Triterpene saponins from clematis chinensis and their potential anti-inflammatory activity

Article abstract of DOI:10.1021/np100057y

Seven new triterpene saponins, clematochinenosides A-G (1-7), together with 17 known saponins (8-24), were isolated from the roots and rhizomes of Clematis chinensis. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products. Compounds 1, 3-7, and 20-24 showed inhibitory activities against COX-1 and COX-2 enzymes.

Full text of DOI:10.1021/np100057y

1234
J. Nat. Prod. 2010, 73, 1234–1239
Triterpene Saponins from Clematis chinensis and Their Potential Anti-inflammatory Activity
Qiang Fu, Ke Zan, Mingbo Zhao, Sixiang Zhou, Shepo Shi, Yong Jiang, and Pengfei Tu*
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking UniVersity Health Science Center, No. 38
Xueyuan Road, Beijing 100191, People’s Republic of China
ReceiVed January 26, 2010
Seven new triterpene saponins, clematochinenosides A-G (1-7), together with 17 known saponins (8-24), were isolated
from the roots and rhizomes of Clematis chinensis. Their structures were elucidated on the basis of spectroscopic evidence
and hydrolysis products. Compounds 1, 3-7, and 20-24 showed inhibitory activities against COX-1 and COX-2 enzymes.
Clematis chinensis Osbeck (Ranunculaceae) is distributed widely
in the south of the People’s Republic of China. According to the
Chinese Pharmacopoeia,1 the roots and rhizomes of C. chinensis,
C. hexapetala Pall., and C. mandshurica Rupr. are collectively
termed “Weilingxian”, a traditional Chinese herbal drug that is
commonly used as an anti-inflammatory, antitumor, and analgesic
agent. However, previous phytochemical investigations have re-
vealed that the chemical constituents of these three species are quite
different. The roots and rhizomes of C. mandshurica and C.
chinensis are rich in saponins,2-13 whereas the major components
of C. hexapetala are flavonoids.14 The aim of the present study
was to explore the similarities in chemical constituents and
bioactivity of tissues from these three species. Previously, we
reported on the isolation and structural elucidation of flavonoids
from C. hexapetala14 and seven bidesmosides of oleanolic acid of
and lignans from C. mandshurica.13,15 Herein we report on the
isolation and structure elucidation of seven new saponins based
upon hederagenin and oleanolic acid from the roots and rhizomes
of C. chinensis and on the inhibitory activity of all compounds
isolated against the cyclooxygenase-1 (COX-1) and cyclooxy-
genase-2 (COX-2) enzymes.
Results and Discussion
A 50% EtOH extract of the dried roots and rhizomes of C.
chinensis (4 kg) was suspended in H2O and successively extracted
with petroleum ether, EtOAc, and n-BuOH. The n-BuOH-soluble
fraction was subjected to column chromatography over porous
polymeric resin (D101), silica gel, and C18 silica gel, to yield
compounds 1-24. Comparison of their NMR and MS data with
reported values revealed the structures of the known compounds
8-24 to be cirensenoside O,16 kizutasaponin K3,9 ciwujianoside
C3,17 kizutasaponin K10,18 hederasaponin B,7 kizutasaponin K12,7
huzhangoside B,7 huzhangoside D,7 clematichinenoside C,10 clem-
1H NMR spectra (Table 1) of these compounds suggested that the
arabinopyranosyl moieties have an R-configuration and the glu-
copyranosyl and ribopyranosyl moieties a ꢀ-configuration. The
R-configurations of the rhamnopyranosyl moieties were determined
from the broad singlets observed for the anomeric protons. The
downfield chemical shift at δC 80.9-88.7 (Agly-3) and the upfield
chemical shift at δC 176.4-176.5 (Agly-28) in the 13C NMR spectra
of 1-7 (Table 2) indicated these compounds to be bisdesmosidic
saponins.
atichinenoside B,9 clematomanshurica saponin C,13 3-O-ꢀ-D-
glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-ribopyranosyl-
(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyranosylhederagenin
28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-
glucopyranoside,19 clematernosides B-E,8 and clematernoside K,8
respectively.
Compounds 1-7 were isolated as white, amorphous powders.
The monosaccharides obtained after aqueous acid hydrolysis of each
compound were identified as glucose, rhamnose, arabinose, and
ribose by TLC comparison with authentic samples. The absolute
configuration of the monosaccharides was 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 Experimental Section). The relatively large
