Acyl glycosides lignans, coumarins, and terpenes from the stems of Erycibe obtusifolia

Article abstract of DOI:10.1016/j.carres.2013.02.006

Nine new acyl glycosides, obtusifosides A-I (1-9), and eight known compounds have been isolated from an EtOH extract of the stems of Erycibe obtusifolia. Their structures were elucidated on the basis of a spectroscopic data analysis (NMR, HRESIMS, and CD) and chemical evidence. The hepatoprotective effects of some of the compounds from d-galactosamine-induced cytotoxicity in HL-7702 hepatic cells were evaluated. Compounds 1, 10, 11, 13, 16, and 17 showed significant hepatoprotective activities compared with the positive control bicyclol at concentrations of 1 × 10^-5 M.

Full text of DOI:10.1016/j.carres.2013.02.006

Carbohydrate Research 372 (2013) 47–54
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Acyl glycosides lignans, coumarins, and terpenes from the stems
of Erycibe obtusifolia
⇑
Zhao-zhen Liu, Zhi-lai Zhan, Fu Liu, Ya-nan Yang, Zi-ming Feng, Jian-shuang Jiang, Pei-cheng Zhang
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 October 2012
Received in revised form 26 January 2013
Accepted 13 February 2013
Available online 24 February 2013
Nine new acyl glycosides, obtusifosides A–I (1–9), and eight known compounds have been isolated from
an EtOH extract of the stems of Erycibe obtusifolia. Their structures were elucidated on the basis of a spec-
troscopic data analysis (NMR, HRESIMS, and CD) and chemical evidence. The hepatoprotective effects of
some of the compounds from D-galactosamine-induced cytotoxicity in HL-7702 hepatic cells were eval-
uated. Compounds 1, 10, 11, 13, 16, and 17 showed significant hepatoprotective activities compared with
the positive control bicyclol at concentrations of 1 ꢀ 10ꢁ5 M.
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Erycibe obtusifolia
Convolvulaceae
Acyl glycosides
Hepatoprotective activity
1. Introduction
2. Results and discussion
Erycibe obtusifolia Benth (Convolvulaceae) is a traditional Chi-
nese medicinal herb, which is mainly distributed in Southeast Asia
and Australia. In China, it grows in the south province, such as
Guangdong, Guangxi, etc. Its roots and stems are used in Chinese
folk medicine to relieve symptoms of rheumatoid arthritis.1 Previ-
ous studies on Erycibe species have led to the isolation and identi-
fication of flavonoids, coumarins, chlorogenic acids, alkaloids, and
several other compounds,2–4 of these, scopoletin and scopolin have
exhibited a strong anti-inflammatory activity and several other
compounds also showed muscarinic agonistic and cytotoxic activ-
ities.5–7 In our previous studies on this species, bis-coumarins, cou-
marin, glucosides, chlorogenic acid derivatives, and other
compounds were isolated from the roots of E. obtusifolia and the
roots and stems of Erycibe hainanesis, and some of acyl glycosides
have showed hepatoprotective activity.8,9 Furthermore, our ongo-
ing study on the n-BuOH fraction of the stems of E. obtusifolia led
to the isolation of nine new acyl diglycosides, obtusifosides A–I
(1–9). Compared with the acyl diglycosides isolated from E. haina-
nesis Merr,9 five 7,90:70,9-diepoxylignane acyl diglycosides were
isolated, in which the acyl groups included not only the syringoyl
and vanilloyl moieties but also the sinapoyl moiety. The hepato-
The EtOH extract of the stems of Erycibe obtusifolia Benth was
suspended in H2O and then sequentially partitioned with
petroleumether, EtOAc, and n-BuOH. The n-BuOH fractions were
subjected to separation using various column chromatographic
techniques resulting in nine new compounds (1–9) and eight
known compounds (10–17) (Fig. 1). Compounds (10–17) were
isolated and identified by comparing experimental and reported
spectroscopic data as scopoletin (10),10 scopolin (11),11 7-O-
[2-O-(5-O-syringoyl-b-
methoxycoumarin (12),9 7-O-[6-O-(5-O-syringoyl-b-
anosyl)-b-
-glucop-yranosyl]-6-methoxycoumarin (13),9 7-O-[6-
O-(5-O-vanilloyl-b- -apiofuranosyl)-b- -glucopyranosyl]-6-meth-
oxycoumarin (14),9 2-O-[6-O-(5-O-syringoyl-b-
-apiofuranosyl)-
b-
-glucopyranosyl]isopropyl alchohol (15),9 3,4,5-trimethoxy-
phenyl-1-O-b- -(5-O-syringoyl)-apiofuranosyl-(1?6)-b- -glucopy-
ranoside (16),12 and 9-O-[6-O-(5-O-syringoyl-b-
-apiofurano-
syl)-b- -glucopyranosyl]-(3S,5R,6R,9R,7E)-3,6,9-trihydroxymeg-
D
-apiofuranosyl)-b-
D
-glucopyranosyl]-6-
D
-apiofur-
D
D
D
D
D
D
D
D
D
astigman- 7-ene (17).9
Compound 1, an amorphous white powder, ½a D20
ꢁ62.9 (c 0.05,
ꢂ
MeOH), was assigned the molecular formula C33H44O17 on the ba-
sis of positive-ion HRESIMS data at m/z 735.2469 [M+Na]+. In the
NMR spectrum of 1 (Tables 1 and 2), the signals [dH 6.64 (2H, s),
dC 137.4, 104.1, 104.1, 133.7, 152.7, 152.7 and dH 6.59 (2H, s), dC
131.8, 103.6, 103.6, 134.9, 148.3, 148.3] indicated the presence of
two 1,3,4,5-tetrasubstituted aromatic rings. In 13C NMR spectrum,
the characteristic signals [dC 85.1, 53.7, 71.2, 85.8, 53.1, 71.2] im-
plied the existence of a 2,6-diaryltetrahydrofuran ring system. In
protective effects of some of the compounds from
D-galactos-
amine-induced cytotoxicity in HL-7702 hepatic cells were
evaluated.
⇑
Corresponding author. Tel.: +86 10 63165231; fax: +86 10 63017757.
