Three new triterpene saponins from Clematis chinensis

Article abstract of DOI:10.1080/10286020.2013.794418

Three new triterpene saponins, clematochinenosides H-J (1-3), were isolated from the roots and rhizomes of Clematis chinensis. Their structures were elucidated on the basis of spectroscopic means including 1D and 2D NMR experiments and hydrolysis products

Full text of DOI:10.1080/10286020.2013.794418

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Three new triterpene saponins from
Clematis chinensis
Qiang Fu b , Ke Zan a , Ming-Bo Zhao a , Si-Xiang Zhou a , She-Po Shi
a , Yong Jiang a & Peng-Fei Tu a
a State Key Laboratory of Natural and Biomimetic Drugs, School
of Pharmaceutical Science, Peking University , Beijing , 100191 ,
China
b Beijing Yiling Pharmaceutical Research Institute , Beijing ,
100027 , China
c Modern Research Center for Traditional Chinese Medicine, Beijing
University of Chinese Medicine , Beijing , 100029 , China
Published online: 10 May 2013.
To cite this article: Qiang Fu , Ke Zan , Ming-Bo Zhao , Si-Xiang Zhou , She-Po Shi , Yong Jiang &
Peng-Fei Tu (2013) Three new triterpene saponins from Clematis chinensis , Journal of Asian Natural
Products Research, 15:6, 610-618, DOI: 10.1080/10286020.2013.794418
To link to this article: http://dx.doi.org/10.1080/10286020.2013.794418
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Journal of Asian Natural Products Research, 2013
Vol. 15, No. 6, 610–618, http://dx.doi.org/10.1080/10286020.2013.794418
Three new triterpene saponins from Clematis chinensis
Qiang Fuab, Ke Zana, Ming-Bo Zhaoa, Si-Xiang Zhouc, She-Po Shia,
Yong Jianga and Peng-Fei Tua*
aState Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Science,
Peking University, Beijing 100191, China; Beijing Yiling Pharmaceutical Research Institute,
b
c
Beijing 100027, China; Modern Research Center for Traditional Chinese Medicine,
Beijing University of Chinese Medicine, Beijing 100029, China
(Received 4 February 2013; final version received 7 April 2013)
Three new triterpene saponins, clematochinenosides H–J (1–3), were isolated from the
roots and rhizomes of Clematis chinensis. Their structures were elucidated on the basis
of spectroscopic means including 1D and 2D NMR experiments and hydrolysis
products.
Keywords: Clematis chinensis; triterpene saponins; clematochinenosides H–J
1. Introduction
m/z 2007.8625, which is in agreement with
the molecular formula C88H144O49. The
aglycone of 1 was identified as hederagenin
The roots and rhizomes of Clematis
chinensis Osbeck (Ranunculaceae) are
used in a traditional Chinese herbal drug
‘Weilingxian’ that is commonly used as an
antiinflammatory, antitumor, and analge-
sic agent [1]. Phytochemical research
reveals that it is rich in triterpene saponins
[2–8]. Our previous study showed that
those saponins with isoferulic acid moiety
showed inhibitory activity against the
cyclooxygenase-1 and cyclooxygenase-2
enzymes [9]. In addition, monodesmosidic
saponins exhibited cytotoxic activities
against cultured tumor cells [10]. As a
continual study on this plant, we report on
the isolation and structural elucidation of
three new saponins clematochinenosides
H–J (Figure 1) from the roots and
rhizomes of C. chinensis.
1
in comparison with the H and 13C NMR
data obtained by 2D NMR experiments
with values of the literature (Table 1) [9].
The downfield chemical shift at dC 80.9
(Agly-3) and the upfield chemical shift at
dC 176.5 (Agly-28) in the 13C NMR
spectrum of 1 indicated these compounds
to be bisdesmosidic saponins. The 1H NMR
spectrum of 1 exhibited 10 anomeric proton
resonances at d 6.27 (1H, br s), 6.21 (1H, d,
J ¼ 8.0 Hz), 5.82 (1H, br s), 5.80 (1H, d,
J ¼ 5.0 Hz), 5.42 (1H, br s), 5.10 (1H,
d, J ¼ 8.0Hz), 5.09 (1H, d, J ¼ 8.0 Hz),
5.04 (1H, d, J ¼ 6.5 Hz), 4.99 (1H, d,
J ¼ 8.0 Hz), and 4.90 (1H, d, J ¼ 8.0 Hz),
respectively (Table 2). The three proton
doublets observed at d 1.67 (3H, d,
J ¼ 6.0 Hz), 1.57 (3H, d, J ¼ 6.0 Hz), and
1.50 (3H, d, J ¼ 6.0 Hz) suggested the
presence of three deoxyhexopyranosyl
units in 1. The monosaccharides obtained
after acid hydrolysis of 1 were identified as
glucose, rhamnose, arabinose, and ribose
by thin-layer chromatography (TLC) in
2. Results and discussion
Compound 1 was isolated as a white,
amorphous powder. The HR-ESI-MS
(positive-ion mode) experiment revealed
a pseudomolecular ion peak [M þ Na]þ at
*Corresponding author. Email: pengfeitu@vip.163.com
q 2013 Taylor & Francis

