A new triterpenoid saponin from Gymnema sylvestre

Article abstract of DOI:10.1080/10286020.2012.726517

Besides four known compounds, a new triterpenoid saponin was isolated from the stems of Gymnema sylvestre. The structure of the new triterpenoid saponin was established as 3β,16β,22α-trihydroxy-olean-12-ene 3-O-β-D-xylopyranosyl-(1 → 6)-β-D-glucopyranosyl-(1 → 6)-β-D-glucopyranoside (1) on the basis of 1D and 2D NMR techniques, including COSY, HMBC, HMQC, and NOESY correlations. Four known compounds 2, 3, 4, and 5 were identified on the basis of spectroscopic data.

Full text of DOI:10.1080/10286020.2012.726517

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A new triterpenoid saponin from
Gymnema sylvestre
c
a
b
b
Man-Qi Zhang , Yue Liu , Sheng-Xu Xie , Tun-Hai Xu , Tong-
c
b
b
Hua Liu , Ya-Juan Xu & Dong-Ming Xu
a
School of Pharmacy, China Pharmaceutical University, Nanjing,
11198, China
2
b
Jilin Academy of Chinese Medicine Sciences, Changchun, 130021,
China
c
Department of Traditional Chinese Medicine Chemistry, School
of Traditional Chinese Medicine, Beijing University of Chinese
Medicine, Beijing, 100102, China
Published online: 23 Oct 2012.
To cite this article: Man-Qi Zhang , Yue Liu , Sheng-Xu Xie , Tun-Hai Xu , Tong-Hua Liu , Ya-Juan
Xu & Dong-Ming Xu (2012) A new triterpenoid saponin from Gymnema sylvestre , Journal of Asian
Natural Products Research, 14:12, 1186-1190, DOI: 10.1080/10286020.2012.726517
To link to this article: http://dx.doi.org/10.1080/10286020.2012.726517
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Journal of Asian Natural Products Research
Vol. 14, No. 12, December 2012, 1186–1190
NOTE
A new triterpenoid saponin from Gymnema sylvestre
a
Man-Qi Zhang , Yue Liu , Sheng-Xu Xie , Tun-Hai Xu *, Tong-Hua Liu , Ya-Juan Xu *
b
b
c
c
b
b
and Dong-Ming Xu
a
b
School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China; Jilin Academy of
c
Chinese Medicine Sciences, Changchun 130021, China; Department of Traditional Chinese
Medicine Chemistry, School of Traditional Chinese Medicine, Beijing University of Chinese
Medicine, Beijing 100102, China
Besides four known compounds, a new triterpenoid saponin was isolated from the
stems of Gymnema sylvestre. The structure of the new triterpenoid saponin was
established as 3b,16b,22a-trihydroxy-olean-12-ene 3-O-b-D-xylopyranosyl-(1 ! 6)-
b-D-glucopyranosyl-(1 ! 6)-b-D-glucopyranoside (1) on the basis of 1D and 2D NMR
techniques, including COSY, HMBC, HMQC, and NOESY correlations. Four known
compounds 2, 3, 4, and 5 were identified on the basis of spectroscopic data.
Keywords: Gymnema sylvestre; triterpenoid saponin; Asclepiadaceae
1
.
Introduction
The plant Gymnema sylvestre R. Br.
Asclepiadaceae) is a vine which grows in
2. Results and discussion
Compound1was obtainedasan amorphous
white powder. The molecular formula,
C H O , was determined on the basis
(
thesouthwesternregion ofChina, including
the Guangdong, Guangxi, and Fujian
provinces. This plant species is also
distributed in India where it is used as a
stomachic, diuretic, and anti-diabetic
remedy. The total saponin fraction of the
leaves had an anti-sweetening effect and
was shown to be able to inhibit glucose
absorption in the small intestine and to
suppress elevated glucose levels in blood
following the administration of sucrose in
rats [1,2]. The antisweet properties of the
extracts as well as some of the saponin
components have also been reported [3–7].
In this study, we report the structure
elucidation of a new triterpenoid saponin,
which was isolated from the stems of
G. sylvestre, by using 1D, 2D NMR
techniques, MALDI/TOF-MS, ESI-MS
analysis as well as chemical methods.
4
7 78 17
of positive MALDI/TOF-MS (m/z 937
þ
þ
[M þ Na] , m/z 953 [M þ K] ) and HR-
þ
MS data (m/z 937.5131 [M þ Na] ).
Further fragment ion peaks in the ESI-
MS/MS were observed at m/z 805 [M-
þ
þ
132 þ Na]
and 481 [M-132-162-
162 þ Na] due to the loss of terminal
pentose and two hexose units. Among the
1
3
47 carbons in the C NMR spectrum, 30
were assigned to the triterpenoid aglycone,
and 17 to the oligosaccharide moiety. Eight
signals of the triterpenoid part were
assigned to methyl carbons at d 15.8, 17.0,
17.1, 24.0, 26.3, 26.8, 28.4, and 33.6, and
the corresponding protons were identified
by a HSQC experiment (Table 1). Three
oxygen-bearing methine carbons were
found at d 66.8, 75.8, and 89.1 and a pair
of olefinic carbons was observed at d 122.8
*Corresponding authors. Email: thxu@yahoo.com; xyj6492@sohu.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.726517
http://www.tandfonline.com

