Cytotoxic triterpenesaponins from Ilex pubescens

Article abstract of DOI:10.1080/10286020.2014.920012

Two new triterpenesaponins, ilexsaponins G and H (1 and 2), together with six known triterpenesaponins (3-8), were isolated from the roots of Ilex pubescens. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products.

Full text of DOI:10.1080/10286020.2014.920012

This article was downloaded by: [University of Winnipeg]
On: 28 August 2014, At: 06:56
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Asian Natural Products
Research
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/ganp20
Cytotoxic triterpenesaponins from Ilex
pubescens
a
a
a
Ling Li , Li-Shi Feng & Yu-Xin He
a
Bioengineering College, Xihua University, Chengdu 610039, China
Published online: 01 Aug 2014.
To cite this article: Ling Li, Li-Shi Feng & Yu-Xin He (2014): Cytotoxic triterpenesaponins from Ilex
pubescens, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.920012
To link to this article: http://dx.doi.org/10.1080/10286020.2014.920012
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“
Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Journal of Asian Natural Products Research, 2014
http://dx.doi.org/10.1080/10286020.2014.920012
Cytotoxic triterpenesaponins from Ilex pubescens
Ling Li, Li-Shi Feng and Yu-Xin He*
Bioengineering College, Xihua University, Chengdu 610039, China
(
Received 14 April 2014; final version received 28 April 2014)
Two new triterpenesaponins, ilexsaponins G and H (1 and 2), together with six known
triterpenesaponins (3–8), were isolated from the roots of Ilex pubescens. Their
structures were elucidated on the basis of spectroscopic evidence and hydrolysis
products. Those compounds showed inhibitory activities against two human colorectal
cancer cell lines HCT 116 and HT-29.
Keywords: Ilex pubescens; triterpenesaponins; ilexsaponin G; ilexsaponin H;
cytotoxicity
1
.
Introduction
3-O-b-D-glucopyranosyl-3b,21a,23-trihy-
droxy-urs-12-en-28-oic acid 21-O-b-D-
glucopyranoside (4) [8], ilexsaponin D
The roots of Ilex pubescens Hook et Arn.
are a traditional Chinese medicine com-
monly used in South China for the
treatment of cardiovascular disease and
hypercholesterolemia [1]. Phytochemical
investigation of its roots and leaves led to
the isolation of glaberides, triterpenesapo-
nins, and phenolics, specifically vomifo-
liol [2–8]. In our previous study, a series
of saponins were isolated from this plant
(
(
5) [9], ilexsaponin E (6) [9], ilexsaponin F
7) [9], and ilexoside XXX (8) [10]. Their
structures were elucidated on the basis of
spectroscopic evidence and hydrolysis
products. The cytotoxicity of all com-
pounds against two colorectal human
cancer cell lines HCT 116 and HT-29
was also evaluated.
Compound 1 was isolated as a white
amorphous powder. The HR-ESI-MS
experiment revealed a pseudomolecular
[
9]; however, their bioactivity has not been
investigated. As a continuation of study on
this plant, we present in this report the
isolation and structural elucidation of two
new triterpenesaponins (1 and 2) (Figure 1)
and six known compounds from the roots
of I. pubescens. All of these compounds
were screened for their cytotoxicity.
2
ion peak at m/z 939.4592 [M 2 H] , in
agreement with the molecular formula
C H O .
The
obtained after aqueous acid hydrolysis of
were identified as glucose and arabinose
monosaccharides
47 72 19
1
by thin layer chromotography (TLC)
comparison with authentic samples. The
absolute configuration of the monosac-
charides was determined to be D for
glucose and L for arabinose by GC analysis
of chiral derivatives of the monosacchar-
ides in the hydrolysate of each compound
(see Section 3). The relatively large
coupling constants (5.0–8.0 Hz) for the
2
.
Results and discussion
The 70% EtOH extract of dried roots of
I. pubescens was subjected to silica gel
column chromatography and prep-HPLC
to obtain two new compounds (1 and 2),
together with six known structures,
3b,21a,23-trihydroxy-urs-12-en-28-oic
acid 21-O-b-D-glucopyranoside (3) [8],
1
anomeric protons in the H NMR spectrum
*Corresponding author. Email: heyuxin66@tom.com
q 2014 Taylor & Francis

2
L. Li et al.
12
13
1
COOR
8
Ara1
OH
2
3
O
O
COOH
24
OH O
O
23
HO
HO
Glc1
HO
OH
OH
Glc2
O
1
R = HO
HO
2
R = H
OH
H
Figure 1. The structures of compounds 1 and 2.
(see Section 3) of 1 suggested that the
arabinopyranosyl moiety has an a-con-
figuration and the glucopyranosyl moiety
dien-24,28-dioic acid by comparison of its
1
13
H and C NMR spectral data obtained in
2D NMR experiments with values
reported in the literature (see Table 1)
1
[8]. The H NMR spectrum of 1 exhibited
1
has a b-configuration. The H NMR
spectrum showed six methyl resonances
at d 1.70, 1.16, 1.20, 1.09, 1.73, (s, 3H
each), and 1.06 (3H, d, J ¼ 7.0 Hz) and an
olefinic proton at d 5.72. There were two
carboxyl carbons at d 174.6 and 180.5 and
two pairs of olefinic carbon signals at d
three anomeric proton resonances at d 5.12
(1H, d, J ¼ 6.0 Hz), 5.15 (1H, d,
J ¼ 8.5 Hz), and 6.28 (1H, d,
J ¼ 8.5 Hz), respectively. The spin–spin
coupling system of individual monosac-
charide units was identified by the analysis
1
3
C
1
26.4, 138.7, 133.6, and 136.0 in the
1
NMR spectrum. Above information indi-
cated the presence of an ursane-type
triterpene skeleton [9]. The aglycone of 1
was identified as 3b-hydroxyurs-12,18-
of 1D TOCSY and 2D NMR spectra. H
NMR spectral data of individual mono-
saccharide units were obtained by selec-
tive irradiation of the anomeric protons in
1
3
Table 1.
Position
5 5
C NMR spectral data for the aglycone moieties of 1 and 2 (125 MHz, in C D N).
1
2
Position
1
2
Position
1
2
1
2
3
4
5
6
7
8
9
39.1
28.1
88.7
48.3
57.1
20.5
35.3
39.6
47.2
37.4
39.2
28.4
88.6
48.3
57.2
20.5
35.4
39.5
47.2
37.6
11
12
13
14
15
16
17
18
19
20
23.9
126.4
138.7
45.1
28.9
35.2
23.8
126.3
138.8
45.0
28.9
35.1
21
22
23
24
25
26
27
28
29
30
26.3
31.0
24.7
180.5
14.2
18.6
22.1
174.6
19.3
18.8
26.2
31.1
24.7
180.4
14.2
18.8
22.1
180.5
19.2
18.9
49.6
49.7
133.6
136.0
34.4
133.6
136.0
34.4
1
0

Journal of Asian Natural Products Research
a series of 1D TOCSY experiments.
3
above evidence, the structure of 1 was
established as 3-O-b-D-glucopyranosyl
(1 ! 2)-a-L-arabinopyranosyl urs-12,18-
diene-24,28-dioic acid 28-O-b-D-gluco-
pyranosyl ester, named ilexsaponin G.
Compound 2 was isolated as a white
amorphous powder. The HR-ESI-MS
experiment revealed a pseudomolecular
1
1
Analysis of the H– H COSY spectrum
resulted in sequential assignment of all
proton resonances of three monosacchar-
ide units, as shown in Table 2. In the
HSQC experiment, proton resonances
were correlated with those of the corre-
sponding carbons, and associated anome-
ric protons were correlated with their
respective carbon atoms from HSQC–
TOCSY data, leading to unambiguous
assignments of the carbons in each
monosaccharide unit (see Table 2). In the
2
ion peak at m/z 777.4066 [M 2 H] , in
agreement with the molecular formula
C H O14. Comparison of the NMR
41 62
spectral data for 2 with those of 1 revealed
that both of compounds are similar except
that the signals due to the D-glucopyrano-
syl linked to C-28 in 1 were absent in 2.
This observation was supported by a
relative downfield shift of C-28 (d 180.5)
HMBC spectrum, the anomeric proton
1
signals at d 5.12 (Ara-H-1), 5.15 (Glc -
H
2
H-1), and 6.28 (Glc -H-1) showed cross-
peaks with the carbon signals at d 88.7
C
1
13
(
Agly-C-3), 81.0 (Ara -C-2), and 174.6
Agly-C-28), respectively. These signals
of 2 in the C NMR spectrum. Thus, the
(
structure of 2 was established as 3-O-b-D-
glucopyranosyl(1 ! 2)-a-L-arabinopyra-
nosyl urs-12,18-diene-24,28-dioic acid,
named ilexsaponin H.
provide ample evidence to determine the
linkages between the sugars, and between
the sugar and the aglycone. From the
1
13
Table 2. H NMR and C NMR spectral data for the sugar moieties of compounds 1 and 2
1
13
(
5 5
500 MHz for H NMR and 125 MHz for C NMR, in C D N).
1
2
Position
d
H
d
C
d
H
d
C
Ara
1
2
3
4
5.12 d (6.0)
5.52 dd (8.0, 6.0)
4.22 dd (8.0, 4.5)
4.27–4.28 m
3.58d (9.0)
103.6
81.0
73.4
68.0
64.7
5.11 d (6.0)
103.5
81.1
73.3
68.0
64.8
5.52 dd (8.0, 6.0)
4.22–4.24 m
4.27–4.29 m
3.58d (9.0)
5
5
a
b
4.21d (9.0)
4.21d (9.0)
1
Glc
1
2
3
4
5
6
6
5.15 d (8.5)
4.03 dd (8.5, 9.0)
4.12 dd (9.0, 9.0)
4.23–4.25 m
3.67–3.69 m
4.33 dd (12.0, 7.5)
4.42 dd (12.0, 2.0)
105.7
76.2
78.1
71.2
78.2
62.4
5.14 d (8.0)
4.01 dd (8.0, 9.0)
4.11 dd (9.0, 9.0)
4.23 dd (9.0, 9.0)
3.67–3.69 m
105.8
76.3
78.0
71.2
78.1
62.3
a
4.33 dd (12.0, 7.0)
4.42 dd (12.0, 2.0)
b
2
Glc
1
2
3
4
5
6
6
6.28 d (8.5)
4.11 dd (8.5, 9.0)
4.22 dd (9.0, 9.0)
4.30–4.32 m
4.02–4.04 m
4.34 dd (12.0, 7.0)
4.47 dd (12.0, 2.0)
95.7
74.3
78.7
71.2
79.1
62.1
a
b

4
L. Li et al.
the solvent, the residue (854 g) was
suspended in H O and extracted with n-
3
.
Experimental
3
.1 General experimental procedures
2
BuOH. The n-BuOH extract (114 g) was
further chromatographed over a macro-
porous resin D101 column eluted initially
with water, then with 30%, 50%, and 70%
EtOH to give fractions A–C (fraction A,
Optical rotations were measured on a
JASCO P-1020 digital polarimeter (JASCO
Corporation, Tokyo, Japan). UV spectra
were obtained on a TU-1901 spectrometer
(Beijing Purkinje General Instrument Co.,
22 g; fraction C, 17 g). Fraction B (34 g)
Ltd, Beijing, China). IR spectra were
obtained on a Perkin-Elmer 577 spec-
trometer (PerkinElmer, Waltham, MA,
USA). NMR spectra were recorded on an
Inova 500 spectrometer (Varian, Palo Alto,
was subjected to a silica gel column using
CHCl –MeOH–H O (90:10:0.1 to
3
2
70:30:5) as an eluent to give fractions
B –B . Fraction B (2.9 g) was subjected
1
9
3
to ODS open column chromatography
MeOH–H O 40:60 to 80:20) to afford
1
CA, USA), operating at 500MHz for H and
(
1
3
2
1
25 MHz for C. HR-ESI-MS data were
obtained on a G1969A TOF-MS instrument
AgilentTechnologiesInc.,SantaClara, CA,
USA). Precoated silica gel GF254 plates
Qingdao Haiyang Chemical Co., Qingdao,
fractions B3-1 –B3-4
.
Fraction B3-1
600 mg) was subjected to prep-HPLC
MeOH–H O, 40:60, UV detection at
(
(
(
2
210 nm), affording 5 (18 mg, 16.7 min), 6
(
(12 mg, 20.7 min), and 7 (5 mg, 18.5 min),
China) were used for TLC. Spots were
visualized by spraying 10% H SO –EtOH
respectively. Fraction B4 (1.7 g) was
subjected to ODS open column chroma-
tography (MeOH–H O; 40:60 to 80:20) to
2
4
followed by heating. Column chromatog-
raphy was carried out on silica gel (Qingdao
Haiyang Chemical Co.) and macroporous
resin D101 (pore size B 13–14nm, 26–60
mesh;BohongresintechnologyCo.,Tianjin,
China). High-performance liquid chroma-
tography (HPLC) separation was carried out
on a Waters 1525 EF system (Waters,
Millford, MA, USA; YMC-pack ODS-A,
2
afford fractions B4-1–B4-5. Fraction B4-2
(
(
310 mg) was subjected to prep-HPLC
ACN–H O, 26:74; UV detection at
2
2
10 nm), affording 1 (26 mg, 14.3 min)
and 2 (29 mg, 18.9 min), respectively.
Fraction B4-5 (95 mg) was subjected to
prep-HPLC (CH CN–H O, 29:71, UV
3
2
detection at 210 nm, affording 8 (17 mg,
2.4 min) and 3 (14 mg, 14.4 min),
respectively.
2
50 mm £ 10 mm, i.d. 5 mm, YMC, Kyoto,
1
Japan). GC analysis was carried out on an
Agilent 6890N gas chromatograph (Agi-
lent, Waldbronn, Germany) using a HP-5
capillary column with an FID detector.
3.3.1 Ilexsaponin G (1)
2
D
2
3
.2 Plant material
White amorphous solid, ½aꢀ þ 36
(
c ¼ 0.20, MeOH); UV (MeOH) l
The roots of I. pubescens were collected in
Guangzhou, Guangdong Province, China,
in June 2010, and identified by Prof. Y.-X.
He, Xihua University. A voucher speci-
men (No. 20100810) has been deposited in
the herbarium of Bioengineering College
of Xihua University.
max
2
1
225 nm; IR (KBr): v
(cm ) 3422,
max
1
720, 1682; H NMR (C D N, 500 MHz):
1
d 1.73 (3H, s, Me-29), 1.70 (3H, s, Me-23),
5
5
1
1
.20 (3H, s, Me-26), 1.16 (3H, s, Me-25),
.09 (3H, s, Me-27), 1.06 (3H, d,
J ¼ 7.0 Hz, Me-30), 3.32 (1H, dd,
J ¼ 12.5, 4.0 Hz, H-3), 5.72 (1H, br t,
1
3
J ¼ 3.0 Hz, H-12). For C NMR spectral
2
3
.3 Extraction and isolation
data, see Tables 1 and 2. HR-ESI-MS: m/z
939.4592 [M 2 H] (calcd for C H O ,
939.4590).
The dried roots (5 kg) of I. pubescens were
extracted with 70% EtOH. After removing
4
7 71 19

Journal of Asian Natural Products Research
5
Table 3. Inhibitory effects of compounds 1–8 against two colorectal human cancer cell lines HCT
16 and HT-29.
1
IC50 value (mM)
Compound
HCT 116
HT-29
1
2
3
12.4 ^ 3.2
3.2 ^ 0.3
2.7 ^ 0.5
10.8 ^ 2.1
3.7 ^ 0.4
2.2 ^ 0.4
4
5
6
5.2 ^ 1.2
13.3 ^ 2.2
6.6 ^ 1.5
4.9 ^ 0.7
15.9 ^ 3.5
7.2 ^ 0.8
7
13.1 ^ 3.5
8.3 ^ 1.9
0.0038 ^ 0.0003
11.9 ^ 2.7
7.1 ^ 1.0
0.0040 ^ 0.0004
8
Paclitaxel
3
.3.2 Ilexsaponin H (2)
(1.0 ml), and then extracted with cyclo-
hexane (1.0 ml, three times). The cyclo-
hexane layer was collected and
concentrated to 1.0 ml for GC analysis.
Separations were carried out on an HP-5
column (28 m £ 0.32 mm). Highly pure He
was used as carrier gas (1.0 ml/min flow
rate), and the FID detector was operated at
2
D
2
White amorphous solid, ½aꢀ þ 38
(
c ¼ 0.20, MeOH); UV (MeOH) l
max
2
1
2
25 nm; IR(KBr) v
(cm ): 3424,1721,
max
1
683; H NMR (C D N, 500 MHz): d 1.72
1
5
5
(
(
(
3H, s, Me-29), 1.71 (3H, s, Me-23), 1.20
3H, s, Me-26), 1.15 (3H, s, Me-25), 1.08
3H, s, Me-27), 1.06 (3H, d, J ¼ 7.0 Hz,
2608C (column temp. 1808C). The reten-
Me-30), 3.34 (1H, dd, J ¼ 12.0, 3.5 Hz, H-
1
3
tion times of the monosaccharide deriva-
tives were as follows: L-Ara, 5.45 min, and
D-Glc, 12.45 min.
3
NMR spectral data, see Tables 1 and 2. HR-
), 5.71 (1H, brt, J ¼ 3.5 Hz, H-12). For C
2
ESI-MS: m/z 777.4066 [M 2 H] (calcd
for C H O , 777.4061).
4
1 61 14
3.5 MTT cytotoxicity assay
The inhibitory activities against two
colorectal human cancer cell lines HCT
3
.4 Acid hydrolysis
A solution of compounds 1 or 2 (5.0 mg
each) in 2 M trifluoroacetic acid (1 ml) was
heated at 1108C for 2 h, and then dried by
116 and HT-29 were carried out according
to a previously described method [11].
Paclitaxel was used as a positive control
N gas. After cooling, the reaction mixture
2
(Table 3). The experiments were carried
out for three times.
was neutralized with Amberlite IRA-400
2
resin (OH form) and the resin was
filtered. After removal of the solvent
under pressure from the filtrate, the residue
was passed through a Sep-Pak C18
cartridge with H O and MeOH. The H O
Acknowledgments
This work was financially supported by the
National Natural Science Fund of China (Grant
No. 81202897); the Chunhui Project of
Ministry of Education (Z2010102); the Chun
hui Project of Ministry of Education
2
2
eluate was concentrated and the residue
was treated with L-cysteine methyl ester
hydrochloride (2.0 mg) in pyridine (1.0 ml)
(
Z2010103); the Open Research Fund of Key
at 608C for 2 h. After dried by N gas, the
2
Laboratory of Food Biotechnology, Xihua
University (SZJJ2014-008); and the Innovation
Fund of Postgraduate, Xihua University
(YCJJ2014106).
residue was treated with N-(trimethylsilyl)
imidazol (0.2 ml) at 608C for 1 h. The
reaction was ended by adding water

6
L. Li et al.
[6] F. Feng, M.X. Zhu, N. Xie, W.Y. Liu, D.J.
References
Chen, and Q.D. You, J. Asian Nat. Prod.
Res. 10, 71 (2008).
[
1] Jiangsu New Medical College, editors,
Directory of Chinese Materia Medica
(
Zhong Yao Da Ci) (Shanghai Scientific
and Technological Press, Shanghai,
977), p. 441.
[7] Y.B. Zhou, J.H. Wang, X.M. Li, X.C. Fu,
Z. Yan, Y.M. Zeng, and X. Li, J. Asian
Nat. Prod. Res. 10, 827 (2008).
1
[
[
[
2] C.X. Zhang, C.Z. Lin, T.Q. Xiong, C.C.
Zhu, J.Y. Yang, and Z.X. Zhao, Fitoter-
apia 81, 788 (2010).
3] K. Hidaka, M. Ito, Y. Matsuda, H. Kohda,
K. Yamasaki, and J. Yamahara, Phyto-
chemistry 26, 2023 (1987).
4] K. Hidaka, M. Ito, Y. Matsuda, H. Kohda,
K. Yamasaki, J. Yamahara, T. Chisaka, Y.
Kawakami, T. Sato, and K. Kagei, Chem.
Pharm. Bull. 35, 524 (1987).
[8] G.B. Xie, S.X. Zhou, Y.N. Lu, L.D. Lei,
and P.F. Tu, Chem. Pharm. Bull. 57, 520
(
2009).
9] L. Li, Y.X. He, M.L. Gou, and C. Dai,
[
J. Asian Nat. Prod. Res. 14, 1169 (2012).
10] K. Amimoto, K. Yoshikawa, and S.
[
[
Arihara, Chem. Pharm. Bull. 40, 3138
(
1992).
11] J. Carmichael, W.G. de Graff, A.F.
Gazdar, J.D. Minna, and J.B. Mitchell,
Cancer Res. 47, 936 (1987).
[
5] L.P. Lin, W. Qu, and J.Y. Liang, Chin.
Chem. Lett. 22, 697 (2011).