Journal of Asian Natural Products Research
611
O
12
13
28
OH
1
O
IF =
MeO
Ara1
OH
3
O
OH
O
O
Rha3
HO
OH
HO
Rha2
23
Glc3
OH
O
24
O
Glc4
OH
Rib1
O
HO
HO
O
O
HO
O
O
O
O
HO
OH
O
O
O
HO
HO
O
O
OH
OH
Glc1
HO
O
R1O
O
OH
HO
OH
O
HO
OH
OR3
Glc2
HO
R2O
OH
Rha1
HO
OH
Glc5
Glc6
HO
HO
HO
HO
HO
HO
O
1 R1 = H, R2 = H, R3 = H
3 R1 = H
R2 =
R3 = IF
Glc6
O
Glc7
O
O
Glc7
HO
HO
HO
HO
HO
HO
2 R1 =
R2 =
R3 = IF
O
OH
OH
OH
Figure 1. The structures of compounds 1–3.
comparison with authentic samples. Mild
alkaline hydrolysis of 1 afforded 1a.
The absolute configuration of the mono-
saccharides was determined to be D for
glucose and ribose and L for rhamnose and
arabinose by the 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 protons in
the 1H NMR spectrum (see Section 3) of 1
suggested that the arabinopyranosyl moiety
has an a-configuration and the glucopyr-
anosyl and ribopyranosyl moieties have a
b-configuration. The a-configurations of
the rhamnopyranosyl moieties were deter-
mined from the broad singlets observed for
the anomeric protons. The large 1JH–1,C–1
values of the rhamnosyl moieties (Rha1,
172 Hz; Rha2, 168 Hz; Rha3, 170 Hz)
confirmed that the anomeric protons were
equatorial (a-pyranoid anomeric form)
[11]. The spin–spin coupling system of
individual monosaccharide units was ident-
ified by the analysis of 1D total correlation
spectroscopy (1D TOCSY) and 2D NMR
spectra. 1H NMR spectral data of individual
monosaccharide units were obtained by the
selective irradiation of the anomeric pro-
tons or methyl groups of rhamnose units in
a series of 1D TOCSY experiments.
1
Analysis of the H–1H correlation spec-
troscopy (1H–1H COSY) spectrum resulted
in a sequential assignment of all proton
resonances of the 10 monosaccharide units
(see Table 2). In the heteronuclear single
quantum coherence (HSQC) experiment,
proton resonances were correlated with
those of the corresponding carbons, and the
associated anomeric protons were corre-
lated with their respective carbon atoms
from HSQC–TOCSY data, leading to
unambiguous assignments of the carbons
in each monosaccharide unit (see Table 3).
Taking into account the known effects of
O-glycosylation, 1 contains one L-arabino-
pyranosyl unit (Ara), one D-ribopyranosyl
unit (Rib), three L-rhamnopyranosyl units
(Rha), and five D-glucopyranosyl units
(Glc). Sugar aglycone and sugar–sugar
linkages were determined by HMBC and
NOESY data analyses. In the HMBC
spectrum, the anomeric proton signals at
dH 6.21 (Glc1-H-1), 4.99 (Glc2-H-1),