coupling constants (5.0-8.0 Hz) for the anomeric protons in the
The molecular formula of 1 was determined as C104H162O57 by
HRESIMS (positive-ion mode) experiments, which revealed a
pseudomolecular ion peak [M + Na]+ at m/z 2345.9679 (calcd
2345.9670). The aglycon of 1 was identified as hederagenin by
1
comparison of the H and 13C NMR data obtained in 2D NMR
experiments with literature values.20 The H NMR spectrum of 1
1
exhibited 11 anomeric proton resonances at δ 6.26 (1H, brs), 6.20
(1H, d, J ) 8.0 Hz), 5.83 (1H, brs), 5.79 (1H, d, J ) 5.0 Hz), 5.43
(1H, brs), 5.31 (1H, d, J ) 8.0 Hz), 5.29 (1H, d, J ) 8.0 Hz), 5.04
* To whom correspondence should be addressed. Tel/Fax: +86-10-
82802750. E-mail: pengfeitu@vip.163.com.
10.1021/np100057y  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/11/2010

Triterpene Saponins from Clematis chinensis
Journal of Natural Products, 2010, Vol. 73, No. 7 1235
Table 1. 1H NMR Data of Compounds 1 and 2 (500 MHz, in C5D5N)
position
1
2
position
1
2
Glc1
1
2
3
4
5a
5b
Glc3
1
2
3
4.29 m
4.29 m
4.28 m
4.28 m
6.20 d (8.0)
4.08 dd (9.0, 8.0)
4.20 dd (9.0, 9.0)
4.28 m
6.21 d (8.0)
4.08 dd (9.0, 8.0)
4.19 dd (9.0, 9.0)
4.28 m
4.90 d (8.0)
3.87 dd (9.0, 8.0)
4.15 m
4.90 d (8.0)
3.86 dd (9.0, 8.0)
4.12 m
5
4.07 m
4.07 m
6a
6b
Glc2
1
2
3
4.66 m
4.31 m
4.66 m
4.31 m
4
5
6a
6b
Glc4
1
4.27 m
3.64 m
4.23 m
4.14 m
4.26 m
3.64 m
4.23 m
4.14 m
4.99 d (8.0)
3.92 dd (9.0, 8.0)
4.14 d (9.0)
4.40 (9.0)
3.66 m
4.94 d (8.0)
3.91 dd (9.0, 8.0
4.19 d (9.0)
4.41 (9.0)
3.62 m
5.29 d (8.0)
5.71 dd (9.0, 8.0)
4.13 m
5.10 d (8.0)
4.06 dd (9.0, 8.0)
4.01 m
4
5
2
3
6a
6b
Rha1
1
2
3
4.19 m
4.05 m
4.16 m
4.07 m
4
5
6a
6b
Glc5
1
2
3
4
5
4.04 m
3.64 brd (10.0)
4.22 m
4.05 m
3.86 m
4.22 m
4.17 m
5.83 brs
4.64 brs
4.54 dd (9.0, 3.5)
4.33 dd (9.0, 9.0)
4.93 m
5.83 brs
4.64 brs
4.53 dd (9.0, 3.5)
4.32 dd (9.0, 9.0)
4.92 m
3.94 m
4.83 d (8.5)
5.09 d (8.0)
3.91 dd (9.0, 8.0)
4.07 dd (9.0, 9.0)
3.89 dd (9.0, 9.0)
4.06 m
4
5
6
3.87 dd (9.0, 8.5)
4.06 dd (9.0, 9.0)
3.82 dd (9.0, 9.0)
4.06 m
4.53 brd (10.0)
3.69 m
1.67 d (6.0)
1.67 d (6.0)
Ara1
1
5.04 d (6.5)
4.56 dd (8.0 6.5)
3.99 dd (8.0 4.0)
4.11 m
5.05 d (6.5)
4.54 dd (8.0 6.5)
4.01 dd (8.0 4.0)
4.10 m
6a
6b
Rha3
1
4.48 brd
3.78 brd
2
3
4
5.43 brs
5.46 brs
5a
5b
Rha2
1
4.22 d (9.5)
3.64 d (9.5)
4.23 d (9.5)
3.67 d (9.5)
2
3
4
5
4.76 brs
4.56 m
4.17 dd (9.0, 9.0)
4.22 m
4.83 brs
4.54 m
4.16 dd (9.0, 9.0)
4.25 m
6.26 brs
6.28 brs
2
3
4
5
4.85 brs
4.64 m
4.39 dd (9.0, 9.0)
4.62 m
4.83 brs
4.67 m
4.39 dd (9.0, 9.0)
4.64 m
6
1.55 d (6.0)
1.55 d (6.0)
Glc6
1
5.31 d (8.0)
4.07 m
5.28 d (8.0)
4.07 m
2
6
1.50 d (6.0)
1.50 d (6.0)
3
4
5
6a
6b
4.20 m
4.22 m
3.95 m
4.47 dd (12.0, 5.0)
4.36 dd (12.0, 5.0)
4.19 m
4.26 m
3.93 m
4.45 dd (12.0, 5.0)
4.34 dd (12.0, 5.0)
Rib1
1
5.79 d (5.0)
4.09 m
4.62 m
5.80 d (5.0)
4.07 m
4.64 m
2
3
4
4.31 m
4.29 m
(1H, d, J ) 6.5 Hz), 4.99 (1H, d, J ) 8.0 Hz), 4.90 (1H, d, J ) 8.0
Hz), and 4.83 (1H, d, J ) 8.0 Hz), respectively. The three-proton
doublets observed at δ 1.67 (3H, d, J ) 6.0 Hz), 1.55 (3H, d, J )
6.0 Hz), and 1.50 (3H, d, J ) 6.0 Hz) suggested the presence of
three deoxyhexopyranosyl units in 1. The spin-spin coupling
system of individual monosaccharide units was identified by
analysis of 1D TOCSY and 2D NMR spectra. 1H NMR spectra of
individual monosaccharide units were obtained by selective irradia-
tion of the anomeric protons or methyl groups of rhamnose units
on mild alkaline hydrolysis, which afforded isoferulic acid and
2 (see below). The correlation observed between Glc4-H-2 and
the carbonyl carbon (δ 166.7) of the isoferuloyl moiety in the
HMBC spectrum indicated that the isoferuloyl moiety is linked
to the sugar chain on Glc4-C-2 (δ 72.7). From the above
evidence, the structure of 1 was established as 3-O-ꢀ-D-
glucopyranosyl-(1f2)-R-L-rhamnopyranosyl-(1f6)-ꢀ-D-glu-
copyranosyl-(1f3)-[(2-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-(1f4)-
ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-
1
in a series of 1D TOCSY experiments. Analysis of the H-1H
rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl
hederagenin
COSY spectrum resulted in sequential assignment of all proton
resonances of the 11 monosaccharide units, including identification
of most of their multiple splitting patterns and coupling constants,
as shown in Table 1. In HSQC experiments, proton resonances
were correlated 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.
Comparison of the carbon chemical shifts thus assigned with
those of reference methyl glycosides,21 taking into account the
known effects of O-glycosylation, indicated that 1 contains one
L-arabinopyranosyl unit (Ara), one D-ribopyranosyl unit (Rib),
three L-rhamnopyranosyl units (Rha), and six D-glucopyranosyl
units (Glc). Sugar-aglycon and sugar-sugar linkages were
determined by HMBC and NOESY data analysis (Tables 1-3).
Further analysis of the NMR data of 1 revealed the presence of
a 3-hydroxy-4-methoxycinnamoyl group (isoferuloyl) (Tables 4
and 5). The occurrence of this substructure in 1 was confirmed
28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-
ꢀ-D-glucopyranoside, which has been named clematochinenoside
A.
The molecular formula of 2 was determined as C94H154O54 by
HRESIMS, which showed a pseudomolecular ion peak [M + Na]+
at m/z 2169.9244 (calcd 2169.9197). Acid hydrolysis of 2 with 2
N aqueous CF3COOH gave hederagenin, L-arabinose, D-glucose,
L-rhamnose, and D-ribose. Comparison of the NMR data for 2 and
1 revealed that the compounds are similar except that signals due
to the isoferuloyl moiety linked to Glc4-C-2 in 1 were absent for
2. This observation was supported by a relative upfield shift of
1
Glc4-H-2 of 2 at δ 4.06 (Glc4-H-2 of 1, δ 5.71) in the H NMR
spectrum and a downfield shift of Glc4-C-1 of 2 at δ 104.3 (Glc4-
C-1 of 1, δ 102.1) in the 13C NMR spectrum. Thus, the structure
of 2 was determined as 3-O-ꢀ-D-glucopyranosyl-(1f2)-R-L-rham-
nopyranosyl-(1f6)-ꢀ-D-glucopyranosyl-(1f3)-ꢀ-D-glucopyranosyl-
(1f4)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-
rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl
hederagenin

1236 Journal of Natural Products, 2010, Vol. 73, No. 7
Fu et al.
