Antioxidant phenolic glycosides from the roots of Illicium dunnianum

Article abstract of DOI:10.1016/j.carres.2012.08.017

Eight new phenolic glycosides, dunnianosides A-H (1-8), and nine known phenolic glycosides (9-17), were isolated from the roots of Illicium dunnianum. The structures of these new compounds were elucidated by spectroscopic methods including 1D and 2D NMR,

Full text of DOI:10.1016/j.carres.2012.08.017

Carbohydrate Research 361 (2012) 206–211
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Antioxidant phenolic glycosides from the roots of Illicium dunnianum
⇑
Jian Bai, Zhen-Feng Fang, Hui Chen, Shi-Shan Yu , Dan Zhang, Huai-Ling Wei, Shuang-Gang Ma, Yong Li,
Jing Qu, Song Xu, Jing-Hong Ren, Fen Zhao, Nan Zhao, Jun-Hong Liu
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, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2012
Received in revised form 1 August 2012
Accepted 27 August 2012
Available online 5 September 2012
Eight new phenolic glycosides, dunnianosides A–H (1–8), and nine known phenolic glycosides (9–17),
were isolated from the roots of Illicium dunnianum. The structures of these new compounds were eluci-
dated by spectroscopic methods including 1D and 2D NMR, HRESIMS, and chemical methods. Compounds
2+
1
–5, 7, and 9 exhibited potent antioxidant activities against Fe -cystine-induced rat liver microsomal
lipid peroxidation, with IC50 values ranging from 3.8 ± 0.6 to 23.0 ± 2.2 lM.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords:
Illicium dunnianum
Illiciaceae
Phenolic glycosides
Antioxidant activities
1
. Introduction
503.1524), which was supported by 13C NMR spectroscopic data.
The IR spectrum displayed absorptions ascribable to hydroxy
ꢀ1
ꢀ1
Illicium dunnianum (Illiciaceae) is a toxic shrub distributed in
(3462 cm ), carbonyl (1688 cm ), and aromatic (1614, 1518,
ꢀ1
1
Southern China and used as a folk medicine for relieving pain
and treating rheumatism.1 Studies on this genus showed its rich
content of sesquiterpenes, prenylated C6–C3 compounds, and neo-
lignans with extensive biological activities including cytotoxic,
and 1463 cm ) groups. The H NMR spectroscopic data (Table 1)
showed two aromatic protons attributed to a 1, 2, 4, 5-tetrasubsti-
H
tuted aromatic ring at d 6.54 (1H, s, H-3) and 6.83 (1H, s, H-6), a
singlet assignable to a symmetrical 1, 3, 4, 5-tetrasubstituted aro-
2
–5
00
00
neurotrophic, anti-inflammatory, and antioxidant activities.
In
matic ring at d
nals at d 3.83 (6H, s, 3 , 5 -OCH
(3H, s, 2-CH ) and 1.90 (3H, s, 5-CH
nal at d 4.75 (1H, d, J = 7.5 Hz, H-1 ). Acid hydrolysis of 1 afforded
a glucose which was confirmed by TLC with an authentic sample of
glucose, and the
-configuration was determined by GC analysis.7
The b-anomeric configuration for the glucose was deduced from
H
7.32 (2H, s, H-2 , 6 ), two aromatic methoxy sig-
0
0
00
our previous paper, six allo-cedrane sesquiterpenes, four seco-
prezizaane-type sesquiterpenes, and two monocyclofarnesane ses-
quiterpenes with anti-inflammatory activities were isolated from
H
3
), two methyl signals at d
H
2.12
3
3
0
), and an anomeric proton sig-
H
6
the roots of this species. Further investigation of bioactive constit-
uents from this plant has led to the isolation and structural eluci-
dation of 17 phenolic glycosides including eight new phenolic
glycosides, dunnianosides A–H (1–8), and nine known ones
D
3
13
its large
showed a group of characteristic signals due to a syringoyl group
1 ,2
J coupling constant (7.5 Hz). The C NMR spectrum
0 0
(9–17) (Fig. 1), along with their antioxidant evaluation.
0
0
00
00
00
00
00
at d
1
C
121.3 (C-1 ), 108.2 (C-2 , 6 ), 148.3 (C-3 , 5 ), 141.7 (C-4 ),
66.5 (C-7 ), 56.7 (3 , 5 -OCH
lations from the anomeric proton H-1 (d
carbon C-1 (d
127.1), and C-3 (d
51.1), C-5 (d 122.5), and C-6 (d
C-1 (d 149.9) and C-5 (d
127.1) and C-4 (d
0
0
00
00
3
) (Table 2). A series of HMBC corre-
2
. Results and discussion
0
H
4.75) to an aromatic
149.9), C-2
1.90) to C-4 (d
120.4), from H-3 (d 6.54) to
6.83) to C-2
C
149.9), from 2-CH
3
(d
H
2.12) to C-1 (d
(d
C
The EtOAc fraction of the ethanol extract was subjected to col-
(d
C
C
117.2), from 5-CH
3
H
C
umn chromatography on silica gel, Sephadex LH-20, ODS, and
HPLC, respectively, to afford eight new phenolic glycosides, dunni-
anosides A–H (1–8), together with nine known phenolic glycosides
1
C
C
H
C
C
122.5), and from H-6 (d
H
(d
C
C
151.1) indicated the 4-hydroxy-2, 5-dimeth-
(
9–17).
Compound 1 was isolated as a white amorphous powder. Its
molecular formula was deduced to be C23 11 by a positive
HRESIMS ion at m/z 503.1533 [M+Na] (calcd for C23 11Na:
ylphenyl 1-O-b-
more, the deshielded 6 -methylene proton [d
J = 12.0, 1.5 Hz, H-6 a) and 4.42 (1H, dd, J = 12.0, 7.0 Hz, H-6 b)] of
D
-glucopyranoside moiety in 1 (Fig. 2). Further-
0
4.71 (1H, dd,
28
H O
H
+
0
0
28
H O
the glucose moiety due to the esterification correlated with the
0
0
carbonyl carbon (d
C
166.5, C-7 ) of the syringoyl group, suggesting
⇑
Corresponding author. Tel.: +86 10 63165326; fax: +86 10 63017757.
