Steroidal saponins from Tribulus terrestris

Article abstract of DOI:10.1016/j.phytochem.2014.08.003

Sixteen steroidal saponins, including seven previously unreported compounds, were isolated from Tribulus terrestris. The structures of the saponins were established using 1D and 2D NMR spectroscopy, mass spectrometry, and chemical methods. They were ident

Full text of DOI:10.1016/j.phytochem.2014.08.003

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Steroidal saponins from Tribulus terrestris
Li-Ping Kang a,b,c,1, Ke-Lei Wu a,b,1, He-Shui Yu a,b, Xu Pang a, Jie Liu b, Li-Feng Han b, Jie Zhang a,
Yang Zhao a, Cheng-Qi Xiong a, Xin-Bo Song b, Chao Liu a, Yu-Wen Cong a, Bai-Ping Ma a,
⇑
a Beijing Institute of Radiation Medicine, Beijing 100850, China
b Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
c State Key Laboratory of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sixteen steroidal saponins, including seven previously unreported compounds, were isolated from Tribulus
terrestris. The structures of the saponins were established using 1D and 2D NMR spectroscopy, mass
Received 5 March 2013
Received in revised form 25 May 2014
Available online xxxx
spectrometry, and chemical methods. They were identified as: 26-O-b-
en-2 ,3b,22 ,26-tetrol-12-one (terrestrinin C), 26-O-b- -glucopyranosyl-(25R)-furost-4-en-22
3,12-dione (terrestrinin D), 26-O-b- -glucopyranosyl-(25S)-furost-4-en-22 ,26-diol-3,6,12-trione
(terrestrinin E), 26-O-b- -glucopyranosyl-(25R)-5 -furostan-3b,22 ,26-triol-12-one (terrestrinin F),
26-O-b- -glucopyranosyl-(25R)-furost-4-en-12b,22 ,26-triol-3-one (terrestrinin G), 26-O-b- -glucopyr-
anosyl-(1?6)-b- -glucopyranosyl-(25R)-furost-4-en-22 ,26-diol-3,12-dione (terrestrinin H), and
24-O-b- -glucopyranosyl-(25S)-5 -spirostan-3b,24b-diol-12-one-3-O-b- -glucopyranosyl-(1?4)-b- -galac-
D
-glucopyranosyl-(25R)-furost-4-
a
a
D
a
,26-diol-
D
a
Keywords:
Tribulus terrestris
Liliaceae
Steroidal saponins
Terrestrinins C–I
Platelet aggregation
D
a
a
D
a
D
D
a
D
a
D
D
topyranoside (terrestrinin I). The isolated compounds were evaluated for their platelet aggregation activities.
Three of the known saponins exhibited strong effects on the induction of platelet aggregation.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
being isolated namely six furostanol saponins (1–6) and one spi-
rostanol saponin (7), along with four known steroidal saponins
Tribulus terrestris L. is an annual creeping herb native to the
Mediterranean region, but it now grows worldwide, especially in
subtropical areas. The plant is used in folk medicine in China, India,
Bulgaria and other countries. The fruits of T. terrestris, a Chinese
traditional medicine termed ‘‘Ji Li’’, are used to treat eye inflamma-
tion, skin irritation, high blood pressure, abdominal distention, and
cardiovascular diseases. A wide range of compounds have been
extracted from this plant: saponins (Tomova et al., 1974; Wang
et al., 1996, 1997, 2009; Cai et al., 2001; Huang et al., 2003;
Kostova and Dinchev, 2005; Su et al., 2009; Xu et al., 2009,
2010a, 2010b; Liu et al., 2010; Chen et al., 2012, 2013), flavonoids
(Bhutani et al., 1969; Saleh et al., 1982), and alkaloids (Wo et al.,
1999). Among the compounds, several studies have demonstrated
that saponins are responsible for the biological activities of T. ter-
restris extracts (Tomova et al., 1981; Bedir et al., 2002; Zhang
et al., 2010). To identify additional active steroidal saponins from
T. terrestris, five known compounds (8, 9, 11, 13, and 14) were
recently reported (Wu et al., 2012). Further phytochemical analysis
of the whole plant extract led to seven new steroidal saponins
(10, 12, 15, and 16) (Fig. 1). Among the identified compounds,
the aglycones of compounds 1, 3, 5, and 7 were observed for the
first time. In the present paper, the isolation and structural elucida-
tion of the seven unreported steroidal saponins are described.
Since the saponin fraction of Chinese T. terrestris shows a signifi-
cant effect on cardiovascular diseases (Kostova and Dinchev,
2005; Yin et al., 2005) and the effect of steroidal saponins on
platelet aggregation was previously reported (Fu et al., 2008;
Cong et al., 2010, 2012; Kang et al., 2012), the activities of isolated
saponins (1, 2, and 4–16) on platelet aggregation were also
evaluated in this paper.
2. Results and discussion
The whole plant of T. terrestris was extracted using 70% aq.
EtOH. The extract was subjected to macroporous resin SP825 col-
umn to afford two saponin-rich fractions. The two fractions were
further separated chromatographically using a series of silica-gel
columns, ODS silica-gel columns, and semi-preparative HPLC to
afford sixteen steroidal saponins. The structures of the seven pre-
viously unreported compounds, termed terrestrinins C–I (1–7),
were elucidated by analysis using HRESIMS and 1D- and
2D-NMR, including COSY, TOCSY, HSQC, HMBC, and ROESY
⇑
Corresponding author. Tel.: +86 10 68210077x930265; fax: +86 10 68214653.