Journal of Asian Natural Products Research
1187
1 13
Table 1. H and C NMR spectral data of compound 1 (recorded at 500/125 MHz in pyridine-d ; d
in ppm, J in Hz).
5
No.
dc
dH (J in Hz)
No.
dc
dH (J in Hz)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
38.9
26.8
89.1
39.7
55.9
18.6
33.1
40.3
47.2
36.1
24.2
122.8
144.0
41.2
36.9
66.8
43.9
44.6
47.2
31.2
42.9
75.8
28.4
17.0
15.8
17.1
26.3
26.8
33.6
24.0
0.90 (m), 1.48 (m)
1.22 (m), 2.30 (m)
3.22 (dd, 11.7, 4.0)
–
0.63 (d, 11.2)
1.20 (m), 1.38 (m)
1.38 (m)
C-3
Glc 1
107.0
75.1
78.5
71.2
77.1
70.0
4.74 (d, 8.4)
3.85 (m)
4.04 (m)
4.16 (m)
3.99 (m)
4.80 (overlapped)
4.66 (d, 11.2)
4.92 (d, 7.6)
3.85 (m)
4.04 (m)
4.12 (m)
4.04 (m)
4.31 (d, 10.4)
4.19 (overlapped)
4.83 (d, 7.2)
3.85 (m)
4.04 (m)
4.04 (m)
2
3
4
5
6
–
1.76 (m)
0
Glc 1
105.5
75.7
78.7
71.7
76.9
69.1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
–
2
3
4
5
6
1.72 (m), 0.89 (m)
5.11 (m)
–
–
1.65 (m), 2.13 (m)
4.53 (br s)
–
2.28 (dd, 13.5, 4.0)
1.76 (m), 1.25 (m)
–
Xyl 1
106.1
74.9
78.2
70.5
67.1
2
3
4
5
3.54 (d, 10.0)
4.53 (overlapped)
1.65 (m), 2.13 (m)
3.48 (m)
1.12 (3H, s)
0.85 (3H, s)
0.75 (3H, s)
0.94 (3H, s)
1.71 (3H, s)
2.30 (3H, s)
0.85 (3H, s)
0.90 (3H, s)
and 144.0. These data, coupled with
and C-28 (d 26.8), in the HMBC spectrum
(Figure 1). The correlations between H-16
(d 4.53), H-27 (d 1.71), H-7 (d 1.38), and H-
5 (d 0.63) in the NOESY spectrum
indicated a b-configuration of the hydroxyl
group at C-16. The correlation between H-
22 (d 3.48) and H-30 (d 0.90) in the NOESY
spectrum indicated an a-configuration of
the hydroxyl group at C-22, which was
corroborated by the splitting pattern of H-
22 (m). Correlations of H-3 (d 3.22) with H-
5 (d 0.63) and H-23 (d 1.12) in the NOESY
spectrum indicated a b-configuration of the
hydroxyl group at C-3. Thus, the aglycon of
1 could be identified as 3b,16b,22a-
trihydroxyolean-12-ene.
1
information from the H NMR spectrum:
eight methyl singlets at d 0.75, 0.85, 0.88,
0.90, 0.94, 1.12, 1.71, and 2.30; three
oxygen-bearing methine protons at d 3.22
(
dd, J ¼ 11.7, 4.0 Hz), 3.48 (m), and 4.53
br s), and an olefinic proton at d 5.11 (m),
(
were characteristic signals of a polyhy-
droxyolean-12-ene triterpene derivative.
The carbon signal at d 66.8, which
correlated with H-15 (d 1.65), H-18 (d
2
.28), and H-28 (d 2.30) in the HMBC
spectrum (Figure 1), could be assigned to
C-16, which was also supported by the
splitting pattern of H-16 (d 4.53, br s). The
proton signal at d 3.48 could be assigned to
H-22 on the basis of its correlations with C-
The sugar part of 1 consisted of three
1 13
residues as evidenced by H and C NMR
1
6 (d 66.8), C-18 (d 44.6), C-21 (d 42.9),

