Hz
H-21 H-10 H-20 H-30 H-40 H-50 H-60
a
3.94, 4.11, 3.00, – 4.75, 1.59, 1.72, 4.03, 4.56, 1.49,
s d, J=5.5 Hz m s bs s d, J=8.8 Hz d, J=8.8 Hz d, J=7.3 Hz
a Interferred signal.
Table 2 13 C NMR spectral data for compounds 1–3, 3a and 5 (C5D5N, 100 MHz) Carbon
1
2
3
3a
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 G-1 G-2 G-3 G-4 G-5 G-6
82.6 84.1 124.6 136.0 41.3 26.0 78.6 47.4 45.5 42.0 111.0 80.5 33.1 49.6 68.7 174.3 21.0 10.3 71.8 16.3 106.4 76.1 78.6 71.6 78.5 62.7
82.8 83.7 124.9 135.7 41.6 26.0 79.3 44.8 45.9 42.3 110.9 78.3 31.9 48.5 65.4 171.2 21.1 10.7 72.3 13.0 106.3 76.2 78.6 71.6 78.5 62.7
82.8 83.8 124.8 135.5 41.2 25.7 71.5 50.0 44.8 42.3 110.3 78.8 41.4 76.6 76.1 172.0 21.1 10.9 67.9 10.1 106.2 76.3 78.5 70.7 78.5 62.7
84.1 72.6 127.1 135.0 41.4 25.9 70.8 50.1 45.0 42.3 110.5 79.1 41.9 76.7 76.5 172.1 21.3 11.1 67.9 10.2
84.8 197.4 126.2 162.2 42.5 25.9 70.9 49.9 45.3 45.7 110.1 78.7 41.1 76.6 75.9 171.9 22.4 10.6 67.7 10.1
100 MHz for 13C NMR) with tetramethylsilane (TMS) as internal standard. MS were recorded on a JEOL JMS-SX 102 spectrometer. Preparative HPLC was carried out on columns of ODS (15020 mm i.d., YMC) and polyamine II (25020 mm i.d., YMC) with a Tosoh refraction index (RI-8) detector. The flow rate was 6 ml/ min. For CC, silica gel G 60 (Merck), RP-l8 (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) 80% MeCN, (VII) 12% MeCN, (VIII) 10% MeCN, (IX) CH2Cl2, (X) CH2C12–MeOH (19:1), (XI) CH2Cl2–MeOH (9:1), (XII) CH2Cl2–MeOH (4:1), (XIII) 40–100% MeOH, (XIV) 50–100% MeOH. 3.2. Plant material The root of Eurycoma harmandiana was collected in May 1998 from Nong Khai Province, north-eastern Thailand. The identification of the plant was confirmed by Professor Vichiara Jirawongse, Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher sample (KKU-0017) is kept in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.
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3.3. Extraction and isolation
3.6. Enzymatic hydrolysis of iandonoside B (3)
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 applied to a silica gel column (systems I, II, III and IV, successively) affording nine fractions. Fraction 5 was subjected to a column of RP18 using system V, then followed by HPLC-polyamine II (system VI) and HPLC–ODS (system VII) to provide compounds 1 (5 mg), 2 (4 mg) and 3 (30 mg). The EtOAc extract (27.5 g) was further separated on a column of RP-18 (system V), and finally purified by HPLC–ODS (system VIII) to give compounds 4 (84 mg) and 5 (21 mg). The CH2C12 extract (90.0 g) was subjected to a column of a silica gel (systems IX, X, XI and XII) affording six fractions. Fraction 2 was repeatedly chromatographed on a silica gel column using gradient system (CH2Cl2 to 5% MeOH in CH2Cl2) to provide six fractions. Fraction 2-2 was further purified by using a RP- 18 column (system XIII) to afford compounds 9 (53 mg). Fraction 2-4 was similarly purified by using a RP18 column (system XIV) to give compound 6 (268 mg). Fraction 2-6 was applied to a column of silica gel (system I), this being followed by HPLC–ODS (system VII) to afford compounds 7 (24 mg) and 8 (13 mg).
Iandonoside B (3) 8 mg was dissolved in 0.5 ml of MeOH. A solution of crude hesperidinase (100 mg in 20 ml of H2O) was added. After shaking at 37 C for 24 h, the mixture was extracted with EtOAc. The EtOAc extract was evaporated to provide a new aglycone, iandonol (3a) 3 mg. The structure was identified by 1H and 13 C NMR spectral analysis.
3.4. Iandonoside A (2) 1 Amorphous powder. [a]20 D +19.1 (MeOH, c 0.3); H 13 NMR: Table 1; C NMR: Table 2; negative HR–FAB– MS, m/z: 557.2258 [M–H] (C26H37O13 requires 557.2234).