E-mail address: pczhang@imm.ac.cn (P.-c. Zhang).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.02.006

48
Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
H3CO
RO
H3CO
O
2
O
1'''
RO
O
O 4
2
1
O
4
O
O
OH
O
1
(S)
O
1'''
OH
OH
O
9'
1''
HO OH
9'
1''
7
OH
H3CO
HO OH
OCH3
7
(S)
OH
H3CO
(R)
(R)
(R)
(R)
(R) 1'
OH
2'
(S) 1'
2'
OCH3
OH
9
9
O
7'
O 7'
1
2
R = H
OH
4'
OCH3
4
5
R = syringoyl
4'
R = syringoyl
OCH3
R= sinapoyl
3 R= sinapoyl
5
4
10
9
6
13
H3CO
10
9
3
6
7
4
H3CO
O
3
HO
7
5
O
RO
3
HO
8
O
O
O
10
9
O
O
O
O
O
1
O
8
O
OH
OH
RO
8
1''
OH
OH
O
1'
RO
7
12
1'
11
HO OH
O
O
O
1''
OH
OH
OH
O
1'
1''
HO OH
HO OH
6
R = sinapoyl
OH
13
R = syringoyl
7
R= sinapoyl
8
R= syringoyl
14 R= vanilloyl
12
R= syringoyl
13
4
1
H3CO
O
5
3
2
HO
HO
10
8
9
H3CO
HO
10
O
O
O
6
11
O
7
O
O
RO
OH
OH
11
O
12
1'
OH
O
1''
O
O
OH
OH
1'
HO OH
OH
R= syringoyl
13
4
1
OH
3
9
OCH3
4
5
HO
2
10
OCH3
8
9
2
6
11
CH3
CH3
1
O
O
RO
7
RO
12
O
O
O
RO
OCH3
O
O
O
O
O
O
1''
1''
HO OH
OH
OH
O
OH
OH
1'
OH
1'
HO OH
HO OH
OH
OH
OH
OH
R= syringoyl
17
R= syringoyl
15
16
R= syringoyl
OCH3
OCH3
OH
OCH3
OH
OCH3
Figure 1. Chemical structures of compounds 1–17.
O
O
O
sinapoyl =
OH
vanilloyl =
syringoyl =
OCH3
addition, four aromatic methoxyl signals [dH 3.75 (6H, s) and 3.74
(6H, s)] were observed in 1H NMR spectrum. Furthermore, two
anomeric protons and carbons signals [dH 4.77 (1H, d, J = 7.0 Hz)
and dH 4.74 (1H, br s)] showed the presence of two glycosyl
groups.13 All these spectroscopic data of 1 were similar to those
C-4 of the (+)-syringaresinol. On the basis of the above evidence,
the structure of 1 was characterized as 4-O-(6-O-b- -apiofurano-
D
syl-b-D
-glucopyranosyl]-(+)-(7S,70S,8R,80R)-40-hydroxy-3,30,5,50-
tetramethoxy-7,90:70,9-diepoxylignane, and the compound was
named obtusifoside A.
of (ꢁ)-syringaresinol.14 Acid hydrolysis of 1 resulted in 1a,
D-apiose
Compound 2 was obtained as an amorphous white powder, ½a D20
ꢂ
and
D
-glucose; the sugar components were identified by GC
ꢁ38.2 (c 0.05, MeOH). Its molecular formula was determined as
analysis.15 Furthermore, the coupling constant (J = 7.0 Hz) of the
anomeric proton of the glucosyl moiety and the chemical shift
(dC 109.0) of the anomeric carbon of the apiosyl moiety demon-
strated that both of the sugar moieties were of the b-anomeric con-
figuration.16 Further NMR analysis suggested that the sequence of
C42H52O21 on the basis of the positive-ion HRESIMS data at m/z
915.2900 [M+Na]+. The NMR data of 2 (Tables 1 and 2) were similar
to those of 1 and showed the presence of a syringaresinol moiety
and a b-D-apiofuranosyl-(1?6)-b-D-glucopyranosyl moiety. How-
ever, the characteristic signals of 2 [dH 7.15 (2H, s), 3.78 (6H, s)
and dC 119.2, 107.1, 147.5, 140.8, 165.4, 56.3] suggested the exis-
tence of a syringoyl moiety.20 Compared with the 13C NMR chem-
the sugar moieties in 1 is D-apiofuranosyl-(1?6)-D-glucopyranose,
which was also confirmed by the key HMBC correlation of glc-C-600
(dC 67.3) with api-H-1000 (dH 4.74). The aglycone (1a) was identified
as (+)-(7S,70S,8R,80R)-4,40-dihydroxy-3,30,5,50-tetramethoxy-7,90:70,9-
diepoxylignane by the NMR (Tables 1 and 2), MS, and CD data
ical shift of the sugar moiety in 1, the C-5000 of b-
D
-apiose in 2 was
observed at a lower field (dC 66.6), suggesting that the syringoyl
group was linked to C-5000 of the b-
-apiofuranosyl moiety. This
D
(positive at 205, 235, and 274 nm) and specific rotation {½a D20
ꢂ
+12.1
association was also confirmed by the HMBC correlation (Fig. 2)
of H-5000 with C-70000of a syringoyl moiety. The HMBC correlation
(Fig. 2) of C-4 (dC 133.7) with glc-H-100 (dH 4.83) indicated that
(c 0.05, CHCl3)}.17–19 The HMBC correlation of C-4 (dC 133.7) with
glc-H-100 (dH 4.77) indicated that the sugar chain was located at

Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
49
Table 1
The 1H NMR (500 MHz) spectral data of the compounds 1–5a (in DMSO-d6, J in Hz)
Position
1
1a
2
3
4
4a
5
2/6
7
8
6.64 s
4.66 d (4.5)
3.07 m
6.59 s
4.66 d (4.5)
3.04 m
6.62 s
4.64 d (5.0)
3.07 m
6.63 s
4.65 d (4.5)
3.07 m
6.63 s
4.35 d (6.5)
2.83 m
6.60 s
4.31 d (7.0)
2.84 m
6.64 s
4.35 d (6.0)
2.85 m
9
3.79 overlap
4.17 overlap
6.59 s
4.60 d (4.0)
3.05 m
3.79 overlap
4.17 overlap
4.77 d (6.5)
3.19 m
3.76 overlap
4.15dd (9.0,6.5)
6.59 s
4.66 d (4.5)
3.04 m
3.78 overlap
4.16 overlap
6.60 s
4.59 br s
3.05 m
3.78 overlap
4.16 overlap
4.78 d (7.0)
3.21 m
4.17 overlap
3.77 overlap
6.58 s
4.58 br s
3.05 m
4.17 overlap
3.77 overlap
4.78 d (6.5)
3.18 m
3.72 overlap
4.12 d (9.0)
6.57 s
4.72 d (5.5)
3.32 m
3.78 overlap
3.08 overlap
4.79 d (7.5)
3.20 m
3.21 overlap
3.11t(8.5)
3.07 m
3.76 d (9.0)
4.09 d (9.0)
6.58 s
4.75 d (4.5)
3.09 d (9.0)
3.72 overlap
3.16 br s
3.73 overlap
4.12 d (9.0)
6.56 s
4.72 d (5.5)
3.32 m
3.78 overlap
3.10 m
4.81 d (6.5)
3.19 m
20/60
70
80
90
3.76 overlap
4.15 dd (9.0, 6.5)
Glc-100
200
30000
3.19 m
3.06 m
3.17 m
3.22 m
3.09 m
3.06 m
3.21 m
3.06 m
3.08 m
3.21 m
3.11t (8.5)
3.07 m
400
500
600a
600b
Api-1000
2000
3.71 overlap
3.37dd (11.5,6.5)
4.74 br s
3.64 m
3.71 m
3.52 d (9.5)
3.26 m
3.78 overlap
3.45dd (11.5,6.5)
4.83 br s
3.71 overlap
3.69 m
3.75 overlap
3.43dd (11.5,6.5)
4.81 br s
3.67 m
3.65 m
3.73 m
4.06 br s
Sinapoyl
7.02 s
7.56 d (16.0)
6.56 d (16.0)
3.75 s
3.74 overlap
3.43dd (11.5,6.5)
4.85 br s
3.72 overlap
3.78 overlap
3.69 m
4.19 br s
Syringoyl
7.23 s
3.76 overlap
3.42dd (11.5,6.5)
4.85 br s
3.72 overlap
3.78 m
4000
a
4
000b
3.69 m
3.67 m
5000a/5000
b
4.16 br s
Syringoyl
7.22 s
4.07 br s
Sinapoyl
7.01 s
7.56 d (16.0)
6.48 d (16.0)
3.74 s
20000/60000
70000
80000
3/5-OCH3
30/50-OCH3
30000/50000-OCH3
3.75 s
3.74 s
3.74 s
3.74 s
3.71 s
3.71 s
3.79 s
3.74 s
3.74 s
3.80 s
3.74 s
3.74 s
3.73 s
3.79 s
3.73 s
3.78 s
a
Signal without multiplicity was overlapped and obtained from HMQC and HSQC.