E-mail address: yushishan@imm.ac.cn (S.-S. Yu).
0
that the O-syringoyl group was attached to C-6 . Therefore, the
0
008-6215/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.08.017

J. Bai et al. / Carbohydrate Research 361 (2012) 206–211
207
R1 3''
R1
3''
O
O
1
''
HO
7''
HO
7''
5
1
''
5
O
O
5''
OH
5''
R2
OH
R2
5'
O
5'
O
1
1
HO
HO
H OH O 3'
1'
3
1'
3
O
O
OCH3
3
'
OH
OH
1
2
3
R1 = OCH3 R2 = OCH3
R1 = OCH3 R2 = H
4 R = H
R2 = H
1
9 R = OCH3 R2 = H
1
R1 = H
R2 = H
10 R = OCH3 R2 = OCH3
1
R1
R1
3
''
3''
O
O
1
''
1''
HO
7''
HO
7''
OCH3
5
5
O
O
5
''
OH
5''
OCH3
R2
5'
O
R2
5'
O
1
1
HO
HO
1'
HO
HO
3
1'
3
O
O
R3
3
'
OH
R2 = H
3'
OH
OCH3
6
R1 = H
R2 = H R3 = OH
5
R1 = H
1
1
1
3 R = OCH3 R2 = H R3 = OH
1
1 R = OCH3 R2 = H
1
1
1
1
1
4 R = OCH3
7 R = OCH3
R
2
= OCH3
3
R = OH
1
2 R = OCH3 R2 = OCH3
2
R
= OCH3
R = OCH3
3
H CO
3
3''
O
7''
O
1
''
HO
5
OH
5
R
OH
R25''
5
'
O
5'
1
O
1
HO
HO
HO
HO
1'
3
3
1
'
O
O
3
OH
3
'
OH
'
R1
8
R = OCH3
7
R1 = OH R2 = OCH3
1
5 R = H
1
6 R = H R2 = H
1
Figure 1. The structures of compounds 1–17.
Table 1
1
a
H
N
M
R
s
p
e
c
t
r
o
s
c
o
p
i
c
d
a
t
a
f
o
r
c
o
m
p
o
u
n
d
s
1
–
8
(
5
0
0
M
H
z
,
J
i
n
H
z
)
Position
1
2
3
4
5
6
7
8
2
3
5
6
6.68 br s
6.37 d (2.0)
6.54 s
6.57 s
6.58 s
6.47 d (2.5)
6.34 dd (8.5, 2.5)
7.06 d (8.5)
4.73 d (7.5)
3.48 overlap
3.54 overlap
3.47 overlap
3.72 m
4.71 dd (12.0,
2.0)
4.28 dd (12.0,
7.5)
6.28 d (3.0)
5.98 dd (9.0, 3.0)
6.92 d (9.0)
4.60 d (8.0)
3.46 overlap
3.48 overlap
3.41 dd (9.0, 9.0) 3.44 overlap
3.72 m
4.73 dd (12.0,
2.0)
4.41 dd (12.0,
7.5)
6.69 s
6.66 d (8.5)
6.61 d (8.5)
4.87 d (7.5)
3.48 overlap
3.55 overlap
3.47 overlap
3.82 m
6.83 s
6.87 s
6.89 s
6.23 d (2.0)
4.95 d (7.5)
3.48 overlap
3.58 overlap
3.50 overlap
3.87 m
6.96 s
0
1
4.75 d (7.5)
3.49 overlap
3.53 overlap
3.48 overlap
3.76 m
4.76 d (7.5)
3.49 overlap
3.53 overlap
3.47 overlap
3.76 m
4.77 d (7.5)
3.50 overlap
3.54 overlap
3.47 overlap
3.76 m
4.70 dd (12.0,
2.0)
4.33 dd (12.0,
7.0)
4.75 d (7.5)
3.46 overlap
3.47 overlap
0
2
0
3
0
4
0
5
3.41 overlap
3.86 d (12.0)
0
6
4.71 dd (12.0,
4.70 d (12.0)
4.71 d (11.5)
4.73 d (12.0)
1
4
7
.5)
.42 dd (12.0,
.0)
4.33 dd (12.0,
7.0)
4.34 dd (11.5,
7.0)
4.29 dd (12.0,
7.0)
3.69 dd (12.0,
8.5)
0
0
0
0
0
0
0
0
2
3
5
6
2
5
2
3
4
5
3
5
7.32 s
7.55 s
7.91 d (8.5)
6.93 d (8.5)
6.93 d (8.5)
7.91 d (8.5)
2.13 s
7.92 d (8.5)
6.95 d (8.5)
6.95 d (8.5)
7.92 d (8.5)
7.91 d (9.0)
7.02 d (9.0)
7.02 d (9.0)
7.91 d (9.0)
7.94 d (8.5)
6.94 d (8.5)
6.94 d (8.5)
7.94 d (8.5)
7.37 s
6.91 d (8.0)
7.59 d (8.0)
2.13 s
7.32 s
2.12 s
1.90 s
7.37 s
-Me
-Me
2.22 s
2.10 s
1.98 s
2.02 s
-OMe
-OMe
-OMe
-OMe
3.77 s
3.73 s
3.70 s
3.73 s
3.76 s
0
0
0
0
-OMe 3.83 s
-OMe 3.83 s
3.86 s
3.88 s
3.88 s
a
6 4
Measured in acetone-d for compounds 1–6 and 8, and in methanol-d for compound 7.
structure of 1 was elucidated as 4-hydroxy-2, 5-dimethylphenyl 1-
the syringoyl group in the NMR spectra of 1. This was confirmed
by HSQC and HMBC experiments of 2. In the HMBC spectrum,
0
O-b-
A (1).
