Cytotoxic triterpene saponins from Clematis mandshurica

Article abstract of DOI:10.1080/10286020.2011.618453

Two new triterpene saponins, mandshunosides A and B (1 and 2), were isolated from the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products. Compounds 1 and 2 showed inhibitory activities against two colorectal human cancer cells HCT 116 (IC₅₀ 2.1μM for 1 and 2.5μM for 2) and HT-29 (IC₅₀ 3.7μM for 1 and 3.3μM for 2).

Full text of DOI:10.1080/10286020.2011.618453

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Cytotoxic triterpene saponins from
Clematis mandshurica
a
a
a
b
Yu-Xin He , Ling Li , Kang Zhang & Zhong-Rong Liu
a
Bioengineering College, Xihua University, Chengdu, Sichuan,
10039, China
6
b
Chengduzhihao Pharmaceutical Co., Ltd, The Life Science and
Technology Park, Chengdu High Tech Zone, Sichuan, 610041, China
Published online: 25 Nov 2011.
To cite this article: Yu-Xin He , Ling Li , Kang Zhang & Zhong-Rong Liu (2011) Cytotoxic triterpene
saponins from Clematis mandshurica , Journal of Asian Natural Products Research, 13:12, 1104-1109,
DOI: 10.1080/10286020.2011.618453
To link to this article: http://dx.doi.org/10.1080/10286020.2011.618453
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Journal of Asian Natural Products Research
Vol. 13, No. 12, December 2011, 1104–1109
Cytotoxic triterpene saponins from Clematis mandshurica
b
a
Yu-Xin He , Ling Li *, Kang Zhang and Zhong-Rong Liu
a
a
a b
Bioengineering College, Xihua University, Chengdu, Sichuan 610039, China; Chengduzhihao
Pharmaceutical Co., Ltd, The Life Science and Technology Park, Chengdu High Tech Zone, Sichuan
10041, China
6
(
Received 11 July 2011; final version received 25 August 2011)
Two new triterpene saponins, mandshunosides A and B (1 and 2), were isolated from
the roots and rhizomes of Clematis mandshurica. Their structures were elucidated on
the basis of spectroscopic evidence and hydrolysis products. Compounds 1 and 2
showed inhibitory activities against two colorectal human cancer cells HCT 116 (IC50
2.1 mM for 1 and 2.5 mM for 2) and HT-29 (IC50 3.7 mM for 1 and 3.3 mM for 2).
Keywords: Clematis mandshurica; triterpene saponins; mandshunoside A; mandshu-
noside B; cytotoxicity
1
.
Introduction
(positive-ion mode) experiment revealed
þ
a pseudo-molecular ion peak [M þ Na]
The root and rhizome of Clematis
mandshurica Rupr. (Ranunculaceae) are
a main source of traditional Chinese
medicine ‘Weilingxian’, which is widely
used as an anti-inflammatory, antitumor,
analgesic agent [1]. Previous phytochem-
ical research revealed that it was rich in
triterpene saponins. The corresponding
aglycones were oleanolic acid (Ole) and
hederagenin (Hed). A trisaccharide chain
can be linked to the aglycone at the C-28,
and a complex long oligosaccharides chain
at the C-3. Some of them showed anti-
inflammatory and antitumor activity [2,3].
As a continuation of study on this plant, we
report on the isolation and structural
elucidation of two new saponins mandshu-
nosides A and B (Figure 1), as well as their
cytotoxicity.
at m/z 1403.6614, corresponding to a
molecular formula of C H O . The
69 104 28
monosaccharides obtained after aqueous
acid hydrolysis of 1 were identified as
glucose, rhamnose, arabinose, and ribose
by TLC comparison with authentic
samples. The absolute configurations of
the monosaccharides were determined to
be D for glucose and ribose and L for
rhamnose and arabinose by GC analysis of
chiral derivatives of the monosaccharides
in the hydrolysate of each compound (see
Section 3). The relatively large coupling
constants (5.0–8.0 Hz) for the anomeric
1
protons in the H NMR spectrum (see
Section 3) of 1 suggested that the
arabinopyranosyl moiety has an a-con-
figuration and the glucopyranosyl and
ribopyranosyl moieties have a b-configur-
ation. The a-configurations of the rham-
nopyranosyl moieties were determined
from the broad singlets for the anomeric
2
.
