- Email: [email protected]
Cycloartane triterpene glycosides from the hairy root cultures of Astragalus membranaceus
Pergamon
0031-9422(94)00500-l
Phymchemntry. Vol. 37. No. 5. pp. 1403 -1407. 1994 Copyright (Q 1994 Elxner Science Lid Pnated ,n Great Bntain All rights reserved 0031-9422194 17.00 + 0.00
CYCLOARTANE TRITERPENE GLYCOSIDES FROM THE HAIRY ROOT CULTURES OF ASTRAGALUS MEMBRANACEUS* MASAO HIROTANI, Yu ZHOU, HEKAI RUI and TSUTOMU FURUYA School of Pharmaceutical Sciences, Kitasato University, Minato-Ku, Tokyo
108, Japan
(Received 20 April 1994) Key Word Index-Astragalus membranaceus; Leguminosae; hairy root; Agrobacterium rhizogenes; agroastragaloside II; astragaloside II; isoastragaloside I; 3-0-/?-D-xylopyranosyl-cycloastragenol.
Abstract-Agroastragaloside II, a new astragaloside was isolated from the hairy root culture of Astragalus membranaceus. Its structure was established as 3-O-~-(2’-O-acetyl)-D-xylopyranosyl-6-O-~-D-~ucopyranosyl-(24S)3fi,6a,l6~,24,25-pentahydroxy-9,19-cyclolanostane on the basis of spectroscopic data. Three known astragalosides, astragaloside II, isoastragaloside I and 3-0$-D-xylopyranosyl-cycloastragenol were also isolated.
INTRODUCTION In the preceding paper [I], we reported the isolation of five triterpeneoligoglycosides: acetylastragaloside I, astragaloside I, astragaloside III, astragaloside IV and agroastragaloside I from the hairy root cultures of Astragalus membranaceus. This paper describes the structural elucidation of four additional triterpeneoligoglycosides: astragaloside II (I), isoastragaloside I (2), 3-O+D-xylopyranosyl-cycloastragenol (3) and a new compound agroastragaloside II (4) from the same hairy root cultures of Astragalus membranaceus on the basis of various spectroscopic data. RESULTS AND DISCUSSION
Compound 1, astragaloside II, analysed for C43H700,5 by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3400 cm-‘) and an ester carbonyl group (I 740 cm- ‘) in the IR spectrum. The base peak at mjz 143 in the mass spectrum of 1 resulted from cleavage between C- I7 and C-20 and suggested the presence of the partial structure A (a 25-hydroxy-20, 24epoxy residue) as in 1. The ‘H NMR spectrum showed signals due to a cyclopropane-methylene at 60.19 and 0.55 (each d, J = 4.1 Hz H,-19). seven tertiary methyls at 60.93, 1.27, 1.31, 1.31, 1.41, I.58 and 1.80, and one acetyl methyl at 62.04, respectively (Table 1). Furthermore, the ‘H NMR spectrum of I clearly showed two anomeric doublets at 64.77 (J = 8.0 Hz) and 4.90 (J = 7.5 Hz) in the downfield region, indicative of the presence of two b-linked sugars [2, 33. The 13CNMR spectrum of 1 displayed a total of 43 carbon signals. Based on a DEPT experiment, ‘H-‘H
*Part Part
101 in the series’studies
100 see ref. [I].
on Plant Tissue Cultures’.
For
and 1)C-‘H COSY spectra and comparison with “CNMR data of related 2 and 3, all signals could be assigned as in Table 2. The sites of attachment of the xylose and glucose moieties of I were determined by means of the HMBC spectrum, to be at C-3 and C-6, respectively [4]. These data led us to the conclusion that 1 was a saponin, which had the cycloartane skeleton and two sugar moieties, xylose and glucose, such as in astragaloside II. Kitagawa et al. [2] reported the structural elucidation of astragaloside II isolated from the extract of Astragali Radix. Thus, the isolation of astragaloside II from the cultured hairy roots has been demonstrated for the first time. Compound 2, isoastragaloside I, analysed for C4SH72016 by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3425 cm- ‘) and an ester carbonyl group (1750 cm- ‘) in the IR spectrum. In the mass spectrum of 2, a base peak at m/z 143 due to the side chain was observed. The ‘H and 13C NMR spectra of 2 were completely identical with those of isoastragaloside I. Thus, 2 was identified as isoastragaloside I. Compound 3, 3-O-~-D-XylOpyranOSy~~yC~OaStragCnO~, analysed for C,,H,,O, by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3425 cn- ‘) in the IR spectrum. Because a base peak was observed at m/z 143, the same as that of 1.3 was also deduced to have a 25-hydroxy-20, 24-epoxy residue in the side chain structure. The ‘HNMR spectrum of 3 clearly showed only one anomeric doublet at 64.93 (J=7.6 Hz) in the downfield region, indicative of the presence of one plinked sugar [2, 33. The 13C NMR spectrum of 3 displayed a total of 35 carbon signals and one anomeric carbon at 6 107.8 was observed. The site of attachment of the xylose moiety of 3 was determined by means of the HMBC spectrum, to be at C-3 [4]. These data led to the conclusion that 3 was also a saponin, which had the cycloartane skeleton and one sugar moiety, xylose, such