E-mail address: mabaiping@sina.com (B.-P. Ma).
1
Co-first author.
http://dx.doi.org/10.1016/j.phytochem.2014.08.003
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

2
L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
S1
OR5
26
OH
21
18
OH
O
25
HO
HO
20
23
R4
12
22
O
24
27
OH
H
17
OH
S2
O
11
9
13
16
15
O
19
H
HO
HO
1
4
OH
R1
R2
14
O
OH
HO
10
8
2
H
H
7
OH
3
5
6
OH
S3
O
R3
R1=-OH R2=OH R3=H,H
HO
HO
O
1
2
3
4
5
6
8
9
R4==O R5=S1 4(5)-ene
OH
HO
O
R1=H
R1=H
R1=H
R1=H
R1=H
R1=H
R1=H
R1=H
R2==O R3=H,H
R2==O R3==O
R2=OH R3=H,H
R2==O R3=H,H
R2==O R3=H,H
R4==O R5=S1 4(5)-ene
R4==O R5=S1 4(5)-ene
R4==O R5=S1 5-alf a-H
R4=OH R5=S1 4(5)-ene
R4==O R5=S3 4(5)-ene
R4==O R5=S1 5-alf a-H
R4==O R5=S1 5-alf a-H
HO
OH
S4
OH
O
O
HO
O
OH
HO
HO
R2=O-S2 R3=H,H
R2=O-S4 R3=H,H
O
O
O
OH
OH
HO
O
10
R2=O-S6 R3=H,H R4=H
R2=O-S4 R3=H,H R4=H
R4=H
R5=S1 5-alfa-H
HO
HO
OH
11 R1=H
R5=S1 5-alf a-H
OH
12 R1=alfa-OH R2=O-S4 R3=H,H
R5=S1 5-alf a-H
S5
OH
O
O
O
R3
R4
HO
O
OH
HO
HO
H
O
O
O
O
OH
OH
OH
HO
H
O
OH
R1
HO
H
H
OH
R2O
S6
H
OH
O
O
HO
O
7
R =H
1
R =S
R ==O
R =O-S
4 1
OH
2
2
3
HO
HO
O
O
13 R =H
R =S
2 4
R ==O
3
R =H
4
O
1
OH
HO
14 R =H
R =S
R =H
3
R =H
4
1
2
6
O
O
HO
HO
15a R =OH R =S
R =H
R =H
4
1
2
4
3
OH
15b R =OH R =S
R =H
3
R =H
4
1
2
5
O
HO
16a R =H
R =S
R =H
R =H
4
1
2
4
3
HO
16b R =H
R =S
R =H
R =H
4
1
2
5
3
OH
Fig. 1. Structures of compounds 1–16.
experiments. The four known compounds were identified as tribul-
uside A (10) (Xu et al., 2007), purpureagitosid (12) (Tschesche
et al., 1974; Kang et al., 2006), a mixture of F-gitonin (15a) and
desglucolanatigonin II (15b) (Inoue et al., 1995; Wang et al.,
1996), and a mixture of desgalactotigonin (16a) and gitonin
(16b) (Wang et al., 1996) by comparison of their NMR and MS data
with those previously reported, and the five previously isolated
et al., 2003; Wu et al., 2012), 25R-tribulosin (14) (Mahato et al.,
1981; Wang et al., 1996; Wu et al., 2012).
Compound 1 was isolated as a white amorphous powder. The
negative HRESIMS showed an [MꢀH]ꢀ ion peak at m/z 623.3433,
corresponding to the molecular formula C33H52O11. The 1H NMR
spectrum of 1 showed two methyl singlets at d 1.17 (s, CH3-18)
and 1.19 (s, CH3-19); two methyl doublets at d 1.54 (d, J = 7.2 Hz,
CH3-21) and 0.99 (d, J = 7.2 Hz, CH3-27); three methine protons
indicative of secondary alcoholic functions at d 4.20 (m, H-2),
4.52 (br d, J = 4.8 Hz, H-3), and 4.87 (m, H-16); and one olefinic
proton at d 5.71 (br s, H-4). The 13C NMR spectrum showed 33
carbons, including two olefinic carbons at d 123.2 (C-4) and
145.7 (C-5) and one ketone group d 212.3 (C-12). The 1H and
13CNMR spectroscopic assignments of the aglycone moiety of 1
compounds (Wu et al., 2012) were identified as 26-O-b-
pyranosyl-(25R)-5 -furostan-3b,22 ,26-triol-12-one-3-O-b-
copyranosyl-(1?4)-b- -galactopyranoside (8) (Chen et al., 2012;
D-gluco-
a
a
D
-glu-
D
Wu et al., 2012), polianthoside D (9) (Jin et al., 2004; Wu et al.,
2012), uttroside B (11) (Zhou et al., 2006; Wu et al., 2012), hecog-
enin-3-O-b-
D
-glucopyranosyl-(1?2)-[b-
D-xylopyranosyl-(1?3)]-
b-D-glucopyranosyl-(1?4)-b-
D-galactopyranoside (13) (Huang
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

Table 1
1H and 13C NMR chemical shifts assignments of the aglycone moiety of saponins 1–7 in pyridine-d5.
