Three new glycosides from the whole plant of Clematis lasiandra Maxim and their cytotoxicity

Article abstract of DOI:10.1016/j.phytol.2014.09.004

Phytochemical investigation on the whole plant of Clematis lasiandra Maxim led to the isolation of two new phenolic glycosides (1 and 2), one new lignanoid glycoside (3), together with three known lignanoid glycosides (4-6). The structures of the new comp

Full text of DOI:10.1016/j.phytol.2014.09.004

Phytochemistry Letters 10 (2014) 168–172
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Three new glycosides from the whole plant of Clematis lasiandra
Maxim and their cytotoxicity
Xiang-Rong Tian a,1, , Jun-Tao Feng a,1, Zhi-Qing Ma a, Na Xie a, Jing Zhang a,
*
b,**
Xing Zhang a, Hai-Feng Tang
a Research & Development Center of Biorational Pesticide, College of Plant Protection, Northwest A&F University, Yangling 712100, PR China
b Institute of Materia Medica, School of Pharmacy, Fourth Military Medical University, Xi’an 710032, PR China
A R T I C L E I N F O
A B S T R A C T
Article history:
Phytochemical investigation on the whole plant of Clematis lasiandra Maxim led to the isolation of two
new phenolic glycosides (1 and 2), one new lignanoid glycoside (3), together with three known lignanoid
Received 10 July 2014
Received in revised form 5 September 2014
Accepted 9 September 2014
Available online 19 September 2014
glycosides (4–6). The structures of the new compounds were elucidated as 4-O-
ethyl-E-caffeate (1), 4-O-
-galactopyranosyl-3-hydroxyl-phenylethene (2) and (8R)-3,30-dimethoxy-
4,40,9,90-tetrahydroxy-50,8-lignan 30-O-
-glucopyranoside (3), on the basis of extensive spectral
b-D-galactopyranosyl-
b
-D
b
-D
analysis and chemical evidence. The characteristic of the polymerized C-50–C-8 type lignanoid aglycone
in glycoside 3 was found from genus Clematis for the first time. Compounds 1–6 were evaluated for their
cytotoxicity against human tumor cell lines HL-60, Hep-G2 and SGC-7901, the new glycosides 1 and 2
showed significant cytotoxicity against those three tumor cell lines with IC50 in the range from 9.73 to
Keywords:
Clematis lasiandra Maxim
Phenolic glycoside
Lignanoid glycoside
Cytotoxicity
22.31
mM, while lignanoid glycosides 3–6 showed weak cytotoxicity to those test cell lines with IC50
value more than 52.71
mM.
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
new and known cytotoxic or anti-myocardial ischemia triterpe-
noid saponins. In the course of our ongoing search for new
The genus Clematis has been taxonomically placed in the
Ranunculaceae, and comprises about 150 species in China (Wang
and Li, 2005). Some species in this genus have long been used as
folk medicine for their analgesic, diuretic, anticancer, anti-
inflammatory and antimicrobial activities (Hao et al., 2013). The
chemical constituents recorded in the literature from genus
Clematis are saponins, flavonoids, coumarins, lignans, etc., and
most of them exist in the form of their glycosides (Sun and Yang,
2009). Clematis lasiandra Maxim is a perennial herbaceous plant
distributed widely in the north and south slopes of Tsinling
Mountains, Shaanxi province of China (Zhang et al., 2010). The
entire plants and rhizome of this species have been used as folk
medicines for a long history to treatment of dehygrosis, antitoxic,
diuretic, analgesic and antipyretic, etc. (Cai, 2000). Our previous
chemical investigations on this genus from C. lasiandra (Tian et al.,
2013), C. argentilucida (Hai et al., 2012; Zhao et al., 2012) and C.
tangutica (Zhang et al., 2013) have led to the isolation of several
bioactive natural products from C. lasiandra, two new phenolic
glycosides (1 and 2), one new lignanoid glycoside (3), together
with three known lignanoid glycosides (4–6) were isolated (Fig. 1).
This paper deals with the experimental details of isolation and
structural elucidation of these compounds and their cytotoxic
assay results.
