- Email: [email protected]
Three saponins from Oxytropis species
Phytochemistry, Vol. 30, No. 8, pp. 2707-2709, Printed in Great Britain.
THREE
1991 0
SAPONINS
RONG-QI
FROM
OX YTROPIS
C031-9422/91 $3.00+0.00 1991 Pergamon Press plc
SPECIES
SUN,* ZHONG-JIAN JIA, DONG-LIANG CHENG
Institute of Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R. China; *Research Institute of Fine Chemicals, East China University of Chemical Technology, Shanghai 200237, P.R. China (Receioed 10 December 1990) Key Word
Index-Oxytropis
ochrocephala; 0. glabra; .O. bicolor, Leguminosae; flavonoids; triterpenoid saponins.
Abstract-Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-j-D-glucoside, rhamnetin-3.O-/3-D-galactoside, apigenin, 3-0-[a-L-rhamnopyranosyl (l-2)-fl-D-glucopyranosyl( l-4).fl-D-glucuronopyranosyl]soyasapogenol B, 3-O-[/3-D-glucopyranosyl( 1*2)-j-Dglucuronopyranosyl]azubisapogenol and a new saponin 3-O-[j?-D-glucopyranosyl(l~2)-~-D-glucopyranosyl]-25-0cl-L-rhamnopyranosyl-(20~,24S’)-3B,168, 20,24,25-pentahydroxy-9,19-cycloanostane.
INTRODUCTION In preceding papers [l-3], we reported some triterpenoid saponins from Oxytropis ochrocephala, 0. glabra and 0. bicolor. As a continuing study, we now report the isolation and identification of two flavonol glycosides 1,2 and a flavone 3 from 0. ochrocephala, two triterpenoid saponins 4 and 5 from 0. glabra and a new saponin 6 from 0. bicolor. RESULTSAND
DISCUSSION
Three flavonoids l-3 were isolated for the first time from the ethyl acetate extract of 0. ochrocephala. The flavonol glycoside 1 on hydrolysis gave isorhamnetin and D-ghcose, identified by paper chromatography. The aglycone was identified as isorhamnetin by UV spectral analysis and a comparison with literature data [4]. The position of sugar in the glycoside was located in C-3 by the comparison of the properties of aglycone and glycoside and spectral analysis showed 1 to be isorhamnetin 3-0-B-D-glucoside. Flavonol glycoside 2 on hydrolysis gave D-galactose and rhamnetin identified by analyses of its mass, ‘H NMR and UV spectra. The attachment of sugar was also located in C-3 by similar methods to those described above. Thus flavonol glycoside 2 is rhamnetin 3-0-p-Dgalactoside. Flavone 3 gave ions at m/z 270 [Ml’, 153 [A, +H]+, 162 [Al]+, 121 [Ba]+, 118 [BI]+ and 93 [B2 -CO]+. In the ‘HNMR spectrum of 3, the signals of H-3, OH-5 and OH-7 were observed at characteristic values [4] at 66.76, 12.94 and 10.77. Compound 3 was identified as apigenin by analysis of its UV and ‘H NMR spectrum. Two triterpenoid saponins 4 and 5 were isolated from the n-butanol extracts of 0. glabra. The FAB mass spectrum of saponin 4 gave ions at m/z 949 [M + Li] ’ and 965 [M + Na] ‘. On acidic hydrolysis 4 liberated an aglycone which was identified as soyasapogenol B by spectral and TLC comparison with an authentic sample [ 11. From the hydrolysate L-rhamnose, D-glucose and Dglucuronic acid were detected by paper chromatography.
