Bioactive constituents from chinese natural medicines. XXXI.1 hepatoprotective principles from sinocrassula indica: Structures of sinocrassosides A8, A9, A10, A11, and A12

Article abstract of DOI:10.3987/COM-08-11385

The methanolic extract from the whole plant of Sinocrassula indica (Crassulaceae) was found to show hepatoprotective effect on D-galactosamineinduced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic extract, five new acylated flavon

Full text of DOI:10.3987/COM-08-11385

HETEROCYCLES, Vol. 75, No. 8, 2008, pp. 1983 - 1995. © The Japan Institute of Heterocyclic Chemistry
Received, 11th March, 2008, Accepted, 14th April, 2008, Published online, 17th April, 2008. COM-08-11385
BIOACTIVE CONSTITUENTS FROM CHINESE NATURAL MEDICINES.
1
XXXI. HEPATOPROTECTIVE PRINCIPLES FROM SINOCRASSULA
INDICA: STRUCTURES OF SINOCRASSOSIDES A , A , A , A , AND
8
9
10
11
A
12
a,b)
a,b)
a)
Haihui Xie, Hisashi Matsuda,
a)
Kiyofumi Ninomiya,
Toshio Morikawa,
,a)
and Masayuki Yoshikawa*
a) Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412,
Japan
b) Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1
Kowakae, Higashi-osaka, Osaka 577-8502, Japan
Abstract —The methanolic extract from the whole plant of Sinocrassula indica
(Crassulaceae) was found to show hepatoprotective effect on D-galactosamine-
induced cytotoxicity in primary cultured mouse hepatocytes. From the methanolic
extract, five new acylated flavonol glycosides, sinocrassosides A (1), A (2), A
8
9
10
(3), A (4), and A (5), were isolated together with 25 compounds. The
11
12
structures of 1—5 were elucidated on the basis of chemical and physicochemical
evidence. Among the isolated compounds, sinocrassosides A (6), A (7), and B
2
1
2
(14) were found to show potent hepatoprotective effect.
Sinocrassula indica (DECHE.) BERGER, a Crassulaceae biennial plant, is distributed on the mountain areas
in China (e.g. Yunnan, Guangxi, Sichuan, Guizhou, and Hunan provinces of China). The whole plant of S.
2
indica has been used for the treatment of hepatitis and otitis media in Chinese traditional medicine.
1,3–25
During the course of our studies on bioactive constituents from Chinese natural medicines,
we found
that the methanolic extract of the whole plant of S. indica inhibited the increase in serum glucose levels in
3
both sucrose- and glucose-loaded rats. Furthermore, from the methanolic extract, 10 acylated flavonol
glycosides, sinocrassosides A —A and B —B (6—15), were isolated together with 11 known
3
3,4
1
7
1
flavonoids (16—26), two megastigmanes (27, 28), L-phenylalanine, and guanosine.
As a continuing
study on the constituents from S. indica, the methanolic extract of this herbal medicine was found to show
hepatoprotective effect on D-galactosamine (D-GalN)-induced cytotoxicity in primary cultured mouse
hepatocytes. From the methanolic extract, we additionally isolated five new acylated flavonol glycosides,
sinocrassosides A (1), A (2), A (3), A (4), and A (5). This paper deals with the isolation and
8
9
10
11
12
structure elucidation of 1—5 as well as hepatoprotective activities of the isolated compounds.
The methanolic extract from the dried whole plant of S. indica (7.7% from the dried plant) was partitioned
into an EtOAc–H O (1:1, v/v) mixture to furnish an EtOAc-soluble fraction (2.5%) and an
2

3'
6'
OH
OH
OH
2'
1'
4'
5'
O
H
O
H
8
O
O
O
HO
O
O
HO
HO
2
3
O
7
9
O
CH3
CH3
10
6
4
O
O
H
O
H
5
O
O
O
O
O
O
OH
O
OH
HO
OH
CH3
HO
HO
HO
O
O
O
H3C
H3C
CH3
H3C
O
OH
OH
HO
O
O
O
O
O
CH3
OH
OH
OH
CH3
HO
H
HO
H
HO
H
HO
HO
H
OH
OH
OH
OH
HO OH
OH
sinocrassoside A (1)
8
sinocrassoside A (2)
9
sinocrassoside A (3)
10
OH
OH
HO
O
O
O
O
O
O
OH
OH
O
O
H
HO
H
HO
H
OH
OH
OH
O
OH
O
CH3
CH3
O
HO
O
H3C
O
O
O
O
O
O
O
O
OH
OH
CH3
CH3
H
HO
H
HO
H
HO OH
HO OH
OH
OH
sinocrassoside A (4)
11
sinocrassoside A (5)
12
Chart 1
aqueous phase. The aqueous phase was further extracted with n-BuOH to give an n-BuOH-soluble
3
fraction (1.7%) and a H O-soluble fraction (3.4%), which was described previously. As shown in Table
2
1, the methanolic extract and EtOAc- and n-BuOH-soluble fractions were found to show hepatoprotective
activities. From the EtOAc- and n-BuOH-soluble fractions, 1 (0.0014%), 2 (0.0035%), 3 (0.0048%), 4
(0.0013%), and 5 (0.0011%), were purified using normal- and reversed-phase silica gel chromatographies
and finally HPLC.
Table 1. Inhibitory Effects of the MeOH Extract from S. indica and Its Fractions on D-GalN-induced Cytotoxicity in
Primary Cultured Mouse Hepatocytes
Inhibition (%)
10 µg/ml
8.5 ± 3.0
0 µg/ml
0.0 ± 3.4
3 µg/ml
3.6 ± 3.4
30 µg/ml
16.9 ± 1.5**
100 µg/ml
37.2 ± 1.9**
MeOH ext.
EtOAc-soluble fraction
n-BuOH-soluble fraction
H O-soluble fraction
2
0.0 ± 1.5
0.0 ± 1.0
0.0 ± 3.2
9.0 ± 2.4
2.7 ± 1.6
5.1 ± 3.4
14.1 ± 3.2*
4.5 ± 2.6
5.1 ± 2.3
32.4 ± 1.7**
16.0 ± 1.0**
4.9 ± 2.5
85.2 ± 5.1**
31.6 ± 4.0**
11.3 ± 2.3
Each value represents the mean±S.E.M. (N=4). Significantly different from the control, *p<0.05, **p<0.01.
Structures of Sinocrassosides A (1), A (2), A (3), A (4), and A (5)
12
8
9
10
11
23
Sinocrassoside A (1) was obtained as a yellow powder and exhibited a negative optical rotation ([α]
8
–
D
44.5° in MeOH). In the UV spectrum of 1, absorption maxima were observed at 266 (log ε 4.33) and 350
–1
(4.26) nm. The IR spectrum of 1 showed absorption bands at 1721, 1655, and 1599 cm assignable to
ester carbonyl and γ-pyrone functions and aromatic ring in addition to strong absorption bands at 3432 and
–1
1074 cm suggestive of glycoside moieties. In the positive- and negative-ion FAB-MS of 1,
+
–
quasimolecular ion peaks were observed at m/z 849 (M+Na) , and 825 (M–H) , and high-resolution