Table 2. 13C NMR Data for the Aglycon Moieties of 1-7 (125
values.4 The sugar sequence of 4a was confirmed on analysis of
its ESIMSn data in the negative mode, which showed fragments at
m/z 1043.6, 881.5, 749.4, 603.3, and 471.2, corresponding to [M
- Glc-H]-, [M - 2Glc-H]-, [M - 2Glc-Rib-H]-, [M - 2Glc-
Rib-Rha-H]-, and [M - 2Glc-Rib-Rha-Ara-H]-, respectively.
MHz, in C5D5N)
position
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
39.0
26.3
80.9
43.5
47.6
18.0
32.6
39.8
48.1
36.8
24.0
39.0
26.3
80.9
43.5
47.6
18.1
32.6
39.8
48.1
36.8
23.8
39.1
26.3
80.9
43.5
47.6
18.1
32.6
39.9
48.2
36.8
23.8
39.0
26.3
81.0
43.5
47.6
18.0
32.7
39.8
48.1
36.8
23.7
39.0
26.3
80.9
43.5
47.6
18.0
32.6
39.8
48.1
36.8
23.7
38.9
26.6
88.7
39.5
55.7
18.5
33.1
39.8
48.0
37.0
23.6
39.1
26.3
80.9
43.6
47.6
18.0
32.7
39.9
48.2
36.8
23.6
1
Comparison of the H and 13C NMR data for 4 with those for the
known compound clematernoside B revealed that these substances
differ only in the aglycon part. Thus, a set of carbon signals at δ
80.9 (Agly-3), 47.6 (Agly-5), and 63.9 (Agly-23) was observed in
the 13C NMR spectrum of 4 instead of δ 88.6 (Agly-3), 55.9 (Agly-
5), and 28.0 (Agly-23) for clematernoside B. On the basis of all
this evidence, the structure of 4 (clematochinenoside D) was assigned
as 3-O-[(3-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-(1f4)-ꢀ-D-glucopyra-
nosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-
R-L-arabinopyranosyl hederagenin 28-O-R-L-rhamnopyranosyl-(1f4)-
ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranoside.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
123.0 123.0 123.0 123.0 123.0 122.8 122.9
144.0 144.0 144.0 144.0 144.0 144.0 144.0
42.0
28.2
23.3
46.9
41.6
46.1
30.6
33.9
32.5
63.7
14.1
16.1
17.4
26.3
42.0
28.2
23.3
46.9
41.6
46.1
30.7
33.9
32.5
63.8
14.0
16.1
17.5
26.3
42.0
28.2
23.3
47.0
41.6
46.1
30.7
33.9
32.5
63.8
14.1
16.1
17.5
26.4
42.0
28.2
23.3
46.9
41.5
46.1
30.6
33.9
32.4
63.9
14.1
16.1
17.5
26.3
42.0
28.2
23.2
46.9
41.5
46.1
30.7
33.9
32.5
63.8
14.0
16.1
17.4
26.3
42.1
28.1
23.3
47.0
41.6
46.2
30.7
33.9
32.5
28.1
17.1
15.6
17.4
26.0
42.1
28.2
23.3
47.0
41.6
46.1
30.7
33.9
32.7
63.8
14.1
16.1
17.5
26.3
The molecular formula of 5 was determined as C86H132O43 from
the pseudomolecular ion peak [M + Na]+ at m/z 1875.7952 (calcd
1875.8035) in the HRESIMS. Analysis of the 1D and 2D NMR
spectroscopic data led to identification of the aglycon as heder-
agenin.20 Acid hydrolysis of 5 yielded hederagenin, isoferulic acid,
L-arabinose, D-glucose, L-rhamnose, and D-ribose. Comparison of
the 1H and 13C NMR spectra of 5 with that of 4 indicated that the
two compounds are similar structurally. The only difference found
was in the linkage site of the isoferuloyl group, which is at Glc4-
C-6 in 5 instead of Glc4-C-3 in 4. This correlation was established
by the relative downfield shift at δ 5.17 and 4.67 for Glc4-H-6 in
176.5 176.5 176.5 176.4 176.4 176.5 176.5
33.0
23.6
1
the H NMR spectrum of 4 (δ 4.18 and 4.49) and the long-range
33.0
23.6
33.0
23.6
33.0
23.6
33.0
23.6
33.1
23.7
33.0
23.6
correlation observed between Glc4-H-6 and the carbonyl carbon
(δ 167.2) of the isoferuloyl group in the HMBC spectrum. Thus,
the structure of 5 (clematochinenoside E) was established as 3-O-
[(6-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-(1f4)-ꢀ-D-glucopyranosyl-
(1f4)-ꢀ-D- ribopyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-
L-arabinopyranosyl hederagenin 28-O-R-L-rhamnopyranosyl(1f4)-
ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranoside.
28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-
glucopyranoside, (clematochinenoside B).
The molecular formula of 3 was determined as C104H162O57 from
the pseudomolecular ion peak [M + Na]+ at m/z 2345.9673 (calcd
2345.9670) in the HRESIMS. 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 1 revealed that the compounds are similar except for the
linkage site of the terminal glucopyranosyl group (Glc6) in the sugar
chain, which is connected to Glc5-C-4 in 3 instead of to Rha3-C-2
in 1 according to HMBC correlations observed between Glc6-H-1
at δ 4.95 and Glc5-C-4 at δ 82.0. This was further supported by
NOE correlations observed between Glc6-H-1 and Glc5-H-4 at
δ 4.12. The proton and carbon signals were assigned unambigu-
ously using 1H, 13C, 1H-1H COSY, 1D TOCSY, HSQC, HMBC,
and NOESY NMR experiments. Therefore, the structure of 3
(clematochinenoside C) was proposed as 3-O-R-L-rhamnopyra-
nosyl-(1f6)-[ꢀ-D-glucopyranosyl-(1f4)]-ꢀ-D-glucopyranosyl-
(1f3)-[(2-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-(1f4)-ꢀ-D-glu-
copyranosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-rhamnopyra-
nosyl-(1f2)-R-L-arabinopyranosyl hederagenin 28-O-R-L-rham-
nopyranosyl-(1f4)-ꢀ-D -glucopyranosyl-(1f6)-ꢀ-D-
glucopyranoside.
