New pregnane and steroidal glycosides from Tribulus terrestris L.

Article abstract of DOI:10.1080/10286020903535419

Three new steroidal saponins were isolated from the fruits of Tribulus terrestris L. Their structures were elucidated by spectroscopic and chemical analysis as 16-(4'-methyl-5'-O-β- d-glucopyranosyl-pentanoxy)-5α- pregn-3β-ol-12,20-dione-3-O-β- d-glucopyr

Full text of DOI:10.1080/10286020903535419

This article was downloaded by: [University of West Florida]
On: 04 January 2015, At: 14:49
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
New pregnane and steroidal glycosides
from Tribulus terrestris L.
a
a
a
a
Tao Liu , Gang Chen , Guo-Qing Yi , Jian-Kun Xu , Tian-Long
a
a
a
Zhang , Hui-Ming Hua & Yue-Hu Pei
a
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University , Shenyang, 110016, China
Published online: 26 Mar 2010.
To cite this article: Tao Liu , Gang Chen , Guo-Qing Yi , Jian-Kun Xu , Tian-Long Zhang , Hui-Ming
Hua & Yue-Hu Pei (2010) New pregnane and steroidal glycosides from Tribulus terrestris L., Journal
of Asian Natural Products Research, 12:3, 209-214, DOI: 10.1080/10286020903535419
To link to this article: http://dx.doi.org/10.1080/10286020903535419
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
Vol. 12, No. 3, March 2010, 209–214
ORIGINAL ARTICLE
New pregnane and steroidal glycosides from Tribulus terrestris L.
Tao Liu, Gang Chen, Guo-Qing Yi, Jian-Kun Xu, Tian-Long Zhang,
Hui-Ming Hua and Yue-Hu Pei*
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China
(
Received 31 October 2009; final version received 7 December 2009)
Three new steroidal saponins were isolated from the fruits of Tribulus terrestris L.
Their structures were elucidated by spectroscopic and chemical analysis as 16b-(4 -
0
methyl-5 -O-b-D-glucopyranosyl-pentanoxy)-5a-pregn-3b-ol-12,20-dione-3-O-b-D-
0
glucopyranosyl-(1 ! 2)-b-D-glucopyranosyl-(1 ! 4)-b-D-galactopyranoside (1),
2
a,3b-dihydroxy-5a-pregn-16-en-20-one 3-O-b-D-glucopyranosyl-(1 ! 4)-b-D-
galactopyranoside (2) and 26-O-b-D-glucopyranosyl-(25R)-5a-furostan-20(22)-en-
a,3b,26-triol-3-O-b-D-glucopyranosyl-(1 ! 4)-b-D-galactopyranoside (3).
Keywords: Tribulus terrestris L.; steroidal saponins; pregnane glycosides
2
1
.
Introduction aglycone and were essentially analogous to
the aglycone of previously reported com-
pound 4 (5a-furostan-20(22)-en-12-one-
Tribulus terrestris (Zygophyllaceae) is an
annual creeping herb growing on roadsides
and hills in China. The fruit of T. terrestris,
known as ‘jili’ in traditional Chinese
medicine, has been used against various
diseases for a long time. Pharmacological
studies have shown that saponins of this
plant are the main active components. This
paper reports the isolation and structural
elucidation of two new pregnane glyco-
sides and one new furostanol saponin based
on extensive spectroscopic analysis [1,2].
3
b,26-diol) [3]. However, the aglycone of 1
was different from 4 in the lack of the
signals assignable to the olefinic group
forming the bond between C-20 and C-22,
and the presence of a ketone carbonyl
carbon signal at d 204.4 and an ester
carbonyl carbon signal at d 173.3. The
HMBC spectrum (Figure 1) exhibited
correlations of the ketone carbonyl carbon
with H-17 at d 3.26 (1H, d, J ¼ 7.7 Hz) and
Me-21 at d 2.25 (3H, s) and correlations of
the ester carbonyl carbon with H-16 at d
2
.
Results and discussion
5.61 (1H, m) and H -23 at d 2.44 and 2.36,
2
Compound 1 was obtained as a white
2
indicating that C-20 and C-22 of 1 were the
carbonyl groups, instead of the olefinic
group in 4. The J value of 7.7 Hz between
H-16 and H-17 suggested the b-configur-
ation of the side chain attached to C-16.
This information was supported by the
NOESY spectra (Figure 1). Acid hydrolysis
0
amorphous powder, ½aꢀ þ 2.3 (c ¼ 1.55,
D
MeOH). Its molecular formula was deter-
mined to be C H O by HR-ESI-MS at
5
1 82 26
þ
1
13
m/z 1133.4990 [MþNa] . The H and C
NMR spectra of 1 (Tables 1 and 2) revealed
the presence of four methyl groups on the
*Corresponding author. Email: peiyueh@vip.163.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286020903535419
http://www.informaworld.com

