Hepatoprotective triterpenes from Sedum sarmentosum

Article abstract of DOI:10.1016/S0031-9422(98)00434-8

The known δ-amyrone, 3-epi-δ-amyrin, δ-amyrin as well as a new hydroperoxy triterpene, 18β-hydroperoxy-olean-12-en-3-one (named sarmentolin) were isolated as hepatoprotective agents from Sedum sarmentosum. Their structures were elucidated by spectral analysis and chemical reactions.

Full text of DOI:10.1016/S0031-9422(98)00434-8

Phytochemistry Vol. 49, No. 8, pp. 2607±2610, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
0031-9422/98/$ - see front matter
PII: S0031-9422(98)00434-8
HEPATOPROTECTIVE TRITERPENES FROM SEDUM
SARMENTOSUM
AIMIN HE, * MINGSHI WANG,} HONGYAN HAO, DECHENG ZHANG and
KUO-HSIUNG LEEk
Department of Chemistry of Natural Drugs, School of Pharmacy, Shanghai Medical University,
Shanghai 200032, P.R. China, Department of Pharmacognosy, School of Pharmacy, Shanghai
Medical University, Shanghai 200032, P.R. China, }Department of Phytochemistry, China
k
Pharmaceutical University, Nanjing 210009, P.R. China and Natural Products Laboratory, Division of
Medicinal Chemistry and Natural Products, School of Pharmacy, The University of North Carolina,
Chapel Hill, NC 27599, U.S.A.
(Received 20 April 1998)
Key Word IndexÐSedum sarmentosum; Crassulaceae; triterpene; sarmentolin; hepatoprotec-
tive activity.
AbstractÐThe known d-amyrone, 3-epi-d-amyrin, d-amyrin as well as a new hydroperoxy triterpene, 18b-
hydroperoxy-olean-12-en-3-one (named sarmentolin) were isolated as hepatoprotective agents from Sedum
sarmentosum. Their structures were elucidated by spectral analysis and chemical reactions. # 1998 Elsevier
Science Ltd. All rights reserved
INTRODUCTION
RESULTS AND DISCUSSION
The ethanol extract from S. sarmentosum was dis-
solved in ethanol±water (1:9) and extracted succes-
Sedum sarmentosum Bunge has been used in folk
medicine for the treatment of chronic viral hepatitis.
Pharmaceutical granules of this plant have been
used in clinics since 1971 [1]. In view of its repu-
tation as an hepatoprotective drug, many investi-
gators have studied its chemical constituents.
Sarmentosin, a water-soluble cyanophoric glycoside,
was considered as the active constituent of the
drug [2, 3]. However, pharmaceutical granules that
lack this compound but still show remarkable
activity. This prompted us to extend our investi-
gations on the plant. In previous papers, we
reported the occurrence of the ¯avanoids [4] and
sterols [5] in the plant. A detailed search for the
hepatoprotective agents from this plant has led to
the isolation of three known triterpenes and a novel
hydroperoxy triterpene, 18b-hydroperoxy-olean-
12(13)-en-3-one, named sarmentolin. In the present
paper, we describe the isolation and structure eluci-
dation of the active triterpenes from S. sarmentosum
and their hepatoprotective activity.
sively
with
petrol
and
ethyl
acetate.
Chromatography of the ethyl acetate extract gave
four triterpenes: d-amyrone (1, 1007 mg), 3-epi-d-
amyrin (2, 110 mg), d-amyrin (3, 600 mg) and sar-
mentolin (4, 50 mg), respectively.
Sarmentolin (4) gave a [M + H]+ peak at m/z
457.3699 on HRCIMS, corresponding to a molecu-
lar formula of C30H48O3, which was also indicated
by the 13C NMR data. It gave a deep red color
with Liebermann±Burchard reagent and the charac-
teristic IR absorption bands (1384, 1353, 1326,
1295, 1264 cm 1) of a pentacyclotriterpene [6]. The
IR and 13C NMR data show that 4 contained a
carbonyl group [1695 cm 1; dc 217.2 (s)], the posi-
tive Zimmermann colour reaction suggested it as a
3-keto group, and a hydroperoxy group [3423, 874,
816 cm 1; dH 6.44 (1H, s) was lost on addition of
D2O and d 87.2 (s, quaternary carbon bonded to
oxygen function), EIMS m/z 438 [M±H2O]+] [7].
The 1H NMR data also showed the presence of
eight tertiary methyl groups (d 0.85, 0.93, 1.00,
1.04, 1.05, 1.08, 1.13, 1.33) and a trisubstituted
*Author to whom correspondence should be sent.
2607