Compound 2 was assigned the molecular formula C22
D
-(6 -O-syringoyl) glucopyranoside and named dunnianoside
0
0
00
long-range correlations of H-2 (d
H
7.55) with C-1 (d
C
122.7), C-
0
0
00
00
26
H O
10 by
3 (d
bon C-7 (d
C
148.2), C-4 (d
C
152.2), C-6 (d
C
124.7), and the carbonyl car-
+
00
00
00
00
positive HRESIMS (m/z 473.1427 [M+Na] , calcd 473.1418). The
UV, IR, and NMR spectra of 2 resembled those of 1. However, the
C
166.5), of H-5 (d
H
6.91) with C-1 (d
C
122.7), C-3 (d
7.59) with C-2 (d
C
0
0
00
00
148.2), and C-4 (d
C
152.2), of H-6 (d
H
C
113.4),
1
13
00
00
0
H and C NMR spectra of 2 (Tables 1 and 2) exhibited a group
of characteristic signals assignable to a vanilloyl group [d 7.55
1H, s, H-2 ), 6.91 (1H, d, J = 8.0 Hz, H-5 ), 7.59 (1H, d, J = 8.0 Hz,
C-4 (d
C
152.2), and C-7 (d
C
166.5), and of H
2
-6 (d
H
4.70, 4.33)
0
0
H
with C-7 (d
C
166.5) indicated the presence of the O-vanilloyl group
0
0
00
0
(
linked at C-6 . Thus, 2 was determined to be 4-hydroxy-2, 5-
0
0
00
00
00
00
0
H-6 ); d
C
122.7 (C-1 ), 113.4 (C-2 ), 148.2 (C-3 ), 152.2 (C-4 ),
dimethylphenyl 1-O-b-
D-(6 -O-vanilloyl)glucopyranoside and des-
0
0
00
00
1
15.6 (C-5 ), 124.7 (C-6 ), 166.5 (C-7 )], replacing signals due to
ignated as dunnianoside B (2).

2
08
J. Bai et al. / Carbohydrate Research 361 (2012) 206–211
Table 2
Compound 5 possessed the same molecular formula as 4,
20 22 10
C H O , deduced from positive HRESIMS (m/z 445.1111
1
3C NMR spectroscopic data for compounds 1–8 (125 MHz)a
+
Position
1
2
3
4
5
6
7
8
[M+Na] , calcd 445.1105). The IR, UV, and NMR spectroscopic data
of 5 closely resembled those of 4, except that a 1, 2, 4-trisubsituted
aromatic ring moiety instead of a 1, 3, 4-trisubsituted one was lo-
1
2
3
4
5
6
149.9 150.0 149.9 152.2 140.6 155.3 140.0 150.5
127.1 127.0 127.0 103.4 152.0 97.6 149.7 126.6
117.2 117.3 117.3 148.5 101.6 151.5 104.4 113.3
151.1 151.2 151.1 142.7 154.9 132.5 155.4 153.8
122.5 122.6 122.5 115.4 107.0 154.3 107.0 124.7
0
cated at the C-1 position in 5. This was confirmed by HMBC corre-
lations from H-3 (d
42.7), and C-5 (d 115.4), from H-5 (d
(d 101.6), and C-4 (d 142.7), from H-6 (d
C-2 (d 152.0), and C-4 (d
to C-2 (d
H
6.47) to C-1 (d
C
140.6), C-2 (d
6.34) to C-1 (d
7.06) to C-1 (d
C
152.0), C-4 (d
C
140.6), C-3
C
1
C
H
120.4 120.2 120.2 109.4 120.7 94.5
104.1 104.0 104.0 103.0 104.0 102.1 105.8 103.8
120.9 120.1
0
0
0
0
0
0
0
0
0
0
0
0
0
140.6),
1
2
3
4
5
6
1
2
3
4
5
6
7
2
5
2
3
4
5
3
5
C
C
H
C
74.7
77.8
71.6
75.1
65.0
74.8
78.0
71.7
75.0
65.0
74.7
77.8
71.6
75.0
64.9
74.7
77.8
71.5
75.1
64.7
74.7
77.7
71.6
75.2
64.6
74.5
77.7
71.3
75.1
64.9
74.9
77.5
72.0
75.9
65.2
74.9
78.1
71.5
77.6
62.8
C
C
142.7), from a methoxy proton (d
H
3.77)
0
C
152.0), and from H-1 (d
H
4.73) to C-1 (d
C
140.6) (Fig. 3).
Accordingly, the structure of 5 was determined to be 4-hydroxy-2-
0
methoxylphenyl 1-O-b-
D
-[6 -O-(p-hydroxybenzoyl)]glucopyrano-
0
0
0
0
0
0
0
side and named dunnianoside E (5).
121.3 122.7 122.4 122.4 122.2 122.3 121.3
108.2 113.4 132.5 132.5 132.5 132.6 108.4
148.3 148.2 116.0 116.0 116.1 116.1 149.0
141.7 152.2 162.6 162.8 163.0 162.8 142.1
148.3 115.6 116.0 116.0 116.1 116.1 149.0
108.2 124.7 132.5 132.5 132.5 132.6 108.4
166.5 166.5 166.5 166.4 166.4 166.6 167.8
Compound 6 was assigned the molecular formula C21
the basis of its positive HRESIMS (m/z 475.1233 [M+Na] , calcd
H
24
+
O
11 on
4
75.1211). Comparison of the NMR spectroscopic data of 6 with
0
those of 5 indicated that they possessed the same 6 -O-(p-hydrox-
ybenzoyl)-b-
spectrum showed two aromatic protons attributed to a 1, 3, 4, 5-
tetrasubstituted aromatic ring at d 6.37 (1H, d, J = 2.0 Hz, H-2)
and 6.23 (1H, d, J = 2.0 Hz, H-6), and two aromatic methoxy signals
D
-glucopyranosyl moiety. In addition, the 1H NMR
-Me
-Me
16.2
15.9
16.0
16.2
16.2
16.1
16.2
16.4
H
-OMe
-OMe
-OMe
-OMe
56.3
56.2
60.9
56.1
56.0
at d
H
3.70 (3H, s, 4-OCH
3
) and 3.73 (3H, s, 5-OCH
3
) (Table 1). HMBC
155.3), from H-2 (d
151.5), C-4 (d 132.5), and C-6 (d
155.3), C-2 (d 97.6), C-4 (d
0
correlations from H-1 (d
H
4.95) to C-1 (d
C
H
0
0
0
0
-OMe
-OMe
56.7
56.7
56.3
56.9
56.9
6
1
.37) to C-1 (d
94.5), from H-6 (d
C
155.3), C-3 (d
C
C
C
H
6.23) to C-1 (d
C
C
C
a
Measured in acetone-d
compound 7.