2
10
T. Liu et al.
1 13
Table 1. H and C NMR (pyridine-d ) spectral data of the aglycone moieties of compounds 1–3.
5
1
2
3
No.
d
C
d
H
d
C
d
H
d
C
d
H
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
36.6
29.7
76.8
34.6
44.4
28.5
31.3
33.7
56.3
36.4
37.5
212.0
57.1
53.9
34.5
73.4
59.1
14.1
11.7
204.4
30.7
173.3
32.2
29.0
33.4
74.7
17.0
45.5
70.4
84.6
34.1
44.9
28.1
31.9
33.2
54.9
37.0
21.5
35.2
46.5
56.3
32.2
144.7
155.4
16.2
13.3
196.3
27.1
45.8
70.5
84.7
34.0
44.7
28.1
32.4
34.4
54.4
36.9
21.6
39.8
43.8
54.7
34.4
84.5
64.6
14.5
13.4
103.7
11.8
152.4
23.7
31.5
33.5
75.0
17.4
3.93 (m)
3.83 (m)
3.95 (m)
3.85 (m)
3.87 (m)
0.98 (m)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
5.61 (m)
3.26 (d, J ¼ 7.7 Hz)
6.58 (m)
4.60 (m)
1.50 (s)
0.62 (s)
0.87 (s)
0.70 (s)
0.68 (s)
0.71 (s)
2.25 (s)
2.22 (s)
1.61 (s)
2.36, 2.44 (m)
3.69, 4.02 (m)
0.90 (d, J ¼ 6.5 Hz)
3.63, 3.95 (m)
1.01 (d, J ¼ 6.5 Hz)
of 1 gave D-glucose and D-galactose in a
ratio of 3:1 on the basis of GC analysis. The
b-anomeric configurations for both glucose
and galactose were judged from their
coupling constants (J1,2 . 7.0 Hz) [1].
The HMBC experiment showed corre-
MeOH). Its molecular formula was deter-
mined to be C H O by HR-ESI-MS at
3
3 52 13
þ
1
13
m/z 679.3305 [MþNa] . The H and
C
NMR spectra of 2 (Tables 1 and 2)
revealed the presence of three methyl
groups on the aglycone and were essen-
tially analogous to the aglycone of
previously reported compound 5 (2a,3b-
dihydroxypregna-5,16-dien-20-one) [4].
However, the aglycone of 2 was different
from 5 in the lack of the signals assignable
to the olefinic group forming the bond
between C-5 and C-6, and a methine
carbon signal at d 44.9 and a methylene
lations between the proton signal at d 4.88
0
(H-1 , galactosyl group) and the carbon
signal at d 76.8 (C-3, aglycone), between
0
0
00
0
H-1 at d 5.14 and C-4 at d 81.1, between
00
0
H-1 at d 5.22 and C-2 at d 86.2, and
0
000
between H-1 at d 4.78 and C-26 at d 74.7.
Accordingly, the structure of 1 was
0
0
determined to be 16b-(4 -methyl-5 -O-b-
D-glucopyranosyl-pentanoxy)-5a-pregn-
1
3
carbon signal at d 28.1 appeared in the
C
3
b-ol-12,20-dione-3-O-b-D-glucopyrano-
syl-(1 ! 2)-b-D-glucopyranosyl-(1 ! 4)-
NMR spectrum. The HMBC spectrum
(Figure 2) showed correlation of the
methine carbon with Me-19 at d 0.70
(3H, s) and correlation of the methylene
carbon with H-5 at d 0.98 (1H, m). All
b-D-galactopyranoside.
Compound 2 was obtained as a white
2
0
amorphous powder, ½aꢀD þ 3.9 (c ¼ 0.89,

