Furostanol saponins from the fruits of Tribulus terrestris

Article abstract of DOI:10.1080/14786419.2012.718773

Two new steroidal saponins were isolated from the fruits of Tribulus terrestris. Their structures were assigned by spectroscopic analysis and colour reaction as 26-O-β-D-glucopyranosyl-(25R)-5α-furostane-12-one- 3β,22α,26-triol-3-O-β-D-glucopyranosyl(1 →

Full text of DOI:10.1080/14786419.2012.718773

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Furostanol saponins from the fruits of
Tribulus terrestris
a
a
a
a
Gang Chen , Lan Su , Sheng-Guang Feng , Xuan Lu , Haifeng
a
a
Wang & Yue-Hu Pei
a
School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University , Shenyang 110016 , China
Published online: 30 Aug 2012.
To cite this article: Gang Chen , Lan Su , Sheng-Guang Feng , Xuan Lu , Haifeng Wang & Yue-Hu
Pei (2013) Furostanol saponins from the fruits of Tribulus terrestris , Natural Product Research:
Formerly Natural Product Letters, 27:13, 1186-1190, DOI: 10.1080/14786419.2012.718773
To link to this article: http://dx.doi.org/10.1080/14786419.2012.718773
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Natural Product Research, 2013
Vol. 27, No. 13, 1186–1190, http://dx.doi.org/10.1080/14786419.2012.718773
Furostanol saponins from the fruits of Tribulus terrestris
Gang Chen, Lan Su, Sheng-Guang Feng, Xuan Lu, Haifeng Wang
and Yue-Hu Pei*
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China
(
Received 7 February 2012; final version received 6 July 2012)
Two new steroidal saponins were isolated from the fruits of Tribulus terrestris.
Their structures were assigned by spectroscopic analysis and colour reaction
as 26-O-�-D-glucopyranosyl-(25R)-5ꢀ-furostane-12-one-3�,22ꢀ,26-triol-3-O-�-D-
glucopyranosyl(1 ! 4)-�-D-galactopyranoside (1); 26-O-�-D-glucopyranosyl-
2
5(R)-5ꢀ-furostan-12-one-3�,22ꢀ,26-triol-3-O-ꢀ-L-rhamnopyranosyl-(1 ! 2)-O-
[�-D-glucopyranosyl-(1 ! 4)]-�-D-galactopyranoside (2).
Keywords: Tribulus terrestris; steroidal saponins; furostanols
1
. Introduction
Tribulus terrestris L., growing in subtropical areas around the world, the fruits of which
were used as the treatment of eye trouble, oedema, abdominal distention, emission,
morbid leucorrhea, sexual dysfunction and veiling in traditional Chinese medicine.
In addition, it was also used as medicine in India, South Africa and Japan. Many
pharmaceutical preparations and food supplements were for sale based on the saponin
fraction of this plant. Such as ‘Tribestane’ and ‘Vitanone’ were used for the treatment of
impotency, ‘tribusaponins’ and ‘Xin-nao-shu-tong’ were used for the treatment of
cardiovascular disease (Bedir, Khan, & Walker, 2002; Dinchev et al., 2008; Kostova
&
&
Dinchev, 2005; Li, Li, Li, & Yang, 2006; Li & Yang, 2006; Neychev, Nikolova, Zhelev,
Mitev, 2007; Yang, Qu, & Sun, 2005; Zhang, Qu, & Zhou, 2006). This article reports the
structural assignment of the new saponins based on extensive spectroscopic analysis and
chemical evidence.
2
. Results and discussion
Two new compounds were isolated from the chloroformic fraction of the alcoholic extract
of T. terrestris L. by HPLC. The structures of the isolated compounds were established
based on 1D and 2D NMR spectral data.