6
for compounds 1–6 and 8, and in methanol-d
4
for
132.5), and C-5 (d
H
C-4 (d 132.5), and from another methoxy proton (d 3.73) to C-5
154.3), from one methoxy proton (d
3.70) to
C
H
C
(
d
C
154.3) indicated that the aglycone of 6 was 3-hydroxy-4, 5-
0
dimethoxyphenoxy unit and 6 -O-(p-hydroxybenzoyl)-b-
D-gluco-
H CO
3
pyranosyl moiety was located at C-1. Therefore, 6 was elucidated
3
''
O
0
as 3-hydroxy-4, 5-dimethoxyphenyl 1-O-b-
D-[6 -O-(p-hydrox-
7
''
HO
1''
5
ybenzoyl)] glucopyranoside and named dunnianoside F (6).
O
5
''
OH
The molecular formula of compound 7 was deduced to be
H CO
5'
O
3
+
HO
HO
3
C H
21 24
O
12 by positive HRESIMS (m/z 491.1165 [M+Na] , calcd
1
O
OH 1'
3
'
491.1160). The NMR spectroscopic data (Tables 1 and 2) of 7 were
closely related to those of 1 except for the signals attributed to the
Figure 2. Key HMBC correlations (H?C) in compound 1.
1
13
aglycone of 7. Furthermore, the H and C NMR spectra showed a
set of ABX coupling assignable to a 1, 2, 4-trisubstituted aromatic
The molecular formula of compound 3 was determined as
ring at d
H-5), and 6.92 (1H, d, J = 9.0 Hz, H-6), and six aromatic carbon sig-
nals at d 140.0 (C-1), 149.7 (C-2), 104.4 (C-3), 155.4 (C-4), 107.0
H
6.28 (1H, d, J = 3.0 Hz, H-3), 5.98 (1H, dd, J = 9.0, 3.0 Hz,
+
C
4
2
(
1
21 24 9
H O by positive HRESIMS (m/z 443.1334 [M+Na] , calcd
1
13
43.1313). The H and C NMR spectroscopic data (Tables 1 and
) indicated the presence of p-hydroxybenzoyl group [d 7.91
C
H
00
(C-5), and 120.9 (C-6), indicating the presence of a 2,4-dihydroxy-
phenoxy moiety. This was confirmed by HMBC correlations of H-3
with C-1, C-2, C-4, and C-5, of H-5 with C-1 and C-3, and of H-6
with C-1, C-2, and C-4. Moreover, HMBC correlation between the
00
00
00
2H, d, J = 8.5 Hz, H-2 , 6 ), 6.93 (2H, d, J = 8.5 Hz, H-3 , 5 ); d
C
00
00
00
00
00
00
22.4 (C-1 ), 132.5 (C-2 , 6 ), 116.0 (C-3 , 5 ), 162.6 (C-4 ), 166.5
C-7 )]. Comparison of the NMR spectroscopic data of 3 with those
0
0
(
0
0
of 1 indicated that the structure of 3 was very close to that of 1, ex-
cept that the syringoyl group in 1 was substituted by the p-hydrox-
ybenzoyl group in 3. Moreover, the 2D NMR experiments resulted
in the assignments of all NMR signals and the linkage of the struc-
anomeric proton H-1 (d
H
4.60) and C-1 suggested that 6 -O-syring-
oyl-b- -glucopyranosyl moiety was attached to C-1. Thus, the
D
structure of 7 was established as 2, 4-dihydroxyphenyl 1-O-b-
(6 -O-syringoyl)glucopyranoside and named dunnianoside G (7).
D-
0
tural units of 3. Consequently, 3 was established as 4-hydroxy-2,
Compound 8 was found to possess the molecular formula
15 22 7
C H O according to a positive HRESIMS ion at m/z 337.1260
0
5
-dimethylphenyl 1-O-b-
D-[6 -O-(p-hydroxybenzoyl)] glucopyran-
+
oside and named dunnianoside C (3).
[M+Na] (calcd 337.1258). The IR spectrum showed the presence
ꢀ
1
ꢀ1
Compound 4 was assigned the molecular formula C20
according to a positive HRESIMS ion at m/z 445.1112 [M+Na]
H
22
O
10
+
of hydroxy (3423 cm ) and aromatic (1518 and 1460 cm )
1
groups. The H NMR spectrum showed the signals for a 1,2,4,5-tet-
rasubstituted aromatic ring at d 6.69 (1H, s, H-3) and 6.96 (1H, s,
3.76 (3H, s, 4-OCH ), two methyls
(
calcd 445.1105). Comparison of the NMR spectroscopic data be-
H
0
tween 3 and 4 indicated that they shared the same 6 -O-(p-hydrox-
H-6), an aromatic methoxy at d
H
3
ybenzoyl)-b-
D
-glucopyranosyl moiety. The remaining seven carbon
0
3''
resonances were identical to those of the aglycone of 6 -O-vanil-
loyltachioside (9), suggesting that the aglycone of 4 was 4-hydro-
xy-3-methoxylphenoxy moiety. HMBC correlation between the
O
8
HO
7''
O
5
1''
OH
5
''
0
anomeric proton H-1 (d
H
4.87) and C-1 (d
C
152.2) indicated that
5'
O
HO
HO
0
1'
3
the 6 -O-(p-hydroxybenzoyl)-b-
D-glucopyranosyl moiety was at-
O
1
3'
OH
tached to C-1. Therefore, 4 was elucidated as 4-hydroxy-3-meth-
OCH3
0
oxylphenyl 1-O-b-
D
-[6 -O-(p-hydroxybenzoyl)] glucopyranoside
and named dunnianoside D (4).