2. Results and discussion
Compound 1 was obtained as a pale yellow solid. The positive
ion mode HR-ESI-MS showed a pseudomolecular ion peak at m/z
393.1164 [M+Na]+ (calcd. for C17H22O9Na, 393.1162), enabled the
determination of the molecular formula as C17H22O9, with the help
of NMR spectral data. The 1H NMR spectrum showed three
aromatic protons at dH 7.07 (1H, dd, J = 8.4, 2.0 Hz, H-6), 7.12 (1H, d,
J = 2.0 Hz, H-2) and 7.22 (1H, d, J = 8.4 Hz, H-5), indicating an ABX
system for orth- and inter-trisubstituted benzene ring. Two olefinic
protons at dH 7.59 (1H, d, J = 16.0 Hz, H-7) and 6.37 (1H, d,
J = 16.0 Hz, H-8) can be determined to trans configuration due to
the large coupling constant (J7,8 = 16.0 Hz) between H-7 and H-8.
The 13C NMR spectrum gave seventeen carbon signals, except for
six aromatic carbons and two olefin carbons, the remainder carbon
signals can be described to one ester carbonyl dC 169.1 (C-9), one
*
Corresponding author. Tel.: +86 29 87092122; fax: +86 29 87092122.
Corresponding author. Tel.: +86 29 84774748; fax: +86 29 84774748.
E-mail addresses: tianxiangrong@163.com, tianxangrong@163.com (X.-R. Tian),
**
tanghaifeng71@163.com (H.-F. Tang).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.phytol.2014.09.004
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

X.-R. Tian et al. / Phytochemistry Letters 10 (2014) 168–172
169
9
6'
9'
7'
5'
4'
8
O
HO
O
1'
2'
7
6
7
8'
1'
6
6
7
5
8
9
2'
1
2
O
5
1
2
HO
1
5
3'
8
4
OCH3
2
O
4
3
4
O
3
HO
3
6''
6'
OH
O
OH
O
OH
O
OH
6''
5''
OH
5'
OCH3
5''
OH
OH
OH
1''
1'
OH
3'
4''
4'
1''
4'' OH
OH3''
2''
2'
3
2
1
3''
2''
OH
OH
OH
Fig. 1. Structures of new compounds 1–3 from Clematis lasiandra.
oxyethyl group
d
C 61.7 (C-10) and 14.7 (C-20), and one sugar unit (dC
were in good agreement, except for the absence of a ester carbonyl
101.7, 72.3, 73.0, 68.7, 76.2, 62.9). These 1H and 13C NMR signals
were assigned with the aid of 1H–1H COSY, HSQC and HMBC
spectra as shown in Table 1, and they suggested that compound 1
was a glycoside with caffeic acid as its aglycone. The position of the
oxyethyl group was determined at C-9 since the observation of
and oxyethyl group in the aglycon in compound 2. The 1H NMR
spectrum of 2 showed one vinyl methenyl proton at dH 6.63 (1H, d,
J = 17.6 Hz, H-7), and two vinylic methine protons at dH 5.13 (1H,
dd, J = 10.9, 17.6 Hz, Ha-8) and 5.63 (1H, dd, J = 10.9, 17.6 Hz, Hb-8),
corresponding to two olefinic carbons at dC 137.9 (C-7) and 112.8
HMBC correlations from
d
H 1.34 (3H, t, J = 7.1 Hz, H3-20) to C-10 (dC
(C-8), with the help of HSQC spectrum. The position of the terminal
vinyl group was assigned at C-1 (dC 134.9) due to HMBC
correlations from H-7 (dH 6.63) to C-1 (dC 134.9), C-2 (dC 114.4)
and C-6 (dC 119.6), and from H2-8 (dH 5.13 and 5.63) to C-1 (dC
61.7), and from dH 4.25 (2H, q, J = 7.1 Hz, H2-10) to C-9 (dC 169.1).
The linkage of the sugar unit was deduced to be attached at C-4 (dC
148.8) due to HMBC correlation from anomeric proton
d, J = 8.0 Hz, Gal-100) to C-4, and NOESY interaction between Gal H-
100 and H-5 (Fig. 2). The presence of the
-galactose in compound 1
was established by methanolysis followed by TLC analysis of the
corresponding 1-O-methyl carbohydrate derivates (Tian et al.,
dH 5.23 (1H,
134.9). Methanolysis of 2 revealed the presence of
comparison with an authentic sample. The -configuration of the
sugar unit was identified by the same method of 1. The
configuration of -galactose in 2 was deduced on the coupling
constant (J = 8.1 Hz) of the anomeric proton, and the linkage of the
-galactose at C-4 was also confirmed by the observation of the
correlation between anomeric proton signal of Gal H-10 dH 5.15
(1H, d, J = 8.1 Hz) to C-4 ( C 146.9) (Fig. 2). Hence, the structure of
compound was confirmed as 4-O- -galactopyranosyl-3-
hydroxyl-phenylethene.