Position
1
2
3
4
5
6
7
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dHJ (Hz)
1
2
40.5 1.71 dd (10.2, 13.8)
2.19 dd (3.0, 13.8)
70.6 4.20 m
35.4 1.42 td (5.1, 13.8)
1.65 o⁄
34.1 2.36 m
2.33 m
35.0 1.68 m
1.80 m
34.1 2.43 m
2.54 m
36.9 0.85 m
1.45 m
32.2 1.63 m
1.98 m
35.8 1.48 dt (4.5, 13.8)
1.81 o
34.3 2.29 o,
2.36 m
35.4 1.43 m
1.66 m
34.2 2.37 m,
2.33 m
36.6 0.71 m
1.30 m
29.8 1.53 m
1.97 m
3
4
73.1 4.52br d (4.8)
123.2 5.71 br s
197.9
124.7 5.84 s
–
198.8
126.5 6.47 s
–
70.3 3.79 m
39.1 1.54 m
1.79 m
198.2
124.2 5.84 br s
–
197.9
124.7 5.83 br s
76.9 3.86 m
34.6 1.30 m
1.80 br d (12.0)
5
6
145.7
31.7 2.03 o
2.29 br t (13.2)
–
168.6
32.4 2.11 m
2.28 m
–
159.1
200.5
–
–
45.0 1.03 m
28.7 1.23 m
1.25 m
170.1
32.8 2.07 o
2.23 dt (4.8, 14.0)
–
168.6
32.4 2.10 m
2.29 m
–
44.4 0.83 m
28.6 1.12 o
7
33.4 1.64 m,
0.75 m
34.9 2.00 m
53.1 1.50 m
31.5 0.88 qd (13.0, 4.0)
1.67 o
34.3 1.96 m
54.7 1.17 o
46.0 2.15 dd (8.8, 16.5)
2.80 dd (4.5, 16.5)
32.5 2.47 m
31.7 0.79 m,
1.59 m
34.4 1.81 m
55.7 0.96 m
32.1 0.82 br q (13.0)
1.65 o
34.3 1.63 o
31.5 1.66 m,
31.7 1.51 m
0.74 m
34.3 1.67 m
55.3 0.87 m
0.83 qd (4.2, 13.2)
34.2 1.96 m
54.7 1.18 m
8
9
51.5 1.79 m
53.1 0.91 br t (10.4)
10
11
39.9
–
38.9
37.4 2.25 m
2.53 t (13.8)
–
39.4
–
36.4
–
38.7
31.2 1.64 o,
1.81 m
–
38.9
37.3 2.25 m,
2.53 t (13.8)
–
36.3
–
38.7 2.48 dd (4.8, 13.8)
2.55 dd (13.2, 13.8)
37.0 2.42 dd (5.5, 14.6)
2.67 t (14.4)
38.1 2.31 dd (5.0, 13.8)
2.45 t (13.8)
37.9 2.20 dd (4.9, 14.0)
2.33 t (13.4)
12
13
14
15
212.3
55.8
55.4 1.28 m
31.7 1.61 m
2.04 o
–
–
211.9
55.5
55.0 1.40 m
31.7 1.64 o
–
–
211.2
55.4
55.4 1.67 m
31.4 1.65 m
2.12 m
–
–
213.1
55.7
55.8 1.41 m
–
–
78.9 3.57 dd (4.8, 10.8) 212.0
46.7 55.5
–
–
212.7
55.3
55.5 1.33 m
31.3 1.49 m
2.03 m
–
–
–
54.4 1.08 m
32.1 1.65 o
2.07 m
55.0 1.40 m
31.7 1.64 o
31.8 1.61 m
2.09 m
2.08 m
2.08 m
16
17
18
19
20
21
22
23
79.7 4.87 m
55.2 2.86 qd (7.2, 8.4)
16.3 1.17 s
21.7 1.19 s
41.2 2.21 m
15.3 1.54 d (7.2)
79.6 4.87 q (7.1)
55.1 2.88 qd (6.6, 8.6)
16.2 1.19 s
16.5 1.08 s
41.3 2.22 m
15.2 1.54 d J = 7.2
79.4 4.89 q (7.0)
55.0 2.93 dd (6.7, 8.5)
16.3 1.24 s
16.8 1.15 s
41.3 2.24 m
15.2 1.57 d (6.9)
79.7 4.88 m
55.1 2.91 dd (6.6, 8.4)
16.3 1.15 s
11.9 0.82 s
41.3 2.22 qd (6.6, 6.0)
15.3 1.56 d (6.6)
81.1 5.01 q (7.2)
63.7 2.31 o
11.3 1.19 s
17.1 1.04 s
41.7 2.50 p (7.8)
15.7 1.61 d J = 6.6
79.5 4.87 q (8.4)
55.1 2.88 dd (6.0 8.6)
16.2 1.20 s
16.5 1.08 s
41.2 2.23 m
15.3 1.55 d (6.6)
80.1 4.44 q (7.0)
53.9 2.69 dd (6.6 8.4)
16.1 0.98 s
11.7 0.65 s
42.8 1.87 m
13.8 1.26 d (7.2)
110.7
–
110.8
–
110.8
–
110.8
–
110.9
–
110.8
–
111.7
40.9 1.95 t (12.6)
2.67 dd (4.8, 13.1)
–
37.1 2.00 o
2.06 o
37.1 2.02 o
2.05 o
37.1 2.06 o
37.1 2.02 o
37.3 2.08 o
37.1 2.05 o
24
28.4 1.66 o
2.03 o
28.4 1.66 o
2.04 o
28.4 1.68 m
2.06 o
28.4 1.65 m
2.04 o
28.5 1.70 m
2.04 m
28.5 1.68 m
2.05 o
81.5 4.01 m
25
26
34.3 1.92 m
75.3 3.61 dd (6.0, 9.0)
3.94 m
34.3 1.93 m
75.3 3.61 dd (6.0, 8.4)
3.94 m
34.5 1.96 m
75.3 3.63 t (5.9, 9.3)
3.97 m
34.3 1.94 m
75.3 3.61 dd (8.4, 6.6)
3.94 m
34.3 1.92 m
75.3 3.63 dd (6.0, 9.0)
3.92 m
34.2 1.93 m
75.3 3.57 dd (6.0, 9.6)
4.03 m
38.1 1.89 m
65.2 3.55 t (11.5)
3.63 dd (5.0, 11.5)
13.5 1.13 d (6.6)
27
17.4 0.99 d (7.2)
17.4 0.98 d (6.6)
17.4 0.99 d (6.7)
17.4 0.98 d (6.6)
17.5 0.98 d (6.6)
17.4 0.99 d (6.6)

4
L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
(Table 1) were established from the analysis of the 1H–1H COSY,