2608
Short Report
Scheme 1.
double bond, indicating the presence of an olean and leaves them una€ected. Therefore, the stereo-
12(13)-ene system [8, 9]. Further evidence was
obtained from the diagnostically important EIMS
fragment ions (m/z 205 and 217) (Scheme 1) associ-
ated with an RDA fragmentation in ring-C [10].
The hydroperoxy group in 4 was assigned at C-
18 by HMBC correlation. Both protons of the C-17
methyl group and the C-12 ole®nic proton corre-
lated to the carbon signal at d 87.2 (s). All proton
chemistry at C-18 was unequivocally established as
18b-hydroperoxy in 4.
Triterpenes bearing a hydroperoxy moiety are
rarely distributed in nature. So far only two com-
pounds have been reported [7, 14]. Sarmentolin 4 is
the third triterpenoid hydroperoxide isolated from
plants.
Identi®cation of three known triterpenes isolated
from S. sarmentosum was achieved by spectroscopic
1
and carbon signals were assigned by DEPT, H±1H
COSY, 13C±1H COSY, HMQC and HMBC
(Table 1).
1
methods (Mp, IR, H NMR, 13C NMR and EIMS)
as well as chemical reactions. Their structures were
elucidated as d-amyrone (1) [15, 16], 3-epi-d-amyrin
(2) [17] and d-amyrin (3) [18]. The C13±C18 double
bond of d-amyrone (1) could not be catalytically
hydrogenated using PtO2 as a catalyst, but the 3-
keto group of 1 could be reduced to give 2 and 3.
On oxidation of 2 and 3 with Jones' reagent, both
of them yield d-amyrone (1).
The stereochemistry at C-18 was deduced from
the e€ect of the hydroperoxy group on the 12±13
ole®nic bond in 4. This e€ect was revealed by the
13C NMR data [11, 12]. If the D/E ring-junction is
cis, as usually present in an olean-12(13)-ene type
triterpene, the 18-hydroperoxy group is equatorial
and quite close to the p electron system of the 12±
13 ole®nic bond. Because of the steric in¯uence due
to the polar character of the hydroperoxy, C-12
was deshielded by 4.0 ppm and C-13 shielded by
5.4 ppm compared to the C-12 and C-13 chemical
shift in b-amyrin [13]. However, if the D/E ring is
trans, the 18-hydroperoxy assumes a-axial con®gur-
It was found that d-amyrone (1) markedly low-
ered the level of serum glutamic±pyruvic transamin-
ase
tetrachloromethane in olive oil [19]. Further
of
mice
treated
with
0.1 ml/kg
research into the hepatoprotective activity of 2±4
ation and is far removed from 12±13 ole®nic bond on liver injury mice is being carried out.

