New acylated triterpene saponins from the roots of Securidaca inappendiculata Hassk

Article abstract of DOI:10.1016/j.phytol.2015.05.022

Two new acylated triterpene saponins, named as securioside C (1), securioside D (2), and one pair of isomers 3/4, the (Z)-isomer securioside E (3) being new, together with a known triterpene saponin polygalasaponin XLIV (4) were isolated from the roots of Securidaca inappendiculata Hassk. Their structures were established by HRESIMS, 1D and 2D NMR experiments and comparison of their NMR data with previous reported data. In addition, Compounds 1-2, 3/4, 4 were evaluated for cytotoxicities against LLC (Lewis lung carcinoma) and MCF-7 (human breast cancer) cell lines. Compounds 1 and 2 exhibited moderate cytotoxic activities against LLC cells with IC₅₀ values of 45.56 μM and 85.98 μM.

Full text of DOI:10.1016/j.phytol.2015.05.022

Phytochemistry Letters 13 (2015) 108–113
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
New acylated triterpene saponins from the roots of Securidaca
inappendiculata Hassk
a
a
a,
b
a
a
Haiyan Zha , Zhi Wang , Xuedong Yang *, Daqing Jin , Licui Hu , Wenfeng Zheng ,
c
d
Lizhen Xu , Shilin Yang
School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, PR China
School of Medicine, Nankai University, Tianjin 300072, PR China
Institute of Medicinal Plant Development, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100094, PR China
Jiangxi University of Traditional Chinese Medicine, No. 56 Yangmin Road, Nanchang 330006, PR China
a
b
c
d
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new acylated triterpene saponins, named as securioside C (1), securioside D (2), and one pair of
isomers 3/4, the (Z)–isomer securioside E (3) being new, together with a known triterpene saponin
polygalasaponin XLIV (4) were isolated from the roots of Securidaca inappendiculata Hassk. Their
structures were established by HRESIMS,1D and 2D NMR experiments and comparison of their NMR data
with previous reported data. In addition, Compounds 1–2,3/4, 4 were evaluated for cytotoxicities against
LLC (Lewis lung carcinoma) and MCF-7 (human breast cancer) cell lines. Compounds 1 and 2 exhibited
Received 16 March 2015
Received in revised form 23 May 2015
Accepted 26 May 2015
Available online xxx
Keywords:
moderate cytotoxic activities against LLC cells with IC50 values of 45.56 mM and 85.98 mM.
Polygalaceae
Triterpene saponin
Cytotoxicity
ã2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1
. Introduction
(Lewis lung carcinoma) and MCF-7 (human breast cancer) cell
lines. Therefore, as part of our ongoing investigation of anti-tumor
constituents, we reported the isolation and the structural
elucidation of two new triterpene saponins, one pair of (E)/(Z) –
isomers (the (Z) – isomer being new), and a known triterpene
saponin with presenegenin as aglycon (see Fig. 1) isolated from the
roots of S. inappendiculata. Cytotoxicities of compounds 1–2, 3/4, 4
were evaluated against LLC and MCF-7 cell lines, respectively.
Securidaca inappendiculata Hassk is a traditional Chinese herbal
medicine, belonging to the Polygalaceae family, mainly distributed
in Yunnan, Guangxi, Guangdong, and Hainan provinces of southern
China and the tropical regions of Asia (Delecti Florae Reipublicae
Popularis Sinicae Agendae Academiae Sinicae,1997). The roots of S.
inappendiculata are used as an anti-inflammatory, antibacterial,
and antirheumatism agent in China (Jiangsu Institute of Botany,
1988). Our previous investigations on this species resulted in the
2. Results and discussion
isolation of benzophenones (Yang et al., 2003b) from the
chloroform fraction, xanthones (Yang et al., 2001a) from the
chloroform/ethyl acetate fractions, xanthone glycosides (Yang
et al., 2002a) and organic acids (Yang et al., 2001b) from the ethyl
acetate/acetone fractions, and oligosaccharide esters (Yang et al.,
Compound 1, obtained as a white amorphous powder, was
96
assigned a molecular formula of C65H O28 which was deduced
+
from quasi-molecular ion peak ([M + Na] ) at m/z 1347.6020 in the
1
positive ion mode HRESIMS. The H NMR spectrum of 1 showed
2
003a) and terpenoic glycosides (Yang et al., 2002b) from the
acetone fraction of ethanol extract. Two saponins (Kuroda et al.,
001) obtained from the water extract of the roots of the plant have
signals for five tertiary methyl groups at
d
H
0.76, 0.88, 1.11, 1.48,
1.90, which correlated with five sp C-atoms at 34.0, 24.3, 19.0,
17.8, 14.6 respectively based on the HSQC spectrum, also an
isolated oxymethylene at 3.78, 4.04 (H -27), an oxymethine
proton at 4.68 (H-2), an olefinic proton at 5.78 (H-12)
3
d
C
2
been reported. Modern pharmacological evaluations revealed that
the saponins from the roots of S. inappendiculata exhibited
macrophage-oriented cytotoxic activity. In our preliminary exper-
iment, the aqueous portion of ethanol extract from the roots of S.