R1
OH
OH
O
H
R2O
O
O
O
O
HO
O
CH3
OR1
1
2
OH
R
R
R2O OH
OH
OH
kaempferol (16):
H
H
1
2
kaempferol 3-O-!-D-glucopyranoside (17):
kaempferol 7-O-"-L-rhamnopyranoside (18):
kaempferol 3-O-!-D-glucopyranosyl-7-O-"-L-
rhamnopyranoside (19):
Glc
H
H
R
R
Rha
sinocrassoside A (6):
1
H
IB
sinocrassoside A (7):
2
H
(S)-2MB
IB
Glc
4
Rha
H
sinocrassoside B (13):
1
OH
1
multiflorin B (20): Rha—Glc
sinocrassoside B (14):
2
OH (S)-2MB
4
21: Rha—Glc
1
Rha
3
Rha—Glc
1
R1
1a: Glc
4
1
4a: Rha—Glc
OH
OH
Glc
OH
O
O
O
HO
O
R2O
O
O
CH3
O
H
H
1
2
OR1
R4O OR3
R
R
OH
HO
quercetin (22):
H
H
H
OH
quercetin 3-O-!-D-glucopyranoside (23):
Glc
O
quercetin 3-O-!-D-glucopyranosyl-7-O-"-L-
OR2
rhamnopyranoside (24):
Glc
1
Rha—Glc H
Rha
HO
4
multinoside A (25):
OH
OR2
OH
OH
1
2
3
4
R
R
R
R
sinocrassoside A (8):
3
H
Ac
H
H
H
OH
OH
HO
O
O
sinocrassoside A (9):
4
H
H
H
IB
H
R1O
sinocrassoside A (10):
5
Ac
IB
sinocrassoside A (11):
6
H
Ac (S)-2MB
H
OH
trans-(3S,5R,6R,9S)-megastigman-7-ene-3,5,6,9-tetraol
2
sinocrassoside A (12):
7
H
Ac
H
H
H
(S)-2MB
(S)-2MB
1
3-O-!-D-glucopyranoside (27): R = Glc, R = H
sinocrassoside B (15):
3
OH
luteolin (26)
trans-(3S,5R,6R,9S)-megastigman-7-ene-3,5,6,9-tetraol
1
2
O
O
O
9-O-!-D-glucopyranoside (28): R = H, R = Glc
Glc: !-D-glucopyranosyl
Ac =
CH3
(S)-2MB =
IB =
CH3
CH3
Rha: "-L-rhamnopyranosyl
CH3
CH3
Chart 2
FAB-MS analysis revealed the molecular formula of 1 to be C H O . Alkaline hydrolysis of 1 with
38 46 21
10% aqueous potassium hydroxide (KOH)–50% aqueous 1,4-dioxane (1:1, v/v) furnished the desacyl-
derivative (1a), together with an organic acid, isobutyric acid (IB), which was identified by HPLC
3,4,13,15,16,21
analysis of its p-nitrobenzyl derivative.
Acid hydrolysis of 1a with 1.0 M hydrochloric acid
(HCl) liberated kaempferol (16), together with L-rhamnose and D-glucose, which were identified by
1,3–8,11–16,18–22,24,25
1
The H- (DMSO-d , Table 2) and
HPLC analysis using an optical rotation detector.
13
6
26
C-NMR (Table 3) spectra of 1a, which were assigned by various NMR experiments, showed signals
assignable to meta-coupled and A B type aromatic protons [δ 6.48, 6.86 (1H each, both d, J = 2.2 Hz, 6,
2 2
8-H), 6.89, 8.08 (2H each, both d, J = 8.9 Hz, 3',5', 2',6'-H)] together with two β-D-glucopyranosyl and a
α-L-rhamnopyranosyl moieties [δ 1.14 (3H, d, J = 5.8 Hz, Rha-6-H ), 4.48 (1H, d, J = 7.6 Hz, 7-O-
3
terminal-Glc-1-H), 5.48 (1H, d, J = 7.3 Hz, 3-O-Glc-1-H), 5.59 (1H, br s, Rha-1-H)]. The connectivities
of glycopyranosyl parts were determined by a heteronuclear multiple-bond correlations (HMBC)
experiment on 1a. Namely, long-range correlations were observed between the following protons and
carbons [3-O-Glc-1-H and 3-C (δ 133.4), Rha-1-H and 7-C (δ 161.2), and 7-O-terminal-Glc-1-H and
C
C

