Cytotoxic triterpenoid saponins from Clematis tangutica

Article abstract of DOI:10.1016/j.phytochem.2016.05.009

Eight previously undescribed oleanane-type triterpenoid saponins, clematangoticosides A-H, together with eight known saponins, were isolated from the whole plants of Clematis tangutica (Maxim.) Korsh. Their structures were elucidated by extensive spectroscopic analysis, in combination with chemical methods (acid hydrolysis and mild alkaline hydrolysis). Clematangoticosides D-G were found to be unusual 23, 28-bidesmosidic glycosides. The cytotoxic activities of all of the isolated saponins were evaluated against the four human cancer cell lines SGC-7901, HepG2, HL-60 and U251MG. Clematoside S, sapindoside B, kalopanax saponin A, and koelreuteria saponin A exhibited cytotoxicity against all of the test cancer cell lines with IC₅₀ values in the range of 1.88–27.20?μM, while clematangoticoside D and F showed selective cytotoxicity against SGC-7901 with IC₅₀ values of 24.22 and 21.35?μM, respectively.

Full text of DOI:10.1016/j.phytochem.2016.05.009

Phytochemistry 130 (2016) 228e237
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Cytotoxic triterpenoid saponins from Clematis tangutica
,
,
Min Zhao a 1, Zhuo-Ma Da-Wa b 1, Da-Le Guo a, Dong-Mei Fang a, Xiao-Zhen Chen a,
Hong-Xi Xu c, Yu-Cheng Gu d, Bing Xia a, Lei Chen a, Li-Sheng Ding a **, Yan Zhou a
,
, *
a Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu
610041, PR China
b Tibet Autonomous Region Institute for Food and Drug Control, Lhasa 850000, PR China
c Shanghai University of Traditional Chinese Medicine, Shanghai 201203, PR China
d Syngenta Jealott’s Hill International Research Centre, Berkshire RG42 6EY, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Eight previously undescribed oleanane-type triterpenoid saponins, clematangoticosides A-H, together
with eight known saponins, were isolated from the whole plants of Clematis tangutica (Maxim.) Korsh.
Their structures were elucidated by extensive spectroscopic analysis, in combination with chemical
methods (acid hydrolysis and mild alkaline hydrolysis). Clematangoticosides D-G were found to be
unusual 23, 28-bidesmosidic glycosides. The cytotoxic activities of all of the isolated saponins were
evaluated against the four human cancer cell lines SGC-7901, HepG2, HL-60 and U251MG. Clematoside S,
sapindoside B, kalopanax saponin A, and koelreuteria saponin A exhibited cytotoxicity against all of the
Received 12 January 2016
Received in revised form
15 May 2016
Accepted 25 May 2016
Available online 1 June 2016
Keywords:
Clematis tangutica
Ranunculaceae
Triterpenoid saponins
Cytotoxic activities
test cancer cell lines with IC50 values in the range of 1.88e27.20
showed selective cytotoxicity against SGC-7901 with IC50 values of 24.22 and 21.35
m
M, while clematangoticoside D and F
M, respectively.
m
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
antifungal and cardioprotective activities from this alpine plant (Du
et al., 2003a; Du et al., 2003b; Zhang et al., 2013a; Zhang et al.,
2013b). However, there is no report on the cytotoxic activity of
the triterpenoid saponins of this plant. As part of an ongoing
research for cytotoxic triterpenoid saponins from Clematis tangu-
tica, an approach based on bioassay-guided fractionation according
to the cytotoxic activities against the human gastric carcinoma
SGC-7901 cell line and HPLC/MS-guided isolation was conducted to
profile the saponins occurring in the n-BuOH fraction of a MeOH
extract of the whole plant. As a result, 16 triterpene glycosides,
including eight previously undescribed saponins (1e8) were iso-
lated, and a new secondary glycoside 8a was obtained by mild
alkaline-hydrolysis of saponin 8. Notably, saponins 4e7 were un-
usual 23, 28-bidesmosidic glycosides which are first reported in a
Clematis species. This paper describes the isolation, structure
elucidation and cytotoxicity evaluation of the isolated saponins.
Clematis L. (Ranunculaceae) is a large genus that consists of 355
species in the world. There are approximately 155 species (93
endemic ones) distributed in China, of which 70 species have been
used as traditional Chinese medicines (Jiang et al., 2007). The tri-
terpenoid saponins isolated from this genus have shown various
bioactivities, including anticancer (Li et al., 2014; Zhao et al., 2014),
antibacterial (Ding et al., 2009), antifungal (Du et al., 2003b), anti-
inflammatory (Fu et al., 2010), cyclooxygenase-2 and TNF-a inhib-
itory (Liu et al., 2009; Shi et al., 2006), anti-proliferative (Gong et al.,
2013), and cardioprotective activities (Zhang et al., 2013a; Zhang
et al., 2013b). Clematis tangutica (Maxim.) Korsh. is a perennial
twining plant that is widespread in Tibet. The whole plant was
called “Ye-Mang-Na-Bao” in traditional Tibetan medicine and was
used to treat skin ulcers and indigestion (Du et al., 2003a; Zhang
et al., 2013b). Previous chemical and pharmacological in-
vestigations have reported some triterpenoid saponins with
2. Results and discussion
The BuOH fraction, obtained from the 75% MeOH extract of the
whole plant of C. tangutica, was submitted to several column
chromatography steps yielding eight new (1e8) and eight known
(9e16) triterpenoid saponins. Elucidation of their structures was
performed by NMR analysis (1H, 13C, 1He1H COSY, NOESY, HSQC,
* Corresponding author.
** Corresponding author.
E-mail addresses: lsding@cib.ac.cn (L.-S. Ding), zhouyan@cib.ac.cn (Y. Zhou).
The authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.phytochem.2016.05.009
0031-9422/© 2016 Elsevier Ltd. All rights reserved.

M. Zhao et al. / Phytochemistry 130 (2016) 228e237
229
HMBC, TOCSY, HSQC-TOCSY), and mass spectrometry (HRESIMS
and ESIMS), in combination with chemical methods (acid hydro-
lysis and mild alkaline hydrolysis) (see Fig. 1).
Saponin 2 was obtained as a white amorphous powder. Its
molecular formula was determined to be C65H106O30 from the
[MþNa]þ peak at m/z 1389.6655 in the HRESIMS spectrum (calcd.
for C65H106O30Na, 1389.6667). By NMR analysis and the hydrolysis
results, saponin 2 possessed the same sugar components and
sequence of sugar chains as 1. The difference was in the aglycone
part (Table 1). The 1H NMR spectrum showed signals for six tertiary
methyl groups at dH 0.82, 0.86, 0.97, 1.01, 1.09, and 1.16 (3H, s), and
an olefinic group at dH 5.41 (br s). Extensive 2D NMR analysis
allowed the identification of the aglycone of 2. This compound was
obtained by the acid hydrolysis method and was identified as
Saponin 1 was obtained as colorless needles. Its molecular for-
mula C65H106O29 was established by the HRESIMS spectrum (m/z
1373.6724 [MþNa]þ, calcd. for C65H106O29Na, 1373.6717) and was
supported by the 13C NMR spectroscopic data. The 13C NMR spec-
trum of 1 displayed 65 carbons, of which 30 were assigned to the
aglycone part and the remaining 35 were assigned to six sugar units
comprising one pentose, two hexoses and three deoxyhexoses. The
NMR spectra showed signals for seven tertiary groups at dH 0.84,
0.85, 0.85, 1.02, 1.05, 1.09, and 1.23 (3H, s), and their corresponding
carbons at dC 23.5, 15.5, 33.0, 16.8, 17.2, 27.8, and 25.6; an olefinic
group at dH 5.42 (br s) and dC 122.3, 144.0; and a carboxy group at dC
176.3, which were characteristic for the oleanane skeleton with a
carboxy group at C-17. Two-dimensional NMR analysis confirmed
the aglycone of 1 to be oleanolic acid and was in full agreement
with the literature data (Magid et al., 2015). Furthermore, the ole-
anolic acid of 1 was obtained by acid hydrolysis, and was identified
by ESIMS and NMR analysis. The chemical values of C-3 (dC 88.6)
and C-28 (dC 176.3) suggested 1 to be a 3, 28-bidesmosidic glyco-
side. An acid hydrolysis of 1 with 2 mol/L CF3COOH afforded sugar
components identified by the GC analysis of their trimethylsilylated
derivatives as L-arabinose, D-glucose and L-rhamnose in a ratio of
1:2:3. Six anomeric protons at dH 4.86 (d, J ¼ 5.3, Ara), 4.95 (d,
J ¼ 7.8, Glc II), 5.84 (br s, Rha II), 6.08 (d, J ¼ 8.1, Glc I), 6.16 (br s, Rha
III), and 6.60 (br s, Rha I) were observed in the 1H NMR spectrum
and their corresponding carbons (dC 104.8, 104.6, 102.5, 94.6, 101.5,
hederagenin (3b,23-dihydroxy-12-oleanen-28-oic acid) by ESIMS
and NMR analysis, consistent with literature data (Kizu and
Tomimori, 1982). The chemical values of C-3 (dC 81.0) and C-28
(
dC 176.3) suggested 2 to be a 3, 28-bidesmosidic glycoside. Based
on the above evidence, structure 2 was elucidated as 3 -O- -L-
rhamnopyranosyl-(1 / 2)- -L-arabinopyranosyl hederagenin 28-
O- -L-rhamnopyranosyl-(1 / 4)- -D-glucopyranosyl-(1 / 6)-[
L-rhamnopyranosyl-(1 2)]- -D-glucopyranoside and was
named clematangoticoside B.