Results and discussion
Compound 1 was isolated as a white
amorphous powder. The HR-ESI-MS
1
protons. The H NMR spectrum showed
*Corresponding author. Email: lily1188@126.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2011.618453
http://www.tandfonline.com

Journal of Asian Natural Products Research
1105
12
13
28
1
COOH
1
OH Ara
O
3
O
R
23
24
1
O
H
O
1
Rha OH
Glc
OH
O
H
1
HO
O
Rib
2
O
O
Glc
1 R = CH OH
2
O
OH
O
MeO
O
O
2
R = CH3
HO
HO
OH H
HO
OH
OH
OH
H
OH H
Figure 1. The structures of compounds 1 and 2.
six tertiary methyl resonances at d 0.83,
spectrum of 1 exhibited five anomeric
proton resonances at d 6.27 (1H, br s), 5.80
(1H, d, J ¼ 5.0 Hz), 5.14 (1H, d,
J ¼ 8.0 Hz), 5.05 (1H, d, J ¼ 6.5 Hz),
and 4.90 (1H, d, J ¼ 8.0 Hz), respectively.
The three-proton doublets observed at 1.51
(3H, d, J ¼ 6.0 Hz) suggested the presence
of a deoxyhexopyranosyl unit in 1. The
spin–spin coupling system of individual
monosaccharide units was identified by
analysis of 1D TOCSY and 2D NMR
0.84, 0.93, 1.06, 1.07, and 1.13 and an
olefinic proton at d 5.37, which were
typical signals of the Ole skeleton. The
1
3
C
resonances at d 123.0 and 145.1 in the
NMR spectrum also suggested that 1
possessed an Ole aglycone. The aglycone
of 1 was identified as Hed by comparison
1
13
of its H and C NMR data obtained in 2D
NMR experiments with those reported in
the literature [4]. The downfield chemical
1
3
1
shift at dC 80.9 (Aglycone-3) in the
C
spectra. H NMR spectral data of individ-
NMR spectrum of 1 (Table 1) indicated
that a sugar chain was linked to C-3 of
aglycone. Alkaline hydrolysis of 1
afforded a prosapogenin (1a), which was
identified as CP . This was achieved by
ual monosaccharide units were obtained
by selective irradiation of the anomeric
protons or methyl groups of rhamnose
units in a series of 1D TOCSY exper-
1
1
iments. The analysis of the H– H COSY
spectrum resulted in sequential assignment
of all proton resonances of the five
monosaccharide units, including the
1
0
1
13
direct comparison of H and C NMR
spectral data of 1a with those of CP
1
0
1
reported in the literature [5]. The H NMR
1
3
Table 1.
No.
5 5
C NMR spectral data for the aglycone moieties of 1 and 2 (125 MHz, in C D N).
1
2
No.
1
2
No.
1
2
1
2
3
4
5
6
7
8
9
39.0
26.3
80.9
43.5
47.6
18.0
32.6
39.8
48.1
36.8
39.0
26.5
88.6
39.4
56.1
18.1
33.0
39.8
48.0
36.8
11
12
13
14
15
16
17
18
19
20
24.0
123.0
145.1
42.0
28.2
23.6
46.7
42.2
46.7
31.1
23.8
122.8
145.1
42.0
28.2
23.2
46.7
42.2
46.7
31.0
21
22
23
24
25
26
27
28
29
30
34.3
33.4
63.7
16.5
15.7
16.5
20.8
180.1
33.0
23.6
34.1
33.5
28.0
17.1
15.6
16.4
20.8
180.1
33.0
23.6
1
0

1
106
Y.-X. He et al.