Figure 3. Key HMBC correlations (H?C) in compound 5.

J. Bai et al. / Carbohydrate Research 361 (2012) 206–211
209
Table 3
spectrophotometer. IR spectra were recorded on a Nicolet 5700
FT-IR spectrometer. NMR spectra were recorded on an INOVA-500
spectrometer at 25 °C. ESIMS was measured on an Agilent 1100 Ser-
ies LC/MSD ion trap mass spectrometer. HRESIMS data were re-
corded on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/
MS spectrometer. Preparative HPLC was performed on a Shimadzu
LC-6AD instrument with an SPD-10A detector, using a YMC-Pack
a
Antioxidant activities of compounds 1–5, 7, and 9
Compound
IC50 (lM)
1
2
3
4
5
7
9
20.6 ± 2.0
6.9 ± 0.8
3.8 ± 0.6
5.4 ± 0.8
8.4 ± 1.3
15.2 ± 1.2
23.0 ± 2.2
23.4 ± 2.5
ODS-A column (250 ꢁ 20 mm, 5
an Agilent 7890A instrument with an FID detector. Sephadex LH-
20 (Amersham Pharmacia Biotech AB, Sweden), ODS (45–70 m,
lm). GC data were recorded on
Vitamin Eb
l
a
Merck), silica gel (160–200 mesh, Qingdao Marine Chemical Co.,
Ltd, China), and diatomite (Sinopharm Chemical Reagent Co., Ltd,
China) were used for column chromatography (CC). TLC was carried
out with glass precoated with silica gel GF254 plates (Qingdao Mar-
ine Chemical Co., Ltd, China). Spots were visualized under UV light
or by spraying with 10% H SO in EtOH–H O (95:5, v/v) followed by
Data are mean ± standard deviation (n = 3). The
other compounds were inactive (IC50 >50
lM).
b
Positive control.
at d
proton at d
H
2.22 (3H, s, 2-CH
3
) and 2.10 (3H, s, 5-CH
3
), and a anomeric
0
13
H
4.75 (1H, d, J = 7.5 Hz, H-1 ). The C NMR spectrum
2
4
2
exhibited signals for six aromatic carbons, one methoxy group
56.0), two methyl groups (d 16.2 and 16.4), and a glycosyl moi-
ety (d 103.8, 78.1, 77.6, 74.9, 71.5, and 62.8). Acid hydrolysis of 8
afforded a d-glucose which was compared with an authentic sam-
ple of -glucose, and the -configuration was confirmed by GC
heating. Solvents [petroleum ether (60–90 °C), CHCl , EtOAc,
3
(d
C
C
MeOH, CH Cl , and EtOH] were of analytical grade and purchased
2
2
C
from Beijing Chemical Company, Beijing, China.
D
D
3.2. Plant material
7
analysis. The large coupling constant (7.5 Hz) of the anomeric
0
proton at d
H
4.75 (H-1 ) indicated that the glucose was in the b-
The roots of I. dunnianum were collected in Guangxi Province,
China, in November 2009, and identified by Professor Song-Ji Wei
of Guangxi College of Traditional Chinese Medicine. A voucher
specimen (No. ID-S-2328) was deposited in the herbarium of the
Department of Medicinal plants, Institute of Materia Medica, Chi-
nese Academy of Medical Sciences.
configuration. The NMR spectroscopic data (Tables 1 and 2) of 8
were very similar to those of 4-methoxy-2, 5-dimethylphenyl
-arabinofuranosyl-(1?6)-b-
absence of signals due to -arabinofuranosyl moiety in 8. This
a-
L
D
-glucopyranoside,9 except for the
a-L
was confirmed by the analysis of HSQC and HMBC experiments.
Consequently, 8 was elucidated as 4-methoxy-2,5-dimethylphenyl
1
-O-b-
D-glucopyranoside and named dunnianoside H (8).
3.3. Extraction and isolation
In addition, the absolute configuration of the glucoses in com-
pounds 2–7 was also assigned as
D
by GC analysis.7 The known
The roots of I. dunnianum (7.5 kg) was air-dried, ground, and ex-
0
8
0
compounds were identified as 6 -O-vanilloyltachioside (9), 4 -hy-
tracted three times (2 h for each time) with EtOH–H O (3 ꢁ 80 L,
2
0
00
00 00
droxy-3 -methoxyphenol-b-
D
-[6-O-(4 -hydroxy-3 ,5 -dimethoxybenz
95:5, v/v) under conditions of reflux (90–95 °C), filtered, and the
oate)]glucopyranoside (10),10 6 -O-vanilloylisotachioside (11), 4-hy
0
8
residue was refluxed with EtOH–H O (2 ꢁ 64 L, 70:30, v/v). The
2
11
droxy-2-methoxyphenyl-6-O-syringyl-b-
D
-glucopyranoside (12), 1-
-glucopyra
noside (13), 1-O-3,4-dimethoxy-5-hydroxyphenyl-(6-O-3,5-dime
combined EtOH extract was evaporated to near dryness under re-
O-3,4-dimethoxy-5-hydroxyphenyl-(6-O-vanilloyl)-b-
D
duced pressure to give the crude extract (850 g), which was ab-
sorbed by diatomite, and then successively extracted with
petroleum ether (60–90 °C) (10 L), CHCl3 (10 L), EtOAc (10 L), and
MeOH (10 L). The EtOAc extract (110 g) was subjected to a silica
1
2
12
thoxygalloyl)-b-
O-b- -glucopyranoside (15),
oldhamioside (17),
D-glucopyranoside (14), 2,5-dimethylphenyl 1-
13
0
14
D
6 -O-vanilloylarbutin (16),
and
1
5
respectively, by the comparison of their
gel CC (50 ꢁ 8 cm, 160–200 mesh) eluted with CHCl –MeOH
3
NMR spectroscopic data with those reported in literatures.