The mass spectrum of the acetate of 4 gave ions at m/z 273 (terminal Rha)Ac]+ and 561 [(Glc-Rha)Ac] ‘, indicating the sequence of sugar moieties. The melting point, [a&, MS and “C NMR spectrum is identical with the known saponin 3-0-[cr-L-rhamnopyranosyl( l-+2)-/?-D-©ranosyl( 1-r4)-/?-D-glucuronopyranosyllsoyasapogenol B [l]; thus they are identical. Saponin 5 exhibited peaks in its FAB mass spectrum at m/z 817 [M+Li]+ and 833 [M+Na]‘, suggesting the molecular formula as C,,H,,O,,. Saponin 5 on hydrolysis with presence of methanol gave D-&tCOSe and Dglucuronic acid as sugar moieties, as well as an aglycone which was identified as azukisapogenol methyl ester by comparison of its [a&,, mass and ’ 3C NMR spectra with that of an authentic sample [S]. However, azukisapogenol, not its methyl ester, was confirmed to be the genuine aglycone of saponin 5 as follows. In the 13CNMR spectrum of 5, two carboxyl signals were observed at 6 173.2 ascribable to C-6 of glucuronic acid and 181.2 due to C-29 of the aglycone. The spectral comparison of its l 3C NMR with that of saponin 7 [S] also supported this conclusion. On methylation saponin 5 and 7 gave a same methyl ether. Thus saponin 5 is 3-0-[j?-D-glucopyranosyl (1+2)-fl-D-glucuronopyranosyl]azukisapogenol. Saponin 6 is a new compound isolated from the n-butanol extracts of 0. bicolor. The FAB mass spectrum of 6 showed peaks at m/z 969 [M + Li] ‘, and 985 [M +Na]+ suggesting a molecular formula of C,sH,,O,,. On hydrolysis 6 gave L-rhamnose and D-glucose as sugar moieties, as well as an aglycone which was identified as (20R,24S)-3P,16B,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane by its spectral and TLC comparison with an authentic sample [3]. The EI mass spectrum of 6 gave ions at m/z 492 [(M - Rha - Glc x 2)] + 474 [(M - Rha 456 [(M-Rha-Glcx2)-H,O -Glcx2)-H,O]+, x2]+, 438 [(M-Rha-Glcx2)-H,Ox3]+, 420 [(M -Rha-Glcx2)-H,Ox4] and 402 [(M-Rha-Glc x 2)-Hz0 x 5]+, Those were quite similar to that of saponin 8 [3]. The EI mass spectrum of the acetate of 6 showed peaks at m/z 273 [(terminal Rha)Ac]‘, 331 [(terminal Glc)Ac]+ and 619 [(Glc-Glc)Ac] ‘. In the 13C NMR of 6, the signals of two sets of glucose and one
2707
2708
R.-Q. SW et al.
Rs
4
R1=H,R2+Glc=a-Rha,R3=OH,R4=Me
5
R’= p - Glc,R2=R3=H,R4=CG,H
7
RLP-Glc,R2=R3=H,R4=C02Me
BH ,
Q’-’
@-Glc-2/3-GlcO
6
R=a-Rha
8
R=H
-%Y
set of rhamnose were observed. The i3CNMR spectral comparisons of 6 with 8 showed that signals due to C-24, C-26 and C-27 of saponin 6 were somewhat displaced upfield, but signal due to C-25 downfield, while other carbon resonances remained almost unchanged. This indicated that the location of the rhamnose of 6 should be limited to OH-25 [6-81. Thus saponin 6 is 3-O-[/?D-gIucopyranosyI( l-+2)-/?-D-glucopyranosyfj-25-0-a-Lrhamnopyranosyl-(20S,24S)-3P,16B,20,24,25-pentahydroxy-9,19-cyclolanostane. EXPERIMENTAL Plant material. Oxytropis plants were collected from Gansu. China. Mps: uncorr. ‘H and i3CNMR with TMS as int. standard, EIMS were obtained at 70eV. Polyamide powder made by ourself and silica gel G (10-40, Qingdao Marine Chemical Factory, China) were used for CC. Solvent systems (A) C,H,-MeOH (5:l); (B) nused as follow, BuOH-EtOH-NH,OH (25%) (7:2:3); (C) CHCI,- MeOH-H,O (6:3:1, lower