Journal of Asian Natural Products Research
211
1 13
Table 2. H and C NMR (pyridine-d ) spectral data of the sugar moieties of compounds 1–3.
5
1
2
3
No.
3-O)-b-D-Gal
d
C
d
H
d
C
d
H
d
C
d
H
(
102.3
4.88
d, J ¼ 7.6 Hz)
103.5
4.92
(d, J ¼ 7.7 Hz)
103.5
4.93
(d, J ¼ 7.6 Hz)
(
(
(
(
2
3
4
5
6
73.4
75.6
81.1
75.3
60.5
73.1
75.8
80.2
76.0
61.0
73.1
75.8
80.2
76.0
61.0
4
Gal )-b-D-Glc
(
105.3
5.14
d, J ¼ 7.6 Hz)
107.2
5.27
(d, J ¼ 7.7 Hz)
107.2
5.27
(d, J ¼ 7.7 Hz)
2
3
4
5
6
86.2
78.2
70.3
77.7
61.6
75.2
78.8
72.3
78.6
63.1
75.2
78.8
72.3
78.6
63.1
2
Glc )-b-D-Glc
(
107.0
5.22
d, J ¼ 7.0 Hz)
2
3
4
5
6
77.0
79.0
71.7
78.5
63.2
(
26-O)-b-D-Glc 104.9
4.78
d, J ¼ 7.8 Hz)
104.9
4.83
(d, J ¼ 7.7 Hz)
2
3
4
5
6
75.2
78.6
71.8
78.5
62.9
75.2
78.7
71.8
78.6
62.9
OH
HMBC
NOESY
O
HO
HO
O
′′′′
1
26
OH
O
21
O
O
H
2
0
7
23
22
1
OH
16
O
H
O
HO
HO
OH
H
2′′
O
1′′
H
H
3
4′
OH
O
O
O
HO
H
O
1′
OH
H
HO
HO
1′′′
OH
Figure 1. The structure and key HMBC and NOESY correlations of compound 1.

2
12
T. Liu et al.
HMBC
NOESY
O
OH
O
H
H
1
9
9
HO
2
HO
HO
OH
O
′′
1
H
H
4
′
3
5
O
OH
HO
O
6
H
1′
H
OH
Figure 2. The structure and key HMBC and NOESY correlations of compound 2.
these evidences indicated that the double
bond between C-5 and C-6 was saturated.
The orientation of C-5 is a because of the
chemical shifts of C-5 (d 44.9), C-9 (d
54.9), and C-19 (d 13.3). For 5a
compounds, the chemical shifts of C-5,
was determined to be C H O by HR-
45 74 19
þ
ESI-MS at m/z 941.7723 [MþNa] .
1
13
Comparing the H and C NMR spectra
of 3 (Tables 1 and 2) with those of reported
steroidal sapogenin [5], we confirm the
aglycone moiety as (25R)-5a-furostane-
C-9, and C-19 appear at ,43–46, ,54–
2
0(22)-en-2a,3b,26-triol. The above
56, and ,11–14 ppm, respectively, and
for 5b compounds, the chemical shifts of
conclusion was also confirmed by the
NOESY spectra (Figure 3). Acid hydroly-
sis of 3 afforded D-glucose and D-galactose
in a ratio of 2:1 on the basis of GC
analysis. The b-anomeric configurations
for both glucose and galactose were
judged from their coupling constants.
The positions of the sugar residues in 3
were defined by the HMBC experiment
these carbons are observed at ,35–36.5,
,
40, and ,24 ppm, respectively [1].
Thus, the aglycone moiety of 2 was
deduced to be 2a,3b-dihydroxy-5a-
pregn-16-en-20-one. The above con-
clusion was also confirmed by the
NOESY spectra (Figure 2). Acid hydroly-
sis of 2 afforded D-glucose and D-galactose
in a ratio of 1:1 on the basis of GC
analysis. The b-anomeric configurations
for both glucose and galactose were
judged from their coupling constants.
The HMBC experiment showed corre-
0
0
(Figure 3). The cross-peaks between H-1
0
at d 4.93 and C-3 at d 84.7, between H-1
0
at d 5.27 and C-4 at d 80.2 indicated that a
sugar moiety, 3-O-b-D-glucopyranosyl-
(
1 ! 4)-b-D-galactopyranoside,
was
lations between the proton signal at d 4.92
0
linked to the aglycone at C-3. Addition-
(H-1 , galactosyl group) and the carbon
signal at d 84.6 (C-3, aglycone), between
0
00
ally, a cross-peak between H-1 at d 4.83
and C-26 at d 75.0 definitively proved that
the other glucose was linked to C-26 of the
aglycone.
0
0
0
H-1 at d 5.27 and C-4 at d 80.2.
In conclusion, the structure of 2 was
elucidated as 2a,3b-dihydroxy-5a-pregn-
In conclusion, the structure of 3 was
elucidated as 26-O-b-D-glucopyranosyl-
1
6-en-20-one 3-O-b-D-glucopyranosyl-
(1 ! 4)-b-D-galactopyranoside.
(
25R)-5a-furostan-20(22)-en-2a,3b,26-
Compound 3 was obtained as a white
powder,
20
amorphous
c ¼ 1.15, MeOH). Its molecular formula
½aꢀ þ 0.16
triol-3-O-b-D-glucopyranosyl-(1 ! 4)-b-
D
(
D-galactopyranoside.