b
a
a
a
b
a-
/
b
Saponin 3 was obtained as a white amorphous powder. Its
molecular formula was determined as C54H88O22 from the [MþNa]þ
peak at m/z 1111.5651 in HRESIMS spectrum (calcd. for
C
54H88O22Na, 1111.5665). The 13C NMR spectrum of 3 displayed 54
carbons, of which 30 were assigned to the aglycone part and the
remaining 24 to four sugar units comprising two hexoses and two
deoxyhexoses. Only the glycosidation shift was observed for C-28
(dC 176.3), confirming the monodesmosidic nature of 3. The acid
and 101.2) were obtained from the HSQC spectrum. The
b
-anomeric
hydrolysis of 3 afforded D-glucose and L-rhamnose in a ratio of 1:1.
The sequence of the 28-O-sugar chain was determined to be the
same as that of 1 and 2 by analysis of their NMR spectra (Tables 2
and 3). On the basis of these evidence, the structure of 3 was
configurations of the glucose and xylose units were determined
3
from their JH1,
coupling constants (7.6e8.1 Hz) (Melek et al.,
H2
2014). The a-configuration of the arabinose unit was determined
from the 3JH1, H2 value (5.3 Hz) and the NOESY correlations between
elucidated as hederagenin 28-O-
D-glucopyranosyl-(1 / 6)-[ -L-rhamnopyranosyl-(1 / 2)]-
glucopyranoside and was named clematangoticoside C.
a-L-rhamnopyranosyl-(1 / 4)-
b-
-D-
H-1 and H-3 and between H-1 and H-5 observed in the 4C1 form
a
b
1
(Chang et al., 2007). For the rhamnose unit, the large JC1,
H1
coupling constant (168.5 Hz for Rha I and Rha III, and 169.0 Hz for
Rha II), the broad singlet of the anomeric proton, the chemical
values of C-3 and C-5, and the strong three-bond HMBC correla-
tions from the anomeric proton to C-3 and C-5 indicated that the
Saponin 4 was obtained as a white amorphous powder. Its
molecular formula was determined as C60H98O27 from [MþNa]þ
peak at m/z 1273.6173 in the HRESIMS spectrum (calcd. for
C60H98O27Na, 1273.6193). The NMR spectroscopic data of 4 were
anomeric proton had an
a
-configuration (Jia et al., 1998). The
almost identical with those of 3 except for an additional hexose unit
(Tables 2 and 3). The acid hydrolysis of 4 afforded D-glucose and L-
rhamnose in a ratio of 3:2. The sequence of the sugar chain at
aglycone C-28 was proved to be the same as that of 3 by comparison
of their 13C and HMBC spectra. The chemical value of C-3 (dC 72.3)
indicated the position was free, while the signal of the oxy-
methylene group C-23 (dC 75.4), which was found 7.4 ppm shifted
linkage sites and the sequence of sugar moieties were deduced
mainly from the analysis of the HMBC and NOESY spectra (Fig. 2).
The HMBC correlations between H-1 of Rha III (dH 6.16) and C-2 of
Ara (dC 75.7), H-1 of Ara (dH 4.86) and C-3 (dC 88.6), and the NOESY
correlations between H-1 of Rha III (dH 6.16) and H-2 of Ara (dH
4.55), H-1 of Ara (dH 4.86) and H-3 (dH 3.19) confirmed the sequence
of the 3-O-sugar chain as O-
arabinopyranosyl. Similarly, the sequence of the 28-O-sugar chain
was determined to be O- -L-rhamnopyranosyl-(1 / 4)- -D-glu-
copyranosyl-(1 / 6)-[ -L-rhamnopyranosyl-(1 / 2)]- -D-gluco-
a
-L-rhamnopyranosyl-(1 / 2)-
a
-L-
downfield compared to that of 3, suggested that an additional b-D-
glucopyranosyl unit was located at the C-23 position of the
hederagenin. This was also confirmed by the HMBC correlation
between H-1 of Glc III (dH 4.90) and C-23 (dC 75.4). On the basis of
a
b
a
b
pyranosyl, which was supported by the HMBC correlations
between H-1 of Rha II (dH 5.84) and C-4 of Glc II (dC 78.0), H-1 of Glc
II (dH 4.95) and C-6 of Glc I (dC 68.8), H-1 of Rha I (dH 6.60) and C-2 of
Glc I (dC 74.7), and H-1 of Glc I (dH 6.08) and C-28 (dC 176.3). This was
further confirmed by the NOESY correlations between H-1 of Rha II
the evidence, the structure of 4 was elucidated as 23-O-
copyranosyl hederagenin 28-O- -L-rhamnopyranosyl-(1 / 4)-
D-glucopyranosyl-(1 / 6)-[ -L-rhamnopyranosyl-(1 / 2)]- -D-
glucopyranoside and was named clematangoticoside D.
Saponin 5 was obtained as a white amorphous powder. Its
molecular formula was determined as C48H78O19 from the [MþNa]þ
peak at m/z 981.5015 in the HRESIMS spectrum (calcd. for
b-D-glu-
a
b-
a
b
(dH 5.84) and H-4 of Glc II (dH 4.39), H-1 of Glc II (dH 4.95) and H-6 of
Glc I (dH 4.26, 4.61), and H-1 of Rha I (dH 6.60) and H-2 of Glc I (dH
4.38). Extensive 2D NMR analysis (1He1H COSY, NOESY, HSQC,
HMBC, TOCSY, and HSQC-TOCSY) enabled the full assignments of
the resonances of each sugar. On the basis of all of the evidence,
C48H78O19Na, 981.5035). Its spectroscopic features and the signals
at dC 75.0 (C-23) and 176.3 (C-28) suggested 5 to be another 23, 28-
bidesmosidic oleanane-type glycoside. The D-glucose units were
established by acid hydrolysis and GC analysis. The sequence of the
structure 1 was elucidated as 3
-L-arabinopyranosyl oleanolic acid 28-O-
(1 4)- -D-glucopyranosyl-(1 6)-[
b-O-a-L-rhamnopyranosyl-(1 / 2)-
a
a
-L-rhamnopyranosyl-
-L-rhamnopyranosyl-
28-O-sugar chain was determined to be O-
b-D-glucopyranosyl-
/
b
/
a
(1 / 6)- -D-glucopyranosyl by the HMBC correlations between H-
b
(1 / 2)]-
A.