(50:1, 20:1, 10:1, 5:1, 1:1, 0:1, v/v) to afford nine fractions E –E .
1
9
Compounds 1–16 were tested for their antioxidant activities
Fraction E (4.5 g) was applied to an ODS CC eluted with a gradient
4
2
+
against rat liver microsomal lipid peroxidation induced by Fe
-
of MeOH–H O (10:90 ? 100:0) to yield fractions E4-1–E4-9. Fraction
2
cystine in vitro. Vitamin E was used as the positive control. As
shown in Table 3, compounds 1–5, 7, and 9 exhibited more signif-
icant antioxidant activities than the positive control Vitamin E
E4-4 (140 mg) was purified by preparative HPLC using CH CN–H O
3
2
(20:80, 7 mL/min) to afford 17 (9.0 mg, Rt 16.1 min). Fraction E4-6
(250 mg) was submitted to a Sephadex LH-20 CC eluted with
CH Cl –MeOH (50:50) to afford fractions E4-6-1–E4-6-5. Fraction
(
IC50 23.4 ± 2.5
3.0 ± 2.2 M, while the other compounds were inactive (IC50
50 M). In compounds 1–3, the antioxidant activity increased
lM), with IC50 values ranging from 3.8 ± 0.6 to
2
2
2
l
E4-6-2 (49 mg) was separated by preparative HPLC using CH CN–
3
>
l
2
H O (20:80, 7 mL/min) to yield 8 (9.0 mg, Rt 40.3 min) and 15
with the decreasing number of methoxy group substituted in the
C-6 benzoxy group. The same activity tendency was shown in
(5.0 mg, Rt 42.6 min). Fraction E4-6-3 (80 mg) was purified by pre-
0
parative HPLC using MeOH–H O (39:61, 7 mL/min) to afford 9
2
compounds 4, 9, and 10. These results suggested that the variation
of the substituent units in the C-6 benzoxy group and aglycone
(17.0 mg, Rt 35.2 min) and 11 (7.2 mg, Rt 38.7 min). Fraction E4-6-4
0
(30 mg) was separated by preparative HPLC using MeOH–H O
2
significantly influenced the activity and that the methoxy group
substituted in the C-6 benzoxy group could cause a considerable
(41:59, 7 mL/min) to yield 10 (3.6 mg, Rt 33.5 min) and 12
(12.5 mg, Rt 36.1 min). Fraction E4-7 (320 mg) was separated by
0
decrease in activity. In conclusion, the active phenolic glycosides
showed potential as candidates of antioxidant agents useful for
folk medicine application in treating rheumatism.
preparative HPLC using CH CN–H O (15:85, 8 mL/min) to afford
14 (45.0 mg, Rt 28.0 min), 13 (20.0 mg, Rt 31.1 min), and 1
(45.0 mg, Rt 33.2 min). Fraction E4-8 (170 mg) was submitted to a
3
2
Sephadex LH-20 CC eluted with CH
fractions E4-8-1–E4-8-4. Fraction E4-8-4 (67 mg) was separated by
preparative HPLC using CH CN–H O (24:76, 7 mL/min) to yield 2
8.5 mg, Rt 23.4 min).
Fraction E (8.4 g) was subjected to an ODS CC eluted with a
gradient of MeOH–H O (10:90 ? 100:0) to give eighteen fractions
5-1–E5-18. Fraction E5-10 (3.1 g) was subjected to a Sephadex LH-20
2 2
Cl –MeOH (50:50) to afford
3
3
. Experimental
3
2
.1. General experimental procedures
Optical rotations were taken on a JASCO P-2000 automatic
(
5
2
digital polarimeter. UV spectra were measured on a JASCO V650
E

2
10
J. Bai et al. / Carbohydrate Research 361 (2012) 206–211
CC eluted with a gradient of MeOH–H
fractions E5-10-1–E5-10-20. Fraction E5-10-10 (50 mg) was separated by
preparative HPLC using 0.1% TFA in CH CN–H O (21:79, 7 mL/min)
to yield 7 (2.7 mg, Rt 26.3 min), 16 (4.3 mg, Rt 28.6 min), 6 (6.3 mg,
Rt 41.9 min), and a mixture E5-10-10-1 (17.1 mg, Rt 31.0 min). The
mixture E5-10-10-1 (17.1 mg) was purified by preparative HPLC
2
O (50:50 ? 100:0) to afford
NMR (500 MHz, CD
3
OD) and 13C NMR (125 MHz, CD
3
OD) data,
+
+
see Tables 1 and 2; ESI-MS m/z 469 [M+H] , 491 [M+Na] , 507
+
3
2
[M+K] , 467 [MꢀH], 503 [M+Cl]; HR ESI-MS m/z 491.1165
+
[M+Na] (calcd for C21
H
24
O
12Na, 491.1160).
2
D
0
Dunnianoside H (8): White amorphous powder; ½
0.16, MeOH); UV (MeOH) kmax (log ) 203 (4.17), 220 (3.73), 285
(3.25) nm; IR (KBr) max 3424, 3325, 3254, 2940, 2881, 1514,
1460, 1380, 1211, 1082, 1044, 996, 898, 854 cm
aꢂ
ꢀ10.4 (c
e
2
using 0.1% TFA in MeOH–H O (42:58, 7 mL/min) to give 4
m
ꢀ1
1
(
(
(
7.9 mg, Rt 31.1 min) and 5 (4.0 mg, Rt 34.5 min). Fraction E5-10-15
;
H NMR
1
3
30 mg) was separated by preparative HPLC using CH
3
CN–H
2
O
(500 MHz, acetone-d
see Tables 1 and 2; ESI-MS m/z 337 [M+Na] , 353 [M+K] , 651
6
) and C NMR (125 MHz, acetone-d ) data,
6
+
+
28:72, 7 mL/min) to yield 3 (5.0 mg, Rt 14.0 min).