the sugar chain was located at C-4 of the syringaresinol. Acid
(2H, s, H-20000/60000), 7.56 (1H, d, J = 16.0 Hz, H-70000), 6.56 (1H, d,
J = 16.0 Hz, H-80000), 3.79 (6H, s, 30000/50000-OCH3), dC C-10000 (124.4),
C-20000/60000 (106.3), C-30000/50000 (148.0), C-40000 (138.4), C-70000 (145.8),
C-80000 (114.6), C-90000 (166.5 C@O)] existed in 3. Acid hydrolysis of
3 resulted in the same aglycon as 1a. Thus, compound 3 was
hydrolysis of 2 resulted in 2a, -apiose and D-glucose. A compari-
son of the NMR, MS, CD, and ½Daꢂ2D0 data of 2a with the data of 1a
(acid hydrolysis of 1 resulted in 1a) suggested that 2a has the same
absolute configuration as 1a. Thus, compound 2 was elucidated to
be 4-O-[6-O-(5-O-syringoyl-b-
D
-apiofuranosyl)-b-
D
-glucopyrano-
characterized as 4-O-[6-O-(5-O-sinapoyl-b-D-apiofuranosyl)-b-D-
syl]-(+)-(7S,70S,8R,80R)-40-hydroxy-3,30,5,50-tetramethoxy-7,90:70,9-
glucopyranosyl]-(+)-(7S,70S,8R,80R)-40-hydroxy-3,30,5,50-tetrameth-
diepoxylignane and was named obtusifoside B.
oxy-7,90:70,9-diepoxylignane and was named obtusifoside C.
Compound 3 was obtained as a yellow powder, ½a D20
ꢂ
ꢁ45.5 (c
Compound 4 was obtained as a white powder, ½a D20
ꢁ57.9
ꢂ
0.05, MeOH). The molecular formula was determined to be
(c 0.05, MeOH). The positive-ion HRESIMS analysis indicated 4
had the same molecular formula as 2, C42H52O21. The NMR data
C
44H54O21 on the basis of the positive HRESIMS data at m/z
941.3043 [M+Na]+. A comparison of the NMR data (Tables 1 and
of 4 also showed the presence of a syringaresinol moiety, a b-
D-
2) of 3 with that of 2 suggested that a sinapoyl moiety [dH 7.02
apiofuranosyl-(1?6)-b- -glucopyranosyl moiety, and a syringoyl
D
Table 2
The 13C NMR (125 MHz) spectral data of the compounds 1–5a (in DMSO-d6)
Position
1
1a
2
3
4
4a
5
Position
1
2
3
4
5
1
2/6
3/5
4
7
8
137.4
104.1
152.7
133.7
85.1
131.4
103.6
147.9
134.8
85.3
137.4
104.0
152.7
133.7
85.1
137.4
104.0
152.7
133.7
85.0
137.4
103.9
152.7
133.6
86.8
131.5
103.6
147.9
134.9
87.1
137.4
104.0
152.7
133.7
86.7
200
74.0
76.4
70.0
75.8
67.3
109.0
75.8
78.8
73.2
63.2
74.0
76.4
70.0
75.8
67.3
108.7
76.9
77.1
73.2
74.0
76.4
69.9
75.7
67.3
108.6
76.4
77.0
73.2
74.0
76.4
70.1
75.8
67.3
108.8
76.9
77.1
73.2
74.0
76.4
70.0
75.7
67.3
108.6
76.4
77.0
73.2
300
400
500
600
53.7
71.2
53.6
71.0
53.7
71.2
53.7
71.2
53.8
70.3
53.9
70.3
53.7
70.3
Api-1000
2000
3000
4000
5000
9
10
131.8
103.6
148.3
134.9
85.8
53.1
71.2
56.0
56.4
131.4
103.6
147.9
134.8
85.3
53.6
71.0
56.0
56.0
131.4
103.7
147.9
134.9
85.4
53.6
71.2
56.1
56.0
131.3
103.7
147.9
134.9
85.3
53.6
71.2
56.1
56.4
128.8
102.9
147.8
134.2
81.4
49.3
69.0
56.1
56.3
128.8
103.0
147.8
134.2
81.5
49.3
68.9
56.0
56.0
128.8
103.7
147.8
134.2
81.4
49.3
69.0
56.1
56.4
20/60
30/50
66.6
65.9
66.6
65.9
40
Syringoyl
119.2
107.1
147.5
140.8
165.4
Sinapoyl
124.4
106.3
148.0
138.4
145.7
114.6
166.5
Syringoyl
119.2
107.1
147.5
140.8
165.4
Sinapoyl
124.4
106.3
148.1
138.4
145.7
114.5
166.5
70
10000
80
20000/60000
3000/5000
40000
90
3/5-OCH3
30/50-OCH3
30000/50000-OCH3
Glc-100
70000
56.3
102.9
56.0
102.9
55.9
102.8
56.0
102.9
80000
102.9
90000
a
13C NMR data (d) were measured in DMSO-d6. The assignments are based on HMQC and HSQC experiments.