Short Report
2609
Table 1. 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectral data of compound 4 and 13C NMR
(100 MHz, CDCl3) spectral data of compounds 1±3 (d values from TMS; J in Hz)
C
4
4
1
2
3
1
2
3
4
5
6
7
8
1.92 (1H, m); 1.42 (1H, m)
2.52 (1H, m); 2.38 (1H, m)
39.3
34.1
217.2
47.2
55.3
19.7
32.2
40.1
46.1
36.7
23.8
125.7
139.6
42.7
25.5
30.7
37.3
87.2
36.6
30.9
33.8
31.5
26.7
21.4
15.3
16.7
24.5
20.1
35.1
28.3
39.3
34.1
218.2
47.2
54.8
19.8
26.5
40.9
50.0
36.9
22.2
26.5
134.1
44.7
25.0
36.5
34.5
133.6
39.6
33.3
35.3
38.7
21.0
26.9
16.3
17.6
21.2
24.1
23.8
32.3
33.6
25.5
76.3
37.6
49.2
18.5
34.8
41.4
50.7
37.5
21.7
26.6
134.6
44.9
25.2
36.8
34.7
133.1
39.5
33.4
35.6
38.7
22.3
28.3
16.2
17.7
21.6
24.2
23.9
32.4
38.8
27.4
79.0
38.8
55.4
18.5
34.9
41.0
50.7
37.3
21.8
26.5
134.3
44.7
25.0
36.7
34.6
133.1
39.4
33.3
35.4
38.7
17.7
28.1
15.5
16.4
21.4
24.1
23.8
32.4
1.33 (1H, t, J = 6.7 Hz)
1.49 (2H, br d, J = 6.7 Hz)
1.57 (1H, m); 1.39 (1H, m)
9
1.69 (1H, t, J = 9 Hz)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
-OOH
2.03 (2H, dd, J = 9, 3.7 Hz)
5.63 (1H, d, J = 3.7 Hz)
1.90 (1H, m); 1.04 (1H, m)
2.01 (1H, m); 0.93 (1H, m)
1.98 (1H, br s); 1.15 (1H, br s)
1.49 (1H, m); 1.24 (1H, m)
1.88 (1H, m); 0.93 (1H, m)
1.08 (3H, s)
1.04 (3H, s)
1.05 (3H, s)
1.00 (3H, s)
1.33 (3H, s)
0.85 (3H, s)
0.93 (3H, s)
1.12 (3H, s)
6.44 (1H, s)
EXPERIMENTAL
according to their TLC behavior. Fractions 11±17
gave d-amyrone (1, 1007 mg), fractions 20±22 gave
3-epi-d-amyrin (2, 110 mg), fractions 39±44 gave d-
amyrin (3, 600 mg), fractions 49±53 were recrystal-
lized from EtOH±CHCl3 sarmentolin to give (4,
50 mg).
General
1
M.p.'s: uncorr; H NMR and 13C NMR: 400 and
100 MHz (TMS as internal standard in CDCl3), re-
spectively. 2D NMR spectra (1H±1H COSY, 13C±
1H COSY, HMQC and HMBC): 400 MHz using
standard pulse sequences; optical rotations: CHCl3;
MS: 70 eV, direct inlet system: CC: silica gel 60
d-Amyrone (1)
Needles, m.p. 202±2038C [a]2D8 +2.28 (CHCl3, c
cm 1: 2960, 2860, 1706, 1383, 1361,
KBr
max
(200±300 mesh); TLC: precoated silica gel (Merck 0.11); IR n
silica gel 60 F-254). Detection of compounds by 1349, 1294, 1263; 1H NMR (CDCl3): d 0.68, 0.88,
spraying with 7.5% H2SO4 EtOH soln followed by 1.00, 1.02, 1.08, 1.16 (each 3H, s), 0.92 (6H, s); 13C
heating at 1058C for 3 min.
NMR (CDCl3): Table 1; MS m/z (rel. int.):
424.3755 [M]+ (C30H48O requires 424.3707) (23.4),
409 (13.8), 205 (100), 189 (20.5), 109 (60.3), 95
(42.9).
Plant material
The fresh herb (S. sarmentosum Bunge) was col-
lected from Ying county, Zhejiang Province, China,
in August 1993. A voucher specimen (No. 930824)
is available for inspection at the Herbarium of
China Pharmaceutical University, Nanjing, China.
Hydrogenation of d-amyrone (1)
A pure sample of 1 (10 mg) was dissolved in
EtOH (5 ml) and hydrogenated with PtO2 at atm.
pres. for 24 h with stirring. The soln was ®ltered
and after removal of the solvent under reduced
pressure the residue was chromatographed by CC
[6 g silica gel 60, 200±300 mesh, eluting with petrol
EtOAc (1:49)] to give 3-epi-d-amyrin (2) and d-
amyrin (3) (1:1, total 7.6 mg).
Extraction and isolation of compounds
The fresh whole herb materials (70 kg) were
extracted successively with 90 and 80% EtOH.
Removal of solvent gave a syrup. The syrup was
dissolved in 10% EtOH and partitioned with petrol,
EtOAc and n-BuOH, respectively. Chromatography
of the EtOAc extract on silica gel (10 Â110 cm)
using a petrol EtOAc gradient gave 180 fractions
3-Epi-d-amyrin (2)
Needles, m.p. 209±2108C [a]2D8 47.58 (CHCl3; c
KBr
max
(1000 ml each) which were collected and combined 0.22); IR n
cm 1: 3385, 3045, 2950, 2865, 1636,