1403
M. HIROTANI ei al.
3HH
H
Table 1. ‘H NMR spectral data of 1-4 (400 MHz, 6, C,D,N) H
1
2
3
4
33
3.39 dd (11.1‘4.1) 1.88 d
3.39dd (11.2, 4.4) 1.87d
3.65 dd ( 11.6, 4.4) 1.77d
3.39 dd (11.1,4.1) 1.91 d
(8.7) 3.78 ddd (8.9, 8.9, 3.8) 4.99 dd ( 14.7. 7.3) 2.53 d
(9.4) 2.74 m 5.02 m
(8.5) 3.80 ddd (8.5, 8.5.4.0) 4.72 m
17a
(9.1) 3.76 ddd (10.0, 9.1, 3.0) 4.97 dd (14.1. 7.1) 2.51 d
2.53 d
1.82 m
H,-IX 19a
(7.5) 1.41 s 0.19d
(7.6) 1.42s 0.19d
(7.7) 1.42 s 0.198
1.40s 0.18d
19b
(4.1) 0.55 d
(4.21 0.55 d
(4.1) 0.57 d
(4.0) 0.546
H,-21
(4.1) t.31 s
(4.2) 1.31 s
(4.1) 1.30s
(4.0) 1.08 d
3.11 dd (21.4,10.0) 3.88dd (8.0, 5.5) 1.58 s 1.31 s 1.80s 1.27 s 0.93 s 4.77 d
3.14dd (20.9, 10.6) 3.89 dd (9.2, 5.2) 1.59 s 1.31 s 1.82 s 1.28 s 0.93 s 4.81 d
3.12dd (21.5,11.1) 3.89 dd (9.2, 5.2) 1.59 s 1.32 s 2.02 s 1.35 s 1.01 s 4.93 d
(8.0) 4.90d
(7.8) 4.94 d
(7.6)
(7.5) 2.04s
(7.4) 1.96s 2.03 s
5a 6P Km
22b 24 H&S H,-27 H,-28 Ha-29 H,-30 H-l’ (xylosef H-l” (glucose) AC AC
as in 3-O-rkxylopyranosyl-cycloastragenol. Kitagawa er al. [2] reported the structural elucidation of 3-0fl-D-xylopyranosyl-cycloastragenol obtained from the Pb(OAc), oxidation followed by NaBH, reduction of
(6.5) 2.31 m 3.96 dd
(11.o, 2.0) 1.495 1.47 s 1.83 s 1.29s 0.98 s 4.80 d (7.7) 4.96 d (7.4) 2.04 s
astragaloside I. The isolation of 3-O-8-D-xylopyranosylcycloastragenol from the extract of Astragah Radix was also been reported [2, 5-j. The isolation of isoastragaloside I and 3-0-/I-D-xylopyranosyl-cycloastragenol from
Cycloartane glycosides Table
C Aglycone
3-o-/?-DXylopyranosyl moiety
6-o-/%0-
Glucopyranosyl moiety
Acetoxyl
1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1’ 2 3 4 5’ I, 1 2” 3” 4” 5” 6”
2. ‘%NMR
in hairy root cultures
data of l-6
1 32.3 30.2 89.1 42.5 52.1 79.5 35.0 46.1 21.4 29.2 26.4 33.6 45.3 46.4 46.4 13.7 58.5 21.4 29.2 87.5 27.3 35.1 26.1 81.9 71.6 28.4 28.5 28.8 16.8 20. I 104.9 75.8 76.4 12.0 67.3 105.4 75.8 19.3 71.5 78.3 63.3 170.3
group 21.5
1405
(100 MHz 6, CSD,N)
2
3
4
5*
6*
32.1 30.0 89.2 42.4 52.6 19.4 35.0 46.0 21.3 29.1 26.3 33.5 45.2 46.4 46.5 13.5 58.4 21.4 29.2 87.4 27.2 35.1 26.6 81.8 71.4 28.3 28.5 28.7 16.7 20.0 104.5 75.5 72.8 12.9 63.2 105.4 75.8 19.3 72.0 78.3 63.2 170.1 170.5 20.9 21.2
32.6 30.5 88.8 42.9 52.2 68.2 38.8 47.2 21.2 29.7 26.4 33.6 45.2 46.3 46.8 73.6 58.5 21.7 30.8 87.4 28.7 35.1 26.6 81.9 71.4 21.3 28.3 29.1 16.8 20.3 107.8 75.8 78.7 71.4 61.2
32.2 30.1 89.1 42.4 52.6 79.3 34.6 45.9 21.6 28.8 26.3 33.3 45.9 47.0 48.1 72.1 57.3 18.7 28.5 28.7 18.5 33.1 28.0 77.2 12.6 25.9 26.6 28.4 16.7 20.0 104.9 75.8 16.4 71.5 61.3 105.3 75.8 79.3 72.0 78.3 63.3 170.2
32.8 31.3 78.3 42.3 54.0 68.3 38.5 47.2 21.4 30.4 26.4 33.3 45.8 46.9 48.4 72.0 51.4 18.2 29.1 28.8 19.0 33.1 28.0 17.2 72.5 25.4 26.5 29.2 16.0 20.2
32.3 28.8 88.6 42.7 52.5 79.1 34.3 45.6 21.5 30.2 26.3 33.2 45.8 46.9 47.9 72.0 57.2 18.4 28.2 28.6 18.5 33.0 27.9 77.1 12.6 25.8 26.5 19.8 28.7 16.7 107.6 75.6 78.5 71.3 67.0 105.2 75.6 79.1 72.0 78.1 63.2
*The data of 5 and 6 (cyclocanthogenin
the cultured hairy roots has been demonstrated for the first time. Compound 4, a new astragaloside derivative, mp 267-269”, analysed for C4JH720L5 by HR-FAB mass spectrometry. The IR spectrum of 4 showed the presence of a hydroxyl group (3420 cm-‘) and an ester carbonyl group (1780 cm-r). The ‘H NMR spectrum of 4 showed two anomeric doublets at 64.80 (J = 7.7 Hz) and 4.96 (J = 7.4 Hz) in the downfield region, indicative of the presence of two /I-linked sugars [2,3]. The upfield region was generally similar to that of 1-3, except for the number
and cyclocanthoside
21.4
E) are cited from ref. [6].