612
Q. Fu et al.
5.82 (Rha1-H-1), 5.04 (Ara1-H-1), 6.27
(Rha2-H-1), 5.80 (Rib1-H-1), 4.90 (Glc3-H-
1), 5.10 (Glc4-H-1), 5.09 (Glc5-H-1), and
5.42 (Rha3-H-1) showed cross-peaks with
the carbon signals at dC 176.5 (Agly-C-28),
69.1 (Glc1-C-6), 78.3 (Glc2-C-4), 80.9
(Agly-C-3), 75.3 (Ara1-C-2), 81.9 (Rha2-C-
3), 76.2 (Rib1-C-4), 81.0 (Glc3-C-4), 88.9
(Glc4-C-3), and 68.8 (Glc5-C-6), respect-
ively. These signals provide ample evidence
to determine the linkages between the
sugars, and the sugar and the aglycone.
From the above evidence, the structure of 1
was established as 3-O-a-L-rhamnopyrano-
syl-(1 ! 6)-b-D-glucopyranosyl-(1 ! 3)-
b-D-glucopyranosyl-(1 ! 4)-b-D-gluco-
pyranosyl-(1 ! 4)-b-D-ribopyranosyl-(1 !
3)-a-L-rhamnopyranosyl-(1 ! 2)-a-L-ara
binopyranosyl-hederagenin-28-O-a-L-rha
mnopyranosyl-(1 ! 4)-b-D-glucopyrano-
syl-(1 ! 6)-b-D-glucopyranoside, which
has been named clematochinenoside H.
Compound 2 was isolated as a white,
amorphous powder. The HR-ESI-MS
(positive-ion mode) experiment revealed
a pseudomolecular ion peak [M þ Na]þ at
m/z 2508.0181, in agreement with the
1
molecular formula C110H172O62. The H
NMR spectrum of 2 showed signals for 12
anomeric protons, along with signals for 3
deoxyhexopyranosyl units. The spectro-
scopic properties of 2 were closely related
to those of 1. When the 13C NMR spectrum
of 2 was compared with that of 1, two sets
of additional 12 signals corresponding to
two terminal b-D-glucopyranosyl groups
appeared, and two HMBC correlations
were observed between the signals at d
5.31 (Glc6-H-1) and d 82.0 (Rha3-C-2),
and d 4.90 (Glc7-H-1) and d 72.0 (Glc5-C-
4). Further analysis of the NMR spectral
data of 2 revealed the presence of a
3-hydroxy-4-methoxycinnamoyl group
(isoferuloyl) (Table 4). The typical protons
of the isoferuloyl moiety resonated at dH
7.52 (1H, d, J ¼ 2.0Hz), 7.09 (1H, dd,
J ¼ 8.5, 2.0 Hz), 6.90 (1H, d, J ¼ 16.0 Hz),
8.10 (1H, d, J ¼ 16.0 Hz), 6.86 (1H, d,
J ¼ 8.5 Hz), and 3.73 (3H, s) and dC 116.4,