D-galactose by
D
D
b-
D
2009). The
D-configuration for Gal was determined by acidic
hydrolysis after comparison of the GC retention times of the
corresponding trimethylsilylated hydrolysate with those of the
authentic samples prepared in the same manner (Ding et al., 2012;
Gerwig et al., 1978), and the
deduced by the coupling constant (J = 8.1 Hz) of the anomeric
D
d
b-configuration for the sugar unit was
2
b-D
proton signal and careful analysis of NOESY spectrum. Based on the
Compound 3 was obtained as a white amorphous powder. The
molecular formula was established as C26H36O11 on the basis of its
positive HR-ESI-MS m/z 547.2159 [M+Na]+ (calcd. for C26H36O11Na,
547.2155). The 1H NMR spectrum of 3 showed one 1,3,4-
trisubstituted benzene ring signals at dH 6.58 (1H, brs, H-2),
6.61 (1H, d, J = 8.0 Hz, H-5) and 6.53 (1H, d, J = 8.0 Hz, H-6), one
10,30,40,50-tetrasubstituted benzene ring signals at 6.68 (1H, brs, H-
above analysis, compound 1 was undoubtedly deduced as 4-O-
-galactopyranosyl-ethyl-E-caffeate.
Compound 2 was obtained as a pale yellow solid. The molecular
b-
D
formula of 2 was deduced as C14H18O7 according to the positive
HR-ESI-MS from the pseudomolecular ion peak at m/z
321.0946 [M+Na]+ (calcd. for C14H18O7Na, 321.0950). Detailed
comparison of the 1H and 13C NMR data of compound 2 with 1
showed that the signals for the sugar moiety of the two compounds
20) and 6.50 (1H, brs, H-60), two methoxyl proton signals at
dH 3.70
(3H, s, 3-OCH3) and 3.84 (3H, s, 30-OCH3), and a glucose (Glc)
Table 1
1H NMR (500 MHz) and 13C NMR (125 MHz) data of compounds 1 and 2 in CD3OD.a
1
2
Position
dC mult.
dH mult. (J in Hz)
Position
dC mult.
dH mult. (J in Hz)
1
2
3
4
5
6
7
8
131.2 s
116.1 d
149.2 s
148.8 s
118.3 d
122.3 d
146.1 d
117.6 d
1
2
3
4
5
6
7
8
134.9 s
114.4 d
148.6 s
146.9 s
118.6 d
119.6 d
137.9 d
112.8 t
7.12 d (2.0)
6.96 d (2.0)
7.22 d (8.4)
7.15 d (8.3)
7.07 dd (8.4, 2.0)
7.59 d (16.0)
6.37 d (16.0)
6.87 dd (8.3, 2.0)
6.63 d (17.6)
a 5.13 dd (10.9, 17.6)
b 5.63 dd (10.9, 17.6)
9
169.1 s
61.7 t
10
4.25 q (7.1)
1.34 t (7.1)
20
14.7 q
101.7 d
72.3 d
73.0 d
68.7 d
76.2 d
62.9 t
Gal-100
200
5.23 d (8.0)
3.67 dd (3.0, 8.0)
4.19 m
Gal-10
102.2 d
72.3 d
73.0 d
68.7 d
76.1 d
62.9 t
5.15 d (8.1)
3.66 m
20
30
40
50
60
300
4.19 brt (2.8)
3.63 dd (2.8, 9.2)
3.88 m
400
3.63 dd (2.9, 9.6)
3.87 m
500
600
a 3.72 dd (5.2, 11.7)
b 3.89 m
a 3.73 dd (5.3, 11.8)
b 3.91 m
a
Assignments aided by HSQC, 1H–1H COSY, HMBC and NOESY experiments.