The molecular formula of 4 was determined as C86H132O43 by
HRESIMS, which showed a pseudomolecular ion peak [M + Na]+
at m/z 1875.8055 (calcd 1875.8035). Studies of 1D and 2D NMR
spectra data led to identification of hederagenin as the aglycon in
4.20 Acid hydrolysis of 4 with 2 N aqueous CF3COOH gave
hederagenin, isoferulic acid, L-arabinose, D-glucose, L-rhamnose,
and D-ribose. Mild alkaline hydrolysis of 4 with 0.1 N KOH
afforded isoferulic acid and 3-O-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-
glucopyranosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-rhamnopyra-
nosyl-(1f2)-R-L-arabinopyranosyl hederagenin 28-O-R-L-rham-
nopyranosyl-(1f4)-ꢀ-D -glucopyranosyl-(1f6)-ꢀ-D -
glucopyranoside.19 Alkaline hydrolysis of 4 afforded a prosapogenin
(4a), which was identified as CP10 by direct comparison of 1H and
13C NMR data obtained in 2D NMR experiments with literature
The molecular formula of 6 was determined as C86H132O42 by
HRESIMS, which showed a pseudomolecular ion peak at m/z
1859.8105 [M + Na]+ (calcd 1859.8085). The aglycon of 6 was
1
identified as oleanolic acid by comparison of its H and 13C 2D
NMR spectroscopic data with reported values.22 Acid hydrolysis
of 6 yielded oleanolic acid, L-arabinose, D-glucose, L-rhamnose,
and D-ribose. Mild alkaline hydrolysis of 6 afforded a prosapogenin
1
(6a), which was identified as CP9 by direct comparison of H and
13C NMR data obtained in 2D NMR experiments with literature
values.4 The sugar sequence of 6a was confirmed on analysis of
its ESIMSn data in the negative mode, which gave fragments at
m/z 1027.6, 865.5, 733.4, 587.4, and 455.3, corresponding to [M
- Glc-H]-, [M - 2Glc-H]-, [M - 2Glc-Rib-H]-, [M - 2Glc-
Rib-Rha-H]-, and [M - 2Glc-Rib-Rha-Ara-H]-, respectively.
1
Comparison of the H and 13C NMR data for 6 with those for 5
revealed that they differ only in the aglycon part. Thus, a set of
carbon signals at δ 88.7 (Agly-3), 55.7 (Agly-5), and 28.1 (Agly-
23) was observed for 6 compared to δ 80.9 (Agly-3), 47.6 (Agly-
5), and 63.8 (Agly-23) for 5. NMR signals due to the sugar moieties
were identical for both compounds, except for signals due to an
arabinopyranosyl moiety connected to the aglycon (Ara-H-1 at δ
4.82, Ara-H-3 at δ 4.25, Ara-C-1 at δ 105.3, Ara-C-3 at δ 74.8).
Thus, the structure of 6 (clematochinenoside F) was determined
unambiguously as 3-O-[(6-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-
(1f4)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D- ribopyranosyl-(1f3)-R-L-
rhamnopyranosyl-(1f2)-R-L-arabinopyranosyl oleanolic acid 28-
O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-
glucopyranoside.
The molecular formula of 7 was determined as C92H142O48 from
the pseudomolecular ion peak at m/z 2037.8506 [M + Na]+ (calcd
2037.8563) in the HRESIMS. Acid hydrolysis of 7 afforded
hederagenin, L-arabinose, D-glucose, L-rhamnose, and D-ribose.

Triterpene Saponins from Clematis chinensis
Journal of Natural Products, 2010, Vol. 73, No. 7 1237
Table 3. 13C NMR Data for the Sugar Moieties of Compounds 1-7 (125 MHz, in C5D5N)
position
1
2
3
4
5
6
7
position
1
2
3
4
5
6
7
Glc1
Glc3
1
2
3
4
95.6
73.8
78.6
70.7
78.0
69.1
95.6
73.8
78.6
70.7
78.0
69.1
95.6
73.8
78.7
70.8
78.0
69.2
95.5
73.8
78.6
70.7
78.0
69.1
95.5
73.8
78.6
70.7
78.0
69.1
95.5
73.8
78.6
70.7
78.0
69.1
95.6
73.8
78.6
70.8
78.0
69.2
1
2
3
4
5
6
102.8
74.3
76.4
81.0
76.3
60.6
103.1
74.2
76.6
80.9
76.4
61.9
102.9
74.2
76.5
81.1
76.2
60.6
103.1
74.2
76.4
81.0
76.4
61.2
103.1
74.2
76.5
81.8
76.4
61.5
103.1
74.2
76.5
81.8
76.4
61.5
102.9
74.2
76.5
81.1
76.4
60.7
5
6
Glc2
Glc4
1
2
3
4
5
1
2
3
4
104.7
75.3
76.3
78.3
77.1
61.2
104.8
75.3
76.4
78.2
77.1
61.2
104.8
75.3
76.5
78.2
77.1
61.2
104.7
75.3
76.4
78.1
77.1
61.2
104.7
75.3
76.4
78.3
77.1
61.2
104.7
75.3
76.4
78.1
77.1
61.2
104.8
75.3
76.3
78.2
77.1
61.2
102.1
72.7
87.3
70.1
77.7
61.9
104.3
73.2
88.9
69.4
77.7
61.4
102.2
72.7
86.5
70.1
77.8
62.0
104.3
72.5
78.9
69.1
78.1
61.9
105.1
72.3
78.6
71.2
75.5
64.2
105.1
72.3
78.6
71.2
75.6
64.2
102.3
72.9
86.4
70.2
77.9
62.2
5
6
6
Rha1
Glc5
1
1
2
3
4
102.6
72.5
72.7
73.9
70.2
18.5
102.6
72.4
72.7
73.9
70.2
18.5
102.7
72.6
72.8
74.0
70.3
18.5
102.7
72.5
72.7
73.9
70.2
18.4
102.7
72.5
72.7
73.9
70.2
18.4
102.7
72.5
72.7
73.9
70.2
18.4
102.7
72.5
72.7
74.0
70.3
18.5
105.8
74.5