Journal of Asian Natural Products Research
213
OH
HMBC
O
NOESY
HO
HO
O
′′′
1
26
OH
O
OH
H
H
H
H
O
HO
O
HO
HO
OH
H
O
′′
1
H
H
4′
3
O
OH
HO
H
1′
OH
Figure 3. The structure and key HMBC and NOESY correlations of compound 3.
3
.
Experimental
3.3 Extraction and isolation
3
.1 General experimental procedures
The fruits of T. terrestris (5 kg) were
extracted with 75% EtOH three times for
Optical rotations were measured on a
Perkin-Elmer 241 polarimeter. IR spectra
were taken on a Bruker IFS-55 infrared
spectrophotometer. The NMR spectral
data were recorded on Bruker AV-600
2 h each. The extract (200 g) was succes-
sively partitioned with CHCl , EtOAc, and
n-BuOH. The n-BuOH-soluble fraction
3
(65 g) was subjected to the silica gel
column, eluted with CHCl –CH OH
1
13
(600 MHz for H and 150 MHz for C) in
C D N with TMS as an internal standard.
3
3
(
100:1–0:1), yielding eight fractions.
5
5
Fraction 2 (4 g) was subjected to HPLC,
eluted with MeOH (50%) by a flow rate of
The HR-ESI-MS data were obtained on the
Micross Mass Autospec-UltimaE TOF
mass spectrophotometer. Chromatography
was performed on silica gel (200–300
mesh; Qingdao Haiyang Chemical Fac-
tory, Qingdao, China), and purified by
HPLC (Shimadzu LC-8A, RID-10A,
Kyoto, Japan). GC analysis was performed
on a Shimadzu GC-2010 gas chromato-
3
ml/min and afforded compound 2
25 mg) at 169 min. Fraction 4 (2 g) was
subjected to HPLC, eluted with MeOH
67%) by a flow rate of 3 ml/min and
(
(
afforded compound 3 (15 mg) at 38.7 min.
Fraction 5 (3 g) was subjected to HPLC,
eluted with MeOH (41%) by a flow rate of
3
ml/min and afforded compound 1
23 mg) at 87 min.
graph equipped with an H flame ioniza-
2
(
tion detector and a DB-5 quartz capillary
column (30 m £ 0.25 mm £ 0.25 mm).
0
0
3.3.1 16b-(4 -Methyl-5 -O-b-D-
glucopyranosyl-pentanoxy)-5a-pregn-3b-
ol-12,20-dione-3-O-b-D-glucopyranosyl-
3
.2 Plant material
(
1 ! 2)-b-D-glucopyranosyl-(1 ! 4)-b-
The fruits of T. terrestris were bought from
Henan Province, China and identified by
Prof. Qishi Sun of Shenyang Pharmaceu-
tical University. A voucher specimen (No.
D-galactopyranoside (1)
2
0
White amorphous powder; ½aꢀ þ 2.3
D
(c ¼ 1.55, MeOH); IR (KBr)
n
max
2
(cm ): 3410, 2927, 1706, 1645, 1372,
1
0
093) is deposited in the School of
1
13
Traditional Chinese Materia Medica of
Shenyang Pharmaceutical University.
1070, 893, 633; H and C NMR spectral
data, see Tables 1 and 2; HR-ESI-MS:

2
14
T. Liu et al.
þ
m/z 1133.4990 [MþNa] (calcd for
hydrochloride (2 mg) was added to the
solution. The mixture was heated at 608C
for 2 h, followed by an equal volume of
acetic anhydride at 908C for another 2 h.
The solution was concentrated in vacuo
dryness and dissolved in MeOH (0.5 ml),
which was analyzed by GC [column, DB-5
quartz capillary column (30 m £ 0.25 mm
C H O Na, 1133.4992).
51 82 26
3
.3.2 2a,3b-Dihydroxy-5a-pregn-16-
en-20-one 3-O-b-D-glucopyranosyl-
(
1 ! 4)-b-D-galactopyranoside (2)
20
D
White amorphous powder; ½aꢀ þ 3.9
(
(
c ¼ 0.89, MeOH); IR (KBr)
n
max
£0.25 mm); H flame ionization detector;
2
2
cm ): 3407, 2932, 1661, 1586, 1380,
1
column temperature, 100–2808C; pro-
grammed increase, 108C/min; carrier gas,
N2 (1.5 ml/min); injector and detector
temperature, 2808C; injection volume,
0.5 ml; split ratio, 10:1]. The derivatives
of D-glucose and D-galactose were detected
1
232, 1164, 1074, 924, 892, 607; H and
1
1
3
C NMR spectral data, see Tables 1 and 2;
þ
HR-ESI-MS: m/z 679.3305 [MþNa]
(
calcd for C H O Na, 679.3306).
33 52 13
[
Rf (min): 26.11 and 26.60, respectively].
3
5
.3.3 26-O-b-D-Glucopyranosyl-(25R)-
a-furostan-20(22)-en-2a,3b,26-triol-3-
The standard monosaccharides were sub-
jected to the same reaction and GC analysis
under the same conditions.
O-b-D-glucopyranosyl-(1 ! 4)-b-D-
galactopyranoside (3)
20
White amorphous powder; ½aꢀ þ 0.16
D
2
(cm ):
1
(
c ¼ 1.15, MeOH); IR (KBr) n
Acknowledgement
max
3
6
421, 2927, 1653, 1380, 1071, 746, 704,
1 13
66; H and C NMR spectral data, see
The authors are grateful to the members of the
Analytical Center in Shenyang Pharmaceutical
University for the measurements of all spectra.
Tables 1 and 2; HR-ESI-MS: m/z 941.4723
þ
[
MþNa]
(calcd for C H O Na,
45 74 19
References
9
41.4722).
[
1] I. Kostova and D. Dinchev, Phytochem.
Rev. 4, 111 (2005).
3
.4 Acid hydrolysis of compounds 1–3
[2] J.L. Li and S.S. Yang, Chin. Arch. Trad.
Chin. Med. 24, 1509 (2006).
Each solution of compounds 1–3 (5 mg) in
M HCl–MeOH (4:1, 5 ml) was refluxed
at 908C for 5 h. Then, the reaction mixture
[
[
[
3] Y. Wang, K. Ohtani, R. Kasai, and
K. Yamasaki, Phytochemistry 45, 811
(1997).
4] Y. Mimaki, K. Watanabe, H. Sakagami,
and Y. Sashida, J. Nat. Prod. 65, 1863
2
was diluted with H O and extracted with
2
CHCl (3£ 20 ml). The water layer was
3
(
2002).
concentrated in vacuo to dryness to give a
residue which was dissolved in pyridine
5] L. Su, G. Chen, S.G. Feng, W. Wang,
Z.F. Li, H. Chen, Y.X. Liu, and Y.H. Pei,
Steroids 74, 399 (2009).
(1 ml), and then L-cysteine methyl ester