1
Table 2. H NMR spectral data for the sugar moieties of compounds 1 and 2 (500 MHz, in C D N).
5
5
No.
1 (J in Hz)
2 (J in Hz)
No.
1 (J in Hz)
2 (J in Hz)
1
1
Ara
1
Glc
1
5.05 d (6.5)
4.54 dd (8.0, 6.5)
4.01 dd (8.0, 4.5)
4.11 m
4.20 d (9.0)
3.62 d (9.0)
4.82 d (6.5)
4.52 dd (8.0, 6.5)
4.16 m
4.16 m
4.22 m
3.76 m
4.90 d (8.0)
3.86 dd (9.0, 8.0)
4.13 m
4.92 d (8.0)
3.85 dd (9.0, 8.0)
4.12 m
2
3
4
2
3
4
5
6a
6b
4.25 m
3.64 m
4.24 m
3.64 m
5a
5
b
4.23 m
4.12 m
4.24 m
4.12 m
2
Rha
1
2
6.27 br s
4.83 br s
4.63 m
6.27 br s
4.92 br s
4.71 m
Glc
1
2
3
5.14 d (8.0)
3.92 m
4.11 m
4.14 m
4.01 m
5.17 m
4.66 m
5.14 d (7.5)
3.92 m
4.11 m
4.13 m
4.01 m
5.16 m
4.65 m
2
4
5
6
4.38 dd (9.0, 9.0)
4.62 m
1.51 d (6.0)
4.39 dd (9.0, 9.0)
4.60 m
1.51 d (6.0)
3
4
5
6a
6b
1
Rib
1
5.80 d (5.0)
4.09 m
4.58 m
5.79 d (5.0)
4.07 m
4.57 m
2
3
4
4.30 m
4.28 m
4.28 m
4.30 m
4.27 m
4.27 m
5a
5b
identification of most of their multiple
splitting patterns and coupling constants,
as shown in Table 2. In the HSQC
experiment, proton resonances were cor-
related with those of the corresponding
carbons, and associated anomeric protons
were correlated with their respective
carbon atoms from HSQC–TOCSY data,
leading to unambiguous assignments of
the carbons in each monosaccharide unit
signals provide ample evidence to deter-
mine the linkages between the sugars, as
well as between the sugar and the
aglycone. Further analysis of the NMR
13
Table 3.
C NMR spectral data for the sugar
moieties of compounds 1 and 2 (125 MHz, in
N).
C D
5 5
No.
1
2
No.
1
2
1
1
Ara
1
Glc
1
(see Table 3). By comparison of the carbon
104.6 105.3
103.1 103.1
chemical shifts thus assigned with those of
reference methyl glycosides [6], and
taking into account the known effects of
O-glycosylation, 1 was found to contain
one L-arabinopyranosyl unit (Ara), one D-
ribopyranosyl unit (Rib), one L-rhamno-
pyranosyl unit (Rha), and two D-glucopyr-
anosyl units (Glc). In the HMBC spectrum,
2
3
4
5
Rha
1
2
3
4
75.3
75.1
69.6
66.2
75.3
74.8
69.4
65.5
2
3
4
5
74.3
76.4
81.8
76.4
61.5
74.2
76.6
81.8
76.4
61.5
2
6
101.3 101.4 Glc
2
71.8
82.0
72.5
69.6
18.4
71.9
82.1
72.5
69.7
18.4
1
2
3
4
5
6
105.1 105.1
72.3
78.6
71.2
75.5
64.2
72.3
78.6
71.2
75.6
64.2
5
6
the anomeric proton signals at d 5.05
H
1
Ara -H-1), 6.27 (Rha -H-1), 5.80 (Rib -
1
1
(
1
Rib
1
1
H-1), 4.90 (Glc -H-1), and 5.14 (Glc -H-
2
104.7 104.7
2
3
4
5
72.6
69.6
76.4
61.5
72.6
69.8
76.4
61.6
1
) showed cross-peaks with the carbon
1
signals at d 80.9 (Agly-C-3), 75.3 (Ara -
C
1
C-2), 82.0 (Rha -C-3), 76.4 (Rib -C-4),
1
and 81.8 (Glc -C-4), respectively. These
1

Journal of Asian Natural Products Research
1107
1 13
Table 4. H and C NMR spectral data for the isoferuloyl moieties of compounds 1 and 2
1
13
(
5 5
500 MHz for H NMR, 125 MHz for C NMR, in C D N).
1
2
1
13
1
13
No.