50
Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
OCH3
1''''
HO
4''''
H3CO
6''
5'''
O
O
H3CO
4
O
O
O
1''
1
OH
O
1'''
O
7
HO OH
9'
1'
OH
H3CO
OH
8
OCH3
OH
2
9
O
4'
OCH3
5
10
9
H3CO
3
HO
7
O
OCH3
OCH3
O
O
O
3
O
O
HO
7
8
HO
4'''
2'''
4'''
10
OH
OH
HO
9
1
2'''
1'
O
O
9'''
O
O
H3CO
O
O
H3CO
O
O
1''
OH
O
1''
OH
1'
HO OH
OH
HO
OH
7
8
Figure 2. Key HMBC correlations of 2, 7, and 8.
moiety. Acid hydrolysis of 4 resulted in 4a,
D
-apiose, and
D
-glucose
ization of the C-70 position.18,21 Furthermore, 4a was identified as
(+)-(7S,70R,8R,80R)-4,40-dihydroxy-3,30,5,50-tetramethoxy-7,90:70,9-
diepoxylignane (epi-syringaresinol) by the NMR, MS, and CD data
[ 210 (ꢁ3.00), 230 (+0.60), 272 (+0.15), 280 (0.00) and 288 (ꢁ0.16)
(Tables 1 and 2). However, the 13C NMR chemical shift differences
of C-7 (
D
dC +1.8), C-70(
D
dC ꢁ3.8), C-80 (
D
dC ꢁ4.3), and C-90 (
DdC
ꢁ2.1) between 4a and 1a suggested a change of stereochemistry
in 4a. In addition, the signal of C-10 in 4a was also shifted upfield
by 2.6 ppm. These trends of 13C NMR data from 1a to 4a have also
been observed between syringaresinol (an equatorial guaiacyl
unit) and epi-syringaresinol (an axial guaiacyl unit) by the epimer-
nm] and specific rotation {½a D20
ꢂ
+17.7 (c 0.05, CHCl3)}.18 Finally,
4 was elucidated to be 4-O-[6-O-(5-O-syringoyl-b-D-apiofurano-
syl)-b-D
-glucopyranosyl]-(+)-(7S,70R,8R,80R)-40-hydroxy-3,30,
Table 3
The 1H NMR (500 MHz) spectral data of the compounds 6–9 (J in Hz)
Position
2
6
7
8a
8b
9a
9b
2.69 d (13.5)
1.65 d (13.5)
2.85 d (13.5)
1.81 dd (13.5, 1.5)
2.69 d (13.0)
1.54 d (13.0)
2.85 d (13.0)
1.69 dd (13.5, 2.5)
3
4
6.28 d (9.5)
7.92 d (9.5)
6.22 d (9.5)
7.75 d (9.5)
2.27 t (13.5)
1.94 br d (13.5)
2.11 m
2.42 t (13.5)
2.11 ddd (13.5, 6.5, 1.5)
2.24 m
2.29 t (13.5)
1.86 br d (13.5)
2.03 m
2.45 t (13.5)
2.03 br d (13.5)
2.17 m
1.87 m
1.73 m
5
7
7.24 s
7.14 s
7.12 s
7.04 s
5.54 d (16.5)
5.71 d (15.5)
1.64 m
8
9
5.73 dd (16.5,6.0)
4.29 m
5.90 dd (15.5, 6.5)
4.44 m
1.47 m
3.63 m
1.61 m
3.78 m
10
11
1.17 d (6.0)
0.84 s
1.33 d (6.5)
0.99 s
1.05 d (6.0)
0.94 (s)
1.18 d (6.0)
0.94 (s)
12
0.78 s
0.92 s
0.79 (s)
0.91 (s)
13
0.75 d (6.0)
4.18 d (8.0)
2.93 t (8.0)
3.11 t (8.0)
3.01 t (8.0)
3.18 m
0.86 d (6.5)
4.37 d (8.0)
3.22 t (8.5)
3.38 m
3.30 t (8.5)
3.28 m
0.86 d (6.0)
4.15 d (7.5)
2.89 t (7.5)
3.11 m
2.97 t (8.0)
3.24 m
1.01 d (6.0)
4.32 d (8.0)
3.17 t (8.0)
3.37 m
3.25 t (8.0)
3.43 m
Glc-10
20
5.07 d (7.0)
3.29 m
3.28 m
3.12 m
3.63 m
5.15 d (7.5)
3.52 t (9.0)
3.61 t (9.0)
3.17 t (9.0)
3.45 m
30
40
50
60a
60b
Api-100
200
400a
400b
500a
500b
3.87 d (11.5)
3.49 m
4.84 br s
3.75 overlap
3.97 d (9.0)
3.73 m
4.13 br s
4.13 br s
Sinapoyl
6.96 s
7.49 d (15.5)
6.45 d (15.5)
3.77 s
3.68 m
3.43 m
5.46 br s
3.75 overlap
4.22 d (10.0)
3.73 d (10.0)
4.04 d (11.0)
4.00 d (11.0)
Sinapoyl
3.82 d (11.5)
3.42 m
4.89 br s
3.81 overlap
3.92 d (9.0)
3.76 d (9.0)
4.21 d (11.0)
4.21 d (11.0)
Syringoyl
3.92 d (11.5)
3.65 dd (11.5, 6.0)
5.07 d (2.5)
4.07 d (2.5)
4.11 d (10.0)
4.02 d (10.0)
4.44 br s
4.44 br s
Syringoyl
7.40 s
3.85 d (11.5)
3.43 m
4.91 br s
3.80 overlap
3.90 d (10.0)
3.77 d (10.0)
4.23 d (11.0)
4.20 d (11.0)
Syringoyl
7.21 s
4.03 m
3.63 dd (11.5, 6.5)
5.10 d (2.5)
4.02 d (2.5)
4.09 d (9.5)
3.93 overlap
4.40 br s
4.40 br s
Syringoyl
7.41 s
2000/6000
7000
8000
6-OCH3
3000/5000-OCH3
6.88 s
7.22 s
7.33 d (16.0)
6.28 d (16.0)
3.78 s
3.78 s
3.78 s
3.80 s
3.93 s
3.79 s
3.93 s
a
1H NMR data (d) were measured in DMSO-d6.
b
1H NMR data (d) were measured in CD3OD-d4. Signal without multiplicity was overlapped and obtained from HMQC and HSQC.

Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
51
Table 4
The 13C NMR (125 MHz) spectral data of the compounds 6–9
Position
6
7
8a
8b
9a
9b
Position
6
7
8a
8b
9
9b
1
2
3
4
5
6
7
8
42.4
51.1
210.4
44.8
35.8
76.0
132.5
132.8
74.4
20.9
24.5
24.3
15.9
100.6
73.7
76.7
70.0
43.9
52.4
214.8
46.1
37.8
78.1
134.6
134.2
78.0
21.5
25.0
25.3
16.5
102.6
75.3
78.6
71.6
43.4
52.7
210.8
45.6
35.9
74.1
30.3
32.7
74.0
19.4
24.6
24.3
15.8
100.6
73.4
76.7
70.4
45.0
53.9
215.4
47.0
38.0
76.3
31.9
34.1
76.7
20.1
25.5
25.2
16.5
102.3
75.1
78.5
71.9
50
75.4
67.9
109.2
76.5
77.0
77.1
60.6
107.9
76.7
77.6
75.6
67.5
109.0
76.8
77.1
76.9
68.7
110.9
77.5
79.1
75.4
67.7
109.0
76.8
77.1
76.9
68.9
110.8
78.2
79.1
160.4
113.4
144.0
109.6
145.9
149.7
103.0
148.9
112.3
160.5
113.1
144.1
109.3
145.8
149.5
102.7
148.8
112.2
60
Api-100
200
300
400
73.5
65.8
73.8
66.8
73.4
66.8
75.1
68.1
73.3
66.8
75.1
68.1
500
Sinapoyl
124.4
106.2
148.0
138.4
145.5
114.6
166.4
55.9
Sinapoyl
124.2
106.2
148.1
138.5
145.3
114.3
166.3
55.9
Syringoyl
118.9
107.1
147.3
140.5
165.5
Syringoyl
120.8
108.4
149.0
142.6
167.9
Syringoyl
118.4
107.1
147.5
141.7
165.2
Syringoyl
120.3
108.5
148.8
142.8
167.8
9
1000
10
11
12
13
Glc-10
20
2000/6000
3000/5000
4000
7000
99.5
73.0
76.7
69.8
97.8
77.2
74.5
70.0
8000
9000
30
40
6-OCH3
3000/5000-OCH3
56.0
56.1
56.0
56.9
56.0
56.9
a
13C NMR data (d) were measured in DMSO-d6.
13C NMR data (d) were measured in CD3OD-d4.
b
5,50-tetramethoxy-7,90:70,9-diepoxylignane and was named
obtusifoside D.
spectrum of 7 suggested that the apiofuranosyl moiety was located
at C-20 of -glucosyl moiety in 7, instead of at C-60 in 6. This finding
D
Compound 5 was obtained as a yellow powder, and the posi-
tive-ion HRESIMS at m/z 941.3049 [M+Na]+ implied that it had
the same molecular formula as 3. The NMR data of 5 (Tables 1
and 2) were also similar to those of 3 except for the signals of
was supported by an HMBC correlation (Fig. 2) of C-100 with H-20.
From these data, 7 was established as 7-O-[2-O-(5-O-sinapoyl-
b-D-apiofuranosyl)-b-D-glucopyranosyl]-6-methoxycoumarin and
was named obtusifoside G.
the aglycone moiety [C-7 (
D
dC +1.7), C-70 (
D
dC ꢁ3.9), C-80 (
D
dC
Compound 8 was obtained as a white powder, ½a D20
ꢁ45.1 (c
ꢂ
ꢁ4.3), and C-90
(D
dC ꢁ2.2)], which suggested the existence of
0.05, MeOH). The positive-ion HRESIMS data at m/z 723.2844
epi-syringaresinol and 5-O-sinapoyl-b-
D
-apiofuranosyl-(1?6)-b-
[M+Na]+, indicated that the molecular formula was C33H48O16
The 1H and 13C NMR spectra of 8 (Tables 3 and 4) displayed sig-
nals assignable to a 5-O-syringoyl-b- -apiofuranosyl-(1?6)-b-
.
D
-glucopyranosyl moiety. Acid hydrolysis of 5 resulted in 5a, the
aglycone 5a was determined to be the same as 4a by a comparison
D
D-
of the NMR, MS, CD, and ½a D20
ꢂ
data between them. Thus, compound
-apiofuranosyl)-
glucopyranosyl moiety. Furthermore, a comparative analysis of
1H and 13C NMR spectra data of 8 with those of ampelopsisiono-
side suggested the presence of a megastigmane skeleton with a
carbonyl group at C-3,23 hydroxyl functional groups at C-6 and
C-9 positions, and a disubstituted double bond. These findings
suggested that the planar structure of the aglycone moiety was
6,9-dihydroxymegastigman-3-on-7-ene. In the 1H NMR spectrum,
the coupling constants (J = 16.5 Hz) of H-7 and H-8 proved the
double bond in C-7/C-8 was E. In addition, HMBC correlations
(Fig. 2) from the anomeric proton H-10 to C-9 and from H-9 to
C-10 indicated that the sugar moiety was attached to C-9. In
the 1H NMR spectrum, the large couplings of H-4ax with H-5
(J = 13.5 Hz) implied that H-5 should be in the axial position
and that the CH3-13 had an equatorial orientation. In addition,
the axial protons at the 2- and 4-positions were shifted down-
field because of the 1,3-diaxial interaction of the hydroxyl group
at the C-6 position. Therefore, the absolute configurations of the
6,9-dihydroxy megastigman-7-ene moieties were determined to
be 5R and 6S.24,25 The absolute configuration of C-9 was reflected
5 was elucidated to be 4-O-[6-O-(5-O-sinapoyl-b-
D
b-D
-glucopyranosyl]-(+)-(7S,70R,8R,80R)-40-hydroxy-3,30,5,50-tetra-
methoxy-7,90:70,9-diepoxylignane and was named obtusifoside E.
Compound 6 was obtained as a yellow powder, ½a D20
ꢁ27.9
ꢂ
(c 0.05, MeOH). The molecular formula was determined to be
C
32H36O17 on the basis of the positive HRESIMS data at m/z
693.2020 [M+H]+. The 1H NMR spectrum of 6 (Table 3) showed
the presence of a 6,7-disubstitutedcoumarin skeleton [dH 6.28
(1H, d, J = 9.5 Hz), 7.92 (1H, d, J = 9.5 Hz), 7.24 (1H, s), 7.14 (1H,
s)].9 A comparison of the 1H and 13C NMR (Tables 3 and 4) data
of 6 with those of 5 indicated the presence of a 5-O-sinapoyl-b-
D-
apiofuranosyl-(1?6)-b- -glucopyranosyl moiety, which was fur-
D
ther confirmed by HMBC correlations of C-60 (dC 67.9) with H-100
(dH 4.84 br s) and C-9000 (dC 166.4) with H-500 (dH 4.13 br s). The
HMBC correlations of C-6 with the methoxy proton (dH 3.77) and
of C-7 with H-10 indicated that the methoxy group and the sugar
chain were located at C-6 and C-7, respectively. Acid hydrolysis
of
coupling constant (J = 7.0 Hz) of the anomeric proton signal of
the -glucosyl moiety as well as the chemical shift (dC 109.2) of
the anomeric carbon of the -apiosyl moiety demonstrated that
both the sugar moieties have b-anomeric configurations.20 The
structure of 6 was characterized as 7-O-[6-O-(5-O-sinapoyl-b-
apiofuranosyl)-b- -glucopyranosyl]-6-methoxycoumarin, and the
6 resulted in D-glucose and D
-apiose.22 Furthermore, the
by chemical shifts. The 13C NMR chemical shifts of C-9 of the b-
D-
D
glucopyranoside of (9R)-and (9S)-3-oxo-a-ionol are dC 77.0 and
D
74.7,23,25 respectively. However, the C-9 chemical shift in 8 was
observed at dC 78.0. This finding suggested that the aglycone
moiety of 8 was a (5R,6S,9R,7E)-6,9-dihydroxymegastigman-3-
D
-
D
on-7-ene moiety. Thus,
8
was established as 9-O-[6-O-(5-O-
-glucopyranosyl]-(5R,6S,9R,7E)-
compound was named obtusifoside F.