78.3
71.8
76.3
68.9
105.8
75.2
78.3
72.0
76.4
68.8
105.2
73.9
76.4
82.0
74.8
68.0
105.5
74.6
78.3
71.5
78.7
62.6
2
3
4
5
5
6
6
Ara1
Rha3
1
1
2
3
4
104.6
75.3
75.2
69.7
66.3
104.6
75.3
75.2
69.7
66.3
104.7
75.3
75.2
69.7
66.4
104.6
75.3
75.1
69.6
66.2
104.6
75.3
75.1
69.6
66.2
105.3
75.3
74.8
69.4
65.5
104.7
75.3
75.2
69.7
66.4
101.3
82.0
72.6
74.5
69.6
18.5
101.3
82.0
72.6
74.7
69.7
18.5
102.7
71.9
72.7
73.9
69.9
18.6
2
3
4
5
5
Rha2
6
1
2
3
4
5
6
101.3
71.8
81.9
72.7
69.6
18.4
101.4
71.9
82.0
72.8
69.6
18.4
101.4
71.9
82.1
72.7
69.8
18.4
101.3
71.8
81.2
72.6
69.7
18.4
101.3
71.8
82.0
72.5
69.6
18.4
101.4
71.9
82.1
72.5
69.7
18.4
101.4
71.9
80.9
72.7
69.7
18.4
Glc6
1
107.2
75.9
78.3
71.2
78.6
62.3
107.2
75.9
78.5
71.2
78.6
62.4
105.2
74.7
78.3
71.6
78.5
62.5
2
3
4
5
Rib1
1
2
3
4
5
6
104.6
72.6
69.6
76.2
61.5
104.6
72.5
69.5
76.3
61.6
104.7
72.5
69.6
76.5
61.5
104.7
72.6
69.6
76.4
61.7
104.7
72.6
69.6
76.4
61.6
104.7
72.6
69.8
76.4
61.7
104.7
72.5
69.7
76.4
61.5
Table 4. 1H NMR Data for the Isoferuloyl Moieties of Compounds 1 and 3-7 (500 MHz, in C5D5N)
position
1
3
4
5
6
7
2
3
5
6.89 d (16)
8.09 d (16)
7.52 d (2)
6.90 d (16)
8.12 d (16)
7.53 d (2)
6.56 d (16)
7.86 d (16)
7.41 d (2)
6.64 d (16)
8.01 d (16)
7.50 d (2)
6.64 d (16)
8.01 d (16)
7.50 d (2)
6.89 d (16)
8.10 d (16)
7.52 d (2)
8
9
6.87 d (8.5)
7.07 dd (8.5, 2)
3.73 s
6.87 d (8.5)
7.09 dd (8.5, 2)
3.73 s
6.89 d (8.5)
6.99 dd (8.5, 2)
3.71 s
6.83 d (8.5)
7.11 dd (8.5, 2)
3.73 s
6.83 d (8.5)
7.11 dd (8.5, 2)
3.73 s
6.87 d (8.5)
7.08dd (8.5, 2)
3.73 s
OCH3
Table 5. 13C NMR Data for the Isoferuloyl Moieties of
Compounds 1 and 3-7 (125 MHz, in C5D5N)
(1f2)-R-L-arabinopyranosyl hederagenin 28-O-R-L-rhamnopyranosyl-
(1f4)-ꢀ-D-glucopyranosyl-(1f6)-ꢀ-D-glucopyranoside.
Compounds 1-24 were assayed for their potential anti-inflam-
matory activity against the COX-1 and COX-2 enzymes. Com-
pounds 1, 3-7, and 20-24 showed inhibitory activities against both
enzymes (Table 6). However, no COX-2 selectivity was evident
for any of the active compounds.
position
1
3
4
5
6
7
1
2
3
4
5
6
7
8
166.7
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
167.2
116.5
145.1
128.3
115.2
148.3
150.8
111.9
121.7
55.7
167.2
115.3
145.8
128.3
115.2
148.3
150.8
111.9
121.7
55.7
167.3
115.8
145.8
128.3
115.2
148.3
150.8
111.9
121.7
55.7
166.8
116.4
145.7
128.6
115.4
148.4
150.9
112.1
121.5
55.8
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 243B digital polarimeter. UV spectra were obtained
on a TU-1901 spectrometer. IR spectra were recorded on an Avater-
360 spectrometer. NMR spectra were recorded on a Bruker INOVA
500 spectrometer with TMS as internal standard. HRESIMS was
measured on a Bruker APEX IV FT-MS (7.0 T) mass spectrometer in
the positive-ion mode. ESIMS was obtained on an Agilent XCT 6320
IT mass spectrometer in the negative-ion mode. HPLC was performed
on an ODS column (Agilent Technologies 250 × 10 mm i.d., 5 µm)
with an Alltech evaporative light scattering detector. GC was employed
on an Agilent 6890N gas chromatograph. Column chromatography was
performed with silica gel (200-300 mesh, Qingdao Haiyang Chemical
9
OCH3
Comparison of the NMR data for 7 with those of 1 revealed that
the Rha3 linked to Glc5-C-6 in 1 was absent in 7. This was supported
by a relative upfield shift of the 13C NMR peak at δ 62.6 for Glc5-
C-6 in 7 compared to δ 68.9 in 1. Thus, the structure of 7
(clematochinenoside G) was determined as 3-O-ꢀ-D-glucopyranosyl-
(1f3)-[(2-O-isoferuloyl)-ꢀ-D-glucopyranosyl]-(1f4)-ꢀ-D-glucopy-
ranosyl-(1f4)-ꢀ-D-ribopyranosyl-(1f3)-R-L-rhamnopyranosyl-

1238 Journal of Natural Products, 2010, Vol. 73, No. 7
Fu et al.
5.05 (1H, d, J ) 8.0 Hz, Ara1-H-1), 4.98 (1H, d, J ) 8.0 Hz, Glc2-
H-1), 4.95 (1H, d, J ) 8.0 Hz, Glc6-H-1), 4.90 (1H, d, J ) 8.0 Hz,
Glc5-H-1), 4.89 (1H, d, J ) 8.0 Hz, Glc3-H-1), 4.56 (1H, dd, J ) 8.0,
6.5 Hz, Ara1-H-2), 4.65 (1H, m, Rha2-H-3), 4.30 (1H, m, Rib1-H-4),
4.26 (1H, m, Glc3-H-4), 4.14 (1H, m, Glc4-H-3), 3.90 (1H, m, Glc5-
H-4), 4.87 (1H, m, Glc5-H-6a), 4.05 (1H, m, Glc5-H-6b), 1.69 (3H, d,
J ) 6.0 Hz, Rha1-H-6), 1.64 (3H, d, J ) 6.0 Hz, Rha3-H-6), 1.51 (3H,
d, J ) 6.0 Hz, Rha2-H-6); 1H NMR data of the isoferuloyl moiety, see
Table 4; 13C NMR data, see Tables 2, 3, and 5; HRESIMS m/z
2345.9673 [M + Na]+ (calcd for C104H162O57Na, 2345.9670).