Journal of Asian Natural Products Research
613

614
Q. Fu et al.
121.4, 112.1, 145.6, 115.4, 150.9, 148.4,
128.6, 166.7, and 55.8. The correlation
observed between Glc4-H-2 at d 5.71 and
the carbonyl carbon at d 166.7 in the
HMBC spectrum indicated that the iso-
feruloyl moiety is linked to the sugar chain
on Glc4-C-2 (d 73.2). Accordingly, the
structure of 2 was determined as 3-O-b-
D-glucopyranosyl-(1 ! 2)-a-L-rhamno-
pyranosyl-(1 ! 6)-[b-D-glucopyranosyl-
(1 ! 4)]-b-D-glucopyranosyl-(1 ! 3)-
[(2-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-
hederagenin-28-O-a-L-rhamnopyranosyl-
(1 ! 4)-b-D-glucopyranosyl-(1 ! 6)-b-D-
glucopyranoside (clematochinenoside I).
The molecular formula of 3 was
determined as C110H172O62 from the
pseudomolecular ion peak at m/z
2508.0181 [M þ Na]þ in the HR-ESI-MS.
Acid hydrolysis of 3 with 2 N aqueous
CF3COOH gave hederagenin, isoferulic
acid, L-arabinose, D-glucose, L-rhamnose,
and D-ribose. Comparison of the NMR data
for 3 and 2 revealed that the compounds are
similar except for the linkage site of the
terminal glucopyranosyl group (Glc7) in the
sugar chain, which is connected to Glc6-C-2
in 3 instead of Rha3-C-2 in 2 according to
HMBC correlations observed between
Glc7-H-1 at d 5.22 and Glc6-C-2 at d
84.4. The proton and carbon signals were
assigned unambiguously using 1H, 13C,
1H–1H COSY, 1D TOCSY, HSQC,
HMBC, and nuclear overhauser effect
spectroscopy (NOESY) NMR experiments.
Therefore, the structure of 3 was proposed
as 3-O-b-D-glucopyranosyl-(1 ! 2)-b-D-
glucopyranosyl-(1 ! 4) [a-L-rhamno-
pyranosyl-(1 ! 6)]-b-D-glucopyranosyl-
(1 ! 3)-[(2-O-isoferuloyl)-b-D-gluco-
pyranosyl]-(1 ! 4)-b-D-glucopyranosyl-
(1 ! 4)-b-ribopyranosyl-(1 ! 3)-a-L-
rhamnopyranosyl-(1 ! 2)-a-L-arabino-
pyranosyl-hederagenin-28-O-a-L-rham-
nopyranosyl-(1 ! 4)-b-D-glucopyrano-