3.5. Iandonoside B (3) 1 Amorphous powder. [a]20 D +4.1 (MeOH, c 0.7); H 13 NMR: Table 1; C NMR: Table 2; negative HR–FAB– MS, m/z: 573.2148 [M–H] (C26H37O14 requires 573.2183).
3.7. Iandonol (3a) 1 Amorphous powder. [a]20 D 72.6 (MeOH, c 0.2); H 13 NMR: Table 1; C NMR: Table 2; positive HR–FAB– MS, m/z: 411.1691 [M–H], (C20H27O9 requires 411.1655).
3.8. Iandonone (5) 1 Amorphous powder. [a]20 D +4.6 (MeOH, c 0.4); H 13 NMR: Table 1; C NMR: Table 2; positive HR–FAB– MS, m/z: 409.1468 [M–H] (C20H25O9 requires 409.1498).
Acknowledgements We would like to thank the Research Center for Molecular Medicine, Hiroshima University for the use of its NMR facilities. References Aono, H., Koike, K., Kaneko, J., Ohmoto, T., 1994. Alkaloids and quassinoids from Ailanthus malabarica. Phytochemistry 37, 579– 584. Chaudhuri, S.K., Kubo, I., 1992. Two quassinoid glucosides from Castela tortuosa. Phytochemistry 31, 3961–3964. Kanchanapoom, T., Kasai, R., Chumsri, P., Hiraga, Y., Yamasaki, K., 2001. Canthin-6-one and b-carboline alkaloids from Eurycoma harmandiana. Phytochemistry 56, 383–386. Kohda, H., Tanaka, O., 1975. Enzymatic hydrolysis of ginseng saponins and their related glycosides. Yakugaku Zasshi 95, 246– 249. Morita, H., Kishi, E., Takeya, K., Itokawa, H., Tanaka, O., 1990. New quassinoids from the roots of Eurycoma longifolia. Chemistry Letters 749–752.
Quassinoids from Eurycoma harmandiana Tripetch Kanchanapooma,b, Ryoji Kasaia, Phannipha Chumsric, Kazuo Yamasakia,* a Institute of Pharmaceutical Sciences, Faculty of Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
b
Received 21 December 2000; received in revised form 12 April 2001 Dedicated to Professor Vichiara Jirawongse on the occasion of his 83rd birthday
Abstract Three quassinoids, iandonosides A and B and iandonone, were isolated from the root of Eurycoma harmandiana, along with five known quassinoids, casteloside B, 13 b, 21-dihydroeurycomanone, chaparrinone, glaucarubolone and ailanquassin B as well as the coumarin, scopoletin. The structural elucidations were based on analyses of spectroscopic data. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Eurycoma harmandiana; Simaroubaceae; Quassinoids, Iandonoside A; Iandonoside B; Iandonone; Iandonol
1. Introduction Eurycoma harmandiana Pierre is a small Simaroubaceous plant (Thai name: Ian-don) distributed in the border regions between Thailand and Laos. In a previous paper (Kanchanapoom et al., 2001), we reported the isolation and structural elucidation of canthin-6-one and b-carboline alkaloids. Further investigation of the root of the same plant afforded nine compounds (1–9), which included three new unusual 15a-OH quassinoids (2, 3, 5) together with five known quassinoids (1, 4, 6–8) and one known coumarin (9). The present paper deals with the structural determination of these compounds.
2. Results and discussion From the ethanolic extract of the roots of E. harmandiana, nine compounds (1–9) were isolated. Five were identified as known quassinoids; casteloside B (1) (Chaudhuri and Kubo, 1992), l3b,21-dihydroeurycomanone (4) (Morita et al., 1990), chaparrinone (6), glaucarubolone (7) (Chaudhuri and Kubo, 1992) and ailanquassin B (8) (Aono et al., 1994) together with one
* Corresponding author. Tel.: +81-82-257-5285; fax: +81-82-2575289. E-mail address: [email protected] (K. Yamasaki).