Compound 1 was obtained as an amorphous solid with a molecular formula of
C H O , as determined by data of the negative-ion HRESI–MS (m/z 933.4684
4
5
74 20
�
1
[
M � H] ). The H-NMR spectrum of 1 showed proton signals for four steroidal methyl
groups at ꢁ 1.12 (3H, s, H-18), 0.67 (3H, s, H-19), 1.54 (3H, d, J ¼ 7.0 Hz, H-21) and
*
Corresponding author. Email: peiyueh@vip.163.com
©
2013 Taylor & Francis

Natural Product Research
1187
0
.98 (3H, d, J ¼ 6.5 Hz, H-27), two methine proton signals at � 3.87 (1H, m, H-3) and 4.86
(
1H, m, H-16) which was indicative of secondary alcoholic functions, two methylene
proton signals at � 4.08 (1H, m, H-26 a) and 3.61 (1H, dd, J ¼ 6.0, 9.2, H-26 b) ascribable
to a primary alcoholic function, and three anomeric protons at � 4.86 (1H, d, J ¼ 7.6 Hz,
0
00
0000
13
H-1 ), 5.28 (1H, d, J ¼ 7.7 Hz, H-1 ), and 4.81 (1H, J ¼ 7.8 Hz, H-1 ). The C-NMR
spectrum displayed signals ascribable to a carbonyl function at � 213.0 (C-12), a hemiacetal
function at � 110.8 (C-22), two secondary alcoholic functions at � 76.9 (C-3) and 79.7
(
C-16), and one primary alcoholic function at � 75.25 (C-26), suggesting the occurrence of
a 3,26-bisdesmosidic furostanol saponin. In the HMBC spectrum, the methyl protons at
1.12 (18-CH ) showed long-range correlation with the carbons at � 213.0 (C-12), 55.6
�
3
(
C-13), 55.8 (C-14) and 55.1 (C-17), indicating the attachment of a carbonyl group at C-12.
1
3
Comparison of the signals from the sterol part of 1 in the C-NMR spectra with those
from the sterol part of 26-O-ꢀ-D-glucopyranosyl-(25R)-5ꢁ-furostane-12-one-
3
ꢀ,22ꢁ,26-triol-3-O-ꢀ-D-glucopyranosyl (1 ! 2)-ꢀ-D-galactopyranoside (Cai et al., 1999)
showed that the sterol part of 1 was the same as that of the compound in the literature,
including the orientations of the C-3 oxygen atom, C-5 hydrogen atom and C-22 hydroxyl
group (3ꢀ, 5ꢁ, 22ꢁ). Acid hydrolysis of 1 with 2 M HCl in CH OH–H O (4 : 1) gave
3
2
glucose and galactose in a ratio of 2 : 1. The ꢀ-anomeric configurations for both the
glucose and galactose were judged from its coupling constants (J1,2 4 7.0 Hz).
The absolute configurations of the sugar units were determined to be D-glucose and
D-galactose on the basis of GC analysis. In the HMBC spectrum, a cross-peak from
0
proton signal at � 4.86 (H-1 , galactose) to the carbon signal at � 76.9 (C-3, aglycone), from
0000
�
aglycone at C-3 and C-26. Similarly, anomeric proton signal at � 5.28 (H-1 , terminal
4.81(H-1 , glucose) to � 75.25 (C-26) were observed, indicating the glycosylation of the
0
0
0
glucose) showed cross-peak with the carbon signal at � 80.1 (C-4 of the galactose). Thus,
the structure of 1 was established to be 26-O-ꢀ-D-glucopyranosyl-(25R)-5ꢁ-furostane-
12-one-3ꢀ,22ꢁ,26-triol-3-O-ꢀ-D-glucopyranosyl (1 ! 4)-ꢀ-D-galactopyranoside, named
Tribulusaponin A.
Compound 2 was obtained as an amorphous solid with a molecular formula of
C H O , as determined by data of the negative-ion HRESI–MS (m/z 1079.5216
5
1
84 24
�
1
[
(
M � H] ). The H-NMR spectrum showed signals for steroid methyl protons at � 1.07
3H, s, H-18), 0.87 (3H, s, H-19), 1.53 (3H, d, J ¼ 7.2, H-21), 0.96 (3H, d, J ¼ 6.6 Hz,
0
H-27), along with signals for four anomeric protons at � 4.87 (1H, d, J ¼ 7.2 Hz, H-1 ), 6.19
00
000
0000
(
1H, br s, H-1 ), 5.16 (1H, d, J ¼ 7.8 Hz, H-1 ), 4.79 (1H, d, J ¼ 7.8 Hz, H-1 ).