inappendiculata showed cytotoxic activities in vitro against LLC
d
H
2
d
H
d
H
2
combined with two sp olefinic carbons at
d
C
128.2 (C-12),139.2 (C-
13), and two carboxylic carbon signals at d 181.0 (C-23), 177.0 (C-
C
13
28) in the C NMR spectrum. These data proved that the aglycone
possessed an olean-12-ene-23,28-dioic acid skeleton (Tables 1 and
2
), and was in full agreement with those of tenuifolin (3-O-
glucopyranosyl presenegenin) in which presenegenin
((2 ,3 ,4 )-2,3,27-trihydroxyolean-12-ene-23,28-dioic acid)
b-D-
*
Corresponding author. Tel.: +86 22 87401152.
E-mail address: yangxd@tju.edu.cn (X. Yang).
b b a
http://dx.doi.org/10.1016/j.phytol.2015.05.022
874-3900/ã 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
1

H. Zha et al. / Phytochemistry Letters 13 (2015) 108–113
109
30
29
2
0
1
9
21
22
1
2
1
8
1
3
1
1
17
25
26
1
4
16
HO
O
28
1
5
O
1
9
1
5
0
2
3
8
7
C
CH OH
2
2
OH
7
4
6
OR1
H C
O
3
HO
HO
O
O
O
COOH
24
23
2
R O
OH
Fuc
Glc
O
H C
3
O
O
R O
3
HO
HO
Rha
OH
Xyl
H
OH
H
OCH
3
OCH3
H
OCH
3
OCH
3
H
Z-DMC =
OCH
3
OCH3
O
E-DMC =
E-TMC=
O
H
O
H
OCH3
OH
OH
OH
OH
O
O
O
HO
HO
HO
HO
OH
Ara
OH
OH
Glc
Gal
R1
R2
R3
H
1
2
E-TMC
E-DMC
Z-DMC
H
Ara
Glc
H
3
Gal
Fig. 1. Structures of compounds 1–3.
was commonly encountered in triterpene saponins isolated from
the family Polygalaceae (Mitaine-Offer et al., 2002; Teng et al.,
b
-fucopyranosyl, one b-xylopyranosyl, and one a-rhamnopyr-
anosyl. After acid hydrolysis, the sugar units were confirmed to be
-glucose, -fucose, -rhamnose, and -xylose by HPLC analysis of
2002; Zhang et al., 1996). The chemical shift values at
d
C
86.3 (C-3)
D
D
L
D
and 177 (C-28) suggested that the saponin was a bisdesmosidic
d
C
their thiocarbamoyl-thiazolidine derivatives compared with au-
thentic samples.
glycoside with saccharide units attached to these positions. Beside
1
these, the H NMR spectrum was also displayed signals of four
The HMBC correlation between
d
H
5.02 (1H, s, Glc-1) and
C
d 86.3
anomeric protons at
1H, d, J = 5.8 Hz), and 5.02 (1H, s), which gave correlations with
four anomeric carbon signals at 102.0, 94.9, 107.7, and 105.7 in
d
H
6.35 (1H, s), 6.17 (1H, d, J = 8.15 Hz), 5.03
(Agly-C-3) and NOE correlation between
4.54 (Agly-H-3) in the NOESY spectrum revealed a linkage between
the aglycon and a glucopyranosyl moiety. A correlation in the
d
H
5.02 (Glc-1) and d
H
(
d
C
the HSQC spectrum respectively. The COSY, TOCSY, HSQC, and
HMBC spectroscopic experiments identified the direct correlations
of protons of sugar ring from the readily identifiable anomeric
protons (Tables 1 and 2). The coupling constant confirmed the
H C
HMBC spectrum between d 6.17 (1H, d, J = 8.15 Hz) (Fuc-1) and d
177 (Agly-C-28) indicated that a fucose was attached to C-28 of the
aglycon. Moreover, in the HMBC spectrum, long-range correlations
were observed between the following protons and carbons:
H
d 4.70
presence of the units of one
b
-glucopyranosyl, one
(Fuc-2) and 102.0 (Rha-1), 4.35 (Rha-4) and 107.7 (Xyl-1),
d
C
d
H
d
C

110
H. Zha et al. / Phytochemistry Letters 13 (2015) 108–113
Table 1
Table 2
1
13
H NMR spectral data (500 Hz, pyridine-d
5
) for compounds 1–3 (
d
ppm).