+
[
2M+Na] , 313 [MꢀH], 627 [2MꢀH]; HR ESI-MS m/z 337.1260
+
3
.4. Identification
22 7
[M+Na] (calcd for C15H O Na, 337.1258).
2
D
0
Dunnianoside A (1): White amorphous powder; ½
aꢂ
ꢀ42.3 (c
3.5. Determination of the absolute configuration of the sugar
moieties
0
.04, MeOH); UV (MeOH) kmax (loge) 202 (4.59), 218 (4.41), 280
(
1
4.03) nm; IR (KBr)
463, 1425, 1337, 1212, 1191, 1117, 1071, 867, 764 cm ; H
m
max 3462, 3333, 2926, 1688, 1614, 1518,
ꢀ1
1
According to the reported method,7 each (2 mg) of the com-
NMR (500 MHz, acetone-d
data, see Tables 1 and 2; ESI-MS m/z 503 [M+Na] , 519 [M+K] ,
6
) and 13C NMR (125 MHz, acetone-d
6
)
pounds 1–8 was hydrolyzed by 2 M HCl–H
After removal of HCl by evaporation and extraction with EtOAc,
the H O extract was evaporated and dried in vacuo to give the
monosaccharide residue. From the residue, glucose was detected
by TLC [CH Cl : MeOH (5:1), Rf 0.43] with authentic sample. The
residue was dissolved in pyridine (1 mL) containing -cysteine
methyl ester hydrochloride (2 mg) and heated at 60 °C for 2 h, then
evaporated under N stream and dried in vacuo. The residue was
2
O at 95 °C for 10 h.
+
+
+
4
79 [M–H], 515 [M+Cl]; HR ESI-MS m/z 503.1533 [M+Na] (calcd
for C23 11Na, 503.1524).
Dunnianoside B (2): White amorphous powder; ½
.05, MeOH); UV (MeOH) kmax (log ) 202 (4.62), 219 (4.38), 264
4.03), 290 (3.89) nm; IR (KBr) max 3382, 3268, 2974, 2926,
893, 1716, 1688, 1601, 1522, 1467, 1410, 1383, 1285, 1202,
2
28
H O
2
D
0
aꢂ
–49.5 (c
2
2
0
(
2
1
e
L
m
2
ꢀ1
1
085, 1067, 882, 872, 762 cm
;
H NMR (500 MHz, acetone-d
6
)
dissolved in 0.2 mL of N-trimethylsilylimidazole and heated at
60 °C for 2 h. The reaction mixture was partitioned between n-hex-
1
3
and C NMR (125 MHz, acetone-d
6
) data, see Tables 1 and 2;
+
+
ESI-MS m/z 473 [M+Na] , 489 [M+K] , 449 [MꢀH], 485 [M+Cl];
ane and H
by GC (Agilent 7890A) under the following conditions: capillary
column HP-5 (30 m ꢁ 0.32 mm ꢁ 0.25 m); detector FID; carrier
gas N , flow rate 1 mL/min; detector temperature 280 °C; injection
2
O (2 mL each), and the n-hexane extract was analyzed
+
HR ESI-MS m/z 473.1427 [M+Na] (calcd for
C
H
22 26
O
10Na,
4
73.1418).
l
2
D
0
Dunnianoside C (3): White amorphous powder; ½
aꢂ
ꢀ35.0 (c
2
0
.14, MeOH); UV (MeOH) kmax (log
e
) 203 (4.41), 258 (4.00) nm;
temperature 250 °C; oven temperature gradient: 100 °C for 2 min,
100 °C ? 280 °C (10 °C/min), 280 °C for 5 min. The same procedure
was applied to authentic sample. By comparison with retention
IR (KBr)
1
(
mmax 3398, 2917, 1703, 1675, 1611, 1514, 1440, 1354,
292, 1207, 1072, 969, 876, 847, 770, 616 cm
500 MHz, acetone-d
ꢀ1
1
;
H
NMR
) and 13C NMR (125 MHz, acetone-d
) data,
time of authentic sample (tR-
20.433 min), -glucose (t
19.845 ꢃ19.854 min) was identified in
the acid hydrolysate of 1–8.
-glucose 19.851 min,
t
R- -glucose
L
6
6
D
+
+
see Tables 1 and 2; ESI-MS m/z 443 [M + Na] , 459 [M + K] , 419
D
R
+
[
M – H], 455 [M + Cl]; HR ESI-MS m/z 443.1334 [M + Na] (calcd
for C21 Na, 443.1313).
Dunnianoside D (4): White amorphous powder; ½
.1, MeOH); UV (MeOH) kmax (log ) 203 (4.42), 258 (4.10) nm;
IR (KBr) max 3402, 2923, 1693, 1610, 1516, 1451, 1281, 1200,
24 9
H O
20
a
ꢂ
–37.3 (c
D
3
.6. Antioxidant activity assays
0
e
m
ꢀ
1
1
The antioxidant activities of compounds 1–16 were assessed by
measuring the inhibitory ratios of malondialdehyde (MDA) in rat
1
(
170, 1075, 969, 848, 802, 770, 698, 614 cm
500 MHz, acetone-d
;
H
NMR
) and 1 C NMR (125 MHz, acetone-d
3
6
6
) data,
2
+
+
+
liver microsomal lipid peroxidation induced by Fe -cystine as de-
see Tables 1 and 2; ESI-MS m/z 423 [M+H] , 445 [M+Na] , 461
1
6
+
scribed previously.
[
[
M+K] , 421 [MꢀH], 457 [M+Cl]; HR ESI-MS m/z 445.1112
+
M+Na] (calcd for C20
H
22
O
10Na, 445.1105).