of tertiary methyl proton signals. Two doublets at 60.18 and 0.54 for cyclopropane methylene protons, and six tertiary methyl signals at 60.98. 1.29, 1.40, 1.47, 1.49 and 1.83 were consistent with a cycloastragenol aglycone moiety except for its side chain structure. In the ‘HNMR spectrum, a signal at 62.04 (3H, s), and a ’ % NMR signal at 6 170.2, showed the presence of one acetyl group in 4 (Tables 1 and 2). Compound 4 did not give the peak at m/z 143 which was observed in the mass spectra of 1-3 due to the partial structure A (a 25hydroxy-20, 24-epoxy residue). Thus, the structure of 4 appeared to have an
M. HIROTANIer al.
14%
acyclic side chain. This was supported by the ‘HNMR spectrum, which showed a new secondary methyl proton signal (I.08 (1,J = 6.5 Hz, H,-21) relative to those of 1-3. Therefore, instead of the epoxide ring seen in l--3, there was a hydroxyl group at C-24. The configuration of the C-24 chiral centre was determined by comparing the ‘-‘C NMR spectra of 4 and of cyclocanthoside E [6] and cycloasgenin B [73. In the 13C NMR spectra of cyclocanthoside E, the C-24 atom, having the S-configuration, resonated at 677.1. In contrast, the C-24 atom, having the R-configuration. resonated at 680.5 in the r3CNMR spectra of cycloasgenin B [7]. The signal of the C-24 atom in the 13C NMR spectrum of4 was located at 677.2. Thus, the experimental facts permit the conclusion that 4 had the 24S-configuration like that of cyclocanthoside E. Also, it was suggested from the ‘H and 13CNMR data that the sugar moieties in 4 were ,&xylopyranose and /Yglucopyrar,Jsc (Tables I and 2). The glycosidation sites of 4 were suggested by the 13C NMR spectrum when compared with that of 5. As shown in Table 2, the signals of oxygenated carbons C-3 and C-6 appeared at lower field and the carbons C-2, C-S and C-7 at higher field than the corresponding signals of the aglycone (5). while the oxymethine carbons C-16, C-24 and C-25 appeared at almost the same chemical shifts. Therefore, the sugar moieties must be located at the C-3 and C-6 positions, respectively. Finally, the position of each sugar moiety was established by the HMBC (Hcteronuclear Multiple Bond correlatton spectrum of 4. The anomeric proton signal at 64.80 (H-l’) showed a long range correlation with the carbon at (i89.1 (C-3). Also. another anomeric proton signal at fi4.96 (H-l”) showed a long range correlation with the carbon at 679.3 (C-6). Consequently, xylose and glucose residues should be attached to the hydroxyl groups at C-3 and C-6 of 4, respectively. These results suggested that 4 was a glycoside similar to cyclocanthoside E which has a xylopyranosyl residue at C-3 and a glucopyranosyl residue at C-6 [6]. Futhermore, the positions of the acetoxy groups of 4 were elucidated from its HOHAHA (Homonuclear Hartmann-Hahn spectrometry) spectrum [8]. In the HOHAHA spectrum of 4, effects from the anomeric proton (4.80 ppm) of xylopyranose were observed on all other protons after a long mixing time and clearly one acetoxyl group as located at C-2’ of the xylose moiety. Thus, the structure of agroastragaloside II was determined as 3-@/3-(2’-f%acctyl)-D-xylopyranosyl-6-O-p-D-glucopyranosyl-(24S)-3&6.x, 16fi,24,25-pentahydroxy-9,19-cyclolanostane.
EXPERIMENTAI. Mps: uncorr.; ‘H and 13CNMR: 400 and 100 MHz; FAB and EIMS: 20 eV: specific rotations: MeOH; CC: silica gel (C-200). IMUSS culture of A. membranaceus hairy root. The methods of isolation and culture of hairy roots of A. memhranaceus were described in the previous paper [ 11. The hairy roots were subcultured every 4 weeks on a modified Gamborg B5 medium [9] changing the concn of
sucrose from 20 to 30 g, CaC1,.2H,O from 150 to 440 mg and MgSO,.7H,O from 500 to 370 mg I _ ‘, respectively, at 25’ in the dark at 60 rpm on a rotary shaker. The mass culture was carried out using 5@Oml Erlenmeyer flask containing 250 ml medium described above. Extraction and sepn procedures. After 4 weeks culture (143 flasks), the hairy roots (7.14 kg fr. wt) were harvested on a nylon mesh and dried at 60’ for 7 days. The dried hairy roots (549 g) were refluxed with 70% EtOH (3 x ). After filtration. the extracts were combined and the solvent evapd under red. pres. The residue was partitioned between n-BuOH and HzO. The n-BuOH fr. was evapd to dryness (crude saponin fr. 30.8 g). The crude saponin fr. was subjected to Diaion HP 20 column and eluted successively with 20, 50, 80 and 100% MeOH. After removal of the solvent, the 100% MeOH eluate (12.3 g) was subjected to CC over silica gel (cu 1 kg Wako gel C-200) and