5
C NMR spectral data (125 Hz, pyridine-d ) for compounds 1–3 (d ppm).
1
2
3
1
2
3
1
2
3
1
2
3
Aglycone- 1.32,
1.31,
2.24
4.68
4.57
2.17
1.33,
2.24
4.72
4.58
2.17
Xyl-1 5.03(d, 5.02(d, 4.96(d,
Aglycone-1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
3-O-Glc-1
2
44.5
70.6
86.3
53.2
52.8
21.7
33.9
41.5
49.6
37.3
23.9
128.2
139.2
48.3
24.1
24.2
47.3
42.3
45.6
31.1
33.6
32.1
181.0
14.6
17.8
19.0
64.8
177
34.0
24.3
105.7
75.6
78.7
71.9
78.7
63.0
94.9
74.6
74.6
75.3
71.2
17.0
44.3
70.1
86.0
52.9
52.5
21.4
33.5
41.1
49.3
37.0
23.5
127.9
139.1
48.0
24.5
24.0
47.0
42.0
45.2
30.8
33.7
32.3
181.2
14.2
17.5
18.9
64.2
176.9
32.8
24.0
105.3
75.1
78.3
71.5
78.3
62.4
94.1
73.8
81.6
74.4
71.1
44.0
70.3
85.8
52.6
52.2
21.4
33.7
41.0
49.1
36.9
23.4
127.6
139.0
47.9
24.4
23.8
46.9
42.0
45.2
30.6
33.6
32.1
181.0
14.2
17.3
18.7
64.4
176.8
33.0
23.8
105.2
74.8
78.0
71.1
77.9
62.6
94.7
73.2
82.7
74.2
70.9
16.5
Rha-1
2
3
4
5
6
Xyl-1
2
3
4
102.0
72.1
72.8
85.3
68.8
18.8
107.7
76.5
79.1
71.2
67.8
101.7
71.9
72.3
84.4
68.7
18.7
107.1
74.2
76.1
78.6
67.0
101.5
71.8
72.9
84.5
68.4
18.7
106.8
75.6
76.5
78.2
64.8
104.3
71.4
75.0
70.1
77.0
62.1
105.6
74.9
78.2
71.2
78.2
62.7
1
2.24
4.68
4.54
2.16
5.8)
6.5)
7.0)
2
3
5
6
2
3
4
5
4.03
4.02
4.15
3.78,
4.04
4.02
4.02
3.46,
4.33
3.98
4.00
4.27
3.44,
4.28
4.94(d,
1.75,1.91 1.75,1.91 1.76,1.93
4
.18
7
9
1.15,1.20 1.11,1.22 1.14,1.25 Gal-1
7
.5)
2.30
1.92,
2.32
1.92,
2.07
5.80
1.63,
2.11
1.62,
2.03
3.19
2.33
1.92, nd
2
3
4.44
3.89
11
5
2
.11
Gal-1
2
3
4
5
1
15
2
5.78
1.63,
5.82
1.62,
2.07
1.65,
2.15
3.21
4
5
4.47
4.13
2
.10
1.62,
.04
3.18
1
6
6
4.33
2
6
18
Glc-1
2
5.10(d,
7.2)
3.95
Glc-1
2
3
4
5
(
d,13.5) (d,13.5) (d,13.5)
1
9
1.26,
.70
1.17
1.27,
1.71
1.21
1.30,
1.75
1.17
1
2
2
2
2
2
1
3
4
5
6
4.14
4.02
3.90
4.39
2
nd, 2.08 nd, 2.08 nd, 2.01
6
4
5
6
1.90(s)
1.48(s)
1.11(s)
1.94(s)
1.53(s)
1.11(s)
1.93(s)
1.53(s)
1.10(s)
Ara-1
2
3
4
5
MC
C(1)
C(2)
C(3)
C(4)
C(5)
C(6)
106.5
72.4
74.6
69.1
67.2
Ara-1
4.97(d,
6
.9)
27
3.78,
3.78,
4.04
0.76(s)
0.89(s)
3.78,
4.04
0.76(s)
0.88(s)
5.04(d,
7.5)
2
4.40
4
.04
2
3
3
9
0
0.76(s)
0.88(s)
5.02 (s) 5.02(d,
.5)
3
4
5
4.04
4.26
3.46,
4.19
130.7
105.8
154.4
141.5
154.4
106.7
167.8
118.1
146.2
56.7
128.0
111.3
149.5
152.4
112.1
123.4
167.4
116.1
146.2
56.1
127.5
115.0
151.4
152.0
111.6
125.8
166.6
116.6
145.1
55.5
-Glc-1
6
3.90
4.14
3
4
5
6
2
3
3.93
4.14
3.92
4.15
MC
H-C
6.84(s) 7.04(s) 7.92(br
(
2)
H-C
5)
H-C
6)
H-C
s)
C(
C(
C(
a
b
g)
)
)
4
5
6