syringoyl-b- -apiofuranosyl)-b-D
D
Compound 7 was obtained as a yellow powder, ½a D20
ꢂ
ꢁ50.7
6,9-dihydroxymegastigman-3-on-7-ene and was named obtusifo-
(c 0.05, MeOH). The molecular formula was determined to be
C
side H.Compound 9 was obtained as a white powder, ½a D20
ꢁ45.1
ꢂ
32H36O17 on the basis of the positive-ion HRESIMS data ([M+H]+,
(c 0.05, MeOH). Its positive HRESIMS data at m/z 725.2987
m/z found 693.2021), indicating that this compound had the same
molecular formula as 6. The NMR spectroscopic data of 7 (Tables 3
and 4) also resembled those of 6. However, 4.2 ppm downfield shift
for C-20 and 7.3 ppm upfield shifts for C-60 in the 13C NMR
[M+Na]+ indicated that the molecular formula was C33H50O16
.
Compared with the NMR spectra data (Tables 3 and 4) in 8, the
NMR spectra data of compound 9 also indicated the presence
of
a 5-O-syringoyl-b-D-apiofuranosyl-(1?6)-b-D-glucopyranosyl

52
Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
Table 5
Hepatoprotective effects of compounds 1, 10, 11, 13, 16, and 17 against D-galactosamine-induced toxicity in HL-7702 cellsa
Compound
Means SD
Cell survival rate (% of normal)
Inhibition (% of control)
Normal
100
39###
47**
46**
46*
Control(GalN)
Bicyclolb
0.446 0.022
0.441 0.029
0.435 0.033
0.441 0.041
0.471 0.025
0.459 0.068
0.434 0.040
13.1
11.5
11.5
13.1
18.0
14.8
11.5
1
10
11
13
16
17
47*
50**
48*
46*
a
###
*
The compounds were tested at 1 ꢀ 10ꢁ5 M.
Comparing to normal: p <0.001.
Comparing to control(GalN): p <0.05.
**
Comparing to control(GalN): p <0.01, Results are expressed as means SD (n = 5).
b
Positive control substance (Beijing Union Pharmaceutical Factory, purity is 98% by HPLC).
moiety and a megastigmane skeleton. However, the signal for a
double bond at C-7 of the megastigmane skeleton in 8 was re-
placed by an additional pair of methylenes [dH 1.64 (2H, over-
lapped, H-7), 1.47 (2H, overlapped, H-8) and dC 30.3 (C-7), 32.7
(C-8)] in 9. This finding was confirmed by the HSQC and HMBC
spectra. The 13C NMR chemical shift of C-9 at dC 76.7 in Metha-
nol-d4 indicated the absolute configuration of C-9 in 9 was R.23
at the Institute of Materia Medica, Chinese Academy of Medical
Sciences, Beijing 100050, China.
3.3. Extraction and Isolation
The dried stems of Erycibe obtusifolia Benth (18.5 kg) were ex-
tracted with 95% EtOH under reflux (30 L ꢀ 1.5 h each). The EtOH
extract was concentrated under reduced pressure to form a residue
(0.96 kg), which was suspended in H2O (2.3 L) and sequentially
partitioned with petroleum ether (3 ꢀ 2.0 L), EtOAc (3 ꢀ 2.0 L),
and n-BuOH (3 ꢀ 2.0 L), successively. After the solvent evaporated
under reduced pressure, the n-BuOH extract (400 g) was subjected
to macroporous resin column chromatography, eluting succes-
sively with H2O, 15% EtOH, 30% EtOH, 50% EtOH, 70% EtOH, and
95% EtOH (30 L each). After removing the solvent, the 50% EtOH
fraction (50 g) was subjected to Sephadex LH-20 (500 g,
90 ꢀ 6 cm) and eluted with a gradient of H2O–MeOH (10:1; 10:3;
Thus, 9 was elucidated as 9-O-[6-O-(5-O-syringoyl-b-
anosyl)-b- -glucopyranosyl]-(5R,6S,9R)-6,9-dihydroxy megastig-
man-3-one and was named obtusifoside I.
The hepatoprotective activities of compounds 1–17 against
D-apiofur-
D
D
-
galactosamine-induced toxicity were examined in HL-7702 cells.
Compounds 1, 10, 11, 13, 16, and 17 showed significant hepatopro-
tective activities (Table 5) compared with the positive control
bicyclol at concentrations of 1 ꢀ 10ꢁ5 M.
3. Experimental
10:5; 10:8; 1:1; 100% MeOH), to obtain ten fractions (A1–A10
)
through a HPLC-DAD analysis. Fraction A2 (200 mg) was subjected
to reversed-phase preparative HPLC, using MeOH–H2O (35:65) as
the mobile phase, to give 1 (50 mg), 2 (30 mg), and 6 (15 mg). Frac-
tion A3 (1.1 g) was subjected to Rp-18 column, eluting with a gra-
dient of increasing MeOH (0–45%) in H2O, to yield five subfractions
(A3-1–A3-5). Subfraction A3-4 (300 mg) was further separated by re-
versed-phase preparative HPLC, using MeOH–H2O (48:52) as the
mobile phase, to elute 3 (30 mg), 4 (20 mg), 5 (50 mg), and 10
(50 mg).
After removing the solvent, the 30% EtOH fraction (200 g) was
also applied to Sephadex LH-20 and eluted with a gradient of
H2O–MeOH (10:1; 10:3; 10:5; 10:8; 1:1; 100% MeOH), to yield
twenty fractions (B1–B20) through a HPLC-DAD analysis. Fraction
B3 (1.2 g) was further chromatographed over Sephadex LH-20 with
H2O as the mobile phase, resulting in six subfractions (B3-1, B3-6).
Subfraction B3-4 (500 mg) was purified by reversed-phase prepara-
tive HPLC, using a mobile phase of MeOH–H2O (35:65) to yield 7
(10 mg), 11 (30 mg), 13 (10 mg), and 14 (10 mg). Fraction B5
(10 g) was subjected to chromatography over Sephadex LH-20
with a gradient of increasing MeOH (0–100%) in H2O as the mobile
phase, to elute five subfractions (B5-1–B5-5). Using MeOH–H2O
(45:55) as the mobile phase, subfraction B5-4 was purified by re-
versed-phase preparative HPLC, resulting in 8 (20 mg), 9 (10 mg),
and 15 (10 mg). Fraction B10 (6 g) was subjected to chromatogra-
phy over Sephadex LH-20 with a gradient of increasing MeOH
(0–100%) in H2O as the mobile phase, resulting in five subfractions
(B10-1–B10-5). Using MeOH–H2O (45:55) as the mobile phase, sub-
fraction B10-2 was eluted by reversed-phase preparative HPLC, to
yield 12 (20 mg), 16 (20 mg), and 17 (20 mg).