170
X.-R. Tian et al. / Phytochemistry Letters 10 (2014) 168–172
8
9'
O
7
7
5'
1'
1
HO
O
1'
1
8
9
2'
O
7
4
8
4
4'
HO
3'
O
1
3
O
3
OH
O
OCH3
OH
O
OH
4
HO
HO
3
OH
HO
OH
O
OCH3
OH
OH
1''
1'
1''
OH
OH
OH
1
3
2
OH
OH
1H-1H COSY
NOESY
Fig. 2. Key 1H–1H COSY, HMBC and NOESY correlations of compounds 1–3.
HMBC
anomeric H-atom signal at dH 4.23 (1H, d, J = 7.8 Hz, Glc H-100). In
(
dC 45.8), which was consistent with those reported for natural
product, (8R)-isodehydrodiconiferyl alcohol-40-O-(600-vanilloyl)-
-glucopyranoside (dC 43.4) (Lee et al., 2009), while was
different from its analog, 3-methoxy-4-hydroxy-5-[(80S)-30-meth-
oxy-40-hydroxyphenylpropyl alcohol]-E-cinnamic alcohol-4-O-
the 13C NMR spectrum, twenty-six carbon signals can be observed.
Apart from six signals due to a glucopyranosyl unit (
d
C 104.6, 75.3,
b-D
78.3, 71.8, 78.0 and 62.9) and two methoxyl carbon signals (dC
56.4 and 56.7), the other eighteen carbons could be ascribed to a
lignanoid skeleton for compound 3. Except twelve carbon signals
belonging to two benzenes, the remaining six carbon resonances in
b
-
D
-glucopyranoside (dC 40.5) (Tan et al., 2004). Furthermore, the
25
specific rotation ([a] D = –21.78) of its aglycon obtained by
the up-field can be classified as five methylenes
d
C 37.6 (C-7), 66.0
methanolysis of 3, was in good accordance with (8R)-isodehy-
25
(C-9), 33.0 (C-70), 32.9 (C-80) and 70.0 (C-90), and one methine dC
45.8 (C-8) by DEPT 135 experiment (Table 2). The 13C NMR data of 3
were similar to those of known compound 5, (7S,8R)-3,30,5-
drodiconiferyl alcohol ([
a
]
D = ꢀ18.08) (Iorizzi et al., 2001; Lee
et al., 2009), while was different from 3-methoxy-4-hydroxy-5-
[(80S)-30-methoxy-40-hydroxyphenylpropyl alcohol]-E-cinnamic
25
trimethoxy-4,9,90-trihydroxy-40,7-epoxy-50,8-lignan 4-O-
copyranoside, except for significant downfield shifts of C-40 (dC
b
-D
-glu-
alcohol ([a] D = +20.08) (Tan et al., 2004). This further confirmed
8R configuration for compound 3. Consequently, the structure of 3
was determined to be (8R)-3,30-dimethoxy-4,40,9,90-tetrahydroxy-
50,8-lignan 30-O-
b-D-glucopyranoside. The new glycoside 3 struc-
143.8, ꢀ3.8 ppm), C-50
(
dC 133.8, ꢀ3.3 ppm), C-7
(dC 37.6,
ꢀ51.4 ppm) and C-8 (
d
C 45.8, ꢀ9.6 ppm). These findings suggested
that the furan ring in 5 was cleavaged between oxygen atom and C-
7. The two phenylpropanoid systems were confirmed to be
polymerized between C-50 and C-8 on the basis of key HMBC
turally characterized with a polymerized C-50–C-8 form lignanoid
aglycone, which was isolated for the first time from genus Clematis.
The three known lignanoid glycosides 4–6 were identified as
(7S,8R)-3,30,5-trimethoxy-4,9,90-trihydroxy-40,7-epoxy-50,8-lig-
correlations from H-8 (
and from H2-7 ( H 2.91 and 3.00) to C-2 (
and C-50 (
C 133.8). Methanolysis of 3 yielded methyl-
which was identified on the basis of Rf value by TLC analysis, and
the -configuration for Glc was confirmed by the retention time by
GC analysis with the same method of 1. The -configuration of
d
H 3.41) to C-7 (
d
C 37.6) and C-50 (
d
C 133.8),
C 122.8)
-glucose,
d
d
C 114.1), C-6 (
d
D
nan 4-O-
dimethoxy-4,9,90-trihydroxy-40,7-epoxy-50,8-lignan 90-O-
copyranoside (5) (Kuang et al., 2009) and (7S,8R)-3,30-dimethoxy-
4, 9,90-trihydroxy-40,7-epoxy-50,8-lignan 9-O-
-glucopyrano-
b
-D
-glucopyranoside (4) (Kuang et al., 2009), (7S,8R)-3,30-
d
b
-
b-D
-glu-
D
b-D
b
D
-
side (6) (Abe and Yamauchi, 1986; Kuang et al., 2009), respectively,
by comparing their spectroscopic data and chemical evidences
with those published in the literature. All of them were isolated for
the first time from the whole plants of C. lasiandra.