phase);(D) EtOAc-EtOH-H,O (8:2: 1); (E) EtOAc-pyridine-HOAc-H,O (5: 5: 1: 3); (F) i-PrOH-nBuOH-H,O (7: 1: 2). Isolation of jauonoids. Air-dried powdered aerial parts of 0. ochrocephala Bunge (2 kg) was extracted with 75% EtOH under reflux x 3. After removal of EtOH. the ag. phase was extracted with EtOAc. The EtOAc phase was coned to dryness under red. pres. to give a residue (8 g). Part of the residue (1 g) was chromatographed repeatedly on a polyamide column with solvent A to give flavonoid 1 (40 mg), 2 (30 mg) and 3 (40 mg). Isolation of saponins.The ethanolic extract of 0. glabra DC. (6 kg) was successively extracted with EtOAc and n-butanol saturated with H,O. The n-butanol extract (3 g) was first chromatographed on a dry column of silica gel (300 g). eluting with solvent B. The sepd frs were then further chromatographed repeatedly on silica gel cohunn wtth solvent C to give saponin 4
Table
1. t3CNMR spectral data of aglycone saponins 4-6 and 8 (in pyridine-d,)
moieties
C
4
5
6
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
39.0 26.7 91.7 44.2 56.8 18.9 33.2 40.4 48.2 36.9 24.4 122.6 145.3 42.9 26 7 29.3 38.2 45.9 46.9 31.0 42.1 75.7 23 1 63.8 16.0 17.4 25.5 20.9 32.9 28.9
39.1 27.0 90.3 42.6 55.8 19.0 32.5 39.7 47.4 36.2 24.0 123.0 144.3 41.5 26.3 28.1 32.5 41.5 46.3 43.6 36.2 30.5 22.5 63.4 15.5 16.6 25.8 28.1 181.2 19.7
33.1 32.0 88.7 41.2 47.5 21.0 25.7 48.0 19.7 26.2 26.3 29.9 46.9 46.6 48.7 75.0 56.3 21.4 30.6 76.8 26.1 412 26.6 78.5 78.0 23.1 24.1 20.2 15.2 25.7
33.7 32.0 88.8 41.2 47.3 21.0 26.3 47.6 19.7 26.2 26.5 29.9 47.2 46.6 49.1 73.5 55.5 21.2 30.2 76.7 26.1 42.7 27.3 79.9 72.8 25.7 25.8 20.4 15.3 26.2
in
Saponins from Oxytropis species Table 2. ‘“C NMR spectral data of sugar moieties in saponins 44 and 8 (in pyridine-de) C
4
5
GlllA 1 2 3 4 5 6
105.4 74.6 17.3 79.8 17.3 173.0
105.6 80.7 75.3 72.8 17.0 173.2
Glc 1 2 3 4 5 6
102.3 78.6 78.1 70.5 78.6 62.0
104.6 73.3 11.9 70.7 77.9 63.4
Glc 1 2 3 4 5 6 Rha 1 2 3 4 5 6
101.8 72.0 72.4 73.1 69.4 18.9
6
8
104.7 83.2 17.7 71.3 78.1 62.6
104.9 83.3 77.8 71.4 78.2 626
105.8 76.8 77.7 71.5 78.0 62.6
105.9 76.9 77.8 71.5 78.1 62.6
98.5 71.0 71.5 73.5 68.0 18.7
GluA = /?-n-Glucuronopyranosyl.
(500 mg), 5 (50 mg) and some other saponins reported earlier [Z]. The crude saponin (3g) from 0. bicolor Bunge (2 kg) was obtained by the similar procedure described above. Part of the crude saponin (2g) was repeatedly chromatographed on silica gel column with solvent C and D to give saponins 6 (50 mg) and 8 (3~mgJ c31. Fhomid 1. Yellow needles, mp 167-169”. EIMS (ml. int.) m/z 478 [M]’ (0.4), 316 [M-Glc]+ (lOO),301 [M-Glc-Me] (8), 287 (30), 153 [At+H]+ (8), 151 [Bz]+ (a), 136 [Bz-Me]+ (9). uv ,q$p nm: 254, 266sh, 354; (NaOMe) 277, 328, 411; (AICI,) 263,301,365,403; (AICI, +HCl) 257,404, (NaOAc) 274, 314, 382; (NaOAc+H,BOs) 268, 359. ‘HNMR [80 MHz, DMSG-d,]: 3.84 (3H, s, OMe), 5.6 (lH, d, J = 7 Hz, anomeric H of n-Glc), 6.21 (lH, d, J=2 Hz, H-6), 6.44 (lH, d, J=2 Hz, H-8), 6.91 (lH, d, J=9 Hz, H-S), 7.93 (lH, brs, H-2’), 8.04 (lH, d, J =9 Hz, H-6’). EIMS (acetate) m/z 773 [M +H]+ (0.5), 442 (5), 400 (6) 358 (lo), 331 [(terminal Glc)Ac]+ (39), 316 (ll), 287 (15), 271 (ll), 257 (8), 211(7), 169 (100) 145 (7), 139 (lo), 127 (20), 109 (55).