elutcd with CHCI, --MeOH solvent system to yield 7 frs (frs A-G). The residue of fr. B (3.9 g) after pptn of astragaloside 1 [I] was subjected to CC (Wakogel C200 300 g; CHCI,--MeOH. 12: I) to furnish two frs: Fr.l and Fr.2. Isolation and identification ofastruguloside II (1). Fr. I was purified by HPLC-I and the fr. containing the peak at IO min was collected. Further purification was achievcd by repeated HPLC-2 and I was isolated from the fr. containing the peak at I5 min. Compound I (90.0 mg), needles mp 251-253’ (from MeOH), [x]:’ + 31.3 (MeOH; ~3.23). FAB-MS m;z: 849 [M + Na] _. HR-FABMS: C,,H,OO,,Na (required 849.4613, [M + Na]’ at mjz 849.4620). EIMS (direct inlet) 20 eV, miz (rel. int.): 472 (5), 454(6), 395 (3), 271 (3), 271 (3), 143 (100). 125 (17). IR vi:: cm-‘: 34OO(OH). 174O(COO), 1240. 1050. ‘H and 13CNMR: Tables I and 2. Isolation and idenrijcation o/isoastragaloside I(2). Fr. I was purified by HPLC-I and the fr. containing the peak at IOmin was collected. Further purification of the collected fr. was achieved by repeated H PLC-2 and 2 was isolated from the fr. containing the peak at 20 min. Compound 2 (39.0 mg). needles mp 217--218’ (from MeOH), [x]F + 21.8 (MeOH; c 1.82). FAB-MS m/z: 891 [M +Na]‘. HR-FAB-MS: C,,H,,0,6Na (required 891.4718, [M+Na]+ at rn.?: 891.4741). EIMS (direct inlet) 20 cV, m/z (rel. int.): 472 (IO), 454 (19) 436 (12). 395 (I?). 175(24), l43(100), 125(82). IR r$!:crn-‘: 3425(OH), 175O(COO), 1230.1055. ‘H and “CNMR: Tables I and 2. Isolarion
and identijicarion
of 3-O-~-D-s~loppanosg~-
(3). Further purification of Fr. 2 was achieved by repeated HPLC-2 and 3 was isolated from the fr. containing the peak at 14.2 min. Compound 3 (6.2 mg), needles mp 2955297’ (from MeOH), [z];’ + 28’(MeOH; ~0.32). FAB-MS m/z: 645 [M + Na]‘. HR-FAB-MS: C,,H,,O,Na (required 645.3979, [M + Na] - at m;‘z 645.3986). EIMS (direct inlet) 20 eV, m/z (rel. int.): 472 (6),454(7), 395 (4). 271 (3). 143 (IOO), I25 (I 1). IR vz’$ cm _ ‘: 3425 (OH), 1050. ‘H and ‘“C NMR: Tables I and 2. cylousrrrrgenol
Isolution
and identification
~Jagroustrugaloside
Fr. C (0.6 g) was purified by HPLC-3
II (4).
and the fr. contain-
Cycloartane glycosides in hairy root cultures
ing the peak collected at 13 min. Furthermore, the collected fr. was purified by HPLC-2, repeatedly, and the fr. containing the peak at 9 min was collected. Purification of the fr. containing the peak at 9 min was achieved by repeated HPLC-4 and 4 was isolated from the fr. containing the peak at 11.5 min. Compound 4 (15.6 mg), needles mp 267-269” (from MeOH), [ali + 48 (MeOH; ~0.22). FAB-MS m/z: 851 [M c NaJ+. HR-FAB-MS: C,,H,,O,,Na (required 851.4769, [M + Na]” at m/z 851.4774). EIMS (direct inlet) 20 eV, m/z (rel. int.): 474 (17),456(42), 438 (37), 311(39), 187(43), 175 (65), 151(45), 126 (47), 114 (lOOk 112 (91). IR vtt; cm-‘: 3420 (OH), 2950, 1780 (COO), 1380, 1260, 1070, 1045. ‘H and i3CNMR: Tables 1 and 2. Conditions for HPLC and data of astragahide derivatiues. HPLC analysis and isolation of astragaloside deriv-
atives were run on a Waters liquid chrmatogram model 510 instrument with a Shimadzu absorbance detector model SPD 2 and a differential refractometer and under the following conditions, respectively. HPLC-I: The column (19 mm x 150 mm) packed with p-Bondasphere 5~ C18-100 A, solvent 90% MeOH in H,O, flow rate 3.0 ml min- ‘. HPLC-2: The column (19 mm x 150 mm} packed with @ondasphere 5 p Cl8100 A, solvent 85% MeOH in HzO, flow rate 6.0 ml min- ‘. HPLC-3: The column (10 mm x 300 mm) packed with Senshu Pak ODS, solvent 80% MeQH in H,O, flow rate 3.0 mlmin-‘. HPLC-4: The column (19 mm x 150 mm) packed with p-Bondasphere 5 p C18-
100 A, solvent 6.0 ml min - I.
1407
80%
MeGH
in
H,O,
flow
rate
Acknowledgement-me
are indebted to the members of the Analytical Centre of this University for NMR and mass spectra.
REFERENCES
1. Hirotani, M., Zhou, Y., Rui, H. and Furuya, T. (1994) P~y~oc~ern~sfry36,665.
2. Kitagawa, I., Wang, H. K., Saito, M., Takagi, A. and Yoshikawa, M. (1983) C/tern. Pharm. Bult. 31, 698. 3. Wang, H. K., He, K., Ji, L.,Tezuka, Y., Kikuchi, T. and Kitagawa, I. (1989) Gem. Harm. Bull. 37, 2041. 4. Summers, M. F., Mar&hi, I.,. G. and Bax, A. (1986) J. Am. Gem. Sot. 108,4285. 5. He, Z.-Q. and Findlay, J. A. (1991) J. Nat. Prod. 54, 810. 6. Isaev. M. I., Imomnazarov, B. A., Fadeev, Yu. M. and Kintya, P. A. (1992) Gem. Nat. Compd 28, 315. 7. Isaev, M. I., Gorovits, M. B., Abdullaev, N. D, and Abubakirov, N. K. (1984) Chem. Nat. Compd 20,691. 8. Davis, D. G. and Bax, A. (1985) J. Am. Gem. Sot. 107, 2820. 9. Gamborg,