known coumarin, scopoletin (9) by physical data and spectroscopic evidence. Iandonoside A was obtained as an amorphous powder and determined as C26H38O13 by HR–FAB mass spectrometry. The 13C NMR spectrum revealed the presence of one b-glucopyranosyl unit together with 20 carbon signals for the aglycone, which suggested a C20 quassinoid skeleton. DEPT experiments indicated the presence of three methyls, two methylenes, 10 methines, as well as five quaternary carbons in the aglycone moiety. The signals at 72.3 (C-20) and 110.9 (C-11) in the 13 C NMR spectrum were characteristic of an 11b,20hemiketal moiety. Comparison of the 1H and 13C NMR spectral data of 2 with those of 1 revealed the same relative arrangement of carbons and protons in rings A and B. The chemical shifts of rings C and D suggested differences in the relative configuration of hydroxyl group at C-15. The complete assignments were established by using HSQC, NOESY, difference NOE spectral experiments (Fig. 1) and the coupling constants in the 1H NMR spectrum (Table 1). Irradiation of the H20 signal ( 3.70) gave rise to NOE enhancements at H13 ( 2.81), H-14 ( 2.10) indicating the presence of aconfiguration of Me-21 ( 1.24) and b-configuration of H-14. The coupling constant of H-12 ( 3.97, d, J=5.0 Hz) confirmed the a-configuration for the hydroxyl group at C-12. The configuration of the hydroxyl group at C-15 was assigned as a by the coupling constant of H-15 ( 4.50, d, J=4.6 Hz), and further support was
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00235-7
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Fig. 1. NOESY and NOE enhancements of 2, 3 and 5.
obtained by irradiation of the signal at H-15 which exhibited the NOE enhancements at H-14 and Me-21 but no enhancement at H-9 ( 3.53). Consequently, the structure of 2 was elucidated as shown. Iandonoside B (3) was obtained as an amorphous powder and determined as C26H38O14 by HR–FAB
mass spectrometry. Inspection of the 13C NMR spectrum showed the presence of one b-glucopyranosyl unit and 20 carbon signals for the aglycone, which indicated it to be a C20 quassinoid glucoside. On enzymatic hydrolysis with crude hesperidinase (Kohda and Tanaka, 1975), 3 gave the new aglycone, named iandonol (3a). On going from 3 to 3a, the chemical shifts were changed for C-2, C-1 and C-3 by +11.2, 1.3 and 2.3 ppm, respectively. This confirmed the location of the glucopyranosyl moiety on C-2. The 1H and 13C NMR spectral data were similar with those of 2. However, the signal for H-14 was absent as well as the signal for H-15 ( 4.73) appeared as singlet. In addition, the carbon signals for C-l4, C-13 and C-15 showed different chemical shifts at 76.6, 41.4 and 76.1 (Table 2), respectively, indicating the replacement of a proton (H-14) by a hydroxyl group. The relative configurations were confirmed by using the coupling constants in the 1H NMR spectrum (Table 1), NOESY and difference NOE spectral experiments (Fig. 1). On irradiation of the signal at 2.95 (H-13), the intensity of H-20 ( 4.53) was enhanced, and irradiation of the H-15 signal ( 4.73) caused as determined increase in the NOE enhancement at Me-21 only ( 1.45). Upon irridiation of the H-9 signal ( 3.88), NOE enhancement was observed for the H-5 a signal ( 2.76) but not for either the H-12 ( 4.17) or H-15 ( 4.73) signals. Thus, the stereochemistry of the hydroxyl groups at C-12 and C-15 were assigned as a, a-configurations, and the also Me-21 has an a-configuration. On the basis of these spectral data, the structure of 3 was determined as the 14b-hydroxy analogue of iandonoside A. Iandonone (5) was obtained as an amorphous powder, with a molecular formula of C20H24O9 by HR– FAB mass spectrometry. The 13C NMR spectrum revealed the presence of 20 carbons, including a -lactone carbonyl ( 171.9), an a,b-unsaturated ketone carbonyl carbon ( 197.4) and two olefinic carbons ( 126.2 and 162.2) indicative of a C20 quassinoid skeleton. Iandonone showed the same chemical shifts for rings B, C and D with those of 3, and the chemical shifts of ring A were in full agreement with those of 4. The stereochemistry of three hydroxyl groups at C-1, C-12 and C-15 were also confirmed by using NOESY difference NOE experiments (Fig. 1) and the coupling constants in the 1H NMR spectrum (Table 1), led to deduce as b,a,aconfigurations, respectively. Therefore, the structure of 5 was elucidated as shown.