glucose in 3 was determined on the coupling constant (J = 7.8 Hz)
of the anomeric proton, and the linkage of the glucose unit
attached at C-90 was confirmed by the observation of the
correlation between anomeric proton signal of Glc H-100
4.23, d, J = 7.8 Hz) to C-90 (dC 70.0) in the HMBC experiment, and
NOESY correlation from Glc H-100 ( H 4.23, d, J = 7.8 Hz) to H2-90 (dH
3.44 and 3.89) (Fig. 2). Thus, the planar structure of 3 was
elucidated as 3,30-dimethoxy-4,40,9,90-tetrahydroxy-50,8-lignan 30-
(
dH
The cytotoxicities of the isolated glycosides 1–6 against human
hepatocellular carcinoma Hep-G2, human leukemia HL-60 and
human gastric carcinoma SGC-7901 cells were evaluated by MTT
assay (Mosmann, 1983). The results of the cytotoxicities were
shown in Table 3. Two new phenolic glycosides (1 and 2)
d
O-
b
-D-glucopyranoside.
characterized with a
b-D-galactose at C-4 in the aglycone exhibited
The stereochemistry of C-8 in the aglycone of 3 was determined
as R configuration based on the 13C NMR chemical shift of C-8
significant cytotoxicity against those three tumor cell lines with
IC50 in the range from 9.73 to 22.31
m
M, while lignanoid glycosides
Table 2
1H NMR (500 MHz) and 13C NMR (125 MHz) data of compound 3 in CD3OD.a
Position
dC mult.
dH mult. (J in Hz)
Position
dC mult.
dH mult. (J in Hz)
1
2
3
4
5
6
7
133.9 s
114.1 d
148.5 s
148.8 s
115.8 d
122.8 d
37.6 t
50
133.8 s
122.4 d
33.0 t
6.58 brs
60
6.50 brs
70
1.83 m
80
32.9 t
70.0 t
2.60 m
6.61 d (8.0)
6.53 d (8.0)
2.91 dd (5.7, 13.7)
3.00 dd (5.7, 13.7)
3.41 m
90
3.44 m, 3.89 m
3.70 s
3-OCH3
30-OCH3
Glc-100
200
56.4 q
56.7 q
104.6 d
75.3 d
78.3 d
71.8 d
78.0 d
62.9 t
3.84 s
4.23 d (7.8)
3.23 t (8.6)
3.38 m
8
9
10
20
30
40
45.8 d
66.0 d
3.77 m
300
129.5 s
110.9 d
145.4 s
143.8 s
400
3.31 m
6.68 brs
500
3.29 m
600
3.69 m, 3.87 m
a
Assignments aided by DEPT, HSQC, 1H–1H COSY, HMBC and NOESY experiments.

X.-R. Tian et al. / Phytochemistry Letters 10 (2014) 168–172
171
Table 3
Cytotoxicities of compounds 1–6 against three cancer cell lines in vitro (IC50
,
m
M).a
Cell line
1
2
3
4
5
6
Adriamycinb
HL-60
20.56 ꢂ 0.32
9.73 ꢂ 0.47
15.12 ꢂ 0.39
22.31 ꢂ 0.54
12.24 ꢂ 1.21
18.72 ꢂ 0.33
>100
87.32 ꢂ 0.74
52.71 ꢂ 0.45
68.27 ꢂ 0.55
90.58 ꢂ 0.82
62.31 ꢂ 1.45
78.92 ꢂ 0.95
87.46 ꢂ 0.65
56.12 ꢂ 0.73
81.47 ꢂ 0.43
0.31 ꢂ 0.17
0.45 ꢂ 0.22
0.35 ꢂ 0.13
Hep-G2
SGC-7901
93.52 ꢂ 0.13
>100
a
IC50 values are means from three independent experiments in which each compound concentration was tested in three replicate wells.