Table 6. Inhibitory Effects of Compounds 1, 3-7, and 20-24
against Cyclooxygenases-(COX-) I and II
IC50 value (µM)
compounda COX-1
COX-2
6.7 21
IC50 value (µM)
compounda COX-1
COX-2
8.8
1
7.8
6.3
8.8
8.9
7.8
7.5
8.0
7.6
8.8
8.3
5.9
0.01
3
4
6.1 22
7.8 23
9.0
8.5
6.7
5
6
7
20
7.9 24
7.2 SC560b
8.9 NS398c
7.6
Clematochinenoside D (4): white, amorphous powder; [R]2D0 -52
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 242 (2.36), 298 (sh), 325
(4.11) nm; IR (KBr) νmax 3385, 2922, 1719, 1635, 1058 cm-1; 1H NMR
data (C5D5N, 500 MHz) δ 1.15 (3H, s, Me-27), 1.11 (3H, s, Me-24),
1.07 (3H, s, Me-26), 0.93 (3H, s, Me-25), 0.85 (3H, s, Me-29), 0.83
(3H, s, Me-30), 4.24 (1H, dd, J ) 12.0, 5.0 Hz, H-3), 4.23 (1H, d, J )
12.0 Hz, H-23a), 3.87 (1H, d, J ) 12.0 Hz, H-23b), 5.36 (1H, brs,
H-12), 6.33 (1H, brs, Rha2-H-1), 6.20 (1H, d, J ) 8.0 Hz, Glc1-H-1),
5.82 (1H, brs, Rha1-H-1), 5.78 (1H, d, J ) 5.0 Hz, Rib1-H-1), 5.27
(1H, d, J ) 7.5 Hz, Glc4-H-1), 5.04 (1H, d, J ) 6.5 Hz, Ara1-H-1),
4.97 (1H, d, J ) 8.0 Hz, Glc2-H-1), 4.92 (1H, d, J ) 8.0 Hz, Glc3-
H-1), 4.55 (1H, dd, J ) 8.0, 6.5 Hz, Ara1-H-2), 4.65 (1H, m, Rha2-
H-3), 4.31 (1H, m, Rib1-H-4), 4.27 (1H, m, Glc3-H-4), 1.68 (3H, d,
J ) 6.0 Hz, Rha1-H-6), 1.51 (3H, d, J ) 6.0 Hz, Rha2-H-6); 1H NMR
data of the isoferuloyl moiety, see Table 4; 13C NMR data, see Tables
2, 3, and 5; HRESIMS m/z 1875.8055 [M + Na]+ (calcd for
C86H132O43Na, 1875.8035).
0.15
a Compounds 2 and 8-19 were inactive (IC50 > 50 µM). b SC560 is a
selective inhibitor of COX-1. c NS398 is a selective inhibitor of COX-2.
Co., Ltd.), D101 porous polymer resin (Tianjin Chemical Industry Co.,
Ltd.), and C18 silica gel (150-200 mesh, Merck; performed by applying
a N2 pressure of 0.5 MPa).
Plant Material. The roots and rhizomes of C. chinensis were
collected in December 2007 in Shaoguan, Guangdong Province,
People’s Republic of 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.
Extraction and Isolation. The dried roots and rhizomes (10 kg) of
C. chinensis were extracted with 50% EtOH (3 × 30 L). After removing
the solvent, the residue (4000 g) was suspended in H2O and successively
extracted with petroleum ether, EtOAc, and n-BuOH. The n-BuOH
extract (230 g) was subjected to D101 porous polymer resin column
chromatography and eluted with H2O and 30% and 80% EtOH,
successively. The fraction that eluted with 80% EtOH (120 g) was
subjected to silica gel column chromatography (15 cm × 160 cm) and
eluted with CHCl3-MeOH (4:1, 2:1, 1:2) to afford fractions 1-3. These
fractions were subjected to C18 silica gel column chromatography (4
cm × 50 cm) and eluted with MeOH-H2O in a gradient of MeOH
(MeOH-H2O, 30:70 f 100:0%, N2 pressure 0.5 MPa) to afford
subfractions 1-1-1-4, 2-1-2-3, and 3-1-3-4. Subfraction 1-1 (0.6 g) was
isolated by preparative HPLC (MeOH-H2O, 68:32, 2.0 mL/min) to
yield compounds 8 (32 mg) and 10 (12 mg). Subfraction 1-3 (1.1 g)
was isolated by preparative HPLC (MeOH-H2O, 65:35, 2.0 mL/min)
to yield compounds 9 (8 mg), 11 (48 mg), and 12 (51 mg). Subfraction
1-4 (2.9 g) was isolated by preparative HPLC (MeOH-H2O, 62:38,
2.0 mL/min) to yield compounds 14 (27 mg) and 16 (86 mg).
Subfraction 2-2 (2.8 g) was isolated by preparative HPLC
(MeOH-H2O, 60:40, 2.0 mL/min) to yield compounds 13 (66 mg)
and 18 (72 mg). Subfraction 2-4 (1.6 g) was isolated by preparative
HPLC (MeCN-H2O, 32:68, 2.0 mL/min) to yield compounds 15 (12
mg), 17 (18 mg), and 19 (55 mg). Subfraction 3-1 (3.7 g) was isolated
by preparative HPLC (MeCN-H2O, 29:71, 2.0 mL/min) to yield
compounds 4 (19 mg), 5 (37 mg), 6 (52 mg), 20 (17 mg), and 22 (24
mg). Subfraction 3-2 (4.3 g) was isolated by preparative HPLC
(MeCN-H2O, 27:73, 2.0 mL/min) to yield compounds 7 (33 mg), 21
(48 mg), 23 (21 mg), and 24 (32 mg). Subfraction 3-4 (5.8 g) was
isolated by preparative HPLC (MeCN-H2O, 24:76, 2.0 mL/min) to
yield compounds 1 (27 mg), 2 (27 mg), and 3 (61 mg).
Clematochinenoside E (5): white, amorphous powder; [R]2D0 -44
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 238 (2.56), 298 (sh), 321
(4.37) nm; IR (KBr) νmax 3420, 2929, 1712, 1632, 1060 cm-1; 1H NMR
data (C5D5N, 500 MHz) δ 1.14 (3H, s, Me-27), 1.08 (3H, s, Me-24),
1.06 (3H, s, Me-26), 0.93 (3H, s, Me-25), 0.84 (3H, s, Me-29), 0.83
(3H, s, Me-30), 4.25 (1H, dd, J ) 12.0, 5.0 Hz, H-3), 4.23 (1H, d, J )
12.0 Hz, H-23a), 3.88 (1H, d, J ) 12.0 Hz, H-23b), 5.37 (1H, brs,
H-12), 6.30 (1H, brs, Rha2-H-1), 6.20 (1H, d, J ) 8.0 Hz, Glc1-H-1),
5.82 (1H, brs, Rha1-H-1), 5.79 (1H, d, J ) 5.0 Hz Rib1-H-1), 5.14
(1H, d, J ) 7.5 Hz, Glc4-H-1), 5.04 (1H, d, J ) 6.5 Hz, Ara1-H-1),
4.96 (1H, d, J ) 8.0 Hz, Glc2-H-1), 4.93 (1H, d, J ) 8.0 Hz, Glc3-
H-1), 4.56 (1H, dd, J ) 8.0, 6.5 Hz, Ara1-H-2), 4.68 (1H, m, Rha2-
H-3), 4.31 (1H, m, Rib1-H-4), 4.26 (1H, m, Glc3-H-4), 1.67 (3H, d, J
1
) 6.0 Hz, Rha1-H-6), 1.51 (3H, d, J ) 6.0 Hz, Rha2-H-6); H NMR
data of the isoferuloyl moiety, see Table 4; 13C NMR data, see Tables
2, 3, and 5; HRESIMS m/z 1875.7952 [M + Na]+ (calcd for
C86H132O43Na, 1875.8035).
