P~y~~~em~stry,Vol. 31, No. 4, pp. 1339 1342,1992 Printed in Great Britain.

TRITERPENOID Servo

003l-9422/92 $5.00+ 0.00 0 1992Pergamon Press plc

SAPONINS

FROM

SOPHORA SUBPROSTRATA

SAKA~OTG, MASA~OR~ KUROYANAGI, AKIRA UENO and SETSYJK~SEKITA*

School of Pharmaceutical Sciences, University of Shizuoka, 395 Yada, Shizuoka-shi 422, Japan; *National Institute of Hygienic Sciences, l-18-1 Kamiyouga Setagayaku, Tokyo IS& Japan ~Rece~~e~in

Key Word Index-Sophora

mbprostrata;

revised form

1 October

1991)

Leguminosae; roots; triterpenoidal saponin; kuzusapogenol A.

Abstract-From Sophora ~bprostr~ta Radix, the roots of So&ma su~prostrat~ six triterpenoidal saponins having soyasapogenol A, 3, sophoradioi and kuzusapo~nol A as aglycones, were isolated as their methyl esters. The structure of a new saponin was established to be 3-O-[ ~-L-rhamnopyranosyl(l~2)“~-galactopyranosyl( l-+2)-F-D~ueuronopyra~osyl~ kuzusapogenol A methyl ester by means of ‘H and 13C NMR spectroscopy and chemical evidence.

INTRODUCI’ION

amphora s~bpr~strata Radix is used as a Chinese folk medicine for the trea~ent of fever, inffammatio~ peptic ulcer and tumours. From this material, alkaloids and many types of fiavonoids have been isolated, but there have been no reports on the saponins of this plant. We isolated a new saponin (6) along with soyasaponin I [ 11, kaikasaponin 111[2J, soyasapo~in II [I], k~asaponin II [23 and soyasaponin A, [3] as their methyl esters. RESULTS AND DISCUSSION

The saponin fraction of the methanol extract of the Radix was methylated with di~omethane and separated by repeated column chromatography and HPLC using a reversed phase column to give six saponins as their methyl esters (14). Saponin 1 showed the pseudo molecular ion at m/z 957 [M+H]+ in the positive FAB mass spectrum, the molecular formula was concluded to be C,,H,,U,,. The ‘HNMR spectrum showed the seven singlet methyl groups and signals at 65.76 (d, J==7.3 Hz) and 6.27 (s) indicating the presence of a P-linked methyl glucuronopyranosyl group and an a-linked rhamnopyranosyl group, respectively. The r3C NMR spectrum showed the presence of three sugar moieties, and also showed the presence of a hydr~xym~thyl group at C-24 (663.8) and a hydroxyl group linked to C-22 (675.8). Saponin 1 was methanolized with 5% HCl-methanol to give methyl sugars, which were ident~~ as methyl galactopyr~o~ side and methyl rh~nopyranoside by HPLC of the methyl sugar benzoates and by microanalysis [4, S]. Thus, saponin I is soyasaponin I methyl ester. The molecular formulae of saponins Z-5 were determined from their FAB mass spectra to be C49Hso017, C49H7s017, ~.J&,O,~ and c,~Hs&i,, respectively. The ‘H and 13C NMR spectra showed that 2 and 4 have the same aglycone, sophoradiol [ 133. The aglycones of 3 and 5 were deduced to be soyasapogenol B and A,

respectively. The sugar parts of these saponins were identified by micro analysis and from the ‘H and ’ 3C NMR spectra. Thus, saponins 2-5 are kaikasaponin III methyl ester, soyasaponi~ V methyl ester, kaikasaponin I methyl ester and soyasaponin A, methyl ester, respectively. Saponin 6 showed the pseudo molecular ion at m/z 989 [M+HJ’ and m/z 1011 fM -tNaJ+. Thus the molecular formula is C,,Hs,O,,. The r3C NMR spectrum showed five carbinyl carbon signals (863.4, 70.0, 70.9, 79.7 and 91.3) of the aglycone part. The ‘HNMR spectrum showed the presence of only six methyl signals. From these data, it was suggested that the aglycone has five hydroxyl groups and two of these are present as hydroxymethyl groups. These facts indicated that the aglycone is k~usa~genol A 16). Therefore, the 13CNMR spectrum (Table 1) of the aglycone moiety was compared to kuzusapogenol A and the two showed identical chemical shifts. The 13CNMR spectrum (Table 2) of 6 showed the same signal pattern for the sugar part as that of 1. Thus, saponin 6 is 3-O-[~“~-rhamnopyranosyl(l42)-~-~” galactopyranosyl(l-+2)+~-glucuronopyranosyl]kuzusapogenol A methyl ester.