1
188
M.-Q. Zhang et al.
OH
Me
OH
O
O
O
O
O
O
H
H
H
H
OH
OH
OH
HO
HO
HO
HO
HO
HO
glc′
glc
xyl
Figure 1. Key HMBC correlations for 1.
spectra which displayed three anomeric
protons at d 4.74 (d, J ¼ 8.4 Hz), 4.92 (d,
J ¼ 7.6 Hz), and 4.83 (d, J ¼ 7.2 Hz),
attached to anomeric carbons at d 107.0,
group at d 4.92 and C-6 of glc group at d
70.0, and between H-1 of xyl group at d
4.83 and C-6 of glc group at d 69.1 (Figure
0
1). Consequently, the structure of com-
pound 1 was concluded to be 3b,16b,22a-
trihydroxy-olean-12-ene 3-O-b-D-xylo-
pyranosyl-(1 ! 6)-b-D-glucopyranosyl-
1
05.5, and 106.1, respectively, in the
HSQC spectrum. Acid hydrolysis and
analysis of COSY, HMQC, and NOESY
experiments allowed the full identification
of the sugar residues as one b-D-xylopyr-
(
1 ! 6)-b-D-glucopyranoside.
Four known compounds 2–5 were
identified as 3-O-b-D-glucopyranosyl
(1 ! 3)-a-L-rhamnopyranosyl chichipe-
genin 28-a-L-rhamnopyranoside (2) [9],
oleanolic acid 3-O-b-D-glucopyranosyl
(1 ! 6)-b-D-glucopyranoside (3) [10],
oleanolic acid 3-O-b-D-xylopyranosyl
anosyl(xyl) and two b-D-glucopyranosyl
0
(glc and glc ) groups. Their anomeric
configurations were determined by coup-
1
3
ling constants and by the comparison of C
NMR spectral data with those in the
literature [8]. The coupling constants of
(
1 ! 6)-b-D-glucopyranosyl(1 ! 6)-b-D-
0
anomeric protons of glc (8.4 Hz), glc
(7.6 Hz), and xyl (7.2 Hz) groups, along
glucopyranoside (4) [3], and 3-O-b-D-
xylopyranosyl(1 ! 6)-b-D-glucopyrano-
syl(1 ! 6)-b-D-glucopyranosyl oleanolic
acid 28-O-b-D-glucopyranosyl ester (5)
with the correlations between H-1 (d 4.74)
and H-5 (d 4.14) of glc group, H-1 (d 4.92)
0
and H-5 (d 4.14) of glc group, in the
NOESY experiment, indicated b-configur-
[
3] by comparison of their physical and
spectroscopic data with those reported in
the literature, respectively.
0
ation for the glc and glc groups, and the
correlations between H-1 (d 4.83), H-3 (d
.15), and H-5 (d 4.18) of the xylose unit in
4
3
.
Experimental
the NOESY experiment confirmed the a-
axial orientation of these protons and the b-
anomeric configuration. In addition, the
long-range correlations were observed
between H-1 of glc at d 4.74 and C-3 of
3
.1 General experimental procedures
Optical rotations were determined on
WZZ-2S auto-polarimeter (Shanghai,
China). Melting points were determined
on an X-6 microscope apparatus and are
0
the aglycone at d 89.1, between H-1 of glc