Clematochinenoside F (6): white, amorphous powder; [R]2D0 -39
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 246 (2.06), 292 (sh), 328
(4.43) nm; IR (KBr) νmax 3408, 2938, 1715, 1062 cm-1; 1H NMR data
(C5D5N, 500 MHz) δ 1.24 (3H, s, Me-23), 1.22 (3H, s, Me-27), 1.10
(3H, s, Me-24), 1.04 (3H, s, Me-26), 0.87 (3H, s, Me-29), 0.87 (3H, s,
Me-30), 0.84 (3H, s, Me-25), 3.25 (1H, dd, J ) 12.0, 5.0 Hz, H-3),
5.37 (1H, brs, H-12), 6.28 (1H, brs, Rha2-H-1), 6.21 (1H, d, J ) 8.0
Hz, Glc1-H-1), 5.82 (1H, brs, Rha1-H-1), 5.78 (1H, d, J ) 5.0 Hz,
Rib1-H-1), 5.14 (1H, d, J ) 7.5 Hz, Glc4-H-1), 4.97 (1H, d, J ) 8.0
Hz, Glc2-H-1), 4.94 (1H, d, J ) 8.0 Hz, Glc3-H-1), 4.82 (1H, d, J )
6.5 Hz, Ara1-H-1), 4.54 (1H, dd, J ) 8.0, 6.5 Hz, Ara1-H-2), 4.65 (1H,
m, Rha2-H-3), 4.29 (1H, m, Rib1-H-4), 4.26 (1H, m, Glc3-H-4), 1.68
(3H, d, J ) 6.0 Hz, Rha1-H-6), 1.51 (3H, d, J ) 6.0 Hz, Rha2-H-6);
1H NMR data of the isoferuloyl moiety, see Table 4; 13C NMR data,
see Tables 2, 3, and 5; HRESIMS m/z 1859.8105 [M + Na]+ (calcd
for C86H132O42Na, 1859.8085).
Clematochinenoside A (1): white, amorphous powder; [R]2D0 -62
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 243 (2.36), 296 (sh), 324
(4.33) nm; IR (KBr) νmax 3400, 2927, 1724, 1633, 1513, 1062 cm-1
;
1H NMR data, see Tables 1 and 4; 13C NMR data, see Tables 2, 3, and
5; HRESIMS m/z 2345.9679 [M + Na]+ (calcd for C104H162O57Na,
2345.9670).
Clematochinenoside G (7): white, amorphous powder; [R]2D0 -66
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 233 (2.56), 299 (sh), 317
(4.03) nm; IR (KBr) νmax 3421, 2928, 1715, 1636, 1062 cm-1; 1H NMR
data (C5D5N, 500 MHz) δ 1.15 (3H, s, Me-27), 1.09 (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), 4.25 (1H, dd, J ) 12.0, 5.0 Hz, H-3), 4.23 (1H, d, J
) 12.0 Hz, H-23a), 3.88 (1H, d, J ) 12.0 Hz, H-23b), 5.37 (1H, brs,
H-12), 6.27 (1H, brs, Rha2-H-1), 6.21 (1H, d, J ) 8.0 Hz, Glc1-H-1),
5.84 (1H, brs, Rha1-H-1), 5.80 (1H, d, J ) 5.0 Hz, Rib1-H-1), 5.29
(1H, d, J ) 7.5 Hz, Glc4-H-1), 5.03 (1H, d, J ) 6.5 Hz, Ara1-H-1),
4.98 (1H, d, J ) 7.5 Hz, Glc2-H-1), 4.86 (1H, d, J ) 8.0 Hz, Glc3-
H-1), 4.85 (1H, d, J ) 8.0 Hz, Glc5-H-1), 4.56 (1H, dd, J ) 8.0, 6.5
Hz, Ara1-H-2), 4.68 (1H, m, Rha2-H-3), 4.31 (1H, m, Rib1-H-4), 4.26
(1H, m, Glc3-H-4), 4.15 (1H, m, Glc4-H-3), 1.69 (3H, d, J ) 6.0 Hz,
Clematochinenoside B (2): white, amorphous powder; [R]2D0 -31
(c 1.0, MeOH); IR (KBr) νmax 3421, 2927, 1734, 1062 cm-1; 1H NMR
data, see Table 1; 13C NMR data, see Tables 2 and 3; HRESIMS m/z
2169.9244 [M + Na]+ (calcd for C94H154O54Na, 2169.9197).
Clematochinenoside C (3): white, amorphous powder; [R]2D0 -58
(c 1.0, MeOH); UV (MeOH) λmax (log ε) 242 (2.33), 296 (sh), 326
(4.27) nm; IR (KBr) νmax 3420, 2927, 1719, 1632, 1513, 1062 cm-1
;
1H NMR data (C5D5N, 500 MHz) δ 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), 4.25 (1H, dd, J ) 12.0, 5.0 Hz, H-3), 4.23
(1H, d, J ) 12.0 Hz, H-23a), 3.88 (1H, d, J ) 12.0 Hz, H-23b), 5.37
(1H, brs, H-12), 6.27 (1H, brs, Rha2-H-1), 6.21 (1H, d, J ) 8.0 Hz,
Glc1-H-1), 5.83 (1H, brs, Rha1-H-1), 5.81 (1H, d, J ) 5.0 Hz, Rib1-
H-1), 5.47 (1H, brs, Rha3-H-1), 5.29 (1H, d, J ) 8.0 Hz, Glc4-H-1),
1
Rha1-H-6), 1.51 (3H, d, J ) 6.0 Hz, Rha2-H-6); H NMR data of the

Triterpene Saponins from Clematis chinensis
Journal of Natural Products, 2010, Vol. 73, No. 7 1239
isoferuloyl moiety, see Table 4; 13C NMR data, see Tables 2, 3, and 5;
HRESIMS m/z 2037.8506 [M + Na]+ (calcd for C92H142O48Na,
2037.8563).
Acknowledgment. We are thankful to the State Administration of
Traditional Chinese Medicine of the People’s Republic of China for
financial support (200707007). We are also grateful to members of the
Analytical Center of Peking University Health Science Center.