Dunnianoside E (5): White amorphous powder; ½
.12, MeOH); UV (MeOH) kmax (log ) 204 (4.17), 258 (3.87) nm;
max 3351, 2941, 1679, 1612, 1519, 1456, 1336, 1291,
2
D
0
Acknowledgments
a
ꢂ
ꢀ25.5 (c
0
e
This project was supported by the Natural Science Foundation
of China (No. 201072234, No. 21132009) and the National Science
and Technology Project of China (No. 2012ZX09301002-002). We
are grateful to the Department of Instrumental Analysis, Institute
of Materia Medica, Chinese Academy of Medical Sciences and Pek-
ing Union Medical College for measuring the IR, UV, NMR, and MS
spectra.
IR (KBr)
1
m
ꢀ1
1
205, 1168, 1087, 977, 954, 849, 835, 802, 773, 616 cm ; H
) and 1 C NMR (125 MHz, acetone-d
3
NMR (500 MHz, acetone-d
data, see Tables 1 and 2; ESI-MS m/z 423 [M+H] , 445 [M+Na] ,
4
6
6
)
+
+
+
61 [M+K] , 421 [MꢀH], 457 [M+Cl]; HR ESI-MS m/z 445.1111
+
[
M+Na] (calcd for C20
H
22
O
10Na, 445.1105).
Dunnianoside F (6): White amorphous powder; ½
.27, MeOH); UV (MeOH) kmax (log ) 206 (4.88), 258 (4.33) nm;
max 3392, 2943, 1686, 1608, 1509, 1438, 1318, 1281,
2
D
0
aꢂ
ꢀ33.5 (c
0
e
References
IR (KBr)
1
tone-d
m
ꢀ1
1
204, 1169, 1105, 1073, 851, 773 cm
;
H NMR (500 MHz, ace-
1. Zhang, J. Zhongguo Zhongyao Zazhi 1989, 14, 36–37.
2. Liu, Y. N.; Su, X. H.; Huo, C. H.; Zhang, X. P.; Shi, Q. W.; Gu, Y. C. Chem.
Biodiversity 2009, 6, 963–989.
3. Ma, S. G.; Tang, W. Z.; Liu, Y. X.; Hu, Y. C.; Yu, S. S.; Zhang, Y.; Chen, X. G.; Qu, J.;
Ren, J. H.; Liu, Y. B.; Xu, S.; Liu, J.; Liu, Y. Y.; Li, Y.; Lue, H. N.; Wu, X. F.
Phytochemistry 2011, 72, 115–125.
1
3
6
) and C NMR (125 MHz, acetone-d ) data, see Tables 1
6
+
+
+
and 2; ESI-MS m/z 453 [M+H] , 475 [M+Na] , 491 [M+K] , 451
+
[
MꢀH], 487 [M+Cl]; HR ESI-MS m/z 475.1233 [M+Na] (calcd for
11Na, 475.1211).
Dunnianoside G (7): White amorphous powder; ½
.049, MeOH); UV (MeOH) kmax (log ) 206 (4.17), 219 (4.10), 279
max 3387, 2921, 2851, 1679, 1617, 1516,
21 24
C H O
2
D
0
4
.
Fang, L.; Du, D.; Ding, G. Z.; Si, Y. K.; Yu, S. S.; Liu, Y.; Wang, W. J.; Ma, S. G.; Xu,
S.; Qu, J.; Wang, J. M.; Liu, Y. X. J. Nat. Prod. 2010, 73, 818–824.
Tang, W. Z.; Liu, Y. B.; Yu, S. S.; Qu, J.; Su, D. M. Planta Med. 2007, 73, 484–490.
aꢂ
ꢀ31.2 (c
0
(
e
5
.
3.69) nm; IR (KBr)
466, 1428, 1338, 1206, 1119, 988, 845, 802, 767, 725 cm
m
6. Bai, J.; Chen, H.; Fang, Z. F.; Yu, S. S.; Wang, W. J.; Liu, Y.; Ma, S. G.; Li, Y.; Qu, J.;
ꢀ1
1
1
;
H
Xu, S.; Liu, J. H.; Zhao, F.; Zhao, N. Phytochemistry 2012, 80, 137–147.

J. Bai et al. / Carbohydrate Research 361 (2012) 206–211
211
7
8
.
.
Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35, 501–506.
Yang, X. W.; Wang, J. S.; Ma, Y. L.; Xiao, H. T.; Zuo, Y. Q.; Lin, H.; He, H. P.; Li, L.
H.; Xiao-Jiang Planta Med. 2007, 73, 1415–1417.
Ma, S. G.; Tang, W. Z.; Yu, S. S.; Chen, X. G.; Liu, Y.; Wang, W. J.; Qu, J.; Xu, S.;
Ren, J. H.; Li, Y.; Lü, H. N. Carbohydr. Res. 2011, 346, 1165–1168.
12. Fu, H. Z.; Li, C. J.; Yang, J. Z.; Shen, Z. F.; Zhang, D. M. J. Nat. Prod. 2011, 74, 1066–
1072.
13. Baugh, P. J.; Kershaw, K.; Phillips, G. O.; Webber, M. G. Carbohydr. Res. 1973, 31,
199–209.
14. Wu, Q. X.; Li, Y.; Shi, Y. P. J. Asian Nat. Prod. Res. 2006, 8, 391–396.
15. Shao, Z. Y.; Zhu, D. Y.; Guo, Y. W. Chin. Chem. Lett. 2004, 15, 52–54.
16. Hu, Y. C.; Ma, S. G.; Li, J. B.; Yu, S. S.; Qu, J.; Liu, J.; Du, D. J. Nat. Prod. 2008, 71,
1800–1805.
9
.
1
0. Hongzhou, J.; Shen, Y.; Eri, Y.; Motoi, C.; Minoru, T. Eurasian J. Forest Res. 2001,
, 49–54.
1. Hiltunen, E.; Pakkanen, T. T.; Alvila, L. Holzforschung 2006, 60, 519–527.
3
1