b
-D-glucopyranoside and was named clematangoticoside
1 of Glc II (dH 5.02) and C-6 of Glc I (dC 69.2), H-1 of Glc I (dH 6.25)
and C-28 (dC 176.3). The HMBC correlation between H-1 of Glc III

230
M. Zhao et al. / Phytochemistry 130 (2016) 228e237
H
H
O
O
H
H
HO
R O
H
H
HO
HO
CH R
O
R O
Glc II
HO
O
O
HO
HO
O
O
O
O
O
O
HO
O
OH
HO
HO
O
Glc II
R2
HO
H C
OH
OH
O
O
Glc III
OR
HO
Glc I
HO
HO
Rha II
OH
R1
Rha
H
H C
OH
O
Glc I
HO
4
5
6
7
Rha
HO
Rha I
OH
H
R2
H
R1
H
Rha
H
1
A
A
H
Rha
2
3
OH
OH
HO
A:
H
O
O
HO
H
Ara
O
HO
HO
Ara
O
O
O
H C
O
H
CH OH
HO
HO
HO
O
Rha III
OH
O
O
O
O
H C
HO
O
HO
O
OH
HO
O
HO
H C
HO
Glc II
O
HO
O
O
O
OH
HO
HO
Rha II
OH
O
HO
Glc I
HO
HO
HO
OH
OH
OH
Xyl
B
8
R =
Rha I
Glc III
OR
Glc IV
8a R = H
HO
HO
O
B:
C:
O
HO
Ara
O
H C
O
O
HO
H
CA
Rha
HO
O
O
HO
HO
OH
OR
H
Xyl
OH
R O
HO
HO
D:
H
R
O
O
O
O
H C
OH
HO
O
R2
R3
D
D
D
H
H
H
H
D
R1
Glc
HO
HO
HO
OH
9
C
E
A
F
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CHO
Glc
OH
O
Rha
10
11
12
13
14
HO
HO
O
HO
HO
O
F:
E:
OH
C
A
O
Glc
O
Rib
OH
HO
H C
HO
O
Ara
O
15 Ara
16
O
Rha
O
H C
HO
HO
O
OH
A
HO
HO
OH Xyl
Rha
OH
Fig. 1. Structures of saponins 1e16 and 8a.
(
dH 4.90) and C-23 (dC 75.0) indicated that the last
b
-D-glucopyr-
Consequently, structure 5 was elucidated as 23-O-
anosyl hederagenin 28-O- -D-glucopyranosyl-(1
b
-D-glucopyr-
anosyl unit was located at the C-23 position of the hederagenin.
b
/
6)- -D-
b

M. Zhao et al. / Phytochemistry 130 (2016) 228e237
231
NMR data of 8 with those of 8a, the H-2 and C-2 signals of the
terminal glucose unit (Glc IV) of 8 were found to be shifted
downfield by approximately 1.78 and 0.2 ppm, respectively, and the
C-1 and C-3 signals of Glc IV were found to be shifted upfield by
approximately 2.4 and 1.8 ppm, respectively. These results sug-
gested that the caffeoyl group (CA) was linked to C-2 of the terminal
glucose unit (Glc IV). This was further confirmed by the HMBC
correlation between H-2 of Glc IV (dH 5.77) and C-1 of CA (dC 166.7).
The sequence of the 3-O-sugar chain was determined to be 3
(2-O-caffeoyl)- -D-glucopyranosyl-(1 / 4)- -D-glucopyranosyl-
(1 4)- -D-xylopyranosyl-(1 3)- -L-rhamnopyranosyl-
(1 / 2)- -L-arabinopyranosyl, which was supported by the HMBC
b-O-
b
b
/
b
/
a
a
correlations between H-1 of Glc IV (dH 5.37) and C-4 of Glc III (dC
81.1), H-1 of Glc III (dH 4.86) and C-4 of Xyl (dC 77.4), H-1 of Xyl (dH
5.26) and C-3 of Rha II (dC 82.9), H-1 of Rha II (dH 6.34) and C-2 of
Ara (dC 75.2), and H-1 of Ara (dH 5.03) and C-3 (dC 80.8). Sequence
the trisaccharide chain at C-28 was determined to be the same as
that of 6 by comparison of their NMR spectroscopic data. Conse-
Fig. 2. Key HMBC and NOESY correlations for saponin 1.
quently, structure 8 was elucidated as 3
glucopyranosyl-(1 / 4)- -D-glucopyranosyl-(1 / 4)-
pyranosyl-(1 / 3)- -L-rhamnopyranosyl-(1 / 2)- -L-arabino-
pyranosyl hederagenin 28-O- -L-rhamnopyranosyl-(1 / 4)- -D-
-D-glucopyranoside and was named
b
-O-(2-O-caffeoyl)-b-D-
b
b
-D-xylo-
a
a
glucopyranoside and was named clematangoticoside E.
a
b
Saponin 6 was obtained as a white amorphous powder. Its
molecular formula was determined as C54H88O23 from the [MþNa]þ
peak at m/z 1127.5627 in the HRESIMS spectrum (calcd. for
glucopyranosyl-(1 / 6)-
b
clematangoticoside H.
Additionally, 8 known oleanane-type glycosides were identified
-O- -D-xylopyranosyl-(1 3)- -L-rhamnopyranosyl-
(1 / 2)- -L-arabinopyranosyl hederagenin 28-O- -L-rhamnopyr-
anosyl-(1 / 4)- -D-glucopyranosyl-(1 / 6)- -D-glucopyranoside
C
54H88O23Na, 1127.5614). Comparison of the NMR spectra of 6 with
as
3
b
b
/
a
those of 5 indicated that 6 differed from 5 in the presence of an
additional deoxyhexose unit (Tables 2 and 3). The acid hydrolysis of
6 afforded D-glucose and L-rhamnose in a ratio of 3:1. The sequence
a
a
b
b
(9) (Panov et al., 2006), hederacholichiside F (10) (Wang et al.,
2013), kizutasaponin K12 (11) (Kizu et al., 1995), clematoside S
(12) (Yan et al., 2009), sapindoside B (13) (Panov et al., 2006),
kalopanax saponin A (14) (Panov et al., 2006), koelreuteria saponin
A (15) (Chirva et al.,1970), and clematibetoside B (16) (Kawata et al.,
2001) by comparing their physicochemical and spectroscopic data
with literature values.
The cytotoxic activities (Table 4) of the isolated saponins 1e16
against the human gastric carcinoma SGC-7901, hepatoma HepG2,
promyelocytic leukemia HL-60, and glioblastoma U251MG cell
lines were determined using the MTT colorimetric assay. Saponins
12e15 possessing a free carboxylic group at C-28 exhibited mod-
erate cytotoxicity against all of the test cancer cell lines with the
of the 28-O-sugar chain was determined to be O-
a-L-rhamnopyr-
anosyl-(1 / 4)- -D-glucopyranosyl-(1 / 6)- -D-glucopyranosyl
b
b
by the HMBC correlations between H-1 of Rha (dH 5.87) and C-4 of
Glc II(dC 77.9), H-1 of Glc II (dH 4.98) and C-6 of Glc I (dC 68.9), H-1 of
Glc I (dH 6.23) and C-28 (dC 176.3). Thus, structure 6 was elucidated
as 23-O-
anosyl-(1 / 4)-
and was named clematangoticoside F.
b
-D-glucopyranosyl hederagenin 28-O-
a-L-rhamnopyr-
b
-D-glucopyranosyl-(1 / 6)- -D-glucopyranoside
b
Saponin 7 was obtained as a white amorphous powder. It was an
isomer of 6 with the molecular formula C54H88O22 according to the
HRESIMS spectrum (m/z 1127.5640 [MþNa]þ, calcd. for
C
54H88O23Na, 1127.5614). The acid hydrolysis result of 7 was the
same as that of 6. After comparison of the NMR spectra of 6 and 7,
the only difference was the location of the rhamnose unit. The
sequence of the 28-O-sugar chain was determined to be O-
values of IC50 in the range of 1.88e27.20
showed selective cytotoxicity against PC-3 with the IC50 values of
24.14 and 21.35 M, respectively. The other tested saponins were
inactive (IC50 > 50 M) against the test cancer cell lines (not shown
mM, while saponins 4 and 6
a-L-
m
rhamnopyranosyl-(1 / 2)-[ -D-glucopyranosyl-(1 / 6)]-
b
b-D-
m
glucopyranosyl, which was supported by the HMBC correlations
between H-1 of Rha (dH 6.61) and C-2 of Glc I (dC 74.6), H-1 of Glc II
in Table 4). All isolated saponins 1e16 were inactive (IC50 > 50 mM)
to the normal human hepatocyte cell line (L02).