Adriamycin as positive control.
b
3–6 possessed a
cytotoxicity to those test cell lines with IC50 value more than
52.71 M. Compared to structural features and cytotoxicities of
b
-
D
-glucose in the aglycone showed relative weak
n-BuOH (5 times ꢁ 6 L), respectively. The n-BuOH extract (180 g)
was separated into 24 fractions (Fr. A–Y) on a silica gel column
using a step gradient elution of CHCl3–MeOH–H2O (30:1:0, 20:1:0,
15:1:0, 10:1:0.1, 9:1:0.1, 8.5:1.5:0.15, 8:2:0.2, 7.5:2.5:0.25, 7:3:0.3
and 6.5:3.5:0.35) according to their TLC profiles. Fractions G and J
were selected to our principal study objectives as their different
TLC profiles compared with triterpenoid saponins, which we
previously obtained from C. lasiandra, C. argentilucida and C.
tangutica. Fr. G (1.2 g) was eluted with CHCl3–MeOH (1:1) on
Sephadex LH-20 to give three subfractions (Fr. G1–Fr. G3). Fr. G2
(167.3 mg) was further purified by semi-preparative HPLC using
MeOH–H2O (34:66) as the mobile phase at a flow rate of 2.0 mL/
min to afford compounds 1 (7.2 mg, tR = 59.5 min) and 2 (6.8 mg,
tR = 28.9 min). Fr. J (4.7 g) was subjected to CC over reversed-phase
silica gel eluting with MeOH–H2O (10:90, 40:60, 70:30, 100:0) to
give four subfractions (Fr. J1–Fr. J4). Fr. J2 (0.56 g) was eluted with
CHCl3–MeOH (1:1) on Sephadex LH-20 to remove pigments, and
then was further purified by semi-preparative HPLC using MeOH–
H2O (32:68) as the mobile phase at a flow rate of 2.0 mL/min to
m
the new lignanoid glycoside 3 with the known ones 4–6, the
normal 40,7-epoxy furan ring in the lignanoid aglycone was
collapsed between oxygen atom and C-7 in the new glycoside 3,
and its cytotoxicities against the test tumor cell lines were rather
lower than the known glycosides 4–6. It indicated that the cleavage
of the 40,7-epoxy furan ring between oxygen atom and C-7 can
decrease their cytotoxicities. In fact, lignanoid glycosides often
showed marginal cytotoxicities (Calis et al., 2005; Kuang et al.,
2009). The results for the weak cytotoxicities of lignanoid
glycosides (4–6) confirmed that these secondary metabolites
may be recognized as non-toxic agent.
3. Experimental
3.1. General
afford compounds
3
(23.8 mg, tR = 56.7 min),
4
6
(7.6 mg,
(26.3 mg,
Melting points were determined on an XT5-XMT apparatus and
uncorrected. Specific rotations were measured on a Perkin-Elmer
343 polarimeter. The ESI-MS and HR-ESI-MS spectra were obtained
on a Micromass Quattro mass spectrometer. 1D and 2D NMR
spectral experiments were measured in CD3OD on Bruker
AVANCE-500 NMR spectrometer with tetramethylsilane (TMS)
tR = 49.3 min),
tR = 71.5 min).
5
(110.5 mg, tR = 59.6 min) and
3.3.1. 4-O-b-D-galactopyranosyl-ethyl-E-caffeate (1)
23
Pale yellow solid (CH3OH), mp 206–207 8C, [
a]
+49.68 (c
D
0.10, MeOH); 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz,
CD3OD) data, see Table 1; Positive ESI-MS m/z 763 [2M+Na]+,
393 [M+Na]+; positive HR-ESI-MS m/z 393.1164 [M+Na]+ (calcd. for
as an internal standard. GC analysis was performed on
a
Finnigan Voyager apparatus using an l-Chirasil-Val column
(0.32 mm ꢁ 25 m) with an initial temperature of 180 8C at the
rate of 5 8C/min. Separations and purifications were performed by
C17H22O9Na, 393.1162).