13
Comparison of the C-NMR data for the aglycon moiety of 2 with those of 1 revealed
that the aglycon moiety was identical to that of 1. Sugars obtained on acid hydrolysis of 2
were identified as D-galactose, D-glucose and L-rhamnose in a ratio of 1 : 2 : 1 on the basis
of thin layer chromatography (TLC) and GC analysis. An ꢁ-anomeric configuration for
the rhamnose unit was concluded from its C-5 chemical shift (� 69.5). The ꢀ-anomeric
configurations for both the glucose and galactose were judged from their coupling
0
constants (J1,2 4 7.0 Hz). The HMBC correlations (Figure 1) from � 4.87 (H-1 of galac-
tose) to � 76.8 (C-3 of aglycone), from � 6.19 (H-1 of rhamnose) to � 77.0 (C-2 of glucose),
from � 5.16 (H-1 of glucose) to � 81.3 (C-4 of galactose) and from � 4.79 (1 of glucose)
to � 75.3 (C-26 of aglycone) indicated that the sugar chain was attached to C-3 of the
aglycone and the rhamnose and glucose were linked at C-2 and C-4 of the inner galactose,
respectively, the other glucose was linked at C-26 of aglycone. Accordingly, the structure
of compound 2 was determined to be 26-O-ꢀ-D-glucopyranosyl-(25R)-5ꢁ-furostane-12-
one-3ꢀ,22ꢁ,26-triol-3-O-ꢁ-L-rhamnopyranosyl-(1 ! 2)-O-[ꢀ-D-glucopyranosyl-(1 ! 4)]-ꢀ-
D-galactopyranoside, named Tribulusaponin B.
0
0
0
000
000
0
0
0

1
188
G. Chen et al.
Figure 1. Structures and HMBC correlations of compounds 1 and 2.
3
3
. Experimental
.1. General details
Optical rotations were obtained using a Perkin Elmer 241MC spectropolarimeter at room
1
13
temperature. NMR analysis was measured on H-NMR (600 MHz) and C-NMR
150 MHz): Bruker DRX-300 and DRX-600 spectrometer with TMS as internal standard.
(
HRESI–MS was measured on a TOF of micromass spectrometer. TLC was carried out on
plates precoated with RP-18 gel (Merck) and silica gel F254 (Qingdao Marine Chemistry
Ltd.). Spots on the plates were visualised by spraying with Ehrich reagent, followed by
heating. Column chromatography (CC) was performed on silica gel (200–300 and 300–400
2
¨
mesh; Qingdao Marine Chemical Factory), MPLC (BUCHI, column 3.5 � 45 cm , 50 mm)
2
and HPLC (Shimadzu LC-8, column 10 � 250 mm , 5 mm). GC analysis was performed on
a Shimadzu GC-2010 gas chromatograph equipped with an H flame ionisation detector.
2
The column was DB-5 quartz capillary column (30 m � 0.25 mm� 0.25 mm) with the
ꢀ
ꢀ
ꢁ1
following conditions: column temperature 160–195 C; programmed increase, 2 C min ;
ꢁ
1
carrier gas, N (1 mL min ); injector and detector temperature, 250 C; injection volume,
ꢀ
2
1 mL; split ratio, 1/10. Standards D-glucose, D-galactose and L-rhamnose were purchased
from National Institute for the Control of Pharmaceutical and Biological Products, China.
3
.2. Plant material
The fruits of T. terrestris L. were bought from Henan Province, China and identified by
Prof. Qi-shi Sun of Shenyang Pharmaceutical University. The voucher specimen is
deposited at our laboratory (no. 200912a).