0. L., Miller, R. A. and Ojima, K. (1968) Exp. Cell Res. 50, 15 1.
0031-9422(94)00500-l
Phymchemntry. Vol. 37. No. 5. pp. 1403 -1407. 1994 Copyright (Q 1994 Elxner Science Lid Pnated ,n Great Bntain All rights reserved 0031-9422194 17.00 + 0.00
CYCLOARTANE TRITERPENE GLYCOSIDES FROM THE HAIRY ROOT CULTURES OF ASTRAGALUS MEMBRANACEUS* MASAO HIROTANI, Yu ZHOU, HEKAI RUI and TSUTOMU FURUYA School of Pharmaceutical Sciences, Kitasato University, Minato-Ku, Tokyo
108, Japan
(Received 20 April 1994) Key Word Index-Astragalus membranaceus; Leguminosae; hairy root; Agrobacterium rhizogenes; agroastragaloside II; astragaloside II; isoastragaloside I; 3-0-/?-D-xylopyranosyl-cycloastragenol.
Abstract-Agroastragaloside II, a new astragaloside was isolated from the hairy root culture of Astragalus membranaceus. Its structure was established as 3-O-~-(2’-O-acetyl)-D-xylopyranosyl-6-O-~-D-~ucopyranosyl-(24S)3fi,6a,l6~,24,25-pentahydroxy-9,19-cyclolanostane on the basis of spectroscopic data. Three known astragalosides, astragaloside II, isoastragaloside I and 3-0$-D-xylopyranosyl-cycloastragenol were also isolated.
INTRODUCTION In the preceding paper [I], we reported the isolation of five triterpeneoligoglycosides: acetylastragaloside I, astragaloside I, astragaloside III, astragaloside IV and agroastragaloside I from the hairy root cultures of Astragalus membranaceus. This paper describes the structural elucidation of four additional triterpeneoligoglycosides: astragaloside II (I), isoastragaloside I (2), 3-O+D-xylopyranosyl-cycloastragenol (3) and a new compound agroastragaloside II (4) from the same hairy root cultures of Astragalus membranaceus on the basis of various spectroscopic data. RESULTS AND DISCUSSION
Compound 1, astragaloside II, analysed for C43H700,5 by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3400 cm-‘) and an ester carbonyl group (I 740 cm- ‘) in the IR spectrum. The base peak at mjz 143 in the mass spectrum of 1 resulted from cleavage between C- I7 and C-20 and suggested the presence of the partial structure A (a 25-hydroxy-20, 24epoxy residue) as in 1. The ‘H NMR spectrum showed signals due to a cyclopropane-methylene at 60.19 and 0.55 (each d, J = 4.1 Hz H,-19). seven tertiary methyls at 60.93, 1.27, 1.31, 1.31, 1.41, I.58 and 1.80, and one acetyl methyl at 62.04, respectively (Table 1). Furthermore, the ‘H NMR spectrum of I clearly showed two anomeric doublets at 64.77 (J = 8.0 Hz) and 4.90 (J = 7.5 Hz) in the downfield region, indicative of the presence of two b-linked sugars [2, 33. The 13CNMR spectrum of 1 displayed a total of 43 carbon signals. Based on a DEPT experiment, ‘H-‘H
*Part Part
101 in the series’studies
100 see ref. [I].
on Plant Tissue Cultures’.
For
and 1)C-‘H COSY spectra and comparison with “CNMR data of related 2 and 3, all signals could be assigned as in Table 2. The sites of attachment of the xylose and glucose moieties of I were determined by means of the HMBC spectrum, to be at C-3 and C-6, respectively [4]. These data led us to the conclusion that 1 was a saponin, which had the cycloartane skeleton and two sugar moieties, xylose and glucose, such as in astragaloside II. Kitagawa et al. [2] reported the structural elucidation of astragaloside II isolated from the extract of Astragali Radix. Thus, the isolation of astragaloside II from the cultured hairy roots has been demonstrated for the first time. Compound 2, isoastragaloside I, analysed for C4SH72016 by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3425 cm- ‘) and an ester carbonyl group (1750 cm- ‘) in the IR spectrum. In the mass spectrum of 2, a base peak at m/z 143 due to the side chain was observed. The ‘H and 13C NMR spectra of 2 were completely identical with those of isoastragaloside I. Thus, 2 was identified as isoastragaloside I. Compound 3, 3-O-~-D-XylOpyranOSy~~yC~OaStragCnO~, analysed for C,,H,,O, by HR-FAB mass spectrometry and showed the presence of a hydroxyl group (3425 cn- ‘) in the IR spectrum. Because a base peak was observed at m/z 143, the same as that of 1.3 was also deduced to have a 25-hydroxy-20, 24-epoxy residue in the side chain structure. The ‘HNMR spectrum of 3 clearly showed only one anomeric doublet at 64.93 (J=7.6 Hz) in the downfield region, indicative of the presence of one plinked sugar [2, 33. The 13C NMR spectrum of 3 displayed a total of 35 carbon signals and one anomeric carbon at 6 107.8 was observed. The site of attachment of the xylose moiety of 3 was determined by means of the HMBC spectrum, to be at C-3 [4]. These data led to the conclusion that 3 was also a saponin, which had the cycloartane skeleton and one sugar moiety, xylose, such