2709
Fhmoid 2. Yellow needles, mp 220-222”. EIMS (rel. int.) m/z316 [M-Gal]+ (lOO), 287 (12), 167 [A,+H]+ (7) 150 [Bi]’ (8), 137 [Bz]’ (23), 109 [Bz-CO]+ (8), (acetate) m/z 773 [M+H]+ (l), 442 (3) 400 (4), 358 (7), 331 [(terminal Gal)Ac]+ (40), 316 (16), 287 (ll), 271 (13) 257 (l), 211 (5) 169 (lOO),145 (8), 139 (7), 127 (25), 109 (60). UV %z$‘“nm: 258,269sh, 292sh, 360, (NaOMe) 272,297sh, 330, 418; (AlCl,) 268, 399, (AlCl, + HCI) 256, 296931, 358; (NaOAc) 256, 291sh, 363, 415; (NaOAc + HsBO,) 259,29lsh, 376. ‘H NMR [80 MHz, DMSO-d,]: 3.86 (3H, s, OMe), 5.4 (lH, d, J=7 Hz, anomeric H of D-Gal), 6.37 (lH, d, J=2 Hi., H-6), 6.69 (lH, d, J=2 Hz, H-8), 6.82 (lH, d, J =8.5Hz,H-5’),7.59(1H,brs,H-2’),7.70(1H,d,J=8.5Hz,H-6’), 9.07 (lH, brs, OH-3’, 9.66 (lH, brs, OH-4, 12.61 (lH, s, OH-5). Fhonoid 3. Amorphous, mp 343-345”. EIMS (rel. int.) m/z 270[M]+ (lOO), 153 [A,+H]+ (30), 152 [At]’ (20), 121 [Bz]’ (27), 118~,]+(16),(acetate)m/z396[M]+(1),354(41),312(33), 270 (100). Saponin 4. Crystal, mp 235-237”, [alA +O.l” (MeOH; c 0.5). FABMS (positive ion mode) m/z 949 IM + Li], 965 [M + Na]+. t3CNMR: (Tables 1 and 2). EIMS (acetate) m/z 561 [(RhaGlc)Ac] +, 273 [(terminal Rha)Ac] +. Soponin 5. Crystals, mp 223-225”, [a];’ + 2.2” (MeGH; c 0.5). FABMS(positiveionmode)m/z817 [M+Li]+, 833 [M +Na]+. t3CNMR: (Tables 1 and 2). According to Hakomori’s method [9] saponin 5 (50mg) was completely methylated to give a permethylate, ‘H NMR [80 MHz, (CD&O]; 0.87, 1.00, 1.06, 1.16, 1.21, 1.28 (3H each, all s, Me x 6), 3.31, 3.37, 3.45 (3H each, all s, OMe x 3), 3.48 (6H, s, OMe x 2), 3.55 (3H, s, OMe), 3.61(6H, s, OMe and COzMe), 3.76 (3H, s, COzMe), 4.58 (lH, d, J =7.0 Hz, anomeric H of a methylated sugar), 4.66 (lH, d, J = 7.5 Hz, anomeric H of another methylated sugar), 5.30 (lH, s, H-12). Saponin 6. Pow&r, mp 212-214”, [a]P +2.0 (MeGH; c 0.3). FABMS (positive ion mode) m/z 969 [M + Li]+ and 985 [M + Na] +. “CNMR: (Tables 1 and 2). EIMS (acetate) m/z 619 [(Glc-Glc)Ac] +, 331 [(terminal Glc)Ac]+, 273 [(terminal Rha)Ac] +. An aglycone with mp 24@242”, [a];’ + 47” (MeOH; c 0.5) identified as (20R,24S)-3/?,16j,25-trihydroxy-20,24-epoxy9,19-cyclolanostane by mmp and TLC comparison with authentic sample was obtained.
REFEBENCES
1. Sun, R. Q., Cheng, D. L., Jia, Z. J. and Zhu Z. Q. (1987) Huaxue Xuebao 6,145.
2 Sun, R. Q. and Jia, Z. J. (1990) Phytochemistry 29,2032 3. Sun, R. Q. and Jia, Z. J. (1991) Pkytochemistry 30 (in press). 4. Harborne, J. B. and Mabry, H. (eds) (1975) The Flauonoids. Chapman & Hall, London. 5. Sun, R. Q., Jia, Z. J. and Zhu, Z. Q. (1988) Chem. J. Chinese Univ. (Eng. Edn) 4(4), 39. 6. Kasai, R., Suzuo, M., Asakawa, J. and Tanak, 0. (1977) Tetrahedron Letters 175.
7. Tori, K, Seo, S., Yoshimura, Y., Arita, H. and Tomita, Y. (1977) Tetrahedron Latter-s 179. 8. Seo, S., Tomita, Y., Tori, K. and Yoshimura, M. (1978) J. Am. Chem Sot. loo, 3331. 9. Hakomori, S. (1964) J. Biockem. 55,205.