HSQC and HMBC experiments. The JH–H COSY correlations for d
absolute configuration was identified as D by GC analysis. The large
coupling constants (3J1,2 > 7 Hz) were consistent with the b config-
uration of the sugar (Agrawal, 1992). The long-range correlation
between d 4.81 (H-1-Glc) and d 75.3 (C-26) in the HMBC spectrum
and the NOE correlation between d 4.81 (H-1-Glc) and d 3.94 (H-
26) revealed the sugar linkage site to be the aglycone moiety.
3
5.71 (H-4)/d 4.52 (H-3)/d 4.20 (H-2)/d 2.19 and 1.71 (H2-1) indi-
cated two hydroxyl groups at the C-2 and C-3 positions of the agly-
cone of 1. The HMBC correlations between the proton signal at d
5.71 (H-4) and the carbon resonances at d 70.6 (C-2), 39.9 (C-10),
and 31.7 (C-6), and between d 1.19 (H3-19) and d 39.9 (C-10),
40.5 (C-1), 53.1 (C-9), and 145.7 (C-5) also supported the presence
of the OH-2 and OH-3 in the aglycone. The b orientation of the oxy-
gen atom at C-2 and C-3 positions was confirmed by the spin-cou-
pling constants between the proton signals of H-2 (d 4.20) and H2-
Thus, the structure of 1 was elucidated as 26-O-b-
D-glucopyrano-
syl-(25R)-furost-4-en-2b,3b,22a,26-tetrol-12-one, named terrestr-
inin C.
Compound 2 was isolated as a white amorphous powder with a
molecular formula of C33H50O10, as determined by HRESIMS (m/z:
605.3347 [MꢀH]ꢀ). A comparison of the NMR and MS data of 2
with those of 1 (Tables 1 and 2) indicated that while they have sim-
ilar structures, they exhibited significant differences in the A ring
of the aglycone. Because the 13C NMR resonances of C-2–C-5 were
markedly shifted [d 34.1 (C-2), 197.9 (C-3), 124.7 (C-4), and 168.6
(C-5) instead of d 70.6 (C-2), 73.1 (C-3), 123.2 (C-4), and 145.7 (C-5)
in compound 1], the lack of two hydroxyl groups at C-2 and C-3
positions and a ketone group as the substituent at the C-3 position
of the aglycone in 2 could be deduced. This was confirmed by the
HMBC correlation between the C-3 (d 197.9) and H2-2 (d 2.36 and
2.33) and H2-1 (d 1.42 and 1.65). Thus, the aglycone of saponin 2
3
1 (3JH-1ax–H-2ax = 10.2 Hz and JH-1eq–H-2ax = 3.0 Hz), and between
the proton resonances of H-3 (d 4.52) and H-2 (3JH-2ax–H-3eq = 4.8 -
Hz), and NOE correlations between H-2 (d 4.20) and H-9 (d 1.50),
whereas H-3 at d 4.52 possessed a NOE correlation with H-2. The
C-25 configuration was deduced to be (R) based on the difference
of the chemical shifts of the geminal protons CH -26
2
(Dab = 0.33 ppm). The Dab was established to be <0.48 ppm in
25R-furostane steroids (Agrawal, 2005). NOE correlations were
observed between H3-19/H-11, H-8/H3-18, H3-18/H-20, H-9/H-
14, H-16/H-17, and H-17/H-21, suggesting the usual trans junction
for the B/C and C/D rings and a cis ring fusion for the D/E rings. The
a
orientation of OH-22 in the aglycone moiety was deduced from
was identified as (25R)-furost-4-en-22
et al., 1998). The sugar unit and its linkage site to the aglycone
were identified as in 1. Thus, 2 was determined to be 26-O-b- -glu-
named
a, 26-diol-3,12-dione (Xu
the semiketal carbon signal at d 110.7 (Fattorusso et al., 2002),
and was further confirmed by the NOE correlation between H-20
(d 2.21) and H-23 (d 2.06). Thus, the aglycone moiety of 1 was
D
copyranosyl-(25R)-furost-4-en-22a,26-diol-3,12-dione,
deduced to be (25R)-furost-4-en-2b,3b,22
a,26-tetrol-12-one,
a
terrestrinin D.