3. Experimental 3.1. General NMR spectra were recorded in C5D5N using a JEOL JNM A-400 spectrometer (400 MHz for 1H NMR and
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T. Kanchanapoom et al. / Phytochemistry 57 (2001) 1205–1208 Table 1 1 H NMR spectral data for compounds 2, 3, 3a and 5 (C5D5N, 400 MHz) Proton
2
3
3a
5
H-1 H-2 H-3 H-5 H-6e H-6a H-7 H-9 H-12 H-13 H-14 H-15 H-18 H-19 H-20
4.02, d, J=8.0 Hz 4.58, m 5.77, bs 2.67, bd, J=14.0 Hz 2.02, dt, J=14.0, 2.7 Hz 1.86, td, J=14.0, 2.7 Hz 4.46 t, J=2.7 Hz 3.53, s 3.97, d, J=5.0 Hz 2.81, m 2.10, dd, J=7.8, 5.1 Hz 4.50, d, J=4.6 Hz 1.43, bs 1.54, s 3.70, d, J=8.6 Hz 4.10, d, J=8.6 Hz 1.24, d, J=7.1 Hz 5.19, d, J=7.6 Hz 4.06, dd, J=8.1, 7.6 Hz 4.19, dd, J=8.5, 8.1 Hz 4.24, dd, J=9.1, 8.5 Hz 3.95, m 4.54, bd, J=11.5 Hz 4.38, dd, J=11.5, 4.7 Hz
4.14, d, J=8.0 Hz 4.57, m 5.80, bs 2.76, bd, J=14.0 Hz 2.13, dt, J=140, 2.7 Hz 1.89, td, J=14.0, 2.7 Hz 5.06, t, J=2.7 Hz 3.88, s 4.17, d, J=4.9 Hz 2.95, m – 4.73, s 1.44, bs 1.61, s 4.03, d, J=9.5 Hz 4.53, d, J=9.5 Hz 1.45, d, J=7.1 Hz 5.18, d, J=7.8 Hz 4.06, dd, J=8.5, 7.8 Hz 4.17, dd, J=9.0, 8.5 Hz 4.20, dd, J=9.0, 8.5 Hz 3.94, m 4.51, dd, J=12.0, 2.6 Hz 4.35, dd, J=12.0, 5.1 Hz
4.11, d, J=7.8 Hz 4.62, m 5.78, bs 2.82, bd, J=13.4 Hz 2.18, dt, J=14.0, 2.5 Hz 1.96, td, J=14.0, 2.5 Hz
4.36, s – 6.11, bs 3.15, bd, J=14.0 Hz 2.31, dt, J=14.0, 2.2 Hz 2.04, td, J=14.0, 2.2 Hz 5.13, bs 4.13, s 4.08, d, J=5.1 Hz 2.97, m – 4.80, s 1.76, bs 1.57, s 4.04, d, J=9.0 Hz 4.53, d, J=9.0 Hz 1.48, d, J=7.1 Hz
H-21 H-10 H-20 H-30 H-40 H-50 H-60
a
3.94, 4.11, 3.00, – 4.75, 1.59, 1.72, 4.03, 4.56, 1.49,
s d, J=5.5 Hz m s bs s d, J=8.8 Hz d, J=8.8 Hz d, J=7.3 Hz
a Interferred signal.
Table 2 13 C NMR spectral data for compounds 1–3, 3a and 5 (C5D5N, 100 MHz) Carbon
1
2
3
3a
5
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 18 19 20 21 G-1 G-2 G-3 G-4 G-5 G-6
82.6 84.1 124.6 136.0 41.3 26.0 78.6 47.4 45.5 42.0 111.0 80.5 33.1 49.6 68.7 174.3 21.0 10.3 71.8 16.3 106.4 76.1 78.6 71.6 78.5 62.7
82.8 83.7 124.9 135.7 41.6 26.0 79.3 44.8 45.9 42.3 110.9 78.3 31.9 48.5 65.4 171.2 21.1 10.7 72.3 13.0 106.3 76.2 78.6 71.6 78.5 62.7
82.8 83.8 124.8 135.5 41.2 25.7 71.5 50.0 44.8 42.3 110.3 78.8 41.4 76.6 76.1 172.0 21.1 10.9 67.9 10.1 106.2 76.3 78.5 70.7 78.5 62.7
84.1 72.6 127.1 135.0 41.4 25.9 70.8 50.1 45.0 42.3 110.5 79.1 41.9 76.7 76.5 172.1 21.3 11.1 67.9 10.2
84.8 197.4 126.2 162.2 42.5 25.9 70.9 49.9 45.3 45.7 110.1 78.7 41.1 76.6 75.9 171.9 22.4 10.6 67.7 10.1
100 MHz for 13C NMR) with tetramethylsilane (TMS) as internal standard. MS were recorded on a JEOL JMS-SX 102 spectrometer. Preparative HPLC was carried out on columns of ODS (15020 mm i.d., YMC) and polyamine II (25020 mm i.d., YMC) with a Tosoh refraction index (RI-8) detector. The flow rate was 6 ml/ min. For CC, silica gel G 60 (Merck), RP-l8 (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) 80% MeCN, (VII) 12% MeCN, (VIII) 10% MeCN, (IX) CH2Cl2, (X) CH2C12–MeOH (19:1), (XI) CH2Cl2–MeOH (9:1), (XII) CH2Cl2–MeOH (4:1), (XIII) 40–100% MeOH, (XIV) 50–100% MeOH. 3.2. Plant material The root of Eurycoma harmandiana was collected in May 1998 from Nong Khai Province, north-eastern Thailand. The identification of the plant was confirmed by Professor Vichiara Jirawongse, Department of Pharmaceutical Botany and Pharmacognosy, Faculty of Pharmaceutical Sciences, Khon Kaen University. A voucher sample (KKU-0017) is kept in the Herbarium of the Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.