H (J in Hz)
C
H (J in Hz)
C
1
2
3
4
5
6
7
8
167.1
116.3
145.6
128.5
115.4
148.4
150.8
112.0
121.4
55.8
166.6
116.4
145.6
128.6
115.4
148.4
150.9
112.1
121.4
55.8
6.89 d (16.0)
8.09 d (16.0)
6.90 d (16.0)
8.12 d (16.0)
7.52 d (2.0)
7.53 d (2.0)
6.87 d (8.5)
7.07 dd (8.5, 2.0)
3.73 s
6.87 d (8.5)
7.09 dd (8.5, 2.0)
3.73 s
9
OCH
3
1
than that of 1. Comparison of H and
13
spectral data of 1 revealed the presence of
a 3-hydroxy-4-methoxycinnamoyl group
C
NMR spectral data for 1 with those for 2
revealed that they differed only in the
aglycone part. The upfield shift of Agly-C-
(
isoferuloyl; Table 4). The typical protons
of the isoferuloyl moiety resonated at dH
.52 (1H, d, J ¼ 2.0 Hz), 7.07 (1H, dd,
J ¼ 8.5, 2.0 Hz), 6.87 (1H, d, J ¼ 8.5 Hz),
.09 (1H, d, J ¼ 16.0 Hz), 6.89 (1H, d,
7
2
3 by 28.0, and the correlation between
signals at d 28.0 (Agly-C-23) and d 1.22
3H, s) in the HSQC spectrum indicated the
8
(
J ¼ 16.0 Hz), and 3.73 (3H, s), and at d
C
presence of the methyl (Agly-C-23). In
1
1
16.3, 121.4, 112.0, 145.6, 115.4, 150.8,
48.4, 128.5, 167.1, and 55.8. The
addition, a set of carbon signals for 2 at d
88.6 (Agly-C-3) and 56.1 (Agly-C-5) were
2
correlation observed between Glc -H-6 (d
observed, different from the corresponding
5
1
.17 and 4.66) and the carbonyl carbon (d
67.1) of the isoferuloyl moiety in the
carbons at d 80.9 (Agly-C-3) and 47.6
(Agly-C-5) in 1. Further comparison of its
HMBC spectrum indicated that the iso-
feruloyl moiety is linked to the sugar chain
2
on Glc -C-6 (d 64.2). From the above
evidence, the structure of 1 was estab-
lished as 3-O-[(6-O-isoferuloyl)-b-D-glu-
copyranosyl]-(1 ! 4)-b-D-glucopyrano-
syl-(1 ! 4)-b-D-ribopyranosyl-(1 ! 3)-
a-L-rhamnopyranosyl-(1 ! 2)-a-L-arabi-
nopyranosyl Hed, which has been named
mandshunoside A.
1
13
H and C NMR spectroscopic data with
those reported values confirmed that the
aglycone of 2 is Ole [7]. Acid hydrolysis of
2
yielded Ole, L-arabinose, D-glucose, L-
rhamnose, and D-ribose. Signals of the
arabinopyranosyl moiety connected to the
aglycone were observed at d 4.82 (Ara-H-
1
) and 105.3 (Ara-C-1) for 2. The
correlation observed between Ara-H-1 at
d 5.05 and Agly-3 at d 88.6 confirmed
that the arabinopyranosyl moiety was
linked to the C-3 of aglycone. Thus,
the structure of 2 was determined as 3-O-
Compound 2 was isolated as a white
amorphous powder. The HR-ESI-MS
(
positive-ion mode) experiment revealed a
þ
pseudo-molecular ion peak [M þ Na] at
m/z 1387.6659, suggesting a molecular
formula of C H O . The spectroscopic
[
(6-O-isoferuloyl)-b-D-glucopyranosyl]-
(1 ! 4)-b-D-glucopyranosyl-(1 ! 4)-b-
D-ribopyranosyl-(1 ! 3)-a-L-rhamnopyr-
anosyl-(1 ! 2)-a-L-arabinopyranosyl Ole
(mandshunoside B).