sapogenin previously unreported. The anomeric regions in the 1H
and 13C NMR spectra of 1 showed one anomeric proton at d 4.81
(d, J = 7.8 Hz), corresponding to the anomeric carbon signal at d
105.0. Glucose was detected after acidic hydrolysis of 1 and its
Compound 3 showed an [MꢀH]ꢀ ion peak at m/z 619.3146 in
the negative HRESIMS, corresponding to the empirical molecular
formula C33H48O11, suggesting that saponin 3 was an oxo deriva-
tive of saponin 2. A detailed comparison of the NMR spectroscopic
Table 2
1H and 13C NMR chemical shifts assignments of the glycosides part of saponins 1–7 in pyridine-d5.
Position
1
2
3
4
5
6
7
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dH J (Hz)
dC
dHJ (Hz)
Glc
Glc
Glc
Glc
Glc
Glc0
Gal0
10
20
30
40
105.0 4.81 d (7.8) 105.0 4.81 d
(7.8)
105.0 4.83 d (7.8) 105.0 4.81 d (8.4) 105.0 4.81 d (7.8) 104.9 4.74 d (7.8) 102.5 4.86 d (7.8)
75.2
78.7
71.8
4.02 dd
(7.8, 8.4)
4.22 o
75.2
78.7
71.8
4.01 m
4.22 o
4.21 0
75.3
78.7
71.7
4.04 t (8.5)
4.25 o
75.2
78.7
71.8
4.01 t (8.4)
4.22 o
75.2
78.7
71.7
4.02 dd
(7.8, 9.0)
4.23 o
75.1
78.6
71.7
3.96 t (8.2)
73.5
75.3
80.1
4.38 dd
(7.8, 9.6)
4.24 o
4.17 dd
(8.7, 8.8)
4.15 dd
(8.7, 9.8)
4.05 m
4.33 dd
(6.0, 11.4)
4.83 dd
(1.8, 11.4)
4.21 o
4.24 o
4.22 o
4.24 o
4.70 br d
(3.0)
4.08 m
4.27 dd
(5.4, 10.7)
4.66 dd
(8.5, 10.7)
500
60
78.5
62.9
3.93 m
4.37 dd
(5.4, 12.0),
4.54 dd
(1.8, 12.0)
78.5
62.9
3.93 m
4.38 m,
4.54 brd
(11.7)
78.5
62.9
3.96 m
4.40 dd
(5.0, 11.7)
4.56 dd
(2.2, 11.7)
78.5
62.9
3.94 m
4.38 dd
(5.4, 12.0)
4.54 dd
(1.8, 12.0)
78.5
62.9
3.93 m
77.3
70.2
75.5
61.1
4.39 dd
(4.8, 11.4)
4.55 br d
(11.4)
Glc00
Glc00
100
200
105.5 5.11 (7.8)
75.3
107.2 5.28 d (7.8)
76.0
4.04 m
4.13 dd
(7.8, 9.0)
4.22 o
4.24 o
3.87 m
4.35 dd
(5.3, 11.7)
4.51 dd
(2.4, 11.7)
300
400
500
600
78.5
71.7
78.5
62.8
4.21 o
4.22 o
78.7
71.8
78.1
62.9
3.92 m
4.37 dd
(5.0, 11.8)
4.51 br d
(10.0)
Glc000
1000
2000
3000
4000
5000
6000
106.5 4.92 d (7.8)
75.7
78.6
72.3
78.5
63.2
4.04 o
4.20 o
4.06 o
4.01 o
4.20 o
4.59 dd
(2.2, 11.6)
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
5
data of 3 with those of 2 indicated that an additional carbonyl was
at d 200.5 (Table 1), this being attached to the A or B rings of the
aglycone. In the HMBC spectrum, long range correlations were
observed between C-6 (d 200.5) and H-4 (d 6.47) and H2-7 (d
2.15 and 2.80), and between H3-19 (d 1.15) and C-1 (d 35.0), C-
10 (d 39.4), C-9 (d 51.5), and C-5 (d 159.1), confirming the 6-one
structure. The difference in the chemical shifts of the geminal
as 26-O-b-
ost-4-en-22
D
-glucopyranosyl-(1?6)-b-
,26-diol-3,12-dione, named terrestrinin H.