THREE
1991 0
SAPONINS
RONG-QI
FROM
OX YTROPIS
C031-9422/91 $3.00+0.00 1991 Pergamon Press plc
SPECIES
SUN,* ZHONG-JIAN JIA, DONG-LIANG CHENG
Institute of Organic Chemistry, Lanzhou University, Lanzhou 730000, P.R. China; *Research Institute of Fine Chemicals, East China University of Chemical Technology, Shanghai 200237, P.R. China (Receioed 10 December 1990) Key Word
Index-Oxytropis
ochrocephala; 0. glabra; .O. bicolor, Leguminosae; flavonoids; triterpenoid saponins.
Abstract-Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-j-D-glucoside, rhamnetin-3.O-/3-D-galactoside, apigenin, 3-0-[a-L-rhamnopyranosyl (l-2)-fl-D-glucopyranosyl( l-4).fl-D-glucuronopyranosyl]soyasapogenol B, 3-O-[/3-D-glucopyranosyl( 1*2)-j-Dglucuronopyranosyl]azubisapogenol and a new saponin 3-O-[j?-D-glucopyranosyl(l~2)-~-D-glucopyranosyl]-25-0cl-L-rhamnopyranosyl-(20~,24S’)-3B,168, 20,24,25-pentahydroxy-9,19-cycloanostane.
INTRODUCTION In preceding papers [l-3], we reported some triterpenoid saponins from Oxytropis ochrocephala, 0. glabra and 0. bicolor. As a continuing study, we now report the isolation and identification of two flavonol glycosides 1,2 and a flavone 3 from 0. ochrocephala, two triterpenoid saponins 4 and 5 from 0. glabra and a new saponin 6 from 0. bicolor. RESULTSAND
DISCUSSION
Three flavonoids l-3 were isolated for the first time from the ethyl acetate extract of 0. ochrocephala. The flavonol glycoside 1 on hydrolysis gave isorhamnetin and D-ghcose, identified by paper chromatography. The aglycone was identified as isorhamnetin by UV spectral analysis and a comparison with literature data [4]. The position of sugar in the glycoside was located in C-3 by the comparison of the properties of aglycone and glycoside and spectral analysis showed 1 to be isorhamnetin 3-0-B-D-glucoside. Flavonol glycoside 2 on hydrolysis gave D-galactose and rhamnetin identified by analyses of its mass, ‘H NMR and UV spectra. The attachment of sugar was also located in C-3 by similar methods to those described above. Thus flavonol glycoside 2 is rhamnetin 3-0-p-Dgalactoside. Flavone 3 gave ions at m/z 270 [Ml’, 153 [A, +H]+, 162 [Al]+, 121 [Ba]+, 118 [BI]+ and 93 [B2 -CO]+. In the ‘HNMR spectrum of 3, the signals of H-3, OH-5 and OH-7 were observed at characteristic values [4] at 66.76, 12.94 and 10.77. Compound 3 was identified as apigenin by analysis of its UV and ‘H NMR spectrum. Two triterpenoid saponins 4 and 5 were isolated from the n-butanol extracts of 0. glabra. The FAB mass spectrum of saponin 4 gave ions at m/z 949 [M + Li] ’ and 965 [M + Na] ‘. On acidic hydrolysis 4 liberated an aglycone which was identified as soyasapogenol B by spectral and TLC comparison with an authentic sample [ 11. From the hydrolysate L-rhamnose, D-glucose and Dglucuronic acid were detected by paper chromatography.