6
9 104 27
properties of 2 were closely related to those
of 1, however, the molecular formula of 2
(C H O ) had one oxygen atom lower
69 104 27

1
108
Y.-X. He et al.
3
.
Experimental
MeOH–H O in a gradient of MeOH
2
(
MeOH–H O, 30:70–100:0%, to afford
2
3
.1 General experimental procedures
subfractions 3-1 to 3-7 (1.2, 3.0, 2.3, 1.4,
.5, 0.8, and 4.8 g, respectively). Subfrac-
Optical rotations were measured on a
Perkin-Elmer 241-MC digital polarimeter.
UV spectra were obtained on a TU-1901
spectrometer. IR spectra were recorded on
a Perkin-Elmer 577 spectrometer. NMR
spectra were recorded on a Bruker INOVA
4
tion 3-6 (0.8 g) was isolated by preparative
HPLC (CH CN–H O, 28:72, 2.0 ml/min)
3
2
to yield compound 1 (41 mg) and com-
pound 2 (37 mg).
5
00 spectrometer with TMS as an internal
3
.3.1 Mandshunosides A (1)
standard. HR-ESI-MS was obtained on a
IonSpec 4.7 T Fourier Transform Mass
Spectrometry in m/z. GC was obtained on
an Agilent 6890N gas chromatograph.
Column chromatography was carried out
on silica gel (200–300 mesh, Qingdao
Haiyang Chemical Co., Ltd, Qingdao,
China) and C18 silica gel (150–200
mesh, Merck, Darmstadt, Germany).
HPLC separation was carried out on an
Octadecylsilanized (ODS) column (YMC-
pack ODS-A, 250 £ 10 mm, i.d. 5 mm
YMC, Kyoto, Japan) with an Agilent
evaporative light scattering detector.
2
D
0
A white amorphous solid, ½aꢀ 242,
(
(
c ¼ 1.0, MeOH); UV (MeOH) l
max
log 1)
240
(2.31),
294
(sh),
1
2
3
3
24(4.20) nm; IR(KBr) nmax (cm ):
1
412, 2931, 1710, and 1632; H NMR
(
C D N, 500 MHz): d 1.13 (3H, s, Me-27),
5 5
1
0
0
.07 (3H, s, Me-24), 1.06 (3H, s, Me-26),
.93 (3H, s, Me-25), 0.84 (3H, s, Me-29),
.83(3H, s, Me-30), 4.25 (1H, dd,
J ¼ 12.0, 5.0 Hz, H-3), 4.23 (1H, d,
J ¼ 12.0 Hz, H-23a), 3.88 (1H, d,
J ¼ 12.0 Hz, H-23b), 5.37 (1H, br s,
1
3
H-12); C NMR spectral data, see Tables
1 13
and 3; H and C NMR spectral data for
1
isoferuloyl moiety, see Table 4.
3
.2 Plant material
The roots and rhizomes of C. mandshurica
were collected in September 2010 in
Jinzhou City, Liaoning Province, China.
The identification of the plant was done by
one of the authors (Y.-X.H.). A voucher
specimen (CM 201009) is maintained in
the herbarium of Bioengineering College
of Xihua University.