(
dH 4.97) and C-6 of Glc I (dC 69.1), H-1of Glc I (dH 6.10) and C-28 (dC
176.3). Consequently, structure 7 was elucidated as 23-O- -D-glu-
copyranosyl hederagenin 28- O- -L-rhamnopyranosyl-(1 / 2)-[
-D-glucopyranoside and was named
b
3. Conclusions
a
b-
D-glucopyranosyl-(1 / 6)]-
b
The phytochemical investigation of the C. tangutica extract
yielded eight previously undescribed triterpenoid saponins (1e8),
and eight known saponins (9e16). All of the isolated saponins were
evaluated for their cytotoxic activities against four human cancer
cell lines, SGC-7901, HepG2, HL-60 and U251MG. The five known
compounds (9, 12, 13, 15, and 16) were isolated from this plant for
the first time. Clematangoticosides D-G (4e7) were found to be
unusual 23, 28-bidesmosidic glycosides which are being reported
for the first time in the genus Clematis. Studies investigated the
cytotoxic activity of the triterpenoid saponins of Clematis species,
including Clematis parviloba (Yan et al., 2009), Clematis lasiandra
(Tian et al., 2013), Clematis mandshurica (He et al., 2011; Li et al.,
2013; Gong et al., 2013), Clematis argentilucida (Zhao et al., 2014),
and demonstrated that the triterpenoid saponins possessing a free
carboxylic group at C-28 showed potent cytotoxicity against many
clematangoticoside G.
Saponin 8 was obtained as a yellowish amorphous powder. Its
molecular formula, C85H130O43, was established by the HRESIMS
spectrum (m/z 1839.8074 [MþH]þ, calcd. for C85H131O43
,
1839.8064) and was supported by the NMR spectroscopic data. The
13C NMR spectrum displayed 85 carbons, of which 30 were
assigned to the aglycone part, 46 to eight sugar units, and the
remaining 9 attributed to a caffeoyl (3,4-dihydroxycinnamoyl)
group. Acid hydrolysis of 8 afforded D-glucose, L-rhamnose, L-
arabinose, and D-xylose in a ratio of 4:2:1:1. The NMR spectro-
scopic data of 8 was similar to those of clematibetoside A (Kawata
et al., 2001) except that the ribose of clematibetoside A was
replaced by a xylose unit. Mild alkaline hydrolysis of 8 yielded a
deacylated saponin (8a) and a caffeic acid. By comparing the 13C

232
M. Zhao et al. / Phytochemistry 130 (2016) 228e237
Table 1
13C (100 MHz in pyridine-d5) and 1H (400 MHz in pyridine-d5) NMR spectroscopic data for the aglycone moieties of compounds 1e8, and 8a.
1
2
3
4
5
6
7
8
8a
dC
dH (J in
dC
dH (J in
dC
dH (J in
dC
dH (J in dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in
dC
dH (J in
dC
dH (J in
Hz)
Hz)
Hz)
Hz)
Hz)
Hz)
Hz)
1
2
3
38.7 0.92 m,
1.47 m
26.3 1.81 m,
2.04 m
88.6 3.19 dd
(11.2, 3.6)
39.3 e
55.8 0.69 d
(10.8)
38.9 1.03 m,
1.55 m
38.7 1.04 m,
1.56 m
38.3 0.88 m, 38.3 0.88 m,
1.47 m 1.46 m
38.3 0.86 m,
1.45 m
27.2 1.79 m,
1.84 m
38.3 0.87 m,
1.47 m
38.8 1.04a,
1.52 m
38.8 1.04a,
1.54 m
26.0 1.97a,
2.17 m
27.5 1.87a,
1.92 m
27.2 1.78 m, 27.2 1.79 m,
27.2 1.79 m,
26.2 1.99 m,
2.20 m
26.1 2.00 m,
2.22 m
1.84a
1.84 m
1.84a
81.0 4.21a
73.4 4.15a
72.3 4.16 m
72.1 4.16 m
72.0 4.17 m
72.3 4.15 m
80.8 4.27a
80.8 4.27a
4
5
43.3 e
42.6 e
48.6 1.43 m
42.6 e
42.6 e
42.6 e
48.4 1.41 m
42.6
e
43.4 e
43.3 e
47.6 1.67a
48.5 1.33 m
48.5 1.41 m
48.5 1.34 m
47.4 1.71a
47.4 1.72a
6
7
18.4 1.28 m,
18.0 1.33 m,
1.67a
32.7 1.62 m,
1.70 m
39.7 e
48.0 1.73 m
36.7 e
18.5 1.39 m,
1.58a
18.4 1.30 m, 18.4 1.32 m,
1.56 m
1.63a
32.8 1.54 m, 32.6 1.30 m,
1.60a
1.56 m
39.6 e
18.3 1.30a,
1.63 m
18.5 1.30 m,
1.56 m
17.9 1.30 m,
1.65 m
32.5 1.21 m,
1.54 m
39.6 e
48.0 1.72 m
36.6 e
17.9 1.32 m,
1.67 m
32.5 1.24 m,
1.56 m
39.7 e
48.0 1.73 m
36.6 e
1.35a
33.0 1.33a,
1.35a
32.9 1.58a,
1.68 m
32.6 1.29a,
1.55 m
32.8 1.54 m,
1.61a
8
9
39.7 e
47.8 1.60 m
39.7 e
48.0 1.73 m
37.0 e
23.6 1.90 m,
1.95 m
39.6 e
39.6 e
39.6
47.9 1.62a
37.0
e
47.9 1.62 m
47.9 1.63a
47.8 1.62 m
36.9 e
10 36.8 e
11 23.6 1.89a,
1.91a
37.0 e
37.0 e
e
23.6 1.89 m,
23.6 1.90 m, 23.5 1.89 m,
1.98 m 2.00 m
23.5 1.88a,
1.99 m
23.6 1.90 m,
1.96 m
23.6 1.88 m,
1.96 m
23.6 1.88 m,
1.94 m
1.96a
12 122.3 5.42 br s 122.4 5.41 br s 122.4 5.45 br s 122.3 5.41 br s 122.6 5.39 br s
122.6 5.37 br s
144.0 e
41.8 e
122.2 5.42 br s 122.7 5.36 br s 122.9 5.37 br s
13 144.0 e
14 42.1 e
15 28.6 1.65 m,
2.04a
144.0 e
42.1 e
28.6 1.54 m,
2.03 m
144.0 e
42.2 e
28.6 1.61 m,
2.03 m
144.1 e
42.1 e
144.1 e
41.9 e
144.2
42.1
e
143.8 e
41.8 e
28.1 1.02 m,
2.24 m