“H and 13&NMR spectra: 300 and 400 MHz s~ctr~rn~t~~. FAB-MS gIycero1 as matrix. CC was carried out on silica gel type 60 (Merck). ~sol~~~~n.The dried roots of S~~~oru s~prostr~t~ (4.5 kg), purchase from Niiya, Shi~u-shi Shizuoka were extracted with MeOH under reflux. The MeOH extract was partitioned between EtOAc and H,O. The Ha0 fraction was applied to Diaion HP 20 column and washed with H,O, followed by elution with 50% MeOH and MeOH, successively. The MeOH eluent showed the presence of saponins on TLC. The MeOH eluent was coned in vacua and dissolved in MeOH and methylated with CHIN,. The methylated reaction soln was coned and separated by silica gel CC usmg a gradient of CHCis-MeQH

1339

1340

S. SAKAMOTO rr al.

HO

St

s2

and CHCIs-MeOH-Hz0 (13 : 7: 2, lower phase), successively. The resultant fractions were further purified by HPLC using reversed phase column {YMC ODS-7, Yamamura and Deverosil C-8-10, Nomura) and 45-50% MeCN aq. solvent system to give the saponins 1 (250 mg), 2 (210 mg), 3 (90 mg), 4 (40 mg), 5 (22 mg) and 6 (27 mg). Soyasaponin I methyl ester (I). Amorphous powder. FAB-MS m/z 957 [M + H]+ C4sHs101s. ‘H NMR (pyridine-d,): 60.69 (s, Me), 0.93 (s, Me), 0.97 (s, Me), 1.20 (s, Me), 1.25 (s, Me). 1.27 (s, Me), 1.42 (s, Me), 1.74 (d, J=6.2 Hz, Rha-6). 3.73 (s, GlcACO,Me), 5.76 (d, J=7.3 Hz, GlcA-I), 6.27 (br s, Rha-I). 13C NMR data are shown in Tables 1 and 2. Karkasaponin 111 methyl ester (2). Amorphous powder. FABMS: m/z 941 LM+H]+ C4aH8,01,. ‘HNMR (pyridine-d,): 60.83 (s, Me), 0.96 (s, Me), 1.13 (s, Me), 1.20 (s. Me), 1.23 (s, Me),

OH

s3

1.25 (s, Me), 1.30 (s, Me), 1.73 (d, J = 6.0 Hz, Rha-6), 3.69 (s, GlcACOzMe), 5.67 (d, 5~7.0 Hz, GlcA-i), 6.23 (hr s, Rha-1). r3C NMR data are shown in Tables 1 and 2. So yasaponiff If methyl ester (3). Amorphous powder. FAB-MS m/z: 927 CM + H]+ C 48H 790 17 ‘H NMR ~py~din~~): 60.73 (s, Me), 0.96 (s. Me). 0.99 Ls. Me). 1.23 fs, Me), 1.28 fs. Me) x 2.1.43 fs. Me), 1.77 id, J= 5.8 Hi Rha-I), 3.;5 (s, &A-dO,Me), 5.58 (d,‘; 3 7.6 Hz, GlcA-I). 632 (hr s, Rha-1). 13C NMR data are shown in Tables 1 and 2. Kaikasapomn I methyl ester (4). Amorphous powder. FAB-MS m/z: 941 [M+H]+ C,,Hs,O,,. ‘HNMR@yridine-d,): 50.83(s, Me), O.%(s, Me) x 2, 1.13 (s, Me). 1.20(s, Me), 1.23 (s, Me), 1.25(s, Me), 1.30 (s, Me), 1.74 (d, J=6.l_ Hz. Rha-6), 3.31 (dd, J=11.4, 3.5 Hz. H-3), 3.70 (s, GIcA-COzMe), 3.84 (dd, J=121.1, 3.6 Hz, H-22), 5.01 (4 J=7.7 HZ, Glc-1), 5.78 (br s, Rha-1). 5.85 (d, J