3
.3. Extraction and purification of 1 and 2
The comminuted fruits of T. terrestris L. (5 kg) were extracted with 75% EtOH for three
times and the extract was evaporated under reduce press to afford a residue (200 g). The
residue was suspended in H O, and then extracted with petroleum benzin, CHCl , EtOAc
2
3
and n-BuOH, respectively. The n-BuOH layer was then concentrated to dryness giving
a crude saponin fr. (65 g). The extract was subjected to CC on silica gel eluted with
CHCl –MeOH (100 : 1–0 : 100), successively. The fraction (CH Cl–MeOH ¼ 100 : 20) was
3
3
subjected to CC on silica gel to gave five fractions. Fraction D (200 mg) was subjected to

Natural Product Research
1189
HPLC eluted with MeOH/H O (55%) to give tribulusaponin A (1, 25 mg, 0.0005%) and
2
B (2, 22 mg, 0.0005%).
3
.3.1. Tribulusaponin A (1)
2
2
White amorphous power; [�] �14.2 (c 0.064, pyridine); HRESI–MS m/z 933.4684
D
�
[
M � H] (Calcd for C H O , 933.4695). IR (KBr) ꢀ
3413 (OH), 2930 (CH), 1709
4
5
74 20
max
C¼O), 1388, 1065, 749, 705 cm�1. H-NMR (600 MHz, pyridine-d ): ꢁ 0.69, m, 1H,
1
(
C1-Ha; 1.31, m, 1H, C1-Hb; 1.52, m, 1H, C2-Ha; 1.97, m, 1H, C2-Hb; 3.87, m, 1H, C3-H;
5
1
1
.28, m, 1H, C4-Ha; 1.80, m, 1H, C4-Hb; 0.86, m, 1H, C5-H; 1.12, m, 2H, C6-H; 0.70, m,
H, C7-Ha; 1.52, m, 1H, C7-Hb; 1.72, m, 1H, C8-H; 0.89, m, 1H, C9-H; 2.20, m, 1H,
C11-Ha; 2.39, m, 1H, C11-Hb; 1.31, m, 1H, C14-H; 1.60, m, 1H, C15-Ha; 2.10, m, 1H,
C15-Hb; 4.86, m, 1H, C16-H; 2.89, m, 1H, C17-H; 1.12, s, 3H, C18-H; 0.67, s, 3H, C19-H;
2
.18, m, 1H, C20-H; 1.54, d, J ¼ 6.7 Hz, 3H, C21-H; 1.93, m, 1H, C23-Ha; 2.05, m, 1H,
C23-Hb; 1.69, m, 1H, C24-Ha; 2.04, m, 1H, C24-Hb; 1.93, m, 1H, C25-H; 3.61, d, J ¼ 6.0,
.2 Hz, 1H, C26-Ha; 4.08, m, 1H, C26-Hb; 0.98, d, J ¼ 6.4 Hz, 3H, C27-H; 4.86, d,
9
0
0
0
0
J ¼ 7.6 Hz, 1H, C1 -H; 4.37, m, 1H, C2 -H; 4.07, m, 1H, C3 -H; 4.69, m, 1H, C4 -H; 4.12,
0
0
0
00
m, 1H, C5 -H; 4.25, m, 1H, C6 -Ha; 4.65, m, 1H, C6 -Hb; 5.28, d, J ¼ 7.7 Hz, 1H, C1 -H;
00 00 00 00
4
1
4
4
7
.10, m, 1H, C2 -H; 4.20, m, 1H, C3 -H; 4.06, m, 1H, C4 -H; 3.95, m, 1H, C5 -H; 4.21, m,
000
00
00
000
H, C6 -Ha; 4.54, m, 1H, C6 -Hb; 4.81, d, J ¼ 7.8 Hz, 1H, C1 -H; 4.04, m, 1H, C1 -H;
13
0
0
00
00
000
000
000
.22, m, 1H, C3 -H; 4.18, m, 1H, C4 -H; 3.94, m, 1H, C5 -H; 4.38, m, 1H, C6 -Ha;
.58, m, 1H, C6 -Hb. C-NMR (150 MHz, pyridine-d ): ꢁ (C-1) 36.7; (C-2) 29.8; (C-3)
6.9; (C-4) 34.6; (C-5) 44.5; (C-6) 28.6; (C-7) 31.7; (C-8) 34.3; (C-9) 55.8; (C-10) 36.3;
5
(
(
2
C-11) 38.0; (C-12) 213.0; (C-13) 55.6; (C-14) 55.8; (C-15) 31.7; (C-16) 79.7; (C-17) 55.1;
C-18) 16.3; (C-19) 11.7; (C-20) 41.3; (C-21) 15.3; (C-22) 110.8; (C-23) 37.1; (C-24)
0 0 0 0
8.4; (C-25) 34.3; (C-26) 75.2; (C-27) 17.5; (C-1 ) 102.5; (C-2 ) 73.5; (C-3 ) 75.5; (C-4 ) 80.1;
0 0 00 00 00 00 00
(C-5 ) 76.0; (C-6 ) 61.1; (C-1 ) 107.1; (C-2 ) 75.3; (C-3 ) 78.7; (C-4 ) 72.3; (C-5 ) 78.5;
(C-6 ) 62.9; (C-1 ) 105.0; (C-2 ) 75.3; (C-3 ) 78.6; (C-4 ) 71.7; (C-5 ) 78.5; (C-6 ) 63.1.