23.1 1.94 m,
2.02 m
143.8 e
e
41.9 e
28.5 1.60a,
1.99 m
28.0 1.12a, 2.29 dd 28.0 1.10a,, 2.28 dd 28.5 1.60a,
28.1 1.04a,
2.25 m
(10.2, 9.5)
(10.8, 9.8)
23.1 1.88 m,
2.00 m
46.8 e
41.4 3.14 dd
(13.6, 3.8)
2.02 m
23.2 2.12 m,
2.16 m
16 23.1 2.11 m,
2.19 m
17 47.0 e
18 41.7 3.10 dd
(13.2, 3.2)
23.2 2.05 m,
2.13 m
47.0 e
41.7 3.09 dd
(13.4, 3.3)
23.2 2.10 m,
2.17 m
47.0 e
41.8 3.11 dd
(13.4, 3.5)
23.2 2.12 m, 23.1 1.90 m,
2.16 m 2.01 m
46.9 e 46.8 e
41.4 3.17 dd
(13.2, 3.8)
23.1 1.90 m,
1.98 m
47.0
41. 8 3.11 dd
(13.5, 4.0)
e
46.8 e
41.4 3.13 dd
(13.5, 3.5)
46.8 e
41.4 3.14 dd
(13.8, 4.0)
41.8 3.08 m
(13.1,
3.2)
19 46.1 1.19 m,
1.74 m
20 30.5 e
21 33.8 1.11 m,
1.34 m
46.1 1.18 m,
1.69 m
30.5 e
33.8 1.12 m,
1.32 m
32.1 1.81 m,
1.91 m
46.2 1.19 m,
1.72 m
46.2 1.19 m, 46.0 1.20 m,
1.72 m 1.70 m
30.4 e 30.5 e
33.8 1.09 m, 33.7 1.07 m,
46.0 1.18 m,
1.68 m
30.5 e
33.7 1.05 m,
1.26 m
46.2 1.19 m,
1.72 m
45.9 1.17 m,
1.67a
45.9 1.19 m,
1.69 m
30.5 e
33.7 1.08 m,
1.29 m
32.3 1.73 m,
1.84 m
30.4 e
30.4
e
30.5 e
33.8 1.11a,
1.33 m
33.8 1.09 m,
1.32 m
33.7 1.05a,
1.26 m
1.32 m
32.0 1.84a,
1.89 m
1.27 m
32.3 1.74 m,
1.87 m
22 32.0 1.83 m,
32.1 1.84 m,
1.92 m
68.0 3.62 m,
4.11 m
32.3 1.72 m,
1.82 m
32.0 1.84a,
1.92 m
32.3 1.69 m,
1.80 m
63.6 3.86 m,
4.25 m
1.90a
23 27.8 1.09 s
63.7 3.64 m,
75.4 3.93a,
75.0 4.01a,
74.9 4.01a,
75.5 3.93a,
63.7 3.87 m,
4.25 m
4.10a
4.41a
4.40a
4.42a
4.41a
24 16.8 1.02 s
25 15.5 0.85 s
26 17.2 1.05 s
27 25.6 1.23 s
28 176.3 e
13.8 0.97 s
16.0 1.01 s
17.3 1.09 s
25.6 1.16 s
176.3 e
12.9 1.02 s
15.9 0.99 s
17.3 1.11 s
25.6 1.20 s
176.3 e
12.9 0.96 s
15.9 0.92 s
17.3 1.06 s
25.7 1.10 s
176.3 e
13.0 0.99 s
15.9 0.94 s
17.4 1.10 s
25.9 1.12 s
176.3 e
13.0 0.98 s
15.8 0.92 s
17.3 1.09s
25.9 1.10 s
176.3 e
12.9 0.96 s
15.9 0.93 s
17.3 1.07 s
25.7 1.11 s
14.0 1.12 s
16.0 0.94 s
17.3 1.07 s
25.8 1.14 s
176.3 e
13.9 1.11 s
15.9 0.96 s
17.3 1.08 s
25.8 1.15 s
176.2 e
176.3
e
29 33.0 0.85 s
30 23.5 0.84 s
32.9 0.82 s
23.6 0.86 s
32.9 0.82 s
23.5 0.85 s
32.9 0.82 s
23.5 0.82 s
32.9 0.84 s
23.4 0.85 s
32.9 0.83 s
23.4 0.84 s
32.9 0.82 s
23.5 0.82 s
32.9 0.84 s
23.4 0.84 s
32.8 0.84 s
23.4 0.84 s
a
Overlapped with other signals.
cancer cell lines. Previous investigations of C. tangutica were
focused on the antifungal and cardioprotective activities. Herein,
reported were four monodesmosidic saponins (12e15) that
possessed moderate cytotoxicity against all of the test cancer cell
lines for the first time. In addition, clematangoticoside D (4) and F
(6) exhibited selective cytotoxicity against SGC-7901. This work
enriched the diversity of triterpenoid saponins of the genus of
Clematis.
Waters Xevo™ TQ mass spectrometer and a Bruker MicrOTOF QII
mass spectrometer, respectively. GC-MS was performed on an
Agilent 6890 GS/5973 MS apparatus using an HP-1 capillary column
(25 m ꢀ 0.32 mm i.d., 0.25
mM, Agilent Tech., USA) with an initial
temperature of 130 ꢁC for 2 min and then ramping to 300 ꢁC by
temperature programming at the rate of 15 ꢁC/min. Semi-
preparative HPLC was performed on a LC 3000 liquid chromatog-
raphy (ChuangXingTongHeng Science And Technology Co., Beijing)
equipped with a Kromasil RP-C18 column (10 ꢀ 250 mm, 5
a LC 3000 UV detector (detection wavelength: 205 nm). Column
chromatography (CC) was performed with silica gel (75 m, Qing-
dao Marine Chemical Factory, China), ODS (50 m, YMC, Japan), and
Sephadex LH-20 (50 m, Amersham Pharmacia Biotech, UK).
mm) and
4. Experimental
m
m
4.1. General experimental procedures
m
Fractions were monitored by TLC detection which was achieved by
spraying the silica gel plates (Qingdao Marine Chemical Factory,
China) with 20% H2SO4eEtOH solution followed by heating. All of
the solvents used were of analytical grade.
Melting points were determined on a BOT-X6 apparatus. Optical
rotations were measured on a Perkin Elmer Model 341 Polarimeter.
IR measurements were obtained on a PerkinElmer one FT-IR
spectrometer (KBr). The 1D and 2D NMR spectra were performed
on a Bruker Ascend 400 spectrometer in pyridine-d5 with TMS as an
internal standard. The ESIMS and HRESIMS were recorded on a

M. Zhao et al. / Phytochemistry 130 (2016) 228e237
233
Table 2
13C NMR spectroscopic data for the glycoside moieties of compounds 1e8, and 8a (100 MHz in pyridine-d5).