1403
M. HIROTANI ei al.
3HH
H
Table 1. ‘H NMR spectral data of 1-4 (400 MHz, 6, C,D,N) H
1
2
3
4
33
3.39 dd (11.1‘4.1) 1.88 d
3.39dd (11.2, 4.4) 1.87d
3.65 dd ( 11.6, 4.4) 1.77d
3.39 dd (11.1,4.1) 1.91 d
(8.7) 3.78 ddd (8.9, 8.9, 3.8) 4.99 dd ( 14.7. 7.3) 2.53 d
(9.4) 2.74 m 5.02 m
(8.5) 3.80 ddd (8.5, 8.5.4.0) 4.72 m
17a
(9.1) 3.76 ddd (10.0, 9.1, 3.0) 4.97 dd (14.1. 7.1) 2.51 d
2.53 d
1.82 m
H,-IX 19a
(7.5) 1.41 s 0.19d
(7.6) 1.42s 0.19d
(7.7) 1.42 s 0.198
1.40s 0.18d
19b
(4.1) 0.55 d
(4.21 0.55 d
(4.1) 0.57 d
(4.0) 0.546
H,-21
(4.1) t.31 s
(4.2) 1.31 s
(4.1) 1.30s
(4.0) 1.08 d
3.11 dd (21.4,10.0) 3.88dd (8.0, 5.5) 1.58 s 1.31 s 1.80s 1.27 s 0.93 s 4.77 d
3.14dd (20.9, 10.6) 3.89 dd (9.2, 5.2) 1.59 s 1.31 s 1.82 s 1.28 s 0.93 s 4.81 d
3.12dd (21.5,11.1) 3.89 dd (9.2, 5.2) 1.59 s 1.32 s 2.02 s 1.35 s 1.01 s 4.93 d
(8.0) 4.90d
(7.8) 4.94 d
(7.6)
(7.5) 2.04s
(7.4) 1.96s 2.03 s
5a 6P Km
22b 24 H&S H,-27 H,-28 Ha-29 H,-30 H-l’ (xylosef H-l” (glucose) AC AC
as in 3-O-rkxylopyranosyl-cycloastragenol. Kitagawa er al. [2] reported the structural elucidation of 3-0fl-D-xylopyranosyl-cycloastragenol obtained from the Pb(OAc), oxidation followed by NaBH, reduction of
(6.5) 2.31 m 3.96 dd
(11.o, 2.0) 1.495 1.47 s 1.83 s 1.29s 0.98 s 4.80 d (7.7) 4.96 d (7.4) 2.04 s
astragaloside I. The isolation of 3-O-8-D-xylopyranosylcycloastragenol from the extract of Astragah Radix was also been reported [2, 5-j. The isolation of isoastragaloside I and 3-0-/I-D-xylopyranosyl-cycloastragenol from
Cycloartane glycosides Table
C Aglycone
3-o-/?-DXylopyranosyl moiety
6-o-/%0-
Glucopyranosyl moiety
Acetoxyl
1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1’ 2 3 4 5’ I, 1 2” 3” 4” 5” 6”
2. ‘%NMR
in hairy root cultures
data of l-6
1 32.3 30.2 89.1 42.5 52.1 79.5 35.0 46.1 21.4 29.2 26.4 33.6 45.3 46.4 46.4 13.7 58.5 21.4 29.2 87.5 27.3 35.1 26.1 81.9 71.6 28.4 28.5 28.8 16.8 20. I 104.9 75.8 76.4 12.0 67.3 105.4 75.8 19.3 71.5 78.3 63.3 170.3
group 21.5
1405
(100 MHz 6, CSD,N)
2
3
4
5*
6*
32.1 30.0 89.2 42.4 52.6 19.4 35.0 46.0 21.3 29.1 26.3 33.5 45.2 46.4 46.5 13.5 58.4 21.4 29.2 87.4 27.2 35.1 26.6 81.8 71.4 28.3 28.5 28.7 16.7 20.0 104.5 75.5 72.8 12.9 63.2 105.4 75.8 19.3 72.0 78.3 63.2 170.1 170.5 20.9 21.2
32.6 30.5 88.8 42.9 52.2 68.2 38.8 47.2 21.2 29.7 26.4 33.6 45.2 46.3 46.8 73.6 58.5 21.7 30.8 87.4 28.7 35.1 26.6 81.9 71.4 21.3 28.3 29.1 16.8 20.3 107.8 75.8 78.7 71.4 61.2
32.2 30.1 89.1 42.4 52.6 79.3 34.6 45.9 21.6 28.8 26.3 33.3 45.9 47.0 48.1 72.1 57.3 18.7 28.5 28.7 18.5 33.1 28.0 77.2 12.6 25.9 26.6 28.4 16.7 20.0 104.9 75.8 16.4 71.5 61.3 105.3 75.8 79.3 72.0 78.3 63.3 170.2
32.8 31.3 78.3 42.3 54.0 68.3 38.5 47.2 21.4 30.4 26.4 33.3 45.8 46.9 48.4 72.0 51.4 18.2 29.1 28.8 19.0 33.1 28.0 17.2 72.5 25.4 26.5 29.2 16.0 20.2
32.3 28.8 88.6 42.7 52.5 79.1 34.3 45.6 21.5 30.2 26.3 33.2 45.8 46.9 47.9 72.0 57.2 18.4 28.2 28.6 18.5 33.0 27.9 77.1 12.6 25.8 26.5 19.8 28.7 16.7 107.6 75.6 78.5 71.3 67.0 105.2 75.6 79.1 72.0 78.1 63.2
*The data of 5 and 6 (cyclocanthogenin
the cultured hairy roots has been demonstrated for the first time. Compound 4, a new astragaloside derivative, mp 267-269”, analysed for C4JH720L5 by HR-FAB mass spectrometry. The IR spectrum of 4 showed the presence of a hydroxyl group (3420 cm-‘) and an ester carbonyl group (1780 cm-r). The ‘H NMR spectrum of 4 showed two anomeric doublets at 64.80 (J = 7.7 Hz) and 4.96 (J = 7.4 Hz) in the downfield region, indicative of the presence of two /I-linked sugars [2,3]. The upfield region was generally similar to that of 1-3, except for the number
and cyclocanthoside
21.4
E) are cited from ref. [6].