3.1. General method
Optical rotations were measured with a JASCO P-2000 polarim-
eter, and CD spectra were recorded on a JASCO J-815 CD spectrom-
eter. IR spectra were recorded on a Nicolet 5700 spectrometer by a
FT-IR microscope transmission method. GC was conducted on an
Agilent 7890A instrument. NMR and 2D-NMR spectra were run
on INOVA 500 spectrometers. HRESIMS data were obtained using
an Agilent 1100 series LC/MSD ion trap mass spectrometer. The
ESI mass spectra were recorded on an Agilent 1100 series LC/
MSD TOF from Agilent Technologies. Column chromatography
was performed with macroporous resin (Diaion HP-20, Mitsubishi
Chemical Corp., Tokyo, Japan), Rp-18 (50 lm, YMC, Kyoto, Japan),
Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden),
and silica gel (200–300 mesh, Qingdao Marine Chemical Inc.,
Qingdao, China). Preparative HPLC was carried out on a Shimadzu
LC-6AD instrument with an SPD-20A detector, using an YMC-Pack
ODS-A column (250 ꢀ 20 mm, 5
lm). HPLC-DAD analysis was
performed using an Agilent 1260 series system (Agilent
Technologies, Waldbronn, Germany) with an Apollo C18 column
(250 ꢀ 4.6 mm, 5
lm, Grace Davison, IL, USA). D-Apiose (J&K,
Beijing, China).
3.2. Plant material
The stems of Erycibe obtusifolia Benth were collected in Hainan
Province, China, in March 2010. The plant material was identified
by Mr. Huanqiang Chen (Jianfengling National Nature Reserve of
Hainan Province). A voucher specimen (ID-21180) was deposited

Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
53
3.4. Identification
added, and the reaction mixture was incubated at 60 °C for 1 h.
After the mixture was concentrated to dryness under a vacuum,
0.5 mL of N-trimethylsilylimidazole was added, and the mixture
was incubated at 60 °C for 1 h. The reaction mixture was parti-
tioned between n-hexane and H2O (2 mL each). The n-hexane ex-
tract was subjected to GC under the following conditions:
Obtusifoside A (1): White powder; mp 150–152 °C; ½a D20
ꢁ62.9 (c
ꢂ
0.05, MeOH); IR mmax 3421, 1677, 1598, 1515, 1461, 1423, 1330,
1220, 1062, 827, 704 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables
1 and 2); and positive-ion HRESIMS m/z 735.2469 [M+Na]+ (calcd
for C33H44O17Na, 735.2471).
capillary column, HP-5 (30 m ꢀ 0.25 mm, with a 0.25
lm film, Dik-
Obtusifoside B (2): White powder; mp 130–132 °C; ½a D20
ꢂ
ꢁ38.2(c
ma); detection, FID; detector temperature, 280 °C; injection tem-
perature, 250 °C; initial temperature 160 °C, raised to 280 °C at
5 °C/min and final temperature maintained for 10 min; and carrier
0.05, MeOH); IR mmax 3441, 1707, 1598, 1515, 1461, 1424, 1334,
1219, 1115, 827, 764 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables
1 and 2); and positive-ion HRESIMS m/z 915.2900 [M+Na]+ (calcd
for C42H52O21Na, 915.2893).
N2 gas. From the acid hydrolysates of 1–5, D-glucose and D-apiose
were confirmed by comparing the retention times of their deriva-
tives with those of authentic sugars derivatized in a similar way,
which showed retention times of 19.07 and 12.81 min, respec-
tively. The constituent sugars of compounds 6–9 were identified
using the same method as for 1–5.
Obtusifoside C (3): Yellow powder; mp 133–135 °C; ½a D20
ꢁ57.9
ꢂ
(c 0.05, MeOH); IR mmax 3421,1701, 1598, 1514, 1460, 1425,
1223, 1117, 830 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables 1
and 2); and positive-ion HRESIMS m/z 941.3043 [M+Na]+ (calcd
for C44H54O21Na, 941.3050).
Obtusifoside D (4): White powder; mp 161–163 °C; ½a D20
ꢂ
ꢁ45.5 (c
3.6. Hepatoprotective effects on cytotoxicity induced by
galactosamine in HL-7702 cells
D-
0.05, MeOH). IR mmax 3429, 1718, 1612, 1597, 1518, 1461, 1423,
1379, 1339, 1216, 824, 734 cmꢁ1; NMR (DMSO-d6, 500 MHz) data
(Tables
1
and 2); and positive-ion HRESIMS m/z 915.2897
Compounds 1–17 were tested for hepatoprotective effects using
an MTT assay in HL-7702 cells. Each cell suspension of 1 ꢀ 105 cells
in 1 mL of Dulbecco’s modified Eagle’s medium containing fetal
calf serum (10%), penicillin (100 units/mL) and streptomycin
(100 lg/mL) was placed in a 96-well microplate and precultured
for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium con-
taining bicyclol and the test samples was added, and the cells were
cultured for 1 h. The cultured cells were exposed to 25 mM D-
galactosamine for 24 h. The medium was then changed to fresh
medium containing 0.5 mg/mL MTT. After an incubation of 4 h,
the medium was removed, and DMSO was added to dissolve
formazan crystals. The optical density (OD) of the formazan
[M+Na]+ (calcd for C42H52O21Na, 91 5.2893).
Obtusifoside E (5): Yellow powder; mp 156–158 °C; ½a D20
ꢁ59.3
ꢂ
(c 0.05, MeOH). IR mmax 3410, 1701, 1598, 1514, 1461, 1338,
1223, 1117, 831 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables 1
and 2); and positive-ion HRESIMS m/z 941.3049 [M+Na]+ (calcd
for C44H54O21Na, 941.3050).
Obtusifoside F (6): Yellow powder; mp 174–176 °C; ½a D20
ꢂ
ꢁ27.9
(c 0.05, MeOH); IR mmax 3399,1708, 1614, 1565, 1514, 1461,
1280, 1114, 1069, 827 cmꢁ1, NMR (DMSO-d6, 500 MHz) data (Ta-
bles 3 and 4); and positive-ion HRESIMS m/z 693.2020 [M+H]+
(calcd for C32H37O17, 693.2025).
Obtusifoside G (7): Yellow powder; mp 143–145 °C; ½a D20
ꢂ
ꢁ50.7
solution was measured on a microplate reader at 492 nm.