1
2
3
4
5
6
7
8
8a
28-O-Glc I
1 94.6
28-O-Glc I
94.6
28-O-Glc I
94.6
28-O-Glc I
94.6
28-O-Glc I
95.5
28-O-Glc I
95.4
28-O-Glc I
94.6
28-O-Glc I
95.4
28-O-Glc I
95.4
2 74.7
3 79.5
4 70.8
5 77.7
6 68.8
74.6
79.3
71.0
77.7
74.9
79.4
70.9
77.7
74.5
79.6
70.9
77.7
73.6
78.6
70.7
77.8
73.6
78.6
70.6
77.8
74.6
79.5
70.9
77.6
73.6
78.5
70.6
77.8
73.6
78.5
70.6
77.8
69.0
68.9
68.9
69.2
68.9
69.1
68.9
69.0
Rha I (at C-2Glc I
)
Rha I (at C-2Glc I
101.3
72.0
72.3
73.6
)
Rha I (at C-2Glc I
101.2
72.0
72.4
73.6
)
Rha I (at C-2Glc I
101.1
72.0
72.4
73.6
)
Rha (at C-2Glc I
101.1
72.0
72.3
73.6
)
Glc II (at C-6Glc I
104.6
75.2
76.3
77.8
)
Glc II (at C-6Glc I
104.6
75.1
76.1
77.8
)
1 101.2
2 72.0
3 72.4
4 73.6
5 69. 5
6 18.5
69. 5
18.5
69.5
18.5
69.5
18.5
69.5
18.5
76.9
61.0
76.9
61.0
Glc II (at C-6Glc I
)
Glc II (at C-6Glc I
)
Glc II (at C-6Glc I
)
Glc II (at C-6Glc I
)
Glc II (at C-6Glc I) Glc II (at C-6Glc I) Glc II (at C-6Glc I) Rha I (at C-4Glc II
)
Rha I (at C-4Glc II
102.5
72.3
72.5
73.5
)
1 104.6
2 75.1
3 76.2
4 78.0
5 76.9
6 61.0
104.6
75.1
76.3
78.2
76.9
61.1
104.6
75.1
76.2
78.0
76.9
61.1
104.7
75.1
76.2
78.0
76.9
61.0
105.1
75.0
78.3
71.3
78.2
62.4
104.7
75.2
76.3
77.9
77.0
105.2
74.9
78.2
71.2
78.1
62.3
102.5
72.4
72.5
73.6
70.1
70.1
18.3
61.0
18.3
Rha II (at C-4Glc II) Rha II (at C-4Glc II) Rha II (at C-4Glc II) Rha II (at C-4Glc II
)
Rha (at C-4Glc II
102.5
72.4
72.6
73.8
)
3-O-Ara
104.5
75.2
74.9
69.3
3-O-Ara
104.4
75.1
74.9
69.3
1 102.5
2 72.4
3 72.5
4 73.8
5 70.1
6 18.3
102.6
72.4
72.5
73.8
70.1
102.6
72.4
72.5
73.8
70.1
18.3
102.5
72.3
72.5
73.8
70.1
70.1
18.3
66.2
66.0
18.3
18.3
3-O-Ara
3-O-Ara
104.2
75.6
75.2
69.1
23-O-Glc III
105.2
75.1
78.5
71.5
23-O-Glc III
105.2
75.1
78.5
71.5
23-O-Glc III
105.2
75.1
78.5
71.5
23-O-Glc III
105.2
75.1
78.5
71.5
Rha II (at C-2Ara
)
Rha II (at C-2Ara)
1 104.8
2 75.7
3 73.8
4 68.6
5 64.7
6
101.1
71.7
82.9
72.8
69.7
18.2
101.1
71.7
82.9
72.8
69.5
18.2
65.5
78.3
62.8
78.3
62.7
78.3
62.7
78.3
62. 8
Rha III (at C-2Ara
)
Rha III (at C-2Ara
)
Xyl (at C-3Rha II
107.1
75.2
76.0
77.4
)
Xyl(at C-3Rha II
107.0
75.0
76.0
77.2
)
1 101.5
2 72.2
3 72.4
4 73.8
5 69.7
6 18.4
101.4
72.2
72.4
73.9
69.6
18.3
64.5
64.6
Glc III (at C-4Xyl
)
Glc III (at C-4Xyl)
1
2
3
4
5
6
102.8
73.8
76.1
81.1
76.0
60.6
102.8
73.8
76.6
80.8
76.3
61.6
Glc IV (at C-4Glc III) Glc IV (at C-4Glc III
)
1
2
3
4
5
6
102.3
74.7
76.2
71.7
78.4
62.2
104.7
74.5
78.0
71.3
78.2
62.2
4.2. Plant material
(75:25, v/v; 10 L ꢀ 3) at room temperature. The combined extracts
were concentrated to dryness under vacuum. The concentrated
extract (320 g) was suspended in H2O and then partitioned suc-
cessively with EtOAc and n-BuOH saturated with H2O. The n-BuOH
Whole plants of C. tangutica were collected from the Duilong
Country of the Tibet Autonomous Region, P. R. China, in September
2014 and identified by Dr. Zhuo-Ma Da-Wa (Tibet Autonomous
Region Institute for Food and Drug control, Lhasa). A voucher
specimen (No. CIB20150607) is deposited at Chengdu Institute of
Biology, Chinese Academy of Sciences, Chengdu, P. R. China.
extract (60 g) was subjected to silica gel CC (75
and eluted with gradient of CH2Cl2eMeOHeH2O (30:1:0;
20:1:0.1; 10:1:0.1; 8:2:0.1; 7:3:0.3; 3:2:0.2; 1:1:0.1) to obtain
fractions A-G. Fraction D (27 g) was subjected to CC (ODS, 50 m,
mm, 8 ꢀ 60 cm, 1 kg)
a
m
4 ꢀ 50 cm, 400 g), and eluted with 30%, 40%, 50%, 60%, 70%, 80%,
90% and 100% MeOH in H2O to give 10 subfractions (a-h). Fraction
4.3. Extraction and isolation
D-g (1.2 g) was submitted to Sephadex LH-20 CC (50
mm,
2 ꢀ 150 cm, 250 g), with MeOHeH2O (70:30, v/v) to yield com-
Air-dried and powdered plant material of C. tangutica (2.5 kg)
was extracted three times (each for 7 days) with MeOHeH2O
pound 1 (262 mg). Fraction D-d (5.5 g) was purified by semi-

234
M. Zhao et al. / Phytochemistry 130 (2016) 228e237
Table 3
1H NMR spectroscopic data for the glycoside moieties of compounds 1e8, and 8a (400 MHz in pyridine-d5).
1
2
3
4
5
6
7
8
8a
28-O-Glc I
1 6.08 d (8.1)
2 4.38 m
3 4.25a
28-O-Glc I
6.07 d (8.1)
4.36 m
4.23a
28-O-Glc I
6.09 d (8.1)
4.39a
28-O-Glc I
6.08 d (8.1)
4.41 m
28-O-Glc I
6.25 d (8.0)
4.11 m
28-O-Glc I
6.23 d (8.1)
4.11 m
28-O-Glc I
6.10 d (8.1)
4.42 m
28-O-Glc I
6.23 d (8.0)
4.10 m
4.18a
28-O-Glc I
6.22 d (8.1)
4.08a
4.24a
4.26a
4.18a
4.18a
4.28a
4.18a
4 4.25a
4.22a
4.24a
4.25a
4.32 m
4.09 m
4.34 m, 4.70 br
d (10.2)
4.30 m
4.28a
4.29 m
4.29 m
4.08 m
4.31a,
5 4.07 m
4.06 m
4.07 m
4.06 m
4.08a
4.06 m
4.08a
6 4.26a,
4.24a,
4.26a, 4.60 br
d (11.0)
4.26a, 4.60 br
d (10.5)
4.31 m, 4.65 br
d (10.7)
4.28a, 4.65 br
d (10.2)
4.31a,
4.61 m
4.60 m
4.65 m
4.66 m
Rha I (at C-2Glc Rha I (at C-2Glc Rha I (at C-2Glc I
)
Rha I (at C-2Glc I
)
Rha (at C-2Glc I
)
Glc II (at C-6Glc Glc II (at C-6Glc
)
)
I
)
I
)
I
I
1 6.60 br s
2 4.78 br s
3 4.55a
6.53 br s
4.76 br s
4.53a
6.57 br s
4.77 br s
4.55a
6.62 br s
4.76 br s
4.55a
6.61 br s
4.76 br s
4.54a
4.98 d (7.6)
3.98 m
4.14a
4.97 d (7.8)
3.98a
4.14a
4.40 dd (9.0,
8.4)
4 4.33a
4.32a
4.15a
4.32a
4.32 m
4.41 m
5 4.57 m