2610
Short Report
1472, 1465, 1372, 1280, 1043, 976; 1H NMR for the measurements of MS and Ms Ping Wang
(CDCl3): d 0.68, 0.81, 0.91, 0.93, 0.99, 1.15 (each (Shanghai Institute of Meteria Medica, Shanghai,
3H, s), 0.84 (6H, s), 3.4 (1H, br, s); 13C NMR China) for performing the 2D NMR spectra.
(CDCl3): Table 1; MS m/z (rel. int.) 426[M]+
(C30H50O) (15.2), 411 (4.4), 218 (43.4), 205 (53.6),
204 (33.9), 190 (45.9), 135 (34.9), 109 (100), 95
(83.5).
REFERENCES
1. Encyclopedia of Chinese Materia Medica (1993)
(Zhong Yao Da Chi Dian), 6th ed., ed. Jiang Su
New Medical College. Shanghai Science and
Technology Publisher, p. 608.
2. Fang, S. D., Yan, X. Q., Li, J. F., Xu, X. Y.
and Xu, R. S., Kexue Tongbao, 1979, 431.
3. Fang, S. D., Yan, X. Q., Li, J. F., Xu, X. Y.
and Xu, R. S., Acta Chim. Sin., 1982, 40, 273.
4. He, A. M. and Wang, M. S., Zhongcapyao,
1997, 28, 517.
5. He, A. M., Wang, M. S., Hao, H. Y. and
Zhang, D. C., J. China Pharm. Univ., 1997, 28,
271.
6. Snatzke, G., Lampert, F. and Tschesche, R.,
Tetrahedron, 1962, 18, 1417.
Oxidation of 3-epi-d-amyrin (2)
To a soln of 2 (20 mg) in Me2CO (7 ml), was
added 7 drops of Jones' reagent within 10 min. The
mixture was kept at 08C for 2 h with stirring and
then it was diluted with H2O and the product
extracted with CHCl3. The CHCl3 extract was
washed, dried and evapd to give a ketone (15.8 mg).
The spectrum of the ketone was identical to that of
d-amyrone.
d-Amyrin (3)
Needles, m.p. 214±215.58C; [a]2D8 52.48 (CHCl3;
KBr
c 0.20); IR n
cm 1: 3411, 2960, 2920, 2860, 1382,
max
1361, 1342, 1294, 1244; 1H NMR (CDCl3): d 0.68,
0.75, 0.91, 0.97, 1.00, 1.14 (each 3H, s), 0.83 (6H,
s), 3.21 (1H, m); 13C NMR (CDCl3): Table 1;
EIMS m/z (rel. int.) 426.3896 [M]+ (C30H50O
requires 426.3864) (26.1), 411 (9.8), 408 (24.2), 229
(12.1), 218 (51.9), 205 (95.4), 189 (84.0), 109 (100),
95 (76.7), 69 (48.2), 55 (48).
7. Nozaki, H., Suzuki, H., Hirayama, T., Kasai,
R., Wu, R. Y. and Lee, K. H., Phytochemistry,
1986, 25, 479.
8. Nakanishi, K., Goto, T., Ito, S., Natori, S. and
Nozoe, S., Natural Products Chemistry, Vol. 1.
Academic Press, New York, 1974, p. 365.
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J. Org. Chem., 1962, 27, 4512.
10. Budzikiewicz, H., Wilson, J. M. and Djerassi,
C., J. Am. Chem. Soc., 1963, 85, 3688.
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G., Tetrahedron Lett., 1974, 27, 2381.
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S. and Row, L. R., J. Indian Chem. Soc., 1984,
60, 1032.
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Kondoh, H., Ornura, S., Zhang, P. L., Rao, C.
and Chen, W. M., Planta Med., 1989, 55, 44.
14. Chavez, M. I. and Delgado, G., Tetrahedron,
1994, 50, 3869.
Oxidation of d-amyrin (3)
A pure sample of 3 (20 mg) was oxidized with
Jones' reagent, processing as in Section 3.7, to give
d-amyrone (16 mg).
Sarmentolin (4)
Needles, m.p. 210±2118C; [a]2D8 +83.68 (CHCl3, c
KBr
0.11); IR n
cm 1: 3423, 3047, 2948, 2855, 1695,
max
1655, 1461, 1384, 1264, 924, 874, 845, 816; 1H
NMR (CDCl3): Table 1; 13C NMR (CDCl3):
Table 1; CIMS m/z (rel. int.) 457.3621 [M + H]+
(C30H48O3H requires 457.3605) (19.2), 439 [M±
H2O + H]+ (70.2), 423 (86.9), 307 (100); EIMS m/z
(rel. int.) 438 [M±H2O]+ (4.4), 423 (16.1), 356 (4.8),
224 (24.6), 217 (6.2), 205 (17.2), 145 (11.7), 109
(13.7), 105 (18.7), 95 (25.9), 70 (100).
15. Wollenweber, E., Z. Naturforsch. C, 1974, 29,
362.
16. Manners, G. D. and Davis, D. G.,
Phytochemistry, 1984, 25, 1059.
17. Sarker, A. and Ganguly, S. N., Indian J.
Chem., 1978, 16B, 742.
AcknowledgementsÐWe wish to thank Dr Xianguo
Zhao (Department of Pharmacognosy, China 18. Tanaka,
Pharmaceutical University, Nanjing, China) for Phytochemistry, 1988, 27, 3579.
botanical identi®cation of S. sarmentosum, Dr 19. Yue, W. C., Jie, Z. N. and Tang, Y., J. China
Yanlong Guo (China Pharmaceutical University) Pharm. Univ., 1996, 27, 425.
R.
and
Matsunaga,
S.,