Journal of Asian Natural Products Research
615

616
Q. Fu et al.
Table 4. 1H NMR and 13C NMR spectral data for the isoferuloyl moieties of compounds 2 and 3
1
(500 MHz for H NMR, 125 MHz for 13C NMR, in C5D5N).
2
3
No.
1H
13C
1H
13C
1
2
3
4
5
6
7
8
166.7
116.4
145.6
128.6
115.4
148.4
150.9
112.1
121.4
55.8
166.6
116.4
145.6
128.6
115.4
148.4
150.9
112.1
121.4
55.8
6.90 d (16.0)
8.10 d (16.0)
6.90 d (16.0)
8.11 d (16.0)
7.52 d (2.0)
7.53 d (2.0)
6.86 d (8.5)
7.09 dd (8.5 2.0)
3.73 s
6.88 d (8.5)
7.09 dd (8.5 2.0)
3.73 s
9
OCH3
syl-(1 ! 6)-b-D-glucopyranoside (clem-
Tianjin, China), and C18 silica gel (150–
200 mesh, Merck, Darmstadt, Germany;
performed by applying a N2 pressure of
0.5 MPa).
atochinenoside J).
3. Experimental
3.1 General experimental procedures
3.2 Plant materials
Optical rotations were measured on a
PerkinElmer 243B digital polarimeter
(PerkinElmer, Waltham, MA, USA). UV
spectra were obtained on a TU-1901
spectrometer (Puxi, Beijing, China). IR
spectra were recorded on an Avater-360
spectrometer (Thermo Scientific, Wal-
tham, MA, USA). NMR spectra were
recorded on a Bruker INOVA 500
spectrometer (Bruker, Billerica, MA,
USA), with tetramethylsilane as an
internal standard. HR-ESI-MS was
measured on a Bruker APEX IV FT-MS
(7.0 T) mass spectrometer (Bruker) in the
positive-ion mode. HPLC was performed
on an octadecylsilanized column (Agilent
Technologies 250 £ 10 mm i.d., 5 mm,
Santa Clara, CA, USA) with an Alltech
evaporative light scattering detector
(Grace, Dearfield, IL, USA). GC was
carried out on an Agilent 6890N gas
chromatograph (Agilent Technologies).
Column chromatography was performed
with silica gel (200–300 mesh, Qingdao
Haiyang Chemical Co., Ltd, Qingdao,
China), D101 porous polymer resin
(Tianjin Chemical Industry Co., Ltd,
The roots and rhizomes of C. chinensis
were collected in September 2007 in
Shaoguan, Guangdong Province, China.
The identification of the plant was
performed by one of the authors (P.-F.T.).
A voucher specimen (CC 200712) is
maintained in the Herbarium of Peking
University Modern Research Center for
Traditional Chinese Medicine.
3.3 Extraction and isolation
The dried roots and rhizomes (2 kg) of C.
chinensis were extracted with 50% EtOH.
After removing the solvent, the residue
(400 g) was suspended in H2O and
extracted with n- BuOH. The n-BuOH
extract (130 g) was subjected to D101
porous polymer resin column chromatog-
raphy and eluted with H2O and 30% and
80% EtOH, successively. The fraction
eluted with 30% EtOH (120 g) was then
subjected to silica gel column chromatog-
raphy and eluted with CHCl3–MeOH (4:1,
1:1) to afford fractions 1 and 2 (31 and
15 g, respectively). Fraction 2 (15 g) was