6 1.77 d (6.1)
4.51a
1.75 d (6.1)
4.54a
1.76 d (6.0)
4.55a
1.76 d (6.0)
4.55a
1.77 d (6.2)
3.63 m
3.64 m
4.07a, 4.20a
4.08a, 4.19a
Glc II (at C-6Glc Glc II (at C-6Glc Glc II (at C-6Glc I
)
Glc II (at C-6Glc I
)
Glc II (at C-6Glc I
5.02 d (7.7)
)
Glc II (at C-6Glc I
4.98 d (7.8)
)
Glc II (at C-6Glc I
)
Rha I (at C-4Glc Rha I (at C-4Glc
)
)
I
)
)
II
I
II
1 4.95 d (7.8)
2 3.91 dd (8.5,
8.2)
3 4.14 m
4 4.39 dd (9.4,
9.0)
4.94 d (7.6)
3.90 dd (8.5,
8.2)
4.13 m
4.36 dd (9.5,
8.8)
4.95 d (7.6)
4.94 d (7.7)
4.97 d (7.8)
3.94a
5.87 br s
4.65 m
5.83 br s
4.65 m
3.91 dd (8.5, 8.3) 3.93a
3.97 dd (9.0, 8.0)
3.93 dd (8.5, 8.2)
4.15a
4.14 m
4.19 m
4.14a
4.43 dd (9.3, 9.0)
3.85 m
4.54a
4.53a
4.31a
4.39 dd (8.9, 8.1) 4.41 dd (8.4, 7.8) 4.20 m
4.28 dd (8.6, 8.0) 4.32a
5 3.64 m
3.64 m
3.65 m
4.09 m,
4.18 m
3.64 m
4.09 m,
4.18 m
3.88 m
4.35 m,
3.64 m
4.08a,
4.17a
4.36 m,
4.46 m
4.96 m
4.94 m
6 4.08a,
4.08a,
1.69 d (6.1)
1.68 d (6.2)
4.18 m
4.18 m
4.47 dd (11.2, 1.8) 4.18a
Rha II (at C-4Glc Rha II (at C-4Glc Rha II (at C-4Glc II) Rha II (at C-4Glc II
)
Rha (at C-4Glc II
)
3-O-Ara
3-O-Ara
)
)
II
II
1 5.84 br s
5.82 br s
5.84 br s
4.66 br s
5.84 br s
4.66 br s
5.87 br s
4.67 br s
5.03 d (6.0)
4.57 m
5.04 d (6.5)
4.56 dd (6.8,
6.6)
2 4.67a
4.66a
3 4.55a
4.52 dd (9.0,
3.5)
4.31 m
4.93 m
4.55 dd (9.1, 3.0) 4.55a
4.54 dd (9.5, 3.2)
3.99 m
4.00a
4 4.32 m
5 4.96 m
4.32 t (9.4)
4.97 m
4.32 t (9.4)
4.97 m
4.33 m
4.98 m
4.11a
4.12a
3.66a, 4.23a
3.67 d (10.8),
4.24a
6 1.68 d (6.2)
1.68 d (6.2)
1.69 d (6.0)
1.68 d (6.2)
1.69 d (6.2)
3-O-Ara
3-O-Ara
23-O-Glc III
23-O-Glc III
23-O-Glc III
23-O-Glc III
Rha II (at C-
Rha II (at C-
2Ara
)
2Ara)
1 4.86 d (5.3)
2 4.55 dd (8.0,
6.0)
5.07 d (6.3)
4.54a
4.90 d (7.7)
4.03 dd (8.3, 8.0) 4.01 dd (8.5, 8.0)
4.90 d (7.7)
4.90 d (7.7)
4.01a
4.90 d (7.7)
4.03
6.34 br s
4.85 m
6.29 br s
4.83 m
3 4.29a
4.09a
4.15 m
4.19a
4.18a
4.18a
4.18a
4.18a
4.18a
4.18a
4.18a
4.68a
4.68a
4.44 dd (9.6,
9.5)
4 4.27 m
4.43 dd (9.3,
9.2)
5 3.82 d (10.0),
3.67 d (10.0),
4.23a
3.95 m
3.90 m
3.92 m
3.95a
4.67a
4.66a
4.30a
6
4.39 m,
4.58 m
4.36 m,
4.37 m,
4.38 m,
1.51 d (6.0)
1.53 d (6.1)
4.53 dd (11.0, 2.2) 4.53 dd (10.5, 2.5) 4.57 m
Rha III (at C-
Rha III (at C-
Xyl (at C-3Rha II) Xyl(at C-3Rha II)
2Ara
)
2Ara)
1 6.16 br s
2 4.75 br s
6.22 br s
4.70 br s
5.26 d (7.6)
3.98 dd (9.2,
7.8)
5.25 d (7.7)
3.98a
3 4.62 m
4 4.33a
4.62 m
4.26 m
4.66a
4.12a
4.12a
4.21a
4.21a
5 4.66a
3.59 m, 4.30a
3.59 m, 4.30a
6 1.62 d (6.1)
1.62 d (6.2)
Glc III (at C-
Glc III (at C-4Xyl)
4Xyl
)
1
2
4.86 d (8.1)
3.94 dd (8.0,
7.8)
4.94 d (7.7)
3.98a
3
4
5
6
4.12a
4.13a
4.27a
4.27a
3.68 m
3.65 m
4.18a, 4.45a
4.18a, 4.45 m
Glc IV (at C-4Glc Glc IV (at C-4Glc
)
)
III
III
1
2
5.37 d (7.8)
5.77 dd (8.7,
8.5)
5.16 d (7.9)
3.99a

M. Zhao et al. / Phytochemistry 130 (2016) 228e237
235
Table 3 (continued )
1
3
2
3
4
5
6
7
8
8a
4.29 dd (9.0,
8.2)
4.20a
4
5
6
4.16a
4.17 m
4.08a
4.00a
4.22a, 4.55 m
4.27a, 4.52 m
a
Overlapped with other signals.
4.3.4. 23-O-
rhamnopyranosyl-(1 / 4)-
rhamnopyranosyl-(1 / 2)]-
b
-D-glucopyranosyl hederagenin 28-O-
-D-glucopyranosyl-(1 / 6)-[
-D-glucopyranoside (4)
White amorphous powder; C60H98O27 ]20D -45 (c 0.1,
a-L-
Table 4
Cytotoxicity of saponins 4, 6, 12e15 against four cancer cell lines (IC50
a
,
mM).
b
a-L-
b
Saponins
SGC-7901
HepG2
HL-60
U251MG
;
[a
4
6
12
13
14
15
24.14 2.10
21.35 1.88
8.18 0.53
10.32 0.48
9.08 0.29
9.01 0.21
0.33 0.05b
>50
>50
>50
>50
>50
>50
MeOH); IR (KBr) ymax 3444, 2934, 1700, 1639, 1534, 1462, 1065,
1046 cmꢂ1; HRESIMS: m/z 1273.6173 [MþNa]þ (calcd. 1273.6193);
for 1H NMR and 13C NMR spectroscopic data, see Tables 1e3.
1.88 0.41
4.24 0.58
7.26 0.78
6.01 0.52
0.18 0.02b
5.21 0.52
6.58 0.42
8.72 0.91
5.34 0.38
0.36 0.05b
17.32 1.25
15.37 1.18
27.20 2.78
21.12 3.16
0.95 0.06c
4.3.5. 23-O-
glucopyranosyl-(1 / 6)-
White amorphous powder; C48H78O19; [
b
-D-glucopyranosyl hederagenin 28-O-
-D-glucopyranoside (5)
]20D þ4 (c 0.1, MeOH);
b-D-
Positive control
b
a
IC50 values are means from three independent experiments (mean
which each compound concentration was tested in three replicate wells.
SD) in
a
b
IR (KBr) ymax 3502, 2947, 2324, 1716, 1525, 1465, 1066, 1046 cmꢂ1
;
Adriamycin as positive control.
Nimustine as positive control.
HRESIMS: m/z 981.5015 [MþNa]þ (calcd. 981.5035); for 1H NMR
c
and 13C NMR spectroscopic data, see Tables 1e3.
preparative HPLC using MeOHeH2O (65:35, v/v; 4 mL/min) to yield
saponins 4 (36 mg, tR 8.5 min), 6 (17 mg, tR 10.7 min), 7 (24 mg, tR
11.8 min), 11 (197 mg, tR 14.2 min), and 5 (22 mg, tR 15.8 min).
Fraction D-e (4.3 g) was purified by semi-preparative HPLC using
MeOHeH2O (60:40, v/v; 2.5 mL/min) to give saponins 13 (19 mg, tR
28.2 min), 14 (23 mg, tR 32.4 min), 15 (11 mg, tR 35.8 min), 8 (32 mg,
tR 39.2 min), 2 (27 mg, tR 42.3 min), and 9 (21 mg, tR 48.0 min).
Fraction D-f (3.6 g) was also purified by semi-preparative HPLC
using MeOHeH2O (66:34, v/v; 2.5 mL/min) to give saponins 10
(14 mg, tR 15.0 min), 3 (18 mg, tR 22.3 min), 16 (35 mg, tR 24.5 min),
and 12 (20 mg, tR 30.6 min).