The mass spectrum of the acetate of 4 gave ions at m/z 273 (terminal Rha)Ac]+ and 561 [(Glc-Rha)Ac] ‘, indicating the sequence of sugar moieties. The melting point, [a&, MS and “C NMR spectrum is identical with the known saponin 3-0-[cr-L-rhamnopyranosyl( l-+2)-/?-D-©ranosyl( 1-r4)-/?-D-glucuronopyranosyllsoyasapogenol B [l]; thus they are identical. Saponin 5 exhibited peaks in its FAB mass spectrum at m/z 817 [M+Li]+ and 833 [M+Na]‘, suggesting the molecular formula as C,,H,,O,,. Saponin 5 on hydrolysis with presence of methanol gave D-&tCOSe and Dglucuronic acid as sugar moieties, as well as an aglycone which was identified as azukisapogenol methyl ester by comparison of its [a&,, mass and ’ 3C NMR spectra with that of an authentic sample [S]. However, azukisapogenol, not its methyl ester, was confirmed to be the genuine aglycone of saponin 5 as follows. In the 13CNMR spectrum of 5, two carboxyl signals were observed at 6 173.2 ascribable to C-6 of glucuronic acid and 181.2 due to C-29 of the aglycone. The spectral comparison of its l 3C NMR with that of saponin 7 [S] also supported this conclusion. On methylation saponin 5 and 7 gave a same methyl ether. Thus saponin 5 is 3-0-[j?-D-glucopyranosyl (1+2)-fl-D-glucuronopyranosyl]azukisapogenol. Saponin 6 is a new compound isolated from the n-butanol extracts of 0. bicolor. The FAB mass spectrum of 6 showed peaks at m/z 969 [M + Li] ‘, and 985 [M +Na]+ suggesting a molecular formula of C,sH,,O,,. On hydrolysis 6 gave L-rhamnose and D-glucose as sugar moieties, as well as an aglycone which was identified as (20R,24S)-3P,16B,25-trihydroxy-20,24-epoxy-9,19-cyclolanostane by its spectral and TLC comparison with an authentic sample [3]. The EI mass spectrum of 6 gave ions at m/z 492 [(M - Rha - Glc x 2)] + 474 [(M - Rha 456 [(M-Rha-Glcx2)-H,O -Glcx2)-H,O]+, x2]+, 438 [(M-Rha-Glcx2)-H,Ox3]+, 420 [(M -Rha-Glcx2)-H,Ox4] and 402 [(M-Rha-Glc x 2)-Hz0 x 5]+, Those were quite similar to that of saponin 8 [3]. The EI mass spectrum of the acetate of 6 showed peaks at m/z 273 [(terminal Rha)Ac]‘, 331 [(terminal Glc)Ac]+ and 619 [(Glc-Glc)Ac] ‘. In the 13C NMR of 6, the signals of two sets of glucose and one
2707
2708
R.-Q. SW et al.
Rs
4
R1=H,R2+Glc=a-Rha,R3=OH,R4=Me
5
R’= p - Glc,R2=R3=H,R4=CG,H
7
RLP-Glc,R2=R3=H,R4=C02Me
BH ,
Q’-’
@-Glc-2/3-GlcO
6
R=a-Rha
8
R=H
-%Y
set of rhamnose were observed. The i3CNMR spectral comparisons of 6 with 8 showed that signals due to C-24, C-26 and C-27 of saponin 6 were somewhat displaced upfield, but signal due to C-25 downfield, while other carbon resonances remained almost unchanged. This indicated that the location of the rhamnose of 6 should be limited to OH-25 [6-81. Thus saponin 6 is 3-O-[/?D-gIucopyranosyI( l-+2)-/?-D-glucopyranosyfj-25-0-a-Lrhamnopyranosyl-(20S,24S)-3P,16B,20,24,25-pentahydroxy-9,19-cyclolanostane. EXPERIMENTAL Plant material. Oxytropis plants were collected from Gansu. China. Mps: uncorr. ‘H and i3CNMR with TMS as int. standard, EIMS were obtained at 70eV. Polyamide powder made by ourself and silica gel G (10-40, Qingdao Marine Chemical Factory, China) were used for CC. Solvent systems (A) C,H,-MeOH (5:l); (B) nused as follow, BuOH-EtOH-NH,OH (25%) (7:2:3); (C) CHCI,- MeOH-H,O (6:3:1, lower