of tertiary methyl proton signals. Two doublets at 60.18 and 0.54 for cyclopropane methylene protons, and six tertiary methyl signals at 60.98. 1.29, 1.40, 1.47, 1.49 and 1.83 were consistent with a cycloastragenol aglycone moiety except for its side chain structure. In the ‘HNMR spectrum, a signal at 62.04 (3H, s), and a ’ % NMR signal at 6 170.2, showed the presence of one acetyl group in 4 (Tables 1 and 2). Compound 4 did not give the peak at m/z 143 which was observed in the mass spectra of 1-3 due to the partial structure A (a 25hydroxy-20, 24-epoxy residue). Thus, the structure of 4 appeared to have an
M. HIROTANIer al.
14%
acyclic side chain. This was supported by the ‘HNMR spectrum, which showed a new secondary methyl proton signal (I.08 (1,J = 6.5 Hz, H,-21) relative to those of 1-3. Therefore, instead of the epoxide ring seen in l--3, there was a hydroxyl group at C-24. The configuration of the C-24 chiral centre was determined by comparing the ‘-‘C NMR spectra of 4 and of cyclocanthoside E [6] and cycloasgenin B [73. In the 13C NMR spectra of cyclocanthoside E, the C-24 atom, having the S-configuration, resonated at 677.1. In contrast, the C-24 atom, having the R-configuration. resonated at 680.5 in the r3CNMR spectra of cycloasgenin B [7]. The signal of the C-24 atom in the 13C NMR spectrum of4 was located at 677.2. Thus, the experimental facts permit the conclusion that 4 had the 24S-configuration like that of cyclocanthoside E. Also, it was suggested from the ‘H and 13CNMR data that the sugar moieties in 4 were ,&xylopyranose and /Yglucopyrar,Jsc (Tables I and 2). The glycosidation sites of 4 were suggested by the 13C NMR spectrum when compared with that of 5. As shown in Table 2, the signals of oxygenated carbons C-3 and C-6 appeared at lower field and the carbons C-2, C-S and C-7 at higher field than the corresponding signals of the aglycone (5). while the oxymethine carbons C-16, C-24 and C-25 appeared at almost the same chemical shifts. Therefore, the sugar moieties must be located at the C-3 and C-6 positions, respectively. Finally, the position of each sugar moiety was established by the HMBC (Hcteronuclear Multiple Bond correlatton spectrum of 4. The anomeric proton signal at 64.80 (H-l’) showed a long range correlation with the carbon at (i89.1 (C-3). Also. another anomeric proton signal at fi4.96 (H-l”) showed a long range correlation with the carbon at 679.3 (C-6). Consequently, xylose and glucose residues should be attached to the hydroxyl groups at C-3 and C-6 of 4, respectively. These results suggested that 4 was a glycoside similar to cyclocanthoside E which has a xylopyranosyl residue at C-3 and a glucopyranosyl residue at C-6 [6]. Futhermore, the positions of the acetoxy groups of 4 were elucidated from its HOHAHA (Homonuclear Hartmann-Hahn spectrometry) spectrum [8]. In the HOHAHA spectrum of 4, effects from the anomeric proton (4.80 ppm) of xylopyranose were observed on all other protons after a long mixing time and clearly one acetoxyl group as located at C-2’ of the xylose moiety. Thus, the structure of agroastragaloside II was determined as 3-@/3-(2’-f%acctyl)-D-xylopyranosyl-6-O-p-D-glucopyranosyl-(24S)-3&6.x, 16fi,24,25-pentahydroxy-9,19-cyclolanostane.
EXPERIMENTAI. Mps: uncorr.; ‘H and 13CNMR: 400 and 100 MHz; FAB and EIMS: 20 eV: specific rotations: MeOH; CC: silica gel (C-200). IMUSS culture of A. membranaceus hairy root. The methods of isolation and culture of hairy roots of A. memhranaceus were described in the previous paper [ 11. The hairy roots were subcultured every 4 weeks on a modified Gamborg B5 medium [9] changing the concn of
sucrose from 20 to 30 g, CaC1,.2H,O from 150 to 440 mg and MgSO,.7H,O from 500 to 370 mg I _ ‘, respectively, at 25’ in the dark at 60 rpm on a rotary shaker. The mass culture was carried out using 5@Oml Erlenmeyer flask containing 250 ml medium described above. Extraction and sepn procedures. After 4 weeks culture (143 flasks), the hairy roots (7.14 kg fr. wt) were harvested on a nylon mesh and dried at 60’ for 7 days. The dried hairy roots (549 g) were refluxed with 70% EtOH (3 x ). After filtration. the extracts were combined and the solvent evapd under red. pres. The residue was partitioned between n-BuOH and HzO. The n-BuOH fr. was evapd to dryness (crude saponin fr. 30.8 g). The crude saponin fr. was subjected to Diaion HP 20 column and eluted successively with 20, 50, 80 and 100% MeOH. After removal of the solvent, the 100% MeOH eluate (12.3 g) was subjected to CC over silica gel (cu 1 kg Wako gel C-200) and elutcd with CHCI, --MeOH solvent