00
000
000
000
000
000
000
3
.3.2. Tribulusaponin B (2)
2
2
White amorphous power; [�] �16.3(c 0.027, pyridine); HRESI–MS m/z 1079.5216
D
�
[
M � H] (Calcd for C H O , 1079.5274). IR (KBr) ꢀ
3412 (OH), 2933 (CH), 1708
5
1
83 24
max
�1
1
(C¼O), 1388, 1065, 749, 700 cm . H-NMR (600 MHz, pyridine-d ): ꢁ 0.71, m, 1H,
C1-Ha; 1.35, m, 1H, C1-Hb; 1.80, m, 1H, C2-Ha; 2.00, m, 1H, C2-Hb; 3.87, m, 1H, C3-H;
5
1
1
.25, m, 1H, C4-Ha; 1.75, m, 1H, C4-Hb; 0.85, m, 1H, C5-H; 1.15, m, 2H, C6-H; 0.72, m,
H, C7-Ha; 1.55, m, 1H, C7-Hb; 1.65, m, 1H, C8-H; 0.90, m, 1H, C9-H; 2.25, m, 1H,
C11-Ha; 2.41, m, 1H, C11-Hb; 1.37, m, 1H, C14-H; 1.58, m, 1H, C15-Ha; 2.05, m, 1H,
C15-Hb; 4.86, m, 1H, C16-H; 2.89, m, 1H, C17-H; 1.07, s, 3H, C18-H; 0.87, s, 3H, C19-H;
2
.18, m, 1H, C20-H; 1.53, d, J ¼ 7.2 Hz, 3H, C21-H; 2.03, m, 2H, C23-H; 1.65, m, 1H,
C24-Ha; 2.04, m, 1H, C24-Hb; 1.91, m, 1H, C25-H; 3.59, d, J ¼ 6.6, 9.6 Hz, 1H, C26-Ha;
0
3
m, 1H, C2 -H; 4.27, m, 1H, C3 -H; 4.59, m, 1H, C4 -H; 4.04, m, 1H, C5 -H; 4.25, m, 1H,
.93, m, 1H, C26-Hb; 0.96, d, J ¼ 6.6 Hz, 3H, C27-H; 4.87, d, J ¼ 7.2 Hz, 1H, C1 -H; 4.52,
0
0
0
0
0
0
00
00
C6 -Ha; 4.60, m, 1H, C6 -Hb; 6.19, br. s, 1H, C1 -H; 4.76, m, 1H, C2 -H; 4.56, m, 1H,
00
00
00
00
C3 -H; 4.30, m, 1H, C4 -H; 4.85, m, 1H, C5 -H; 1.68, d, J ¼ 6.1 Hz, 3H, C6 -H. 5.16, d,
0
00
000
000
000
J ¼ 7.8 Hz, 1H, C1 -H; 4.04, m, 1H, C1 -H; 4.17, m, 1H, C3 -H; 4.05, m, 1H, C4 -H;
0
00
000
000
3
C1 -H; 4.04, m, 1H, C1 -H; 4.22, m, 1H, C3 -H; 4.21, m, 1H, C4 -H; 3.96, m, 1H,
.96, m, 1H, C5 -H; 4.18, m, 1H, C6 -Ha; 4.58, m, 1H, C6 -Hb; 4.79 d, J ¼ 7.8 Hz, 1H,
0
000
0000
0000
0000
0000
0000
0000
13
C5 -H; 4.38, m, 1H, C6 -Ha; 4.55, m, 1H, C6 -Hb. C-NMR (150 MHz, pyridine-d5):
(C-1) 36.7; (C-2) 29.8; (C-3) 76.9; (C-4) 34.6; (C-5) 44.5; (C-6) 28.6; (C-7) 31.7; (C-8) 34.3;
C-9) 55.8; (C-10) 36.3; (C-11) 38.0; (C-12) 213.0; (C-13) 55.6; (C-14) 55.9; (C-15) 31.7;
ꢁ
(