Triterpenoid saponins from Sophora s~&prostrata Table 1. “CNMR

spectral data of the aglycone moieties of compounds ld (pyridine-d,)

C

1

2

3

4

5

6

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

38.7 26.5 91.6 44.0 56.3 18.6 33.4 40.0 48.0 36.5 24.1 122.8 145.4 42.5 26.7 28.7 38.1 45.4 46.9 31.0 42.4 75.8 23.1 63.8 15.9 17.0 25.8 28.1 33.4 21.2

39.0 26.5 90.3 39.8 56.1 18.6 33.2 40.1 48.1 36.9 23.9 122.9 145.3 42.5 26.5 28.7 38.1 45.4 46.9 31.0 42.4 75.8 28.2 16.8 15.7 17.2 25.8 28.7 33.4 21.2

38.8 26.5 91.4 44.1 56.3 18.6 33.4 40.0 48.0 36.6 24.1 122.8 145.3 42.5 26.8 28.7 38.1 45.4 46.9 31.0 42.4 75.8 23.0 63.7 15.9 17.0 25.8 28.7 33.4 21.2

38.9 26.4 89.6 39.6 55.9 18.8 33.2 40.0 48.0 36.9 23.8 122.8 145.3 42.5 26.6 28.7 38.1 45.4 46.8 30.9 42.3 75.8 28.2 16.9 15.7 17.1 25.9 28.7 33.2 21.2

39.1 26.5 91.2 43.8 55.9 18.9 32.8 40.1 47.1 36.5 24.0 122.7 144.5 42.0 26.5 27.3 38.5 43.8 47.2 36.3 74.6 79.6 32.9 63.5 15.7 16.8 26.5 22.2 31.5 18.4

38.9 26.3 91.3 43.7 55.9 18.7 32.7 39.9 47.6 36.2 23.8 122.5 144.9 41.8 26.4 27.0 38.4 43.0 40.9 40.8 70.0 79.7 22.6 63.6 15.4 16.6 26.4 22.0 70.9 17.1

= 5.8 Hz, GlcA-1). 13CNMR data are shown in Tables 1 and 2 So~asa~ni~ A, methyl ester (5). Amorphous powder. FABMS m/z: 973 [M-i-H]” C49H810,9. ‘HNMR (pyridine-d,): 60.75 (s, Me), 0.92 (s, Me), 1.23 (s, Me), 1.25 (s, Me), 1.27 (s, Me), 1.41 (s, Me), 1.42 (s, Me), 1.75 (d, J =6.2 Hz, Rha-6), 3.74 (s, GlcACO,Me), 6.11 (br s, Rha-1). 13CNMR data are shown in Tables 1 and 2. 3-O-[a-L-rhamnopyranosyl(1-+2)-&n-gaZactopyranosyl(l -t2)D-gluCUrOnOpyrUnOSy~~#ZUSUpOgenol A methyl ester (6). Amorphous powder. FAB-MS m/z: 989 [M + H]+ C,,H,,02e. 1011 [M +Na]+ C,,H,,O,eNa. ‘HNMR (pyrite-dab 60.61 (s, Me), 0.85 (s, Me), 1.22 (s, Me), 1.26 (s, Me), 1.34 (s, Me), 1.39 (s, Me), 1.73 (d, J =6.0 Hz, Rha-6), 3.73 (s, GlcA-CO,Me), 6.11 (br s, Rha-1). 13CNMR data are shown in Tables 1 and 2. Acid hydrolysis of soyasaponin I methyl ester (1). A soln of 1 (1OOmg) in aq. 10% H,SO,-MeGH (1:2, 15ml) was heated under relhtx for 12 hr. The reaction soln was extracted witb EtOAc. The EtOAc layer was washed with satd NaHCO, and H,O, successively. After concn, the reaction product was purified by HPLC (ODS column, 85% Me&N) to give soyasapogenol B (2.7 mg), which was identical with the authentic sample. Methanolysis ofsaponins l-6. A soln of each saponin (co 1 mg) in 5% HCl-MeOH (3 ml) was refluxed for 2 hr. The reaction soln PBYTO