Journal of Asian Natural Products Research
617
subjected to C18 silica gel column
chromatography and eluted with MeOH–
H2O in a gradient of MeOH (MeOH–H2O,
15:85–60:40%) to afford subfractions 2-1
to 2-4 (2.5, 3.2, 1.7, and 3.2 g, respect-
ively). Subfraction 2-2 (1.0 g) was isolated
by preparative HPLC (MeCN–H2O,
28:72, 2.0 ml/min) to yield compounds 1
(52 mg). Subfraction 2-4 (1.5 g) was
isolated by preparative HPLC (MeCN–
H2O, 30:70, 2.0 ml/min) to yield com-
pounds 2 (31 mg) and 3 (37 mg).
[M þ Na]þ (calcd for C110H172O49Na,
2508.0198).
3.3.3 Clematochinenoside J (3)
20
White amorphous solid, ½aꢀD 255 (c 1.0,
MeOH); UV (MeOH) lmax (log 1) 243
(2.33), 295 (sh), 324 (4.28) nm; IR(KBr)
n
max (cm21): 3402, 2932, 1726, and 1630;
1H NMR (C5D5N, 500 MHz): d 1.15 (3H,
s, Me-27), 1.10 (3H, s, Me-24), 1.07 (3H,
s, Me-26), 0.93 (3H, s, Me-25), 0.85 (3H, s,
Me-29), 0.84 (3H, s, Me-30), 4.26 (1H,
dd, J ¼ 12.0, 5.0 Hz, H-3), 3.89 (1H, d,
J ¼ 12.0 Hz, H-23a), 4.22 (1H, d,
J ¼ 12.0 Hz, H-23b), 5.37 (1H, br s, H-
12); 1H NMR spectral data for sugar
moieties, see Table 2; 13C NMR spectral
3.3.1 Clematochinenoside H (1)
20
White amorphous solid, ½aꢀD 232 (c 1.0,
MeOH); IR (KBr) nmax (cm21): 3422,
1
2927, 1730, and 1632; H NMR (C5D5N,
data, see Tables 1 and 3; 1H NMR and 13
C
500 MHz): d 1.14 (3H, s, Me-27), 1.08
(3H, s, Me-24), 1.06 (3H, s, Me-26), 0.93
(3H, s, Me-25), 0.85 (3H, s, Me-29), 0.84
(3H, s, Me-30), 4.24 (1H, dd, J ¼ 12.0,
5.0 Hz, H-3), 3.85 (1H, d, J ¼ 12.0 Hz, H-
23a), 4.21 (1H, d, J ¼ 12.0 Hz, H-23b),
data for sugar moieties, see Table 2; 13C
NMR spectral data, see Tables 1 and 3.
HR-ESI-MS: m/z 2007.8625 [M þ Na]þ
(calcd for C88H144O49Na, 2007.8668).
NMR spectral data for isoferuloyl moiety,
see Table 4. HR-ESI-MS: m/z 2508.0181
[M þ Na]þ (calcd for C110H172O49Na,
2508.0198).
1
5.36 (1H, br s, H-12); H NMR spectral
3.4 Acid hydrolysis
Each compound (5 mg) was hydrolyzed
with 2 N aqueous CF3COOH (10 ml) at
1108C for 2 h. The reaction mixture was
diluted with H2O (20 ml) and extracted
with EtOAc (3 £ 10 ml). The combined
EtOAc extract was evaporated under
reduced pressure and analyzed by TLC.
Hederagenin was detected as a product of
1–3, and isoferulic acid as a product of 2
and 3. The aqueous layer was repeatedly
evaporated with MeOH under vacuum
until the solvent was completely removed.
The residue was dissolved in anhydrous
pyridine (100 ml) and then mixed with a
pyridine solution of L-cysteine methyl
ester hydrochloride (100 ml). After warm-
ing at 608C for 1 h, hexamethyldisilazane
(100 ml) and trimethylsilyl chloride (40 ml)
were added, and the mixture was warmed
at 608C for another 30 min. The mixture
was filtered through a 0.45 mm membrane
to remove the precipitate and analyzed
by GC. Separations were carried out on a
3.3.2 Clematochinenoside I (2)
20
White amorphous solid, ½aꢀD 258 (c 1.0,
MeOH); UV (MeOH) lmax (log 1) 243
(2.34), 295 (sh), 324 (4.28) nm; IR (KBr)
n
max (cm21): 3402, 2931, 1727, and 1632;
1H NMR (C5D5N, 500 MHz): d 1.15 (3H, s,
Me-27), 1.10 (3H, s, Me-24), 1.07 (3H,
s, Me-26), 0.93 (3H, s, Me-25), 0.85 (3H, s,
Me-29), 0.84 (3H, s, Me-30), 4.26 (1H,
dd, J ¼ 12.0, 5.0 Hz, H-3), 3.89 (1H, d,
J ¼ 12.0 Hz, H-23a), 4.22 (1H, d,
J ¼ 12.0 Hz, H-23b), 5.37 (1H, br s, H-
12); 1H NMR spectral data for sugar
moieties, see Table 2; 13C NMR spectral
data, see Tables 1 and 3; 1H NMR and 13
C
NMR spectral data for isoferuloyl moiety,
see Table 4. HR-ESI-MS: m/z 2508.0181

618
Q. Fu et al.
HP-5 column (28 m £ 0.32 mm). Highly
pure helium was employed as carrier gas
(1.0 ml/min flow rate), and the flame
ionization detector was operated at 2608C
(column temp 1808C). The retention
times of the monosaccharide derivatives
were as follows: L-Rha, 5.41 min;
D-Glc, 11.63 min; D-Rib, 5.21 min; L-Ara,
4.97 min.
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Pure compound 1 (15 mg) was refluxed in
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Acknowledgements
We are grateful to members of the Analytical
Center of Peking University Health Science
Center for the measurements of the mass
spectra and elemental analysis.
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