2
2
3
4
4.13
3.91
4.29
4.13
3.90
4.24
3.89
4.26
4.29,
6.94(d, 6.93(d,
28-O-Fuc-1
(
8.5)
6.84(s) 7.05(d, 7.50(d,
8.5) 8.4)
6.49(d, 5.91(d,
(d,16.0) 16.0) 12.6)
7.95 7.85(d, 6.88(d,
(d,16.0) 16.0) 12.6)
8.4)
2
3
4
5
6
MeO-C(3)
MeO-C(4)
MeO-C(5)
(
61.0
56.7
56.4
55.9
6.61
4
.43
(
b)
16.1
8-Fuc-1 6.17(d,
.15)
6.13(d,
8.3)
4.73
6.13(d,
7.7)
4.77
H-C
(g)
8
4.70
4.48
5.75
MeO- 3.81(s) 3.81(s) 3.68(s)
C(3)
MeO- 3.90(s) 3.83(s) 3.86(s)
C(4)
MeO- 3.81(s)
C(5)
H H
H-6 of benzene and signals at d 3.81 (6H, s) and d 3.90 (3H, s)
4.51
5.95
4.47
5.97
belong to the three methoxy groups of benzene. Further
investigations based on the HMBC and HSQC spectra allowed
the complete assignments of all protons and carbons of the
trisubstituted benzene ring (Tables 1 and 2) which were in good
agreement with those described in literatures (Elbandy et al.,
5
6
4.13
1.34(d,
4.11
1.30(d,
6.0)
4.13
1.26(d,
6.0)
6.36(br
s)
4.82(br
s)
4.63
4.24
4.45
1.71(d,
5.5)
6
.0)
2
004; Mitaine-Offeret al., 2010). The downfield shifts observed in
the HSQC spectrum for the H-4 and C-4 resonances of fucose at
.75 and 75.3 established the position at C-4 of fucose to be
acylated by the 3,4,5-trimethoxycinnamic acid. For the above
evidences, the structure of was elucidated as 3-O-
glucopyranosyl presenegenin 28-O-
-xylopyranosyl-(1 !4)-
-rhamnopyransyl-(1 !2)-4-O-[(E)-3,4,5-trimethoxycinna-
moyl]- -fucopyranosyl ester.
Compound 2, obtained as a white amorphous powder, was
assigned a molecular formula of C69 31 which was deduced
Rha-1
2
6.35(s)
6.28(s)
d
H
5
d
C
4.79
4.81
3
4
5
6
4.68
4.35
4.55
1.79(d,
4.64
4.28
4.44
1.74(d,
5.5)
1
b-D-
b-D
a-L
b-D
5
.5)
H O
102
+
which revealed the (1 !2) linkage between rhamnose and fucose,
and a xylopyranosyl unit was linked to rhamnose by a (1 !4)
linkage. Above conclusions also can be confirmed by the NOE
from the quasi-molecular ion peak ([M + Na] ) at m/z 1449.6368 in
the positive ion mode HRESIMS. After acid hydrolysis, the sugar
units were confirmed to be
-xylose, and -arabinose by HPLC analysis of their thiocarbamoyl-
thiazolidine derivatives compared with authentic samples. Com-
parison of the spectroscopic data of 2 (Tables 1 and 2) with those of
showed they have the same structure of 3-O-
presenegenin 28-O-
-xylopyranosyl-(1 !4)-
nosyl-(1 !2)- -fucopyranosyl ester, except for two differences
D-glucose, D-fucose, L-rhamnose,
correlations between
Fuc-2), 5.03 (1H, d, J = 5.8 Hz) (Xyl-1) and
Fig. 2).