3
.3.2 Mandshunoside B (2)
2
D
0
A white amorphous solid, ½aꢀ 237
(c ¼ 1.0, MeOH); UV (MeOH) lmax
(log 1) 242 (2.34), 294 (sh), 326
2
1
(4.24) nm; IR(KBr) nmax (cm ): 3406,
2933, 1714, and 1630; H NMR (C D N,
5 5
1
500 MHz): d 1.22 (3H, s, Me-23), 1.21(3H,
s, Me-27), 1.09 (3H, s, Me-24), 1.03 (3H, s,
Me-26), 0.86 (3H, s, Me-29), 0.85 (3H, s,
Me-30), 0.82 (3H, s, Me-25), 3.22 (1H, dd,
J ¼ 12.0, 5.0 Hz, H-3), 5.36 (1H, br s, H-
3
.3 Extraction and isolation
The dried roots and rhizomes (5 kg) of C.
mandshurica were extracted with 50%
EtOH. After removing the solvent, the
1
3
12); C NMR spectral data, see Tables 1
1
13
and 3; H and C NMR spectral data for
isoferuloyl moiety, see Table 4.
residue (1262 g) was suspended in H O and
2
extracted with EtOAc and n-BuOH. The n-
BuOH extract (120 g) was subjected to
silica gel column chromatography and
eluted with CHCl –MeOH–H O (10:1:0,
3
.4 Acid hydrolysis
A solution of compound 1 or compound 2
(5.0 mg each) in 2 M trifluoroacetic acid
(1 ml) was heated at 1108C for 2 h, and
3
2
4
:1:0.2, 1:2:0.5) to afford fractions 1–3
(
(
29, 31, and 18 g, respectively). Fraction 3
18 g) was subjected to C18 silica gel
then dried by N gas. After cooling, the
2
column chromatography and eluted with
reaction mixture was neutralized with

Journal of Asian Natural Products Research
1109
2
Amberlite IRA-400 resin (OH form) and
the resin was filtered. After removal of the
solvent under pressure from the filtrate, the
residue was passed through a Sep-Pak C18
cartridge with H O and MeOH. The H O
Hydrolysis of 1 afforded a known
compound identified as CP .
1
0
3.6 MTT cytotoxicity assay
2
2
The bioassay was carried out according to
a previously described method, against
two human colorectal cancer cell lines
eluate was concentrated and the residue
was treated with L-cysteine methyl ester
hydrochloride (2.0 mg) in pyridine (1.0 ml)
(HCT 116 and HT-29) [8]. Paclitaxel was
at 608C for 2 h. After drying by N gas, the
2
used as a positive control with IC50 values
of 3.5 nM (HCT 116) and 3.4 nM (HT-29).
residue was treated with N-(trimethylsily-
l)imidazol (0.2 ml) at 608C for 1 h. The
reaction was stopped by adding water
(
(
1.0 ml), and extracted with cyclohexane
1.0 ml, three times). The cyclohexane
Acknowledgements
This work was financially supported by the
Research Fund of Key Laboratory of TCM
Biotechnology, Xihua University, and the Open
Research Fund of Key Laboratory of Food
Biotechnology, Xihua University (SZJJ2009-
layer was collected and concentrated to
.0 ml for GC analysis. Separations were
carried out on HP-5 column
1
a
(
28 m £ 0.32 mm). Highly pure He was
0
06).
employed as carrier gas (1.0 ml/min flow
rate), and the Flame Ionization Detector
detector operated at 2608C (column temp
References
[
1] Editorial Committee of Chinese Pharma-
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1
808C). The retention times of the
monosaccharide derivatives were as fol-
lows: L-Rha, 5.75 min; D-Glc, 11.90 min;
D-Rib, 5.43 min; and L-Ara, 5.12 min. The
retention times of standard monosacchar-
ide derivatives were L-Rha, 5.74 min;
D-Glc, 11.89 min; D-Rib, 5.43 min; and
L-Ara, 5.11 min.
(
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3
.5 Alkaline hydrolysis
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Pure compound 1 (10 mg) was refluxed in
5
1
[
[
[
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% KOH solution (pH 12–13) at 908C for
h. The reaction mixtures were neutral-
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were extracted with n-BuOH, and the
organic layers of pure compounds
were analyzed by NMR spectroscopy.
4
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