system to yield 7 frs (frs A-G). The residue of fr. B (3.9 g) after pptn of astragaloside 1 [I] was subjected to CC (Wakogel C200 300 g; CHCI,--MeOH. 12: I) to furnish two frs: Fr.l and Fr.2. Isolation and identification ofastruguloside II (1). Fr. I was purified by HPLC-I and the fr. containing the peak at IO min was collected. Further purification was achievcd by repeated HPLC-2 and I was isolated from the fr. containing the peak at I5 min. Compound I (90.0 mg), needles mp 251-253’ (from MeOH), [x]:’ + 31.3 (MeOH; ~3.23). FAB-MS m;z: 849 [M + Na] _. HR-FABMS: C,,H,OO,,Na (required 849.4613, [M + Na]’ at mjz 849.4620). EIMS (direct inlet) 20 eV, miz (rel. int.): 472 (5), 454(6), 395 (3), 271 (3), 271 (3), 143 (100). 125 (17). IR vi:: cm-‘: 34OO(OH). 174O(COO), 1240. 1050. ‘H and 13CNMR: Tables I and 2. Isolation and idenrijcation o/isoastragaloside I(2). Fr. I was purified by HPLC-I and the fr. containing the peak at IOmin was collected. Further purification of the collected fr. was achieved by repeated H PLC-2 and 2 was isolated from the fr. containing the peak at 20 min. Compound 2 (39.0 mg). needles mp 217--218’ (from MeOH), [x]F + 21.8 (MeOH; c 1.82). FAB-MS m/z: 891 [M +Na]‘. HR-FAB-MS: C,,H,,0,6Na (required 891.4718, [M+Na]+ at rn.?: 891.4741). EIMS (direct inlet) 20 cV, m/z (rel. int.): 472 (IO), 454 (19) 436 (12). 395 (I?). 175(24), l43(100), 125(82). IR r$!:crn-‘: 3425(OH), 175O(COO), 1230.1055. ‘H and “CNMR: Tables I and 2. Isolarion
and identijicarion
of 3-O-~-D-s~loppanosg~-
(3). Further purification of Fr. 2 was achieved by repeated HPLC-2 and 3 was isolated from the fr. containing the peak at 14.2 min. Compound 3 (6.2 mg), needles mp 2955297’ (from MeOH), [z];’ + 28’(MeOH; ~0.32). FAB-MS m/z: 645 [M + Na]‘. HR-FAB-MS: C,,H,,O,Na (required 645.3979, [M + Na] - at m;‘z 645.3986). EIMS (direct inlet) 20 eV, m/z (rel. int.): 472 (6),454(7), 395 (4). 271 (3). 143 (IOO), I25 (I 1). IR vz’$ cm _ ‘: 3425 (OH), 1050. ‘H and ‘“C NMR: Tables I and 2. cylousrrrrgenol
Isolution
and identification
~Jagroustrugaloside
Fr. C (0.6 g) was purified by HPLC-3
II (4).
and the fr. contain-
Cycloartane glycosides in hairy root cultures
ing the peak collected at 13 min. Furthermore, the collected fr. was purified by HPLC-2, repeatedly, and the fr. containing the peak at 9 min was collected. Purification of the fr. containing the peak at 9 min was achieved by repeated HPLC-4 and 4 was isolated from the fr. containing the peak at 11.5 min. Compound 4 (15.6 mg), needles mp 267-269” (from MeOH), [ali + 48 (MeOH; ~0.22). FAB-MS m/z: 851 [M c NaJ+. HR-FAB-MS: C,,H,,O,,Na (required 851.4769, [M + Na]” at m/z 851.4774). EIMS (direct inlet) 20 eV, m/z (rel. int.): 474 (17),456(42), 438 (37), 311(39), 187(43), 175 (65), 151(45), 126 (47), 114 (lOOk 112 (91). IR vtt; cm-‘: 3420 (OH), 2950, 1780 (COO), 1380, 1260, 1070, 1045. ‘H and i3CNMR: Tables 1 and 2. Conditions for HPLC and data of astragahide derivatiues. HPLC analysis and isolation of astragaloside deriv-
atives were run on a Waters liquid chrmatogram model 510 instrument with a Shimadzu absorbance detector model SPD 2 and a differential refractometer and under the following conditions, respectively. HPLC-I: The column (19 mm x 150 mm) packed with p-Bondasphere 5~ C18-100 A, solvent 90% MeOH in H,O, flow rate 3.0 ml min- ‘. HPLC-2: The column (19 mm x 150 mm} packed with @ondasphere 5 p Cl8100 A, solvent 85% MeOH in HzO, flow rate 6.0 ml min- ‘. HPLC-3: The column (10 mm x 300 mm) packed with Senshu Pak ODS, solvent 80% MeQH in H,O, flow rate 3.0 mlmin-‘. HPLC-4: The column (19 mm x 150 mm) packed with p-Bondasphere 5 p C18-
100 A, solvent 6.0 ml min - I.
1407
80%
MeGH
in
H,O,
flow
rate
Acknowledgement-me
are indebted to the members of the Analytical Centre of this University for NMR and mass spectra.
REFERENCES
1. Hirotani, M., Zhou, Y., Rui, H. and Furuya, T. (1994) P~y~oc~ern~sfry36,665.
2. Kitagawa, I., Wang, H. K., Saito, M., Takagi, A. and Yoshikawa, M. (1983) C/tern. Pharm. Bult. 31, 698. 3. Wang, H. K., He, K., Ji, L.,Tezuka, Y., Kikuchi, T. and Kitagawa, I. (1989) Gem. Harm. Bull. 37, 2041. 4. Summers, M. F., Mar&hi, I.,. G. and Bax, A. (1986) J. Am. Gem. Sot. 108,4285. 5. He, Z.-Q. and Findlay, J. A. (1991) J. Nat. Prod. 54, 810. 6. Isaev. M. I., Imomnazarov, B. A., Fadeev, Yu. M. and Kintya, P. A. (1992) Gem. Nat. Compd 28, 315. 7. Isaev, M. I., Gorovits, M. B., Abdullaev, N. D, and Abubakirov, N. K. (1984) Chem. Nat. Compd 20,691. 8. Davis, D. G. and Bax, A. (1985) J. Am. Gem. Sot. 107, 2820. 9. Gamborg,
0. L., Miller, R. A. and Ojima, K. (1968) Exp. Cell Res. 50, 15 1.