31:4-9

1341

was neutrabz.ed with Ag,CO, and filtered. The filtrate was evapd to dryness. To the reaction mixture in pyridine (1 ml), pbromobenzoyl chloride (excess) was added and stirred at 90” overnight. The reaction soln was poured into Hz0 and extracted with hexane. The hexane soln was washed with H,O and coned. The methylsugar per-p-bromobenzoates were analysed by HPLC (ODS column, 90% Me&N soln), and identified by comparison with the authentic samples. Determination of the A-values. Per-p-bromobenzoates of methyl sugars were purified on analytical scale by HPLC. The collected peaks were coned and MeCN added. The UV and CD spectra of the MeCN solns were measured. The concn of the benzoates were calculated from the absorbance and the reported extinction coefficiences (57200 for tri-p-bromobenzoate and 76400 for tetra-p-bromobenzoate). The A-values wkre calculated from the CD data of p-bromobenzoate of methyl sugars. The Avalues of the p-bromobenzoates were identical with the authentic A-values.

Acknowledgements--We are grateful to Prof. I. Kitagawa for providing soyasaponin I and soyasapogenol B. We also thank Dr M. Uchida of our University for rn~u~rn~t of FAB-MS.

1342

S. SAKAMOTO et al.

Table

GlcA-1 2 3 4 5 6 OMe Gal-l 2 3 4 5 6 Ara-1 2 3 4 5 Glc-1 2 3 4 5 6 Rha-1 2 3 4 5 6

2. ‘“C NMR spectral

data of the sugar moieties

of compounds

l-6 (pyridine-d,)

1

2

3

4

5

6

105.8 78.5 76.7 74.6 77.2 171.0 51.7 102.1 78.0 73.0 71.4 76.8 61.8

105.7 79.0 76.4 74.6 76.8 171.3 52.1 103.1 79.3 72.9 70.7 76.6 62.1

105.8 78.2 76.7 74.6 77.2 171.0 52.3

106.0 77.5 75.3 74.2 77.0 171.1 52.2

105.4 78.2 76.3 74.3 76.8 170.4 52.1 101.7 77.6 72.3 71.0 76.5 61.5

105.5 77.9 76.3 74.2 76.8 170.8 52.3 101.7 77.6 72.4 71.0 76.4 61.5

102.4 72.3 72.7 73.5 69.3 18.4

102.4 72.2 72.4 73.4 69.2 18.3

102.2 77.9 76.0 70.8 67.2

102.8 72.6 73.0 73.8 69.6 19.0

102.4 72.6 72.9 73.4 69.6 18.5

-

102.8 72.6 73.0 74.0 69.6 19.0

REFERENCES 1. Kitagawa, I., Hui, K. W., Taniyama, T. and Yoshikawa, M. (1988) Chem. Pharm. Bull. 36, 153. 2. Kitagawa, I., Taniyama, T., Hui, K. W., Hori, K. and Yoshikawa, M. (1988) Yakugaku Zasshi 108, 538. 3. Caralyn, L., Curl, C. L., Keith, R., Price, G. and Fenwick, R. (1988) J. Nat. Prod. 51, 122.

101.1 79.2 73.2 70.4 75.8 63.2 102.7 72.1 72.6 72.8 66.9 18.4

4. Liu, H.-W. and Nakanishi, K. (1982) J. Am. Chem. Sot. 104, 1178. 5. Nakamshi, K., Kuroyanagi, M., Nambu, H., Oltz, E. M., Takeda, R., Verdine, G. L. and Zask, A. (1984) Pure Appl. Chem. 56, 1031. 6. Kinjo, J., Miyamoto, I., Murakami, K., Kido, K., Tomimatsu, T., Yamasaki, M. and Nohara, T. (1985) Chem. Pharm. Bull. 33, 1293.