d
H
6.35 (1H, d, J = 1.0 Hz, Rha-1) and
H
d 4.70
D
L
(
d
H
d
H
4.35 (Rha-4) (see
1
1
The H- H COSYexperiment allowed us to identify the E-olefinic
1
b
-D-glucopyranosyl
protons of 3,4,5-trimethoxycinnamoyl moiety, which appeared as
two double peaks at
J = 16.0 Hz). Single peak signal at
b
-D
a-L-rhamnopyra-
d
H
7.95 (1H, d, J = 16.0 Hz) and 6.61 (1H, d,
6.84 (2H, s) belongs to H-2 and
b-D
d
H

H. Zha et al. / Phytochemistry Letters 13 (2015) 108–113
111
HO
CH OH
2
2
8
OH
O
3
O
O
C
HO
HO
COOH
OH
H CO
3
Glc
H
O
H CO
3
H CO
H
3
O
H C
3
1
O
O
HO
Fuc
O
O
H C
3
HO
HO
O
O
HO
OH
OH
Rha
Xyl
Fig. 2. Key HMBC and NOESY correlations for compound 1.
located at the saccharide chain linked to C-28 of aglycone. The first
superimposable on those of known compound 4, namely poly-
galasaponin XLIV (Zhang et al., 1998). Except for the signals of 4,
compounds 3/4 also contain signals of the Z-4,5-dimethoxycinna-
difference was at the fucopyranosyl moiety. The 13C NMR spectrum
showed the deshielded signal of the Fuc-3 position at
C
d 81.6 in
comparison with the signal of the free Fuc-3 position of 1. The
signals of an arabinopyranosyl moiety can be deduced from the
NMR data at the same time. In the HMBC spectrum, a cross-peak
moyl moiety at
belong to Z-olefinic protons, and the substituted benzene ring
protons at 7.92 (br. s H-2), 7.50 (d, J = 8.4 Hz, H-6), and 6.93 (d,
H
d 5.91 (d, J = 12.6 Hz) and 6.88 (d, J = 12.6 Hz) which
d
H
d
H
between
between
d
H
4.51 (Fuc-3) and
4.97 (1H, d, J = 6.9 Hz, Ara-1) and d
d
C
106.5 (Ara-1), and a correlation
4.51 (Fuc-3) in the
J = 8.4 Hz, H-5). The relative NMR experiments and HPLC intensities
indicated that 3/4 are a mixture of E- and Z-3,4-dimethoxycinna-
moyl-substituted triterpene saponins. All attempts to separate 3
and 4 by semi-preparative HPLC were unsuccessful. This phenom-
enon of isomerization referred to the effect of light on the 3,4-
methoxycinnamoyl group in aqueous methanolic solution has
already been observed for the saponins isolated from Polygalaceae
(Mitaine-Offer et al., 2003). On the basis of above results, the
d
H
H
NOESY spectrum, proved that an arabinose was linked to the C-
position of the fucose. The HMBC correlations and NOESY
3
correlations of other sugars also can be observed (see Fig. 2).
Another difference was at the acyl group linked to the C-4 position
of the fucose. The acyl group was substituted by an E-3,4-
dimethoxycinnamoyl group instead of an E-3,4,5-trimethoxycin-
1
namoyl group. The H NMR spectrum showed two double peaks at
structure of 3 was elucidated as 3-O-
negenin 28-O-
-galactopyranosyl-(1 !4)-
(1 !4)- -glucopyranosyl-
-fucopyranosyl
b
-D-glucopyranosyl prese-
d
H
6.49 and 7.85, coupling constant of them is 16.0 Hz, which
belong to the E-olefinic protons, a single peak at 7.04 that
belongs to H-2 of benzene, and two double peaks at 6.94 (d,
b
-D
b-D-xylopyranosy-
d
H
a
-
L
-rhamnopyranosyl-(1 !2)-[
b-D
d
H
(1 !3)]-4-O-[(Z)-3,4-dimethoxycinnamoyl]-
b-D
J = 8.5 Hz) and 7.05 (d, J = 8.5 Hz) which belong to H-5 and H-6 of
benzene. The HMBC and HSQC spectrum confirmed the complete
assignments of all protons and carbons of the disubstituted
benzene ring (Tables 1 and 2) which were in good agreement with
those described in literature (Mitaine-Offer et al., 2003). For the
ester.