phase);(D) EtOAc-EtOH-H,O (8:2: 1); (E) EtOAc-pyridine-HOAc-H,O (5: 5: 1: 3); (F) i-PrOH-nBuOH-H,O (7: 1: 2). Isolation of jauonoids. Air-dried powdered aerial parts of 0. ochrocephala Bunge (2 kg) was extracted with 75% EtOH under reflux x 3. After removal of EtOH. the ag. phase was extracted with EtOAc. The EtOAc phase was coned to dryness under red. pres. to give a residue (8 g). Part of the residue (1 g) was chromatographed repeatedly on a polyamide column with solvent A to give flavonoid 1 (40 mg), 2 (30 mg) and 3 (40 mg). Isolation of saponins.The ethanolic extract of 0. glabra DC. (6 kg) was successively extracted with EtOAc and n-butanol saturated with H,O. The n-butanol extract (3 g) was first chromatographed on a dry column of silica gel (300 g). eluting with solvent B. The sepd frs were then further chromatographed repeatedly on silica gel cohunn wtth solvent C to give saponin 4
Table
1. t3CNMR spectral data of aglycone saponins 4-6 and 8 (in pyridine-d,)
moieties
C
4
5
6
8
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
39.0 26.7 91.7 44.2 56.8 18.9 33.2 40.4 48.2 36.9 24.4 122.6 145.3 42.9 26 7 29.3 38.2 45.9 46.9 31.0 42.1 75.7 23 1 63.8 16.0 17.4 25.5 20.9 32.9 28.9
39.1 27.0 90.3 42.6 55.8 19.0 32.5 39.7 47.4 36.2 24.0 123.0 144.3 41.5 26.3 28.1 32.5 41.5 46.3 43.6 36.2 30.5 22.5 63.4 15.5 16.6 25.8 28.1 181.2 19.7
33.1 32.0 88.7 41.2 47.5 21.0 25.7 48.0 19.7 26.2 26.3 29.9 46.9 46.6 48.7 75.0 56.3 21.4 30.6 76.8 26.1 412 26.6 78.5 78.0 23.1 24.1 20.2 15.2 25.7
33.7 32.0 88.8 41.2 47.3 21.0 26.3 47.6 19.7 26.2 26.5 29.9 47.2 46.6 49.1 73.5 55.5 21.2 30.2 76.7 26.1 42.7 27.3 79.9 72.8 25.7 25.8 20.4 15.3 26.2
in
Saponins from Oxytropis species Table 2. ‘“C NMR spectral data of sugar moieties in saponins 44 and 8 (in pyridine-de) C
4
5
GlllA 1 2 3 4 5 6
105.4 74.6 17.3 79.8 17.3 173.0
105.6 80.7 75.3 72.8 17.0 173.2
Glc 1 2 3 4 5 6
102.3 78.6 78.1 70.5 78.6 62.0
104.6 73.3 11.9 70.7 77.9 63.4
Glc 1 2 3 4 5 6 Rha 1 2 3 4 5 6
101.8 72.0 72.4 73.1 69.4 18.9
6
8
104.7 83.2 17.7 71.3 78.1 62.6
104.9 83.3 77.8 71.4 78.2 626
105.8 76.8 77.7 71.5 78.0 62.6
105.9 76.9 77.8 71.5 78.1 62.6
98.5 71.0 71.5 73.5 68.0 18.7
GluA = /?-n-Glucuronopyranosyl.
(500 mg), 5 (50 mg) and some other saponins reported earlier [Z]. The crude saponin (3g) from 0. bicolor Bunge (2 kg) was obtained by the similar procedure described above. Part of the crude saponin (2g) was repeatedly chromatographed on silica gel column with solvent C and D to give saponins 6 (50 mg) and 8 (3~mgJ c31. Fhomid 1. Yellow needles, mp 167-169”. EIMS (ml. int.) m/z 478 [M]’ (0.4), 316 [M-Glc]+ (lOO),301 [M-Glc-Me] (8), 287 (30), 153 [At+H]+ (8), 151 [Bz]+ (a), 136 [Bz-Me]+ (9). uv ,q$p nm: 254, 266sh, 354; (NaOMe) 277, 328, 411; (AICI,) 263,301,365,403; (AICI, +HCl) 257,404, (NaOAc) 274, 314, 382; (NaOAc+H,BOs) 268, 359. ‘HNMR [80 MHz, DMSG-d,]: 3.84 (3H, s, OMe), 5.6 (lH, d, J = 7 Hz, anomeric H of n-Glc), 6.21 (lH, d, J=2 Hz, H-6), 6.44 (lH, d, J=2 Hz, H-8), 6.91 (lH, d, J=9 Hz, H-S), 7.93 (lH, brs, H-2’), 8.04 (lH, d, J =9 Hz, H-6’). EIMS (acetate) m/z 773 [M +H]+ (0.5), 442 (5), 400 (6) 358 (lo), 331 [(terminal Glc)Ac]+ (39), 316 (ll), 287 (15), 271 (ll), 257 (8), 211(7), 169 (100) 145 (7), 139 (lo), 127 (20), 109 (55).