1
190
G. Chen et al.
(
(
(
7
7
C-16) 79.7; (C-17) 55.1; (C-18) 16.3; (C-19) 11.7; (C-20) 41.3; (C-21) 15.3; (C-22) 110.8;
0 0
C-23) 37.1; (C-24) 28.4; (C-25) 34.3; (C-26) 75.2; (C-27) 17.5; (C-1 ) 100.0; (C-2 ) 77.0;
0 0 0 0 00 00 00 00
C-3 ) 76.4; (C-4 ) 81.3; (C-5 ) 75.3; (C-6 ) 61.1; (C-1 ) 102.3; (C-2 ) 72.5; (C-3 ) 72.8; (C-4 )
00 00 000 000 000 000 000
4.1; (C-5 ) 69.5; (C-6 ) 18.7; (C-1 ) 107.2; (C-2 ) 75.7; (C-3 ) 78.9; (C-4 ) 72.2; (C-5 )
0
00
0000
0000
0000
0000
0000
8.6; (C-6 ) 63.1; (C-1 ) 105.0; (C-2 75.2; (C-3 ) 78.7; (C-4 ) 71.8; (C-5 ) 78.5;
0
000
(
C-6 ) 62.9.
3
.4. Acid hydrolysis of compounds 1 and 2
A solution of compounds 1 and 2 (5 mg) in 2 M HCl–MeOH (4 : 1, 5 mL) was refluxed at
�
9
0 C for 6 h. After cooling, the reaction mixture was diluted to 20 mL and extracted with
CH Cl (3 ꢀ 20 mL). The aqueous layer was concentrated to appropriate volume (1 mL)
2
2
and examined by TLC (silica gel) with the solvent system CHCl /MeOH/H O (65 : 35 : 10)
2
3
for sugar analysis. R values of D-glucose, D-galactose and L-rhamnose were 0.25, 0.25
f
and 0.42, respectively. The remaining aqueous layer was concentrated to dryness to give a
residue and dissolved in pyridine (1 mL), and then L-cysteine methyl ester hydrochloride
�
(
2 mg) was added to the solution. The mixture was heated at 60 C for 2 h, equal volume of
�
acetic anhydride was added, followed by heating at 90 C for another 2 h. Then, the
solution was concentrated to dryness and taken up in MeOH (0.5 mL), which was analysed
by GC (Column: DB-5 quartz capillary column (30 m ꢀ 0.25 mm, 0.25 mm)), H flame
2
�
�
ꢁ1
ionisation detector, column temperature: 160–280 C, programmed increase: 5 C min
�
,
carrier gas: N (1.5 mL min ), injector and detector temperature: 280 C, injection volume:
ꢁ
1
2
1
mL, split ratio: 10/1. The derivatives of L-rhamnose, D-glucose and D-galactose were
detected. Room temperature: 23.87, 28.04 and 28.63 min, respectively. The standard
monosaccharides were subjected to the same reaction and GC analysis under the same
condition.
Acknowledgement
The authors are grateful to members of the Analytical Group in Shenyang Pharmaceutical
University for measurements of all spectra.
References
Bedir, E., Khan, I.A., & Walker, L.A. (2002). Biologically active steroidal glycosides from Tribulus terrestris.
Pharmazie, 57, 491–493.
Cai, L.F., Jing, F.Y., Zhang, J.G., Pei, F.K., Xu, Y.J., Liu, S.Y., & Xu, D.M. (1999). Studies on the chemical
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