Saponins, acylated bidesmosidic presenegenin glycosides by
methoxy-substituted cinnamic acids, isolated from the roots of S.
inappendiculata showed potent selective cytotoxic activity against
M-CSF-stimulated macrophages (Kuroda et al., 2001; Yui et al.,
2001). Compounds 1–2, 3/4, 4 were evaluated for their cytotoxic
activity in vitro against LLC (Lewis lung carcinoma) and MCF-7
(human breast cancer) cell lines using the MTT assay method.
Compounds 1 and 2 exhibited moderate cytotoxic activity against
above evidence, the structure of 2 was elucidated as 3-O-
glucopyranosyl presenegenin 28-O-
-xylopyranosyl-(1 !4)-
-rhamnosyl-(1 !2)-( -arabinopyranosyl)-4-O-[(E)-3,4-
dimethoxycinnamoyl]- -fucopyranosyl ester.
Compounds 3/4 were isolated as a white amorphous powder.
The molecular formula was determined to be C76 37 on the
basis of the positive ion mode HRESIMS (m/z 1641.7035 for quasi-
b-D-
b-D
a
-L
a-L
b-D
LLC cells with an IC50 value of 45.56, 85.98
m
M respectively using
M), and did not show
M). Compounds 3/4
114
H O
cisplatin as a positive control (IC50 = 26.67
m
cytotoxicity against MCF-7 cells (IC50 > 100
m
+
1
13
molecular ion [M + Na] ). The H NMR and C NMR signals of 3/4
Tables 1 and 2) assigned from 2D NMR spectra were almost
(as a mixture) and 4 were inactive against all cell lines tested
(IC50 > 100 M). It is reported that the cinnamoyl group at the
(
m

112
H. Zha et al. / Phytochemistry Letters 13 (2015) 108–113
position 4 of the fucopyranosyl residue was important for the
cytotoxicity (Yui et al., 2001). Compounds 1 and 2 all have a
cinnamoyl group at the position 4 of the fucopyranosyl residue.
The structural difference between two compounds mainly was at
min) and purified by semi-preparative HPLC (
p
NAP, 250 ꢀ10 mm,
5
m
m, 4 mL/min) to afford two new compounds 1–2, named as
securioside C (11 mg), securioside D (18 mg), one pair of isomers 3/
4 (17 mg) (the (Z)—isomer (3) being new, named as securioside E),
and a known compound 4, Polygalasaponin XLIV (20 mg). As
securioside A and B were already isolated from this plant (Kuroda
et al., 2001), we adopted this nomenclature to keep consistency.
the position of Fuc-3, compound 2 has a more
a-L-arabinopyr-
anosyl than compound 1. So, the -arabinopyranosyl maybe led
a-L
to the cytotoxicity decrease of compound 2, which need to be
supported by more research results.
3
.4. Acid hydrolysis of the saponins and HPLC analysis
3. Experimental
Each saponin (2 mg) was hydrolyzed with 2 M aq. CF
3
COOH
ꢁ
3.1. General
(2 mL) for 2 h at 90 C. After extraction with CH
2
Cl
2
(3 ꢀ 2 mL), the
aqueous layer was repeatedly evaporated to dryness with MeOH
Optical rotations were recorded on an AUTOPOL II Polarimeter
from Rudolph. UV spectra were determined on Agilent Cary 60 UV–
vis spectrometer. IR spectra were recorded on Bruker TENSOR
7 spectrometer. The high resolution electrospray ionization mass
until neutral, and then analyzed by TLC over silica gel (CHCl
MeOH:H
the residue was dissolved in pyridine (0.1 mL) containing
3
:
2
O, 8:5:1) by comparison with authentic samples. Then,
L
ꢁ
-
2
cysteine methyl ester hydrochloride (0.5 mg) and heated at 60 C
spectroscopy (HRESIMS) was measured with a Bruker MicroTOF-
QII spectrometer. The 1D and 2D NMR spectra ( H- H COSY, TOCSY,
NOESY, HSQC, and HMBC) were performed on Bruker spectrometer
for 1 h. A 0.1 mL solution of o-tolyl isothiocyanate (0.5 mg) in
pyridine was added to the mixture, which was heated at 60 C for
1
1
ꢁ
1 h. The reaction mixture was directly analyzed by reversed-phase
ꢁ
(
500 MHz) using tetramethylsilane (TMS) as internal standard.
HPLC at 35 C with isocratic elution of 25% MeCN in 50 mM H
3
PO
4
Vacuum liquid chromatography (VLC) was carried out using Silica
gel (100–200 mesh, Qingdao Marine, China). Analytical TLC was
carried out with GF254 plates (Qingdao Marine, China). The spray
reagent for saponins was anisaldehyde reagent (0.5 mL Anisalde-
for 50 min and subsequent washing of the column with 90% MeCN
at a flow rate 0.8 mL/min and detected at 250 nm (Tanaka et al.,
2007). Identification of
rhamnose for 1; -glucose,
-arabinose for 2; and -glucose,
D
-glucose,
-xylose,
-xylose,
D
-xylose,
-fucose,
D-fucose, and L-
D
D
D
L-rhamnose, and
hyde, 10 mL Acetic acid, 85 mL MeOH and 5 mL H
2
SO
4
). Medium
L
D
D
D-fucose, L-rhamnose,
pressure liquid chromatography (MPLC) was performed on Dr
Flash II (Suzhou Lisui, China) equipped with a UV3000 UV–vis
Detector and a High Techsil glass column (500 ꢀ 50 mm, RP-18,
and -galactose for 3/4 were carried out by co-injection of the
D
hydrolysate with standard thiazolidine samples prepared in a same
way from standard sugars, giving peaks at t 22.6 min (
galactose), 26.2 min ( -glucose), 29.4 min ( -arabinose), 30.5 min
-xylose), 35.5 min ( -fucose), and 44.8 min ( -rhamnose).
R
D-
50
m
m). Preparative HPLC and semi-preparative HPLC were carried
D
L
out on a LC3000 P instrument (Beijing Chuangxin Tongheng, China)
(D
D
L
equipped with a P3000 pump, UV3000 UV–vis Detector, a YMC-
Pack ODS-A column (250 ꢀ 20 mm, 5
mm) or a Cosmosil pNAP
m) respectively. Analytical HPLC was
3
.5. MTT cytotoxicity bioassays
column (250 ꢀ10 mm, 5
m
performed on a LC3000 instrument (Beijing Chuangxin Tongheng,
China) equipped with a P3000 gradient pump, UV3000 UV–vis
detector, and CXTH-3000 chromatography workstation using a
Sharpsil-T C18 column (250 ꢀ 4.6 mm, 5
Cytotoxicities of compounds 1–2, 3/4, 4 against LLC and MCF-
cells were measured using methylthiazole tetrazolium (MTT)
7
assay (Mosmann, 1983). IC50 values were determined as concen-
tration of a compound resulting in cell growth inhibition by 50%
and cisplatin was used as a positive control. All samples were
assayed in triplicate.
mm).
3.2. Plant material
The roots of S. inappendiculata were collected from Yunnan
3
.6. Securioside C
Province, People’s Republic of China, in 2010, collected and
identified by Hong Wang, plant taxonomist of the Xishuangbanna
Tropical Botanical Garden, Chinese Academy of Sciences. A voucher
specimen (Y201011) was deposited in School of Pharmaceutical
Science and Technology, Tianjin University.
D
A white amorphous powder, [a] —12 (c = 0.08, MeOH); UV
(
MeOH)
l
max (log
e): 206 (5.97), 226 (5.92), 302 (6.07) nm; IR n
max
3
393, 2937, 1740, 1710, 1679, 1637, 1456, 1073, 837. HRESIMS m/z
+
1
1347.6020 [M + Na] (calcd. for C65
H
96
O
28Na 1347.6088); H NMR
13
(
pyridine-d
5
, 500 MHz) and C NMR (pyridine-d , 125 MHz) data,
5
3
.3. Extraction and isolation
see Tables 1 and 2.