1
Table 2. H-NMR (500 MHz) Data of 1—3 and 1a
1
(J Hz)
6.53 (d, 2.1)
6.92 (d, 2.1)
8.09 (d, 8.9)
6.90 (d, 8.9)
12.62 (br s)
1a
(J Hz)
6.48 (d, 2.2)
6.86 (d, 2.2)
8.08 (d, 8.9)
6.89 (d, 8.9)
12.62 (br s)
2
(J Hz)
6.54 (d, 2.1)
6.92 (d, 2.1)
8.09 (d, 8.9)
6.90 (d, 8.9)
12.61 (br s)
3
position
6
8
2',6'
3',5'
5-OH
δ
δ
δ
δ (J Hz)
H
H
H
H
6.23 (d, 2.1)
6.43 (d, 2.1)
7.76 (d, 8.9)
6.94 (d, 8.9)
12.59 (br s)
(3-O-Glc)
(3-O-Glc)
5.48 (d, 7.3)
(3-O-Glc)
5.49 (d, 7.0)
(3-O-Rha)
5.17 (br s)
4.06 (br s)
3.74 (dd, 3.4, 9.5)
3.37 (dd, 9.2, 9.5)
3.31 (dq, 9.2, 6.1)
0.86 (d, 6.1)
1
2
3
4
5
6
5.49 (d, 7.3)
3.21 (dd, 7.3, 8.9)
3.19 (dd, 8.6, 8.9)
3.10 (m)
3.10 (m)
3.34 (dd, 6.0, 12.5)
3.58 (dd, 2.0, 12.5)
3.21 (dd, 7.3, 8.9)
3.19 (dd, 8.9, 8.9)
3.10 (dd, 8.9, 9.2)
3.09 (m)
3.34 (dd, 6.0, 11.6)
3.57 (dd, 2.0, 11.6)
3.20 (dd, 7.0, 8.3)
3.18 (dd, 8.3, 8.9)
3.10 (m)
3.10 (m)
3.35 (dd, 6.5, 11.2)
3.58 (dd, 2.1, 11.2)
(3-O-Rha4—1Glc)
4.36 (d, 7.9)
1
2
3
4
5
6
3.02 (dd, 7.9, 8.5)
3.20 (dd, 8.5, 9.5)
3.07 (dd, 9.2, 9.5)
3.35 (m)
4.02 (dd, 7.3, 11.6)
4.30 (dd, 1.8, 11.6)
(7-O-Rha)
5.70 (br s)
(7-O-Rha)
5.59 (br s)
4.11 (br s)
(7-O-Rha)
5.70 (br s)
1
2
3
4
5
6
5.29 (dd, 1.9, 3.7)
4.00 (dd, 3.7, 9.5)
3.50 (dd, 9.2, 9.5)
3.61 (dq, 9.2, 6.1)
1.18 (d, 6.1)
5.29 (dd, 1.9, 3.7)
4.00 (dd, 3.7, 9.5)
3.50 (dd, 9.5, 9.5)
3.61 (dq, 9.5, 6.1)
1.18 (d, 6.1)
3.77 (dd, 3.1, 8.9)
3.50 (dd, 8.9, 9.2)
3.52 (dq, 8.9, 5.8)
1.14 (d, 5.8)
(7-O-Rha3—1Glc)
4.46 (d, 7.6)
(7-O-Rha3—1Glc)
4.48 (d, 7.6)
(7-O-Rha3—1Glc)
4.46 (d, 7.6)
1
2
3
4
5
6
3.00 (dd, 7.6, 8.9)
3.19 (dd, 8.6, 8.9)
3.03 (dd, 9.5, 9.6)
3.17 (m)
3.08 (dd, 7.6, 8.9)
3.20 (dd, 8.9, 8.9)
3.10 (dd, 8.9, 9.2)
3.17 (m)
3.00 (dd, 7.6, 8.9)
3.19 (dd, 8.9, 9.6)
3.01 (dd, 8.6, 8.9)
3.17 (m)
3.42 (dd, 6.0, 11.5)
3.47 (dd, 6.0, 12.2)
3.40 (dd, 6.5, 11.4)
3.66 (dd, 2.0, 11.5)
3.68 (dd, 2.0, 12.2)
3.68 (dd, 2.4, 11.6)
acyl
2
3
2.62 (qq, 6.7, 6.7)
1.14 (d, 6.7)
2.45 (ddq, 7.0, 7.0, 7.0)
1.50 (ddq, 7.0, 14.0, 7.4)
1.64 (ddq, 7.0, 14.0, 7.4)
0.93 (dd, 7.4, 7.4)
2.47 (qq, 7.0, 7.0)
1.05 (d, 7.0)
4
5
1.15 (d, 6.7)
1.06 (d, 7.0)
1.12 (d, 7.0)
Measured in DMSO-d
6
Rha-3-C (δ 80.6)]. Consequently, the structure of 1a was determined to be kaempferol 3-O-β-D-
C
glucopyranosyl-7-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside. The proton and carbon signals
1
in the H- (DMSO-d , Table 2) and C-NMR (Table 3) spectra of 1 were found to be similar to those
13
26
6
of 1a, except for the signals due to an isobutyryl group [δ 1.14, 1.15 (3H each, both d, J = 6.7 Hz, IB-3, 4-
13
H ), 2.62 (1H, qq, J = 6.7, 6.7 Hz, IB-2-H)]. Comparison of the C-NMR data for 1 with those for 1a
3
indicated an acylation shift at the Rha-2-position of 1 [1: δ 94.7 (Rha-1-C), 70.5 (Rha-2-C), 77.7 (Rha-3-
C
C); 1a: δ 98.1 (Rha-1-C), 69.5 (Rha-2-C), 80.6 (Rha-3-C)]. In the HMBC experiment of 1, a long-range
C
correlation was observed between the Rha-2-proton [δ 5.29 (1H, dd, J = 1.9, 3.7 Hz)] and the isobutyryl
ester carbonyl carbon (δ 175.8). Thus the position of the isobutyryl group in 1 was clarified. On the
C
basis of those findings, the structure of sinocrassoside A was determined as kaempferol 3-O-β-D-
8
glucopyranosyl-7-O-β-D-glucopyranosyl-(1→3)-α-L-(2-O-isobutyryl)-rhamnopyranoside (1).
25
Sinocrassoside A (2) was also isolated as a yellow powder with negative optical rotation ([α]
9
–52.6°
D
in MeOH). The UV spectrum of 2 showed absorption maxima at 266 (log ε 4.31) and 350 (4.24) nm.

13
Table 3. C-NMR (125 MHz) Data of 1—5 and Related Compounds (1a and 4a)
1a
1
2
3
4
4a
δ
5
δ
C
Position
δ
δ
δ
δ
δ
C
C
C
C
C
C
2
3
4
5
6
156.7
133.4
177.6
160.8
99.4
156.7
133.4
177.5
160.8
99.4
156.7
133.5
177.6
160.8
99.5
157.2
134.3
177.6
161.1
98.7
157.8
134.6
177.8
160.8
99.3
157.8
134.5
177.8
160.8
99.3
157.8
134.6
177.8
160.8
99.4
7
8
160.6
94.6
161.2
94.6
160.6
94.7
164.3
93.7
162.9
94.6
162.9
94.6
162.7
94.3
9
10
1'
2',6'
3',5'
4'
155.8
105.9
120.6
130.9
115.1
160.1
(3-O-Glc)
100.7
74.1
155.8
105.6
120.5
130.9
115.1
160.2
(3-O-Glc)
100.6
74.1
155.8
105.9
120.7
130.9
115.1
160.1
(3-O-Glc)
100.7
74.1
156.5
104.0
120.4
130.5
115.4
159.9
(3-O-Rha)
101.8
69.7
156.0
105.7
120.2
130.6
115.4
160.1
(3-O-Rha)
101.8
69.6
156.0
105.7
120.2
130.6
115.5
160.1
(3-O-Rha)
101.8
69.6
156.1
105.8
120.2
130.5
115.3
160.1
(3-O-Rha)
101.8
69.6
1
2
3
4
5
6
76.3
69.8
77.5
60.8
76.3
69.8
77.5
60.7
76.3
69.8
77.5
60.8
70.2
81.7
68.7
70.2
81.8
68.8
17.1
70.2
81.8
68.6
17.2
70.2
81.8
68.9
17.1
17.2
(3-O-Rha4-1Glc)
104.3
74.2
(3-O-Rha4-1Glc) (3-O-Rha4-1Glc)
(3-O-Rha4-1Glc)
104.6
74.4
1
2
3
4
5
6
104.3
74.2
76.2
70.0
73.7
104.6
74.3
76.5
69.7
76.9
76.2
70.0
73.7
63.4
76.5
69.8
73.9
63.4
63.5
60.7
(7-O-Rha)
94.7
70.5
(7-O-Rha)
98.1
69.5
(7-O-Rha)
94.7
70.5
(7-O-Glc)
99.8
73.0
(7-O-Glc)
99.6
72.8
(7-O-Glc)
99.5
72.8
1
2
3
4
5
6
77.7
70.7
69.3
80.6
70.4
68.9
77.8
70.6
69.3
76.3
69.5
77.1
60.5
76.3
69.4
77.1
60.5
76.0
69.8
76.9
60.9
17.8
17.8
17.8
(7-O-Rha3-1Glc)
104.2
73.9
(7-O-Rha3-1Glc)
104.5
73.9
(7-O-Rha3-1Glc)
104.4
73.9
1
2
3
4
76.0
70.0
76.1
69.8
76.0
70.1
5
6
76.6
61.2
76.7
60.9
76.5
61.4
acyl
1
2
3
4
175.8
33.3
18.5
18.8
175.4
40.1
26.2
11.1
16.1
176.0
33.1
18.6
18.6
175.8
33.1
18.6
18.6
174.1
42.0
63.1
13.3
5
Measured in DMSO-d
6
–1
The IR spectrum of 2 showed absorption bands at 3432, 1728, 1655, 1599, and 1079 cm ascribable to
hydroxyl, ester carbonyl, γ-pyrone, and ether functions and aromatic ring. The molecular formula
C H O of 2, was determined by the quasimolecular ion peaks in positive- and negative-ion FAB-MS
38 48 21
+
–
[m/z 863 (M+Na) and m/z 839 (M–H) ] and by high-resolution FAB-MS. Alkaline hydrolysis of 2 with
10% KOH–50% aqueous 1,4-dioxane (1:1, v/v) furnished 1a together with S-(+)-2-methylbutyric acid
3,4
[(S)-2MB], which was identified by HPLC analysis using an optical rotation detector.
The proton and
1
carbon signals in the H- (DMSO-d , Table 2) and C-NMR (Table 3) spectra of 2 were very similar to
13
26
6
those of 1 {an aglycone part [δ 6.54, 6.92 (1H each, both d, J = 2.1 Hz, 6, 8-H), 6.90, 8.09 (2H each, both
d, J = 8.9 Hz, 3',5', 2',6'-H)], two β-D-glucopyranosyl and a α-L-rhamnopyranosyl moieties [δ 1.18 (3H, d,
J = 6.1 Hz, Rha-6-H ), 4.46 (1H, d, J = 7.6 Hz, 7-O-terminal-Glc-1-H), 5.49 (1H, d, J = 7.0 Hz, 3-O-Glc-
3