4.3.6. 23-O-
rhamnopyranosyl-(1 / 4)-
glucopyranoside (6)
b
-D-glucopyranosyl hederagenin 28-O-
a
-L-
b
-D-glucopyranosyl-(1 / 6)-b-D-
White amorphous powder; C54H88O23; [a]20D -11 (c 0.1, MeOH);
IR (KBr) ymax 3393, 2942, 1725, 1457, 1075 cmꢂ1; HRESIMS: m/z
1127.5627 [MþNa]þ (calcd. 1127.5614); for 1H NMR and 13C NMR
spectroscopic data, see Tables 1e3.
4.3.7. 23-O-
rhamnopyranosyl-(1 / 2)-[
glucopyranoside (7)
b
-D-glucopyranosyl hederagenin 28- O-
a
-L-
b-D-glucopyranosyl-(1 / 6)]-b-D-
White amorphous powder; C54H88O23
; [a]20D -22 (c 0.1,
MeOH); IR (KBr) ymax 3393, 2942, 1725, 1457, 1075 cmꢂ1; HRESIMS:
m/z 1127.5640 [MþNa]þ (calcd. 1127.5614); for 1H NMR and 13C
NMR spectroscopic data, see Tables 1e3.
4.3.1. 3
oleanolic acid 28-O-
glucopyranosyl-(1 / 6)-[
b
-O-
a
-L-rhamnopyranosyl-(1 / 2)-
a
-L-arabinopyranosyl
-D-
-L-rhamnopyranosyl-(1 / 2)]-
a-L-rhamnopyranosyl-(1 / 4)-
b
a
b-D-
glucopyranoside (1)
Colorless needles (CH3OH); C65H106O29; mp 212e214 ꢁC; [
a
]20D
4.3.8. 3
glucopyranosyl-(1 / 4)-
rhamnopyranosyl-(1 / 2)-
O- -L-rhamnopyranosyl-(1 / 4)-
D-glucopyranoside (8)
Yellowish amorphous powder; C85H130O43; [
b
-O-(2-O-caffeoyl)-
-D-xylopyranosyl-(1 / 3)-
-L-arabinopyranosyl hederagenin 28-
-D-glucopyranosyl-(1 / 6)-
b
-D-glucopyranosyl-(1 / 4)-
b-D-
-44 (c 0.1, MeOH); IR (KBr) ymax 3444, 2926, 1635, 1061 cmꢂ1
;
b
a-L-
HRESIMS: m/z 1373.6724 [MþNa]þ (calcd. 1373.6717); for 1H NMR
a
and 13C NMR spectroscopic data, see Tables 1e3.
a
b
b-
a]20D -30 (c 0.1,
4.3.2. 3
hederagenin 28-O-
glucopyranosyl-(1 / 6)-[
b
-O-
a
-L-rhamnopyranosyl-(1 / 2)-
a
-L-arabinopyranosyl
-D-
-L-rhamnopyranosyl-(1 / 2)]-
MeOH); IR (KBr) ymax 3435, 2933, 1630, 1063 cmꢂ1; HRESIMS: m/z
1839.8074 [MþH]þ (calcd. 1839.8064); for 1H NMR and 13C NMR
spectroscopic data, see Tables 1e3; the 1H and 13C NMR data for
the caffeoyl moiety: 166.7 (C-1), 114.7 (C-2)/6.69 (d, J ¼ 15.8, H-2),
146.2 (C-3)/8.07 (d, J ¼ 15.8, H-3), 126.7 (C-4), 115.6 (C-5)/7.46 (br s,
H-5), 147.5 (C-6), 150.3 (C-7), 116.5 (C-8)/7.17 (d, J ¼ 7.8, H-8), 121.8
(C-9)/7.10 (d, J ¼ 7.8, H-9).
a-L-rhamnopyranosyl-(1 / 4)-
b
a
b-D-
glucopyranoside (2)
White amorphous powder; C65H106O30; [a]20D -19 (c 0.1,
MeOH); IR (KBr) ymax 3435, 2930, 1623, 1057 cmꢂ1; HRESIMS: m/z
1389.6655 [MþNa]þ (calcd. 1389.6667); for 1H NMR and 13C NMR
spectroscopic data, see Tables 1e3.
4.3.9. 3
(1 / 4)-
(1 / 2)-
rhamnopyranosyl-(1 / 4)-
b
-O-
-D-xylopyranosyl-(1 / 3)-
-L-arabinopyranosyl hederagenin 28-O-a
b
-D-glucopyranosyl-(1 / 4)-
-L-rhamnopyranosyl-
-L-
-D-glucopyranosyl-(1 / 6)-
b-D-glucopyranosyl-
4.3.3. Hederagenin 28-O-
glucopyranosyl-(1 / 6)-[
glucopyranoside (3)
a
-L-rhamnopyranosyl-(1 / 4)-
b
-D-
-D-
b
a
a-L-rhamnopyranosyl-(1 / 2)]-
b
a
b
b
-D-
White amorphous powder;
C54H88O22; [a]20D -22 (c 0.1,
glucopyranoside (8a)
MeOH); IR (KBr) ymax 3431, 2932, 1727, 1623, 1061, 1038 cmꢂ1
;
White amorphous powder; C76H124O40; [a]20D -42 (c 0.1,
HRESIMS: m/z 1111.5651 [MþNa]þ (calcd. 1111.5665); for 1H NMR
MeOH); IR (KBr) ymax 3437, 2923, 1623, 1055 cmꢂ1; HRESIMS: m/z
1677.7750 [MþH]þ (calcd. 1677.7747); for 1H NMR and 13C NMR
and 13C NMR spectroscopic data, see Tables 1e3.

236
M. Zhao et al. / Phytochemistry 130 (2016) 228e237
spectroscopic data, see Tables 1e3.
compared to the untreated controls. Dose-response curves were
plotted for the samples, and the IC50 values were determined as the
mean SD.
4.4. Acid hydrolysis and GC analysis
Each saponin (3 mg) was hydrolyzed with 2 mol/L CF3COOH
(5 mL) at 100 ꢁC for 3 h. After partitioning between CHCl3 and H2O,
the aqueous phase was evaporated to dryness under vacuum. The
residue was dissolved in a solution of anhydrous pyridine (0.1 mL)
and 1-(trimethylsilyl)-imidazole (0.1 mL). The mixture was stirred
at 60 ꢁC for 5 min and dried with a stream of N2. The residue was
partitioned between hexane (1 mL) and H2O (1 mL), and the hexane
layer was analyzed by GC using an L-Chirasil-Val column (Masullo
et al., 2014). The configurations of the sugar units were estab-
lished by comparing retention times with those of the trime-
thylsilylated derivatives prepared in the same manner from the
standard sugars. Retention times for authentic samples after being
treated in the same way were detected at 8.85 and 9.87 min (L-
arabinose), at 9.47 and 10.30 min (L-rhamnose), at 10.98 and
12.00 min (D-xylose), and at 14.72 min (D-glucose), respectively.
Each saponin (10 mg) was hydrolyzed with 1 mol/L CF3COOH
(30 mL) at 80 ꢁC for 6 h. The mixture was neutralized with NaOH.
After partitioning between CHCl3 and H2O, the CHCl3 phase was
concentrated and purified by semi-preparative HPLC to give the
aglycone. The aglycone of 1 was proved to be oleanolic acid, and the
aglycones of 2e8 were proved to be hederagenin.
Acknowledgments
This work was financially supported grants from the National
Natural Sciences Foundation of China (21402157, 21402187,
21572219 and 21572221) and a Syngenta Postgraduate Fellowship
awarded to Min Zhao.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2016.05.009.
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purities > 95%, analyzed by HPLC) against SGC-7901 (human gastric
carcinoma cell line), HepG2 (human liver hepatoma cancer cell
line), HL-60 (human promyelocytic leukemia cancer cell line) and
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into each well, and the plate was incubated for another 4 h. DMSO
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m
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1
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m
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m
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