D
-glucopyranosyl-(25R)-fur-
a
Compound 7 was isolated as a white amorphous powder. It
showed an [MꢀH]ꢀ ion peak at m/z 931.4561 in the negative HRE-
SIMS, corresponding to a molecular formula of C45H72O20. Its 1H
NMR spectrum showed four methyl signals at d 0.65 (s, CH3-19),
0.98 (s, CH3-18), 1.13 (d, J = 6.6 Hz, CH3-27), and 1.26 (d,
J = 7.2 Hz, CH3-21); two methylene proton resonances of CH2OR
at d 3.55 (t, J = 11.5 Hz, Hax-26) and 3.63 (dd, J = 5.0, 11.5 Hz,
Heq-26); and three methine proton signals of CHOR at d 3.86 (m,
H-3), 4.01 (m, H-24), and 4.44 (q, J = 7.0 Hz, H-16). The hydroxy
group at the C-24 position was confirmed by the HMBC corrections
between the proton resonances at d 1.95 and 2.67 (H2-24), 1.89 (H-
25), 3.55 and 3.63 (H2-26), and 1.13 (H3-27) and the carbon signal
at d 81.5 (C-24). The C-24S and C-25S configurations were deduced
from the proton multiplicities of H-23 and H-26, with J values of
12.6 Hz (H-24/H-23ax), 4.8 Hz (H-24/H-23 eq), 11.5 Hz (H-25/H-
26ax), and 5.0 Hz (H-25/H-26 eq). The NOE correlations from H-
23ax to H-20 and H-24 and from H-26ax to H-16 and H-24 in
the ROESY spectrum were consistent with the C-22R, C-24S, and
H2-26 protons (
to be R. The analysis of the 1D- and 2D-NMR experiments allowed
the aglycone of 3 to be identified as (25R)-furost-4-en-22 ,26-diol-
DdH = 0.34 ppm) proved the configuration of C-25
a
3,6,12-trione, an aglycone reported for the first time. The sugar
unit and its linkage site were identified as in compound 1 (Table 2).
Thus, the structure of 3 was established as 26-O-b-
D-glucopyrano-
syl-(25R)-furost-4-en-22a,26-diol-3,6,12-trione, termed terrestri-
nin E.
Compound 4 displayed an [MꢀH]ꢀ ion peak at m/z 607.3497 in
the negative HRESIMS, indicating
a molecular formula of
C33H54O10. A detailed comparison of the NMR spectroscopic data
of 4 with those of 1 indicated they shared similar aglycone and
sugar moieties, except at the A and B rings of the aglycone moiety.
The lack of one hydroxy group at position C-2 and a double bond at
position C-4(5) of the aglycone in compound 4 was deduced from
the chemical shift of its C-1–C6 (d 36.9, 32.2, 70.3, 39.1, 45.0, and
28.7 instead of d 40.5, 70.6, 73.1, 123.2, 145.7, and 31.7 in com-
pound 1) (Table 1). The chemical shift of C-19 at d 11.9 indicated
C-25S configurations (Su et al., 2009). The
was identified by the chemical shift of C-19 at d 11.7 (Agrawal
et al., 1995) and the NOE correlations from H-5 to H-3 and H-9.
Therefore, the aglycone of 7 was identified as (25S)-5
a orientation of H-5
a-spirostan-
3b,24b-diol-12-one. The nature of the monosaccharides was iden-
an
of the 1D- and 2D-NMR spectroscopic data of 4, its structure was
defined as 26-O-b- -glucopyranosyl-(25R)-5 -furostan-3b,22 ,26-
a
orientation for H-5 (Agrawal et al., 1995). Through analysis
tified as D-galactose and D-glucose from acid hydrolysis and GC
analysis. The 1H and 13C NMR spectra showed three anomeric pro-
tons at d 4.86 (d, J = 7.8 Hz), 5.28 (d, J = 7.8 Hz), and 4.92 (d,
J = 7.8 Hz), corresponding to the anomeric carbon signal at d
102.5, 107.2, and 105.0 (Table 2). Complete assignment of the
sugar protons and carbons by COSY, HSQC and HMBC experiments
D
a
a
triol-12-one, named terrestrinin F.
Compound 5 showed an [MꢀH]ꢀ ion peak at m/z 609.3638 in
the negative HRESIMS, corresponding to a molecular formula of
C
33H54O10. A detailed comparison of the NMR spectroscopic data
showed the presence of two b-D-glucopyranosyl units and one b-D-
of 5 with those of 2 established that they shared similar aglycone
and sugar moieties, except that the C and D rings of the aglycone
lack a ketone group but possess an additional hydroxy group at
position C-12. The long range correlations between the proton at
d 3.57 (H-12) and the carbon signals at d 11.3 (C-18), 46.7 (C-13),
54.4 (C-14), and 63.7 (C-17) in the HMBC spectrum and the
cross-peak correlations between d 3.57 (H-12) and d 1.64 and
1.81 (H2-11) in the 1H–1H COSY spectrum indicated that the
hydroxy group was located at the C-12 position of the aglycone.
The b orientation of OH-12 was confirmed by the spin-coupling
constant between the proton resonances of 12-H and 11-H2
galactopyranosyl unit. The HMBC corrections between H-10 (d 4.86)
and C-3 (76.9), H-100 (d 5.28) and C-40 (d 80.1), and H-1000 (d 4.92)
and C-24 (d 81.5) allowed deduction of the sugar sequencing and
the linkage sites. Therefore, the structure of 7 was assigned as
24-O-b-
3-O-b- -glucopyranosyl-(1?4)-b-
restrinin I.