2709
Fhmoid 2. Yellow needles, mp 220-222”. EIMS (rel. int.) m/z316 [M-Gal]+ (lOO), 287 (12), 167 [A,+H]+ (7) 150 [Bi]’ (8), 137 [Bz]’ (23), 109 [Bz-CO]+ (8), (acetate) m/z 773 [M+H]+ (l), 442 (3) 400 (4), 358 (7), 331 [(terminal Gal)Ac]+ (40), 316 (16), 287 (ll), 271 (13) 257 (l), 211 (5) 169 (lOO),145 (8), 139 (7), 127 (25), 109 (60). UV %z$‘“nm: 258,269sh, 292sh, 360, (NaOMe) 272,297sh, 330, 418; (AlCl,) 268, 399, (AlCl, + HCI) 256, 296931, 358; (NaOAc) 256, 291sh, 363, 415; (NaOAc + HsBO,) 259,29lsh, 376. ‘H NMR [80 MHz, DMSO-d,]: 3.86 (3H, s, OMe), 5.4 (lH, d, J=7 Hz, anomeric H of D-Gal), 6.37 (lH, d, J=2 Hi., H-6), 6.69 (lH, d, J=2 Hz, H-8), 6.82 (lH, d, J =8.5Hz,H-5’),7.59(1H,brs,H-2’),7.70(1H,d,J=8.5Hz,H-6’), 9.07 (lH, brs, OH-3’, 9.66 (lH, brs, OH-4, 12.61 (lH, s, OH-5). Fhonoid 3. Amorphous, mp 343-345”. EIMS (rel. int.) m/z 270[M]+ (lOO), 153 [A,+H]+ (30), 152 [At]’ (20), 121 [Bz]’ (27), 118~,]+(16),(acetate)m/z396[M]+(1),354(41),312(33), 270 (100). Saponin 4. Crystal, mp 235-237”, [alA +O.l” (MeOH; c 0.5). FABMS (positive ion mode) m/z 949 IM + Li], 965 [M + Na]+. t3CNMR: (Tables 1 and 2). EIMS (acetate) m/z 561 [(RhaGlc)Ac] +, 273 [(terminal Rha)Ac] +. Soponin 5. Crystals, mp 223-225”, [a];’ + 2.2” (MeGH; c 0.5). FABMS(positiveionmode)m/z817 [M+Li]+, 833 [M +Na]+. t3CNMR: (Tables 1 and 2). According to Hakomori’s method [9] saponin 5 (50mg) was completely methylated to give a permethylate, ‘H NMR [80 MHz, (CD&O]; 0.87, 1.00, 1.06, 1.16, 1.21, 1.28 (3H each, all s, Me x 6), 3.31, 3.37, 3.45 (3H each, all s, OMe x 3), 3.48 (6H, s, OMe x 2), 3.55 (3H, s, OMe), 3.61(6H, s, OMe and COzMe), 3.76 (3H, s, COzMe), 4.58 (lH, d, J =7.0 Hz, anomeric H of a methylated sugar), 4.66 (lH, d, J = 7.5 Hz, anomeric H of another methylated sugar), 5.30 (lH, s, H-12). Saponin 6. Pow&r, mp 212-214”, [a]P +2.0 (MeGH; c 0.3). FABMS (positive ion mode) m/z 969 [M + Li]+ and 985 [M + Na] +. “CNMR: (Tables 1 and 2). EIMS (acetate) m/z 619 [(Glc-Glc)Ac] +, 331 [(terminal Glc)Ac]+, 273 [(terminal Rha)Ac] +. An aglycone with mp 24@242”, [a];’ + 47” (MeOH; c 0.5) identified as (20R,24S)-3/?,16j,25-trihydroxy-20,24-epoxy9,19-cyclolanostane by mmp and TLC comparison with authentic sample was obtained.
REFEBENCES
1. Sun, R. Q., Cheng, D. L., Jia, Z. J. and Zhu Z. Q. (1987) Huaxue Xuebao 6,145.
2 Sun, R. Q. and Jia, Z. J. (1990) Phytochemistry 29,2032 3. Sun, R. Q. and Jia, Z. J. (1991) Pkytochemistry 30 (in press). 4. Harborne, J. B. and Mabry, H. (eds) (1975) The Flauonoids. Chapman & Hall, London. 5. Sun, R. Q., Jia, Z. J. and Zhu, Z. Q. (1988) Chem. J. Chinese Univ. (Eng. Edn) 4(4), 39. 6. Kasai, R., Suzuo, M., Asakawa, J. and Tanak, 0. (1977) Tetrahedron Letters 175.
7. Tori, K, Seo, S., Yoshimura, Y., Arita, H. and Tomita, Y. (1977) Tetrahedron Latter-s 179. 8. Seo, S., Tomita, Y., Tori, K. and Yoshimura, M. (1978) J. Am. Chem Sot. loo, 3331. 9. Hakomori, S. (1964) J. Biockem. 55,205.