column chromatography (CC) on silica gel H (10–40
Marine Chemical Inc., Qingdao, China), reversed-phase silica gel
(Lichroprep RP-18, 40–63 m, Merck Inc., Darmstadt, Germany)
and Sephadex LH-20 (GE Inc., USA). HPLC was carried out on a
Shimadzu LC-10ATVP liquid chromatograph equipped with a SPD-
10ADVP (UV–Vis) detector at 206 nm using a YMC-Pack R&D ODS-
mm, Qingdao
3.3.2. 4-O- -galactopyranosyl-3-hydroxyl-phenylethene (2)
b
-D
m
25
Pale yellow solid (CH3OH), mp 181–182 8C, [
a
]
+47.88 (c
D
0.10, MeOH); 1H NMR (500 MHz, CD3OD) and 13C NMR (125 MHz,
CD3OD) data, see Table 1; Positive HR-ESI-MS m/z
321.0946 [M+Na]+ (calcd. for C14H18O7Na, 321.0950).
A
column (250 mm ꢁ 10 mm i.d.) for semi-preparation. TLC
detection was achieved by spraying the silica gel plates (Qingdao
Marine Chemical Inc., Qingdao, China) with 20% H2SO4 in EtOH
followed by heating.
3.3.3. (8R)-3,30-dimethoxy-4,40,9,90-tetrahydroxy-50,8-lignan 30-O-
b-
D-glucopyranoside (3)
25
White amphous powder (CH3COCH3), mp 242–243 8C, [
a]
D
ꢀ89.18 (c 0.35, MeOH); 1H NMR (500 MHz, CD3OD) and 13C NMR
(125 MHz, CD3OD) data, see Table 2; positive ESI-MS m/z
1071 [2M+Na]+, 547 [M+Na]+; positive HR-ESI-MS m/z
547.2159 [M+Na]+ (calcd. for C26H36O11Na, 547.2155).
3.2. Plant material
The whole plants of Clematis lasiandra Maxim were collected in
the north of Tsinling Mountain, Shaanxi Province of China in
September 2009, and were identified by Prof. Ji-Tao Wang from
Shaanxi University of Chinese Medicine. A voucher specimen (No.
20090904) was deposited in the Department of Pharmacy, Xijing
Hospital, Fourth Military Medical University, Xi’an, Shaanxi, PR
China.
3.4. Methanolysis of glycosides 1–3
Each glycoside (3 mg) was dissolved in 5% HCl–MeOH (3 mL)
and refluxed for 12 h (80 8C). The reaction mixture was evaporated
under reduced pressure to remove residual HCl. The resulting
residue was partitioned between H2O and benzene. The benzene
layer of glycoside 3 was concentrated in vacuo to yield major (8R)-
3.3. Extraction and isolation
3,30-dimethoxy-4,40,9,90-tetrahydroxy-50,8-lignan with the optical
The air-dried whole plants of C. lasiandra (7.2 kg) were crushed
and then extracted with 70% EtOH (3 times ꢁ 56 L, 6 h/time) under
reflux at room temperature. The concentrated EtOH extract
(1.4 kg) was suspended in H2O and then partitioned successively
with petroleum ether (3 times ꢁ 6 L), EtOAc (3 times ꢁ 6 L) and
25
rotation of [
a
]
D ꢀ21.78. The H2O layer of glycosides 1 and 2 were
-galactosylpyranoside
give methyl-
glucopyranoside with Rf value of 0.63, which were subjected to
concentrated to afford the major methyl-
with Rf value of 0.54, while glycoside
b-D
3
b-D-

172
X.-R. Tian et al. / Phytochemistry Letters 10 (2014) 168–172
co-TLC analysis with authentic sugars by developed with n-BuOH–
Pyridine–H2O (6:4:3) and detected with aniline phthalate spray
(Ding et al., 2012; Tian et al., 2009).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytol.2014.09.004.
3.5. Acid hydrolysis of glycosides 1–3
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Acknowledgments
This work was financially supported by grants from Natural
Science Foundation of Shaanxi Province, China (No. 2014JQ3091)
and National Natural Science Foundation of China (No.
81274029). The authors are thanks for Mr. Hong-Li Zhang and
Mrs. Hui-Min Wang for the measurements of NMR and MS
spectra.
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