D
-glucopyranosyl-(25S)-5
a
-spirostan-3b,24b-diol-12-one-
D
D-galactopyranoside, named ter-
In a previous study, pennogenin glycosides with spirostanol
structures were reported as strong platelet agonists (Fu et al.,
2008; Cong et al., 2010, 2012), and it was also found that some sap-
onins with sarsasapogenin have strong antiplatelet activities
(Zhang et al., 1999; Kang et al., 2012). Structure–activity relation-
ship analysis suggested that steroidal saponins exhibited either
agonistic or inhibitory activities on platelet aggregation based on
the difference in their structures. So, the activities of the isolated
saponins on platelet aggregation were evaluated. Using U46619
(a TxA2 analog) -induced rat platelet aggregation as positive con-
trol, the inductive activity of isolated saponins on platelet aggrega-
tion was evaluated. The effect of compounds 1, 2, and 4–16 on
platelet was further investigated. The results showed that com-
pounds isolated from T. terrestris exhibited diverse platelet activi-
ties (Table 3). The screening concentration of compounds was
(3JH-12ax–H-11ax = 10.8 Hz and JH-12ax–H-11eq = 4.8 Hz) and the NOE
correlations between H-12/H-9/H-14/H-11/H-17 in the ROESY
spectrum. Therefore, the aglycone of
(25R)-furost-4-en-12b,22 ,26-triol-3-one (Hamed et al., 2004;
Mimaki et al., 1998). Through analysis of the 1D- and 2D-NMR
spectroscopic data of 5, its structure was defined as 26-O-b-
3
5 was elucidated as
a
D
-
glucopyranosyl-(25R)-furost-4-en-12b,22a,26-triol-3-one, named
terrestrinin G.
Compound 6 was isolated as a white amorphous powder. Its
negative HRESIMS showed an [MꢀH]ꢀ ion peak at m/z 767.3849,
corresponding to a molecular formula of C39H60O15. Comparing
the NMR and MS data with those of 2, compound 6 was deter-
mined to have the same aglycone as 2 but with an additional sugar
residue. The 1H NMR spectrum showed two anomeric protons at d
4.74 (d, J = 7.8 Hz) and 5.11 (d, J = 7.8 Hz), corresponding to two
anomeric carbon signals at d 104.9 and 105.5 in the 13C NMR spec-
trum. The 1H and 13C NMR assignments of the sugar moiety of 6
(Table 2) were established by analysis of the 1H–1H COSY, HSQC
and HMBC experiments. The HMBC cross peaks of d 4.74 (H’-1-
Glc) with d 75.3 (C-26), and d 5.11 (H’’-1-Glc) with d 70.2 (C-60) per-
mitted deduction of the sequence of the sugars and their linkage
site to the aglycone moiety. Thus, the structure of 6 was elucidated
250
platelet aggregation, the induction rate were 73%, 72%, and 74%,
respectively. With the concentration decreased to 25 M, com-
lmol/L. Compounds 13–16 exhibited agonistic activity on
l
pounds 13, 15, and 16 also exhibited significant inductive effects
on platelet aggregation. The compounds 1, 2, and 4–12 exhibited
weak (or no) inhibitory effects on U46619-induced platelet aggre-
gation. Then the inhibitory activity was evaluated on U46619-
induced platelet aggregation. Here, some steroidal saponins
extracted from Chinese T. terrestris exhibited stronger agonistic
activities on rat platelet aggregation, which indicates they may
have a role on hemostasis.
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

6
L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
tubes. Tubes 68–119 were purified by semi-preparative HPLC with
3. Experimental
MeCN–H2O (28:72) to yield 9 (9.5 mg) and 11 (11.4 mg). Fr. C
(30 g) was subjected to CC on a silica-gel column with a gradient
mixture of CHCl3–MeOH–H2O (9:1:0.01, 5:1:0.01, and 2:1:0.01)
as the eluent, and 287 tubes were obtained. Tubes 81–113 was
purified by CC on ODS silica gel with MeCN–H2O (48:52) to give
50 tubes. Tubes 10–21 were purified by semi-preparative HPLC
with MeCN–H2O (53:47) to yield 13 (196.3 mg) and tubes 30–35
were purified by semi-preparative HPLC with MeCN–H2O (50:50)
to yield 16 (38.1 mg). Tubes 114–142 were purified by CC on
ODS silica gel with MeCN–H2O (40:60) to give 100 tubes, and tubes
73–86 were purified by recrystallization to yield 15 (31.5 mg).
Tubes 231–250 were purified by recrystallization to yield 14
(172.4 mg).