Dried roots of S. inappendiculata (9.85 kg) were extracted three
times with 95% EtOH for 2 h each time, to give residue (1.25 kg) on
removal of the solvent. The residue was suspended in H O (5.0 L)
and partitioned with dichloromethane sat. H O and ethyl acetate
sat. H
O successively (each 3 ꢀ 3.0 L). The aqueous portion was
applied to D101 macroporous resin column chromatography
O, 30% EtOH, 60% EtOH,
and 95% EtOH, respectively. The 60% EtOH eluted part (180 g) was
subjected to vacuum liquid chromatography (VLC, 40 ꢀ14 cm)
using silica-gel (100–200 mesh), and eluted with a gradient of
3.7. Securioside D
2
D
A white amorphous powder, [a] —9.8 (c = 0.05, MeOH); UV
2
(MeOH)
lmax (log e): 206 (5.86), 226 (5.91), 308 (6.04) nm; IR n
max
2
3429, 2935, 1723, 1710, 1680, 1632, 1454, 1070, 841; HRESIMS m/z
+
1
(
10.6 ꢀ 94.0 cm) and then washed with H
2
1449.6368 [M + Na] (calcd. for C69
H
102
O
31Na 1449.6405); H NMR
13
(pyridine-d
5
, 500 MHz) and C NMR (pyridine-d , 125 MHz) data,
5
see Tables 1 and 2.
CHCl
3
2
:MeOH:H O (85:15:2, 75:20:5, 68:24:8, 60:32:8, 50:42:8,
3.8. Compounds 3/4
low phase) to afford 30 fractions, namely C1 to C30. Then, fraction
C25 (14.0 g) was separated by MPLC (RP-18, 500 ꢀ 50 mm, 35 mL/
A white amorphous powder, [
a
]
D
—2.73 (c = 0.092, MeCN:
): 206 (4.86), 226 (4.91), 325
nmax 3500, 2937, 1723, 1710, 1678, 1632, 1424, 1055,
min) eluted with MeCN: 0.02% CF
3
COOH-H
2
O (30:70, 32:6, 34:66)
H
2
O = 3:7); UV (MeOH)
(5.08) nm; IR
114
801; HRESIMS at m/z 1614.6838 [M + Na] (calcd. for C76H O37Na
lmax (log e
to yield 5 subfractions. Subfraction C25-2 (2.0 g) were further
separated by preparative HPLC (RP-18, 250 ꢀ 20 mm, 5 m, 12 mL/
+
m

H. Zha et al. / Phytochemistry Letters 13 (2015) 108–113
113
1
614.6937); 1H NMR (pyridine-d
5
,
500 MHz) and 13C NMR
Mitaine-Offer, A.-C., Miyamoto, T., Khan, I.A., Delaude, C., Lacaille-Dubois, M.-A.,
002. Three new triterpene saponins from two species of Carpolobia. J. Nat.
Prod. 65, 553–557.
2
(
pyridine-d , 125 MHz) data, see Tables 1 and 2.
5
Mitaine-Offer, A.-C., Miyamoto, T., Laurens, V., Delaude, C., Lacaille-Dubois, M.-A.,
Acknowledgments
2003. New acylated triterpene saponins from Polygala arenaria. Helv. Chim. Acta
8
6, 2404–2413.
Mitaine-Offer, A.-C., Miyamoto, T., Khan, I.A., Delaude, C., Lacaille-Dubois, M.-A.,
010. Acylated triterpene saponins from the roots of Securidaca
This work was supported by the grant of the National Scientific
and Technological Major Special Project for Significant New Drug
Creation, People's Republic of China (No. 2011ZX09201).
2
longepedunculata. Phytochemistry 71, 90–94.
Teng, R., Wu, Z., He, Y., Wang, D., Yang, C., 2002. Revised structures of arillatanosides
A–C from Polygala arillata. Magn. Reson. Chem. 40, 424–429.
Tanaka, T., Nakashima, T., Ueda, T., Tomil, K., Kouno, I., 2007. Facile discrimination of
aldose enantiomers by reversed-phase HPLC. Chem. Pharm. Bull. 55, 899–901.
Yang, X.D., Xu, L.Z., Yang, S.L., 2001a. Xanthones from the stems of Securidaca
inappendiculata. Phytochemistry 58, 1245–1249.
Yang, X.D., An, N., Xu, L.Z., Yang, S.L., 2001b. Organic acid constituents from the stem
of Securidaca inappendiculata Hassk. Chin. J. Chin. Mater. Med. 26 (4), 258–260.
Yang, X.D., Xu, L.Z., Yang, S.L., 2002a. New xanthone glycosides from Securidaca
inappendiculata. J. Asian Nat. Prod. Res. 4 (2), 141–145.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.05.022.
Yang, X.D., Xu, L.Z., Yang, S.L., 2002b. Studies on the chemical constituents of
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