Acid Hydrolysis of Compounds 1-7. Each compound (5 mg) was
hydrolyzed with 2 N aqueous CF3COOH (10 mL) at 110 °C for 8 h in
a sealed tube. 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. Oleanolic
acid was detected as a product of 6, hederagenin as a product of 1-5
and 7, and isoferulic acid as a product of 1 and 3-7. The aqueous
layer was repeatedly evaporated with MeOH under vacuum until the
solvent was completely removed. The residue was dissolved in
anhydrous pyridine (100 µL) and then mixed with a pyridine solution
of L-cysteine methyl ester hydrochloride (100 µL). After warming at
60 °C for 1 h, hexamethyldisilazane (100 µL) and trimethylsilyl chloride
(40 µL) were added, and the mixture was warmed at 60 °C for another
30 min. The mixture was filtered through a 0.45 µm membrane to
remove the precipitate and analyzed by GC. Separations were carried
out on a HP-5 column (28 m × 0.32 mm). Highly pure He was
employed as carrier gas (1.0 mL/min flow rate), and the FID detector
operated at 260 °C (column temp 180 °C). 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.
Supporting Information Available: 1D and 2D NMR spectra for
compounds 1-7 and structures of known compounds 8-24. This
material is available free of charge via the Internet at http://pubs.acs.org.
References and Notes
(1) Editorial Committee of Chinese Pharmacopeia. Chinese Pharmaco-
poeia (2005 ed.); Chemical & Industry Press: Beijing, 2005; p 175.
(2) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1979, 27, 2388–2393.
(3) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1980, 28, 2827–2830.
(4) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1980, 28, 3555–3560.
(5) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1982, 30, 859–865.
(6) Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1982, 30, 3340–3346.
(7) Kizu, H.; Shimana, H.; Tomimori, T. Chem. Pharm. Bull. 1995, 43,
2189–2194.
(8) Kawata, Y.; Kizu, H.; Tomimori, T. Chem. Pharm. Bull. 1998, 46,
1891–1900.
(9) Shao, B. P.; Qin, G. W.; Xu, R. S.; Wu, H. M.; Ma, K. Phytochemistry
1995, 38, 1473–1479.
Mild Alkaline Hydrolysis of Compounds 1 and 4. Each compound
(6 mg) was dissolved in 0.1 N KOH (2 mL) and allowed to hydrolyze
at room temperature for 1 h. The reaction mixture was neutralized with
dilute HCl and analyzed by HPLC. Isoferulic acid and 2 were detected
as products of 1 (MeCN-H2O, 24:76, 1.0 mL/min), and isoferulic acid
and 3-O-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-(1f4)-ꢀ-D-
ribopyranosyl-(1f3)-R-L-rhamnopyranosyl-(1f2)-R-L-arabinopyrano-
syl hederagenin 28-O-R-L-rhamnopyranosyl-(1f4)-ꢀ-D-glucopyranosyl-
(10) Shao, B. P.; Qin, G. W.; Xu, R. S.; Wu, H. M.; Ma, K. Phytochemistry
1996, 42, 821–825.
(11) He, M.; Zhang, J. H.; Hu, C. Q. Yaoxue Xuebao 2001, 36, 278–280.
(12) Mimaki, Y.; Yokosuka, A.; Hamanaka, M.; Sakuma, C.; Yamori, T.;
Sashida, Y. J. Nat. Prod. 2004, 67, 1511–1516.
(13) Shi, S. P.; Jiang, D.; Dong, C. X.; Tu, P. F. J. Nat. Prod. 2006, 69,
1591–1595.
(14) Dong, C. X.; Wu, K. S.; Shi, S. P.; Tu, P. F. J. Chin. Pharm. Sci.
2006, 15, 15–20.
(1f6)-ꢀ-D-glucopyranoside were detected as products of
(MeCN-H2O, 29:71, 1.0 mL/min).
4
(15) Shi, S. P.; Jiang, D.; Dong, C. X.; Tu, P. F. Z. Naturforsch., B: Chem.
Sci. 2006, 61, 1299–1303.
Alkaline Hydrolysis of Compounds 4 and 6. Pure compounds 4
and 6 (10-15 mg each) were refluxed in 5% KOH solution (pH 12-13)
at 90 °C for 1 h. The reaction mixtures were neutralized with 5% HCl
solution and then concentrated to dryness. The residues were extracted
with n-BuOH, and the organic layers of pure compounds were analyzed
by NMR spectroscopy. Hydrolysis of 4 and 6 afforded two known
compounds that were identified as CP10 and CP9, respectively.4
In Vitro Anti-inflammatory Assay. Potential anti-inflammatory
activity was determined according to the literature.23 In a 78 µL reaction
system, Tris-HCl buffer (0.1 M, pH 8.0), 4.4% heme final concentration,
and COX-1 or COX-2 (Cayman, Ann Arbor, MI) were added
successively. After a 5 min preincubation at 25 °C, N,N,N′,N′-
tetramethyl-p-phenylenediamine and arachidonic acid (Cayman) were
added. SC560 and NS398 (Cayman) were used as positive controls.
After a second 5 min preincubation at 25 °C, enzyme activity was
monitored on a Greiner 384 microplate reader (TECAN Safire2,
Switzerland) by following the rate of change in absorbance (A) at 590
nm.
(16) Wang, G. S.; Chen, X. P; Xu, J. D; Murayama, T.; Shoji, J. Yaoxue
Xuebao 1996, 31, 940–944.
(17) Shao, C. J.; Kasai, R.; Xu, J. D.; Tanaka, O. Chem. Pharm. Bull. 1988,
36, 601–608.
(18) Kizu, H.; Hirabayashi, S.; Suzuki, M.; Tomimori, T. Chem. Pharm.
Bull. 1985, 33, 3347–3478.
(19) Kawata, Y.; Kizu, H.; Miyaichi, Y.; Tomimori, T. Chem. Pharm. Bull.
2001, 49, 635–638.
(20) Jhoo, J. W.; Sang, S.; He, K.; Cheng, X.; Zhu, N.; Stark, R. E.; Zheng,
Q. Y.; Rosen, R. T.; Ho, C.-T. J. Agric. Food. Chem. 2001, 49, 5969–
5974.
(21) Breitmaier, E.; Voelter, W. Carbon-13 NMR Spectroscopy, 3rd ed.;
VCH: Weinheim, 1987; pp 381-391.
(22) Seebacher, W.; Simic, N.; Weis, R.; Saf, R.; Kunert, O. Magn. Reson.
Chem. 2003, 41, 636–638.
(23) Duan, W. G.; Zhang, L. Y. Prostaglandins Leukotrienes Essent. Fatty
Acids 2006, 74, 317–321.
NP100057Y