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3.3. Extraction and isolation
3.6. Enzymatic hydrolysis of iandonoside B (3)
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 applied to a silica gel column (systems I, II, III and IV, successively) affording nine fractions. Fraction 5 was subjected to a column of RP18 using system V, then followed by HPLC-polyamine II (system VI) and HPLC–ODS (system VII) to provide compounds 1 (5 mg), 2 (4 mg) and 3 (30 mg). The EtOAc extract (27.5 g) was further separated on a column of RP-18 (system V), and finally purified by HPLC–ODS (system VIII) to give compounds 4 (84 mg) and 5 (21 mg). The CH2C12 extract (90.0 g) was subjected to a column of a silica gel (systems IX, X, XI and XII) affording six fractions. Fraction 2 was repeatedly chromatographed on a silica gel column using gradient system (CH2Cl2 to 5% MeOH in CH2Cl2) to provide six fractions. Fraction 2-2 was further purified by using a RP- 18 column (system XIII) to afford compounds 9 (53 mg). Fraction 2-4 was similarly purified by using a RP18 column (system XIV) to give compound 6 (268 mg). Fraction 2-6 was applied to a column of silica gel (system I), this being followed by HPLC–ODS (system VII) to afford compounds 7 (24 mg) and 8 (13 mg).
Iandonoside B (3) 8 mg was dissolved in 0.5 ml of MeOH. A solution of crude hesperidinase (100 mg in 20 ml of H2O) was added. After shaking at 37 C for 24 h, the mixture was extracted with EtOAc. The EtOAc extract was evaporated to provide a new aglycone, iandonol (3a) 3 mg. The structure was identified by 1H and 13 C NMR spectral analysis.
3.4. Iandonoside A (2) 1 Amorphous powder. [a]20 D +19.1 (MeOH, c 0.3); H 13 NMR: Table 1; C NMR: Table 2; negative HR–FAB– MS, m/z: 557.2258 [M–H] (C26H37O13 requires 557.2234).
3.5. Iandonoside B (3) 1 Amorphous powder. [a]20 D +4.1 (MeOH, c 0.7); H 13 NMR: Table 1; C NMR: Table 2; negative HR–FAB– MS, m/z: 573.2148 [M–H] (C26H37O14 requires 573.2183).
3.7. Iandonol (3a) 1 Amorphous powder. [a]20 D 72.6 (MeOH, c 0.2); H 13 NMR: Table 1; C NMR: Table 2; positive HR–FAB– MS, m/z: 411.1691 [M–H], (C20H27O9 requires 411.1655).
3.8. Iandonone (5) 1 Amorphous powder. [a]20 D +4.6 (MeOH, c 0.4); H 13 NMR: Table 1; C NMR: Table 2; positive HR–FAB– MS, m/z: 409.1468 [M–H] (C20H25O9 requires 409.1498).
Acknowledgements We would like to thank the Research Center for Molecular Medicine, Hiroshima University for the use of its NMR facilities. References Aono, H., Koike, K., Kaneko, J., Ohmoto, T., 1994. Alkaloids and quassinoids from Ailanthus malabarica. Phytochemistry 37, 579– 584. Chaudhuri, S.K., Kubo, I., 1992. Two quassinoid glucosides from Castela tortuosa. Phytochemistry 31, 3961–3964. Kanchanapoom, T., Kasai, R., Chumsri, P., Hiraga, Y., Yamasaki, K., 2001. Canthin-6-one and b-carboline alkaloids from Eurycoma harmandiana. Phytochemistry 56, 383–386. Kohda, H., Tanaka, O., 1975. Enzymatic hydrolysis of ginseng saponins and their related glycosides. Yakugaku Zasshi 95, 246– 249. Morita, H., Kishi, E., Takeya, K., Itokawa, H., Tanaka, O., 1990. New quassinoids from the roots of Eurycoma longifolia. Chemistry Letters 749–752.