Inhibition (%) was obtained by the following formula: Inhibition
(%) = [(OD(sample) ꢁ OD(control))/(OD(normal)ꢁOD(control))] ꢀ 100.
(c 0.05, MeOH). IR mmax 3403, 1700, 1597, 1515, 1462, 1424,
1122, 1057, 824 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables 3
and 4); and positive-ion HR-ESIMS m/z 693.2021 [M+H]+ (calcd
for C32H37O17, 693.2025).
3.6.1. Statistical analysis
Obtusifoside H (8): White powder; mp 117–119 °C; ½a D20
ꢂ
ꢁ45.1 (c
Student’s t-test for unpaired observations between normal or
control and tested samples was carried out to identify statistical
differences; p values less than 0.05 were considered significantly
different.
0.05, MeOH). IR mmax 3435, 1698, 1610, 1515, 1462, 1335, 1220,
1112, 764 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables 3 and
4); and positive-ion HRESIMS m/z 723.2844 [M+Na]+ (calcd for
C
33H48O16Na, 723.2835).
Obtusifoside I (9): White powder; mp 102–104 °C; ½a D20
ꢁ45.1 (c
ꢂ
Acknowledgments
0.05, MeOH). IR mmax 3421, 1697, 1610, 1515, 1463, 1335, 1221,
1115, 765 cmꢁ1; NMR (DMSO-d6, 500 MHz) data (Tables 3 and
4); and positive-ion HRESIMS m/z 725.2987 [M+Na]+ (calcd for
The research described in this publication was supported by the
Research Fund for the Doctoral Program of Higher Education of
China (No. 20111106110031) and National Science and Technology
Project of China (No. 2011ZX09307-002-01).
C33H50O16Na, 725.2991).
3.5. Acid hydrolysis of the glycoside and determination of the
absolute configuration of the monosaccharides and aglycone
(1–5)
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2013.
02.006.
Compound 1 (20 mg) was dissolved in 1 M HCl (dioxane–H2O,
1:1, 2 mL) and heated at 60 °C for 2 h in a water bath. The mixture
was concentrated in a vacuum; the resulting residue was sus-
pended with H2O and extracted with EtOAc three times. The agly-
cone (1a, 3.2 mg) was obtained as a solid in the EtOAc layer by
Prep-HPLC. Compounds 2–5 were hydrolyzed using the same
method as described for 1 to give pure aglycones of 2a (2.3 mg),
3a (2.6 mg), 4a (3.3 mg), and 5a (1.6 mg). Compounds 2a and 3a
were determined to be the same as 1a. Compound 4a was deter-
mined to be the same as 5a.
References
1. Hsu, H. Y.; Lin, C. C.; Chen, J. Y.; Yang, J. J.; Zhang, R. J. Ethnopharmacol. 1998, 62,
101–105.
2. Song, W.; Jin, R. L. China J. Chin. Mater. Med. 1997, 22, 359–360.
3. Morikawa, T.; Xu, F. M.; Matsuda, H.; Yoshikawa, M. Chem. Pharm Bull. 2006, 54,
1530–1534.
4. Wang, P.; Yao, T. R.; Chen, Z. N. Acta Bot. Sin. 1989, 31, 616–619.
5. Matsuda, H.; Morikawa, T.; Xu, F. M.; Ninomiya, K.; Yoshikawa, M. Planta Med.
2004, 70, 1201–1209.
6. Yao, T. R.; Chen, Z. N. Acta Pharm. Sin. 1979, 14, 731–734.
7. Ye, H. Z.; Fan, Y. X.; Liu, Z. W.; Jin, Y. S. Chin. Trad. Herbal Drugs 1981, 12, 5–7.
8. Liu, J.; Feng, Z. M.; Xu, J. F.; Wang, Y. H.; Zhang, P. C. Phytochemistry 2007, 68,
1775–1780.
The aqueous layer was evaporated under a vacuum, diluted
repeatedly with H2O, and evaporated in a vacuum to produce a
neutral residue. The residue was dissolved in anhydrous pyridine
(1 mL). L-Cysteine methyl ester hydrochloride (2 mg) was then

54
Z. Liu et al. / Carbohydrate Research 372 (2013) 47–54
9. Song, S.; Li, Y. X.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C. J. Nat. Prod. 2010, 73, 177–
18. Greger, H.; Hofer, O. Tetrahedron 1980, 36, 3551–3558.
19. Xiong, L.; Zhu, C. G.; Li, Y. R.; Tian, Y.; Sheng, L.; Yuan, S. P.; Chen, N. H.; Yang, Y.
C.; Shi, J. G. J. Nat. Prod. 2011, 74, 1188–1200.
20. Mee, J. J.; Kang, S. S.; Yu, J. J.; Choi, J. S. Chem. Pharm. Bull. 2004, 52, 1501–1503.
21. Ito, A.; Kasai, R.; Yamasaki, K.; Duc, N. M.; Nham, N. T. Phytochemistry 1994, 37,
1455–1458.
22. Wang, J. S.; Di, Y. T.; Yang, X. W.; Li, S. L.; Wang, Y. H.; Hao, X. J. Phytochemistry
2006, 67, 486–491.
23. De Marino, S.; Borbone, N.; Zollo, F.; Ianaro, A.; Meglio, P. D.; Iorizzi, M. J. Agric.
Food Chem. 2004, 52, 7525–7531.
184.
10. Peng, W.; Han, T.; Liu, Q. C.; Qin, L. P. Zhongguo Zhongyao Zazhi 2011, 36, 578–
581.
11. Xu, Y.; Wu, C. L.; Liu, Y.; Zhang, Z. F. Zhongcaoyao 2010, 41, 1957–1960.
12. Silva, V. C.; Bolzani, V. S.; Young, M. M.; Lopes, M. N. J. Braz. Chem. Soc. 2007, 18,
1405–1409.
13. Yuan, J. Z.; Li, W.; Koike, K.; Chen, Y. J.; Nikaido, T. Heterocycles 2003, 60, 1633–
1637.
14. Kinjo, J.; Fukui, K.; Higuchi, H.; Nohara, T. Chem. Pharm. Bull. 1991, 39, 1623–
1625.
24. Inada, A.; Nakamura, Y.; Konishi, M.; Murata, H.; Kitamura, F.; Nakanishi, T.
Chem. Pharm. Bull. 1991, 39, 2437–2439.
25. Pabst, A.; Barron, D.; Semon, E.; Schreier, P. Phytochemistry 1992, 31, 1649–
1652.
15. Abe, F.; Yamauchi, K. Phytochemistry 1989, 28, 1737–1741.
16. Kitagawa, I.; Sakagami, M.; Hashiuchi, F.; Zhou, J. L.; Yoshikawa, M.; Ren, J.
Chem. Pharm. Bull. 1989, 37, 551–553.
17. Deyama, T. Chem. Pharm. Bull. 1983, 31, 2993–2997.