1-H), 5.70 (1H, br s, Rha-1-H)]} except for the signals due to an acyl moiety [δ 0.93 (3H, dd, J = 7.4, 7.4
Hz, (S)-2MB-4-H ), 1.12 (3H, d, J = 7.0 Hz, (S)-2MB-5-H ), 1.50, 1.64 (1H each, both ddq, J = 7.0, 14.0,
3
3
7.4 Hz, (S)-2MB-3-H ), 2.45 (1H, ddq, J = 7.0, 7.0, 7.0 Hz, (S)-2MB-2-H)]. The position of (S)-2-
2
methylbutyryl part in 2 was confirmed by the HMBC experiment, which showed a long-range correlation
between the Rha-2-proton[δ 5.29 (1H, dd, J = 1.9, 3.7 Hz)] and the (2S)-2-methylbutyryl ester carbonyl
carbon (δ 175.4). Consequently, the structure of sinocrassoside A was determined as kaempferol 3-O-
C
9
β-D-glucopyranosyl-7-O-β-D-glucopyranosyl-(1→3)-α-L-[2-O-(2S)-2-methylbutyryl]rhamnopyranoside
(2).
26
Sinocrassoside A (3) was obtained as a yellow powder with negative optical rotation ([α]
10
–88.9° in
D
–1
MeOH). The IR spectrum of 3 showed absorption bands at 1736, 1655, 1610 cm ascribable to ester
–1
carbonyl and γ-pyrone functions and broad bands at 3432 and 1068 cm , suggestive of glycoside moiety.
In the positive- and negative-ion FAB-MS of 3, quasimolecular ion peaks were observed at m/z 687
+
(M+Na) and m/z 663 (M–H) , and high-resolution FAB-MS analysis revealed the molecular formula of 3
–
to be C H O . On alkaline hydrolysis of 3 with 10% KOH–50% aqueous 1,4-dioxane (1:1, v/v),
31 36 16
27
multiflorin B (20) was obtained together with isobutyric acid, which was identified by HPLC analysis of
1
13
26
its p-nitrobenzyl derivative. The H- (DMSO-d , Table 2) and C-NMR (Table 3) spectra of 3 showed
6
signals assignable to two methyls [δ 1.05, 1.06 (3H each, both d, J = 7.0 Hz, IB-3, IB-4-H )], a methane
3
[δ 2.47 (1H, qq, J = 7.0 Hz, IB-2-H)], meta-coupled and A B -type aromatic protons [δ 6.23, 6.43 (1H
2 2
each, both d, J = 2.1 Hz, 6, 8-H), 6.94, 7.76 (2H each, both d, J = 8.9 Hz, 3',5', 2',6'-H)], together with a
rhamnopyranosyl moiety [δ 0.86 (3H, d, J = 6.1 Hz, Rha-6-H ), 5.17 (1H, br s, Rha-1-H)], and a
3
13
glucopyranosyl part [δ 4.36 (1H, d, J = 7.9 Hz, Glc-1-H)]. Comparison of the C-NMR data for 3 with
27
those for 20 revealed an acylation shift around the Glc-6-position of 3 [3: δ 73.7 (Glc-5-C), 63.4 (Glc-
C
6-C); 20: δ 76.9 (Glc-5-C), 61.1 (Glc-6-C)]. Furthermore, in the HMBC experiment of 3, long-range
C
correlation was observed between the Glc-6-protons [δ 4.02 (1H, dd, J = 7.3, 11.6 Hz), 4.30 (1H, dd, J =
1.8, 11.6 Hz)] and the isobutyryl ester carbonyl carbon (δ 176.0). Consequently, the position of the
C
isobutyryl ester moiety in 3 was determined and the structure of sinocrassoside A was elucidated as
10
kaempferol 3-O-β-D-(6-O-isobutyryl)glucopyranosyl-(1→4)-α-L-rhamnopyranoside (3).
Sinocrassosides A (4) and A (5) were obtained as a yellow powder with negative optical rotations (4:
11
12
26
26
[α]
–10.5°; 5: [α]
–11.3°, both in MeOH). The molecular formulas of 4 (C H O ) and 5
37 46 21
D
D
(C H O ) were determined from the positive- and negative-ion FAB-MS and by high-resolution FAB-
37 46 22
MS. Alkaline hydrolysis of 4 and 5 with 10% KOH–50% aqueous 1,4-dioxane (1:1, v/v) gave the
common desacyl-derivative (4a), together with isobutyric acid from 4 and 3-hydroxyisobutyric acid
(3HIB) from 5, which was identified by HPLC analysis, respectively. Acid hydrolysis of 4a with 1.0 M
1
HCl liberated 16, L-rhamnose, and D-glucose, which were identified by HPLC analysis. The H- (DMSO-
13 26
d , Table 4) and C-NMR (Table 3) spectra of 4a indicated the presence of the following functions: a
6
kaempferol part [δ 6.46, 6.76 (1H each, both d, J = 1.8 Hz, 6, 8-H), 6.92, 7.78 (2H, d, J = 8.9 Hz, 3',5',
2',6'-H)], an α-L-rhamnopyranosyl moiety [δ 0.92 (3H, d, J = 6.1 Hz, Rha-6-H ), 5.27 (1H, br s, Rha-1-
3
H)], and two glucopyranosyl groups [δ 4.30 (1H, d, J = 7.9 Hz, 3-O-terminal-Glc-1-H), 5.07 (1H, d, J =
7.1 Hz, 7-O-Glc-1-H)]. In the HMBC experiment of 4a, long-range correlations were observed

1
Table 4. H-NMR (500 MHz) Data of 4, 5 and 4a
between the following
proton and carbon pairs
4
(J Hz)
6.46 (d, 1.8)
6.77 (d, 1.8)
7.78 (d, 8.6)
6.94 (d, 8.6)
12.59 (br s)
4a
(J Hz)
6.46 (d, 1.8)
6.76 (d, 1.8)
7.78 (d, 8.9)
6.92 (d, 8.9)
12.58 (br s)
5
δ (J Hz)
Position
6
8
2',6'
3',5'
5-OH
δ
δ
H
H
H
[Rha-1-H and 3-C (δ
C
6.45 (d, 2.1)
6.76 (d, 2.1)
7.77 (d, 8.5)
6.94 (d, 8.5)
12.60 (br s)
134.5), 3-O-terminal-
Glc-1-H and Rha-4-C (δ
C
81.8), and 7-O-Glc-1-H
(3-O-Rha)
(3-O-Rha)
(3-O-Rha)
5.21 (br s)
4.06 (br s)
3.76 (dd, 3.4, 9.5)
3.38 (dd, 9.2, 9.5)
3.31 (dq, 9.2, 6.1)
0.93 (d, 6.1)
1
2
3
4
5
6
5.19 (br s)
4.06 (br s)
3.74 (dd, 3.4, 9.5)
3.38 (dd, 9.2, 9.5)
3.31 (dq, 9.2, 6.1)
0.87 (d, 6.1)
5.27 (br s)
4.06 (br s)
3.69 (dd, 3.4, 9.5)
3.41 (dd, 9.2, 9.5)
3.32 (dq, 9.2, 6.1)
0.92 (d, 6.1)
and 7-C (δ 162.9)].
C
Thus, the structure of 4a
w a s c l a r i f i e d a s
kaempferol
3-O-β-D-
(3-O-Rha4—1Glc)
4.35 (d, 7.9)
3.02 (dd, 7.9, 8.6)
3.19 (dd, 8.6, 9.2)
3.07 (dd, 9.2, 9.5)
3.35 (m)
4.02 (dd, 7.4, 11.6)
4.29 (dd, 1.8, 11.6)
(7-O-Glc)
(3-O-Rha4—1Glc)
4.30 (d, 7.9)
2.98 (dd, 7.9, 8.6)
3.17 (dd, 8.6, 9.2)
3.06 (dd, 9.2, 9.5)
3.05 (m)
3.69 (dd, 5.5, 11.2)
3.76 (dd, 1.8, 11.2)
(7-O-Glc)
(3-O-Rha4—1Glc)
4.31 (d, 7.9)
3.00 (dd, 7.9, 8.6)
3.15 (dd, 8.6, 9.2)
3.06 (dd, 9.2, 9.5)
3.75 (m)
3.95 (dd, 6.0, 11.6)
4.43 (dd, 1.8, 11.6)
(7-O-Glc)
glucopyranosyl-(1→4)-
α-L-rhamnopyranosyl-7-
O-β-D-glucopyranoside.
1
2
3
4
5
6
The proton and carbon
1
signals in the
H-
1
2
3
4
5
6
5.07 (d, 7.4)
5.07 (d, 7.1)
5.07 (d, 7.4)
(DMSO-d , Table 4) and
6
3.30 (dd, 7.4, 9.5)
3.32 (dd, 9.2, 9.5)
3.17 (dd, 9.2, 9.3)
3.47 (m)
3.29 (dd, 7.1, 9.5)
3.31 (dd, 9.2, 9.5)
3.17 (dd, 8.3, 9.2)
3.47 (m)
3.30 (dd, 7.4, 9.5)
3.32 (dd, 9.2, 9.5)
3.17 (dd, 8.3, 9.2)
3.47 (m)
13
C-NMR (Table 3)
26
spectra of 4 and 5 were
3.45 (dd, 5.5, 11.2)
3.44 (dd, 5.5, 11.2)
3.45 (dd, 5.5, 11.2)
3.71 (dd, 1.8, 11.2)
3.62 (dd, 1.8, 11.2)
3.71 (dd, 1.8, 11.2)
found to be similar to
those of 4a, except for
the signals due to an acyl
group: [4: δ 1.05, 1.05
(3H each, both d, J = 7.0
acyl
2
3
2.48 (qq, 7.0, 7.0)
1.05 (d, 7.0)
2.51 (ddq, 7.0, 7.0, 7.0)
3.36 (dd, 7.0, 12.8)
3.50 (dd, 7.0, 12.8)
0.98 (d, 7.0)
4
5
1.05 (d, 7.0)
Measured in DMSO-d
6
Hz, IB-3, IB-4-H ), 2.48
3
(1H, qq, J = 7.0, 7.0 Hz, IB-2-H); 5: δ 0.98 (3H, d, J = 7.0 Hz, 3HIB-4-H ), 2.51 (1H, ddq, J = 7.0, 7.0, 7.0
3
Hz, 3HIB-2-H), 3.36, 3.50 (1H each, both dd, J = 7.0, 12.8 Hz, 3HIB-3-H )]. In the HMBC experiment of
2
4 and 5, long-range correlation was observed between the 6-protons of the 7-O-terminal-glucopyranosyl
part [4: δ 3.45 (1H, dd, J = 5.5, 11.2 Hz), 3.71 (1H, dd, J = 1.8, 11.2 Hz); 5: δ 3.45 (1H, dd, J = 5.5, 11.2
Hz), 3.71 (1H, dd, J = 1.8, 11.2 Hz)] and the acyl ester carbonyl carbon (4: δ 175.8; 5: δ 174.1).
C
C
Consequently, the structures of sinocrassosides A and A were elucidated as kaempferol 3-O-β-D-(6-O-
11
12
isobutyryl)glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-β-D-glucopyranoside and kaempferol
3-O-β-D-(6-O-3-hydroxyisobutyryl)glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-β-D-gluco-
pyranoside, respectively.
Protective Effects on D-GalN-induced Cytotoxicity in Primary Cultured Mouse Hepatocytes
Since the methanolic extract of S. indica was found to show protective effect on D-GalN-induced
cytotoxicity in primary cultured mouse hepatocytes (Table 1), the inhibitory effects of the constituents
3
from this herbal medicine were also examined. As shown in Table 5, sinocrassosides A (6, IC = 19.2
1
50
3
3
3
µM), A (7, 11.6 µM), and B (14, 23.2 µM), kaempferol 3-O-β-D-glucopyranoside (17, 16.8 µM),
2
2

3
kaempferol 7-O-α-L-rhamnopyranoside (18, 12.5 µM), and kaempferol 3-O-α-L-rhamnopyranosyl-
3
(1→4)-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (21, 21.2 µM), were found to show strong
inhibitory activity. The activities of 3- or 7-O-mono-glycosides (6, 7, 14, 17, 18, 23) were stronger than
those of corresponding aglycones, kaempferol (16, 51.8 µM) and quercetin (22, 48.0 µM).
Table 5. Inhibitory Effects of Constituents from S. indica on D-GalN-induced Cytotoxicity in
Primary Cultured Mous Hepatocytes
Inhibition (%)
10 µM
IC
50
(µM)
0 µM
3 µM
1.7 ± 1.4
-6.3 ± 1.0
5.1 ± 3.3
5.1 ± 1.2
6.5 ± 2.4
-5.8 ± 2.4
3.5 ± 1.8
2.9 ± 1.0
7.4 ± 4.3
4.1 ± 4.3
3.0 ± 2.0
4.0 ± 0.8
10.4 ± 1.8
3.8 ± 2.1
-1.4 ± 2.1
15.0 ± 3.4*
1.3 ± 1.7
3.1 ± 1.6
2.3 ± 2.9
5.9 ± 1.5
3.0 ± 2.5
2.5 ± 2.7
10.4 ± 5.4
7.7 ± 0.9
0.9 ± 1.4
-1.1 ± 2.0
6.8 ± 1.8
30 µM
100 µM
sinocrassoside A (2)
0.0 ± 3.1
0.0 ± 2.6
0.0 ± 2.9
0.0 ± 0.8
0.0 ± 2.3
0.0 ± 2.4
0.0 ± 2.2
0.0 ± 1.3
0.0 ± 1.0
0.0 ± 3.3
0.0 ± 2.2
0.0 ± 1.1
0.0 ± 4.1
0.0 ± 1.1
0.0 ± 2.3
0.0 ± 2.3
0.0 ± 1.1
0.0 ± 1.4
0.0 ± 3.2
0.0 ± 0.8
0.0 ± 0.4
0.0 ± 1.9
0.0 ± 2.6
0.0 ± 0.7
0.0 ± 1.8
0.0 ± 5.2
0.0 ± 2.1
8.3 ± 3.8
14.8 ± 3.1*
12.9 ± 2.2
85.9 ± 5.7**
18.9 ± 3.7**
74.5 ± 1.7**
9
sinocrassoside A (3)
-5.7 ± 3.9
13.0 ± 3.5*
22.4 ± 3.4*
59.4
19.2
11.6
42.9
38.6
38.2
86.1
23.2
32.9
51.8
16.8
12.5
72.9
10
#
sinocrassoside A (6)
-7.0 ± 6.6
1
sinocrassoside A (7)
99.8 ± 9.3** 13.1 ± 9.3
2
sinocrassoside A (9)
18.9 ± 2.7** 46.8 ± 1.9** 126.2 ± 6.3**
4
sinocrassoside A (10)
-0.5 ± 2.4
14.8 ± 1.9*
7.3 ± 1.6*
16.2 ± 2.1*
14.8 ± 3.8
13.7 ± 4.5
29.5 ± 4.4** 94.2 ± 2.4**
39.2 ± 3.9** 78.7 ± 1.1**
32.9 ± 1.1** 51.4 ± 0.9**
63.3 ± 6.4** -13.1 ± 2.8
53.3 ± 8.4** 108.5 ± 10.0**
39.6 ± 2.1** 64.5 ± 6.6**
5
sinocrassoside A (12)
7
sinocrassoside B (13)
1
#
sinocrassoside B (14)
2
sinocrassoside B (15)
3
kaempferol (16)
kaempferol 3-O-Glc (17)
kaempferol 7-O-Rha (18)
19
multiflorin B (20)
21
quercetin (22)
quercetin 3-O-Glc (23)
24
multinoside A (25)
luteolin (26)
27
24.6 ± 2.0** 72.4 ± 3.5** 102.5 ± 2.7**
28.5 ± 4.9*
8.1 ± 0.6*
-0.3 ± 2.4
80.8 ± 11.2** 124.8 ± 7.5**
24.6 ± 1.5** 59.7 ± 3.1**
3.2 ± 2.4
14.6 ± 4.1*
26.3 ± 2.8** 55.0 ± 0.7** 86.9 ± 4.0**
21.2
48.0
32.0
7.4 ± 3.0
12.1 ± 0.9*
9.0 ± 3.7
8.1 ± 2.3*
6.9 ± 2.7
0.8 ± 1.8
8.9 ± 3.3
21.5 ± 3.0** 79.7 ± 2.7**
30.1 ± 3.6** 104.1 ± 7.3**
11.6 ± 0.9*
22.7 ± 1.2** 47.3 ± 1.6**
38.8 ± 1.3** -42.7 ± 0.4
16.7 ± 1.7**
#
11.2 ± 3.3*
28.8 ± 2.4** 38.3 ± 2.7**
8.2 ± 3.2
28
sinocrassoside B
4
28
sinocrassoside B
16.3 ± 0.6** 28.6 ± 3.1** 66.9 ± 1.6**
55.2
63.6
95.0
5
28
sinocrassoside C
8.2 ± 0.2
3.5 ± 3.0
11.1 ± 1.0*
26.5 ± 3.6** 63.5 ± 6.2**
15.9 ± 1.4** 51.5 ± 0.9**
20.4 ± 1.3** 30.6 ± 2.0**
1
28
sinocrassoside D
sinocrassoside D
1
28
3
a,29
silybin
0.0 ± 0.3
4.8 ± 1.1
7.7 ± 0.7
45.2 ± 8.8** 77.0 ± 5.5**
38.8
Each value represents the mean±S.E.M. (N=4). Significantly different from the control, *p<0.05, **p<0.01.
a
Commercial silybin was purchased from Funakoshi Co., Ltd. (Tokyo, Japan).
Cytotoxicity
#
EXPERIMENTAL
The following instruments were used to obtain physical data: specific rotations, Horiba SEPA-300 digital
polarimeter (l = 5 cm); UV spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100
spectrophotometer; EI-MS and high-resolution MS, JEOL JMS-GCMATE mass spectrometer; FAB-MS
1
and high-resolution MS, JEOL JMS-SX 102A mass spectrometer; H-NMR spectra, JEOL EX-270 (270
13
MHz) and JNM-LA500 (500 MHz) spectrometers; C-NMR spectra, JEOL EX-270 (68 MHz) and JNM-
LA500 (125 MHz) spectrometers with tetramethylsilane as an internal standard; HPLC detector,
Shimadzu RID-6A refractive index and SPD-10Avp UV-VIS detectors; and HPLC column, YMC-Pack

ODS-A (YMC Co., Ltd. 250 × 4.6 mm i.d. and 250 × 20 mm i.d.) columns were used for analytical and
preparative purposes, respectively.
The following experimental conditions were used for chromatography: normal-phase column
chromatography; Silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh), reversed-phase column
chromatography; Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–200 mesh); TLC, pre-
coated TLC plates with Silica gel 60F
(Merck, 0.25 mm) (normal-phase) and Silica gel RP-18 F
254
254S
254S
(Merck, 0.25 mm) (reversed-phase); HPTLC, pre-coated TLC plates with Silica gel RP-18 WF
(Merck, 0.25 mm) (reversed-phase) and detection was achieved by spraying with 1% Ce(SO ) -10%
4 2
aqueous H SO , followed by heating.
2
4
Plant Material
3
This item was described in a previous report.
Extraction and Isolation
Fractions 6-1 (9980 mg) and 7-2 (340 mg) obtained from the EtOAc-soluble fraction and fractions 4-8
(970 mg) and 5-8 (1310 mg) from the n-BuOH-soluble fraction of the methanolic extract from the whole
3
plant of S. indica (9.9 kg, cultivated in Guangxi province, China) as reported previously.
Fraction 6-1 (1180 mg) from the EtOAc-soluble fraction was purified by HPLC [MeOH-H O (52:48,
2
v/v)] to give sinocrassoside A (3, 14.7 mg, 0.0048%) together with sinocrassosides A (8, 4.6mg,
10
3
0.0015%), A (9, 10.0 mg, 0.0032%), and B (15, 11.3 mg, 0.0037%), kaempferol 3-O-β-D-
4
3
glucopyranoside (17, 14.0 mg, 0.0046%), and kaempferol 3-O-β-D-glucopyranosyl-7-O-α-L-
rhamnopyranoside (19, 32.9 mg, 0.0070%). Fraction 7-2 (340 mg) from the EtOAc-soluble fraction was
subjected to HPLC [MeOH-H O (60:40, v/v)] to give sinocrassoside A (2, 22.3 mg, 0.0006%). Fraction
2
9
4-8 (970 mg) from the n-BuOH-soluble fraction was further purified by HPLC [MeOH-H O (55:45, v/v)]
2
to give sinocrassosides A (1, 72.0 mg, 0.0014%) and A (2, 150.4 mg, 0.0029%). Fraction 5-8 (300 mg)
9
8
from the n-BuOH-soluble fraction was further purified by HPLC [MeOH-H O (40:60, v/v)] to give
2
sinocrassosides A (4, 15.8 mg, 0.0013%) and A (5, 13.1 mg, 0.0011%) together with kaempferol 3-O-
11
12
β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (19, 17.9 mg, 0.0015%), kaempferol 3-O-β-D-
glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-α-L-rhamnopyranoside (21, 9.6 mg, 0.0008%),
quercetin 3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside (24, 35.3 mg, 0.0030%), and multinoside
A (25, 27.6 mg, 0.0023%).
23
Sinocrassoside A (1): a yellow powder, [α]
8
–44.5° (c 2.00, MeOH). High-resolution positive-ion
D
+
FAB-MS: Calcd for C H O Na (M+Na) : 849.2429. Found: 849.2438. UV [MeOH, nm (log ε)]: 266
38 46 21
–1
(4.33), 350 (4.26). IR (KBr): 3432, 1721, 1655, 1599, 1074 cm . H-NMR (500 MHz, DMSO-d ) δ:
1
6
13
given in Table 2. C-NMR (125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 849
6
C
+
(M+Na) . Negative-ion FAB-MS m/z: 825 (M–H) .
–
25
Sinocrassoside A (2): a yellow powder, [α]
9
–52.6° (c 1.00, MeOH). High-resolution positive-ion
D
+
FAB-MS: Calcd for C H O Na (M+Na) 863.2586. Found: 863.2581. UV [MeOH, nm (log ε)]: 266
38 48 21

–1
(4.31), 350 (4.24). IR (KBr): 3432, 1728, 1655, 1599, 1079 cm . H-NMR (500 MHz, DMSO-d ) δ:
1
6
13
given in Table 2. C-NMR (125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 863
6
C
+
(M+Na) . Negative-ion FAB-MS m/z: 839 (M–H) .
–
26
Sinocrassoside A (3): a yellow powder, [α]
10
–88.9° (c 1.00, MeOH). High-resolution positive-ion
D
+
FAB-MS: Calcd for C H O Na (M+Na) 687.1904. Found: 687.1901. UV [MeOH, nm (log ε)]: 265
31 36 16
–1
(4.31), 347 (4.16). IR (KBr): 3432, 1736, 1655, 1610, 1068 cm . H-NMR (500 MHz, DMSO-d ) δ:
1
6
13
given in Table 2. C-NMR (125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 687
6
C
+
(M+Na) . Negative-ion FAB-MS m/z: 663 (M–H) .
–
26
Sinocrassoside A (4): a yellow powder, [α]
11
–10.5° (c 0.70, MeOH). High-resolution positive-ion
D
+
FAB-MS: Calcd for C H O Na (M+Na) 849.2429. Found: 849.2436. UV [MeOH, nm (log ε)]: 266
37 46 21
–1
(4.40), 343 (4.23). IR (KBr): 3432, 1719, 1655, 1603, 1074 cm . H-NMR (500 MHz, DMSO-d ) δ:
1
6
13
given in Table 4. C-NMR (125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 849
6
C
+
–
(M+Na) . Negative-ion FAB-MS m/z: 825 (M–H) .
26
Sinocrassoside A (5): a yellow powder, [α]
12
–11.3° (c 1.00, MeOH). High-resolution positive-ion
D
+
FAB-MS: Calcd for C H O Na (M+Na) 865.2378. Found: 865.2381. UV [MeOH, nm (log ε)]: 265
37 46 22
–1
1
(4.31), 344 (4.13). IR (KBr): 3432, 1719, 1655, 1605, 1075 cm . H-NMR (500 MHz, DMSO-d ) δ:
6
13
given in Table 4. C-NMR (125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 865
6
C
+
(M+Na) . Negative-ion FAB-MS m/z: 841 (M–H) .
–
Alkaline Hydrolysis of 1—5
A solution of 1—5 (each 5.0 mg) in 10% aqueous potassium hydroxide (KOH)—50% aqueous 1,4-
dioxane (1:1, v/v, 1.0 mL) was stirred at 37 °C for 1 h. Removal of the solvent under reduced pressure
gave a reaction mixture. A part of the reaction mixture was dissolved in (CH ) Cl (2.0 mL) and the
2 2
2
solution was treated with p-nitrobenzyl-N-N'-diisopropylisourea (10 mg), then the whole was stirred at
80 °C for 1 h. The reaction solution was subjected to HPLC analysis [column: YMC-Pack ODS-A, 250 ×
4.6 mm i.d.; mobile phase: MeOH–H O (70:30, v/v); detection: UV (254 nm); flow rate: 0.9 mL/min] to
2
identify the p-nitrobenzyl esters of 3-hydroxyisobutyric acid (a, t 10.5 min) from 5, isobutyric acid (b, t
R
R
11.5 min) from 1, 3, and 4, and 2-methylbutyric acid (c, t 16.9 min) from 2, which were carried out by
R
comparison of their retention time with those of commercially obtained standard samples. The rest of the
+
reaction mixture was neutralized with Dowex HCR W2 (H form) and the resin was removed by filtration.
Evaporation of the solvent from the filtrate under reduced pressure yielded a product. A part of the
product was subjected to HPLC analysis [column: YMC-Pack ODS-AQ, 250 × 4.6 mm i.d.; mobile phase:
CH CN–0.1% aqueous H PO (20:80, v/v); detection: optical rotation [Shodex OR-2 (Showa Denko Co.,
3
3
4
Ltd., Tokyo, Japan)]; flow rate: 1.0 mL/min] to identify S-(+)-2-methylbutyric acid [c', t 15.4 min
R
(positive)] from 2, which was carried out by comparison of its retention time and optical rotation with that
of commercially obtained standard sample. The rest of the product was subjected to HPLC [MeOH–H O
2
(50:50 v/v)] to give kaempferol 3-O-β-D-glucopyranosyl-7-O-β-D-glucopyranosyl-(1→3)-α-L-
27
rhamnopyranoside (1a, 2.0 mg from 1, 2.0 mg from 2), multiflorin B (20, 1.5 mg form 3), kaempferol 3-
O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-β-D-glucopyranoside (4a, 1.2 mg from 4, 1.4

mg from 5).
Kaempferol 3-O-β-D-glucopyranosyl-7-O-β-D-glucopyranosyl-(1→3)-α-L-rhamnopyranoside (1a): a
26
yellow powder, [α]
–52.5° (c 1.00, MeOH). High-resolution positive-ion FAB-MS: Calcd for
D
+
C H O Na (M+Na) 863.2586. Found: 863.2581. UV [MeOH, nm (log ε)]: 266 (4.31), 350 (4.24).
39 48 21
–1
IR (KBr): 3432, 1655, 1599, 1075 cm . H-NMR (500 MHz, DMSO-d ) δ: given in Table 2. C-NMR
1
13
6
+
(125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 863 (M+Na) . Negative-ion
6
C
–
FAB-MS m/z: 839 (M–H) .
Kaempferol 3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-7-O-β-D-glucopyranoside (4a): a
21
yellow powder, [α]
–98.1° (c 0.20, MeOH). High-resolution positive-ion FAB-MS: Calcd for
D
+
C H O Na (M+Na) 779.2011. Found: 779.2007. UV [MeOH, nm (log ε)]: 265 (4.17), 343 (4.02).
33 40 20
–1
IR (KBr): 3432, 1655, 1603, 1074 cm . H-NMR (500 MHz, DMSO-d ) δ: given in Table 4. C-NMR
1
13
6
+
(125 MHz, DMSO-d ) δ : given in Table 3. Positive-ion FAB-MS m/z: 779 (M+Na) . Negative-ion
6
C
–
FAB-MS m/z: 755 (M–H) .
Acid Hydrolysis of 1a and 4a
A solution of 1a, and 4a (1.0 mg both) in 1 M HCl (2.0 mL) was heated under reflux for 3 h. After
cooling, the reaction mixture was extracted with EtOAc. The EtOAc-soluble fraction was subjected to
HPLC analysis under the following conditions, respectively: HPLC column, YMC-Pack ODS-A, 4.6 mm
i.d. × 250 mm (YMC Co., Ltd., Ktoyo, Japan); detection, UV (254 nm); mobile phase, MeOH-1%
aqueous AcOH (60:40, v/v); flow rate 1.0 mL/min]. Identification of kaempferol (22, t 15.1 min) present
R
in the EtOAc-soluble fraction was carried out by comparison of their retention time with that of authentic
sample, respectively. On the other hand, the aqueous layer was subjected to HPLC analysis under the
following conditions, respectively: HPLC column, Kaseisorb LC NH -60-5, 4.6 mm i.d. × 250 mm
2
(Tokyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Shodex OR-2 (Showa Denko Co., Ltd.,
Tokyo, Japan); mobile phase, CH CN-H O (85:15, v/v); flow rate 0.8 mL/min]. Identification of L-
3
2
rhamnose (i) and D-glucose (ii) present in the aqueous layer was carried out by comparison of their
retention time and optical rotation with those of an authentic samples. t : (i) 7.8 min (negative optical
R
rotation) and (ii) 13.9 min (positive optical rotation), respectively.
Bioassay Method
Protective Effect on Cytotoxicity Induced by D-GalN in Primary Cultured Mouse Hepatocytes
The hepatoprotective effects of the constituents were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-
44
diphenyltetrazolium bromide (MTT) colorimetric assay using primary cultured mouse hepatocytes.
Hepatocytes were isolated from male ddY mice (30—35 g) by collagenase perfusion method. The cell
4
suspension at 4×10 cells in 100 µL William's E medium containing fetal calf serum (10%), penicillin G
(100 units/mL), and streptomycin (100 µg/mL) was inoculated in a 96-well microplate, and precultured
for 4 h at 37 ˚C under a 5% CO atmosphere. The fresh medium (100 µL) containing D-GalN (2 mM) and
2
a test sample were added and the hepatocytes were cultured for 44 h. The medium was exchanged with
100 µL of the fresh medium, and 10 µL of MTT (5 mg/mL in phosphate buffered saline) solution was

added to the medium. After 4 h culture, the medium was removed, 100 µL of isopropanol containing 0.04
M HCl was then added to dissolve the formazan produced in the cells. The optical density (O.D.) of the
formazan solution was measured by microplate reader at 570 nm (reference: 655 nm). Inhibition (%) was
obtained by following formula.
Inhibition (%) = [(O.D.(sample)—O.D.(control))/(O.D.(normal)—O.D.(control))] × 100
Statistics
Values were expressed as means±S.E.M. For statistical analysis, one-way analysis of variance followed by
Dunnett’s test was used. Probability (P) values less than 0.05 were considered significant. ID values
50
were estimated based on linear regressions of probit-transformed values of inhibition (%).
ACKNOWLEDGMENTS
M. Yoshikawa and H. Matsuda were supported by the 21st COE Program, Academic Frontier Project, and
a Grant-in Aid for Scientific Research from MEXT (the Ministry of Education, Culture, Sports, Science
and Technology of Japan). T. Morikawa and N. Ninomiya were supported by High-tech Research Center
Project (2007-2011) and a Grant-in Aid for Scientific Research from MEXT.
REFERENCES AND NOTES
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7. J. Tao, T. Morikawa, I. Toguchida, S. Ando, H. Matsuda, and M . Yoshiakwa, Bioorg. Med. Chem.,
2002, 10, 4005.
8. T. Morikawa, J. Tao, H. Matsuda, M. Yoshikawa, J. Nat. Prod., 2003, 66, 638.
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1
13
26. The H- and C-NMR spectra of 1—5, 1a, and 4a were assigned with the aid of distortionless
1
1
enhancement by polarization transfer (DEPT), homocorrelation spectroscopy ( H- H COSY),
heteronuclear multiple quantum coherence (HMQC), and HMBC experiments.
27. K. Yamasaki, R. Kasai, Y. Masaki, M. Okihara, and O. Tanaka, Tetrahedron Lett., 1977, 14, 1231.
28. These new compounds were also isolated from the methanolic extract of S. indica. The detail of
isolation and structure elucidation of these compounds were described in ref. 30.
29. M. Yoshikawa, F. Xu, T. Morikawa, K. Ninomiya, and H. Matsuda, Bioorg. Med. Chem. Lett., 2003,
13, 1045.
30. T. Morikawa, H. Xie, T. Wang, H. Matsuda, and M. Yoshikawa, Chem. Pharm. Bull., submitted.