3.1. General methods
The NMR spectra were recorded on a Bruker DRX-500 spec-
trometer (500 MHz for 1H NMR and 125 MHz for 13C-NMR, Kar-
lsruhe, Germany) and a Varian UNITYINOVA 600 spectrometer
(600 MHz for 1H NMR and 150 MHz for 13C-NMR, Palo Alto, USA)
in pyridine-d5, and 2D NMR experiments were performed using
standard microprograms. HRESIMS was recorded on a Synapt MS
(Waters Corporation, Milford, MA, USA). The optical rotations were
measured with a Perkin-Elmer 343 polarimeter (PerkinElmer, Wal-
tham, MA, USA). HPLC was performed on a Waters 2695 series;
XBP C18 and XBP C18-2 columns (5
lm, 4.6 ꢁ 250 mm, Agela, Tian-
jin, China); Alltech 2000 ELSD detector (Alltech Corporation, Deer-
field, USA). TLC was performed on silica gel GF254 plates (Qingdao
marine Chemical, China). Column chromatography (CC) was per-
formed on macroporous resin SP825 (Mitsubishi Chemicals, Japan),
silica gel (Qingdao Haiyang Chemical Co., Ltd., China), and ODS-A
3.4. Compound 1
White amorphous power; ½a D20
ꢂ
+ 10.0 (c 0.05, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 659.3
[M+Cl]ꢀ, 623.3 [MꢀH]ꢀ, 605.3 [MꢀHꢀH2O]ꢀ, 443.3 [MꢀHꢀH2-
OꢀGlc]ꢀ, 425.3 [MꢀHꢀ3 ꢁ H2OꢀGlc]ꢀ, and 407.3 [MꢀHꢀ3 ꢁ H2-
OꢀGlc]ꢀ; HRESIMS (negative) m/z: 623.3433 [MꢀH]ꢀ (calc. for
silica gel (120 Å, 50 lm, YMC, Japan). U46619 (P98%) and PGE1
(P98%) was purchased from Cayman. The male Wistar rats were
housed separately and acclimatized in a temperature controlled
(25 2 °C) and illumination-controlled (12-h light/dark cycle)
room for at least 5 days prior to the experiments. The animals were
fed with standard animal food and water.
C
33H51O11, 623.3431).
3.5. Compound 2
3.2. Plant material
White amorphous power; ½a D20
ꢂ
+ 48.0 (c 0.05, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 641.3
[M+Cl]ꢀ, 605.3 [MꢀH]ꢀ, 587.3 [MꢀHꢀH2O]ꢀ, and 425.3 [MꢀHꢀH2-
OꢀGlc]ꢀ; HRESIMS (negative) m/z: 605.3347 [MꢀH]ꢀ (calc. for
Fresh whole T. terrestris L. plants were collected from Beijing,
China in May 2010. The plant was identified by Prof. Li-juan Zhang
(Tianjin University of Traditional Chinese Medicine). A voucher
specimen (No. 100501) is deposited in the Herbarium of the Beijing
Institute of Radiation Medicine, Beijing.
C
33H49O10, 605.3326).
3.6. Compound 3
3.3. Extraction and isolation
White amorphous power; ½a D20
ꢂ
+ 40.5 (c 0.04, pyridine); For 1H-
NMR (pyridine-d5, 500 MHz) and 13C-NMR (pyridine-d5, 125 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 665.3
[MꢀHCOO]ꢀ, 655.3 [M+Cl]ꢀ, 619.3 [MꢀH]ꢀ, 601.3 [MꢀHꢀH2O]ꢀ
and 439.3 [MꢀHꢀGlc]ꢀ; HRESIMS (negative) m/z 619.3146
[MꢀH]ꢀ (calc. for C33H47O11, 619.3118).
The whole plant material of T. terrestris L. (20 kg) was extracted
using EtOH–H2O (7:3, 40 L ꢁ 2, each for 1 h), under conditions of
reflux, this being continued for. The combined extract was evapo-
rated under reduced pressure, and applied to a macroporous resin
SP825 column which was eluted with a gradient mixture of EtOH-
H2O (5:95, 60:40, and 90:10; 10 L of each solvent) to yield three
factions (A–C). Fr. B (60 g) was subjected to silica-gel CC with a gra-
dient mixture of CHCl3–MeOH–H2O (15:1:0.01, 65:25:4, and
2:1:0.01) as the eluent, and four fractions were obtained (B1ꢀB4).
Fr. B1 was purified repeatedly by silica gel CC with a gradient mix-
ture of CHCl3–MeOH–H2O (10:1:0.01, and 3:1:0.01) as the eluent,
and a total of 280 tubes were collected. Tubes 48–57 were purified
by semi-preparative HPLC with MeCN–H2O (25:75) to yield 4
(6.9 mg) and 8 (12.9 mg). Tubes 63–88 were subjected to ODS sil-
ica-gel CC eluted with MeCN–H2O (24:76) and semi-preparative
HPLC with MeCN–H2O (26:74) to yield 5 (4.8 mg). Tubes 121–
164 were purified by semi-preparative HPLC with MeCN–H2O
(25:75) to yield 1 (7.8 mg) and 3 (3.1 mg). Fr. B2 was subjected
to an ODS silica-gel CC eluted with MeCN–H2O (23:77) and 112
tubes were collected. Tubes 56–63 were purified by semi-prepara-
tive HPLC with MeCN–H2O (26:74) to yield 6 (6.4 mg). Tubes 89–
92 were purified by semi-preparative HPLC with MeCN–H2O
(26:74) to yield 2 (59.0 mg) and 10 (180.4 mg). Fr. B3 was subjected
to ODS silica-gel CC eluted with MeCN–H2O (23:77) and 40 tubes
were collected. Tubes 6–8 were purified by semi-preparative HPLC
with MeCN–H2O