Journal of Asian Natural Products Research
1189
uncorrected (Beijing, China). IR spectra
were measured as films on KBr pellets
using a Bruker VERTEX70 spectrometer
subjected to neutral resin to remove most of
color material and then evaporated to
dryness to give crude saponins (27 g). Part
of the crude saponins (27 g) was chromato-
graphed on silica gel (200–300 mesh) with
(Bruker Corporation, Ettlingen, Germany).
NMR spectra were obtained on a Bruker
AV600 instrument (Bruker Company,
Rheinstetten, Switzerland), using tetra-
methylsilane as internal standard. The
HR-MS and MS/MS were recorded on
autoflex III MALDI/TOF/TOF-MS instru-
ment (Bruker Company, Ettlingen,
Germany). HPLC (Shimadzu Company,
Kyoto, Japan) was carried out using an
octadecylsilane bonded silica column (HC-
ODS, 250 £ 4.6 mm). Column chromatog-
raphy was carried out on silica gel (200–
CHCl –MeOH gradients 20:1 to 10:15 and
3
finally with MeOH (500 ml per part), to
give Frs 1–7. Fr. 4 (5.0 g) was subjected to
repeated column chromatography on silica
gel (200 mesh, 520 g) with CHCl –n-
3
BuOH–H O gradients (8:2:0.2 to 7:3:0.5,
2
250 ml per part) to afford Frs 4-1–4-6. Fr.
4-2 (330 mg) was subjected to HPLC
eluting with 45% MeOH (flow velocity
5 ml/min, evaporative light-scattering
detection, retention time 18 min) to give
compounds 1 (43 mg) and 2 (28 mg). Fr. 4-3
(380 mg) was further purified on an ODS
3
00 mesh, Qingdao Oceanic Chemical
Industry, Qingdao, China), reversed silica
gel (20 £ 20 cm, MERCK KGaA, Darm-
stadt, Germany), and macroporous resin
D21 made in Shandong Lu Neng Gel Co.
column using MeOH–H O (30:70 to
2
70:30) as eluent to afford compounds 3
(45 mg), 4 (30 mg), and 5 (35 mg).
(Shandong, China). Spots were detected
after spraying with 10% H SO .
2
4
3.3.1 Compound 1
Amorphous powder, mp 200–2018C;
25
3
.2 Plant material
½
aꢀ þ 6.3 (c 3.2, MeOH); IR (KBr) n
D
max
1
2
1
The stems of G. sylvestre were purchased
from the Company of Chinese Medicinal
Materials in Nanning, Guangxi Province,
China, in September 2009, and were
identified by Professor Minglu Deng,
Changchun College of Traditional Chinese
Medicine. A voucher specimen (No.
3326, 2940, 1051, and 1168 cm
NMR (500 MHz, pyridine-d ) and
;
H
3
1
C
NMR (125 MHz, pyridine-d ) spectral
5
5
data are given in Table 1. ESI-MS: m/z
þ
þ
937 [M þ Na] , 953 [M þ K] , 805
þ
[M þ Na-132] , and 481 [M þ Na-132-
þ
162-162] . HR-TOF-MS: m/z 937.5131
[M þ Na] (calcd for C H O Na,
þ
2
01008) has been deposited in the phyto-
4
7
78 17
chemistry laboratory of Jilin Academy of
Chinese Medicine Sciences.
937.5137).
3
.4 Acid hydrolysis
3
.3 Extraction and isolation
Compound 1 (10 mg) was dissolved in
1 mol/l HCl in MeOH–H O (1:1) and
The dried and powdered stems (9.5 kg) of
G. sylvestre were exhaustively extracted
with 75% EtOH, and then heated on steam
bath to remove EtOH. The extract (105 g)
was partitioned between EtOAc and
2
refluxed for 2 h. The reaction mixture
(10 ml) was analyzed by analytical HPLC
using gradient elution of 15–85% aqueous
CH CN containing 0.1% acetic acid, at a
3
H O. The water solution was chromato-
2
flow rate of 1 ml/min, over a 35 min run.
D-Glucose (rt 17.15 min) and D-xylose
(rt 17.90 min) were identified by comparing
their retention times with those of authentic
samples, while L-glucose and L-xylose
graphed on 1.5 kg D21 macroporous resin,
eluting with water until the elute was
colorless, and then with 50% EtOH (6
liters). The 50% EtOH solution was further

1
190
M.-Q. Zhang et al.
showed different retention times of 17.09
and 17.86 min, respectively.
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4] W.C. Ye, X. Liu, Q.W. Zhang, C.T. Che,
and S.X. Zhao, J. Nat. Prod. 64, 232
(2001).
Acknowledgments
The authors are grateful to the International
Science & Technology Cooperation Program of
China (No. 2010DFB33260) and the Coopera-
tion Program of Beijing Municipal Education
Commission and the Program for New Century
Excellent Talents in University (NCET-08-
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Y.H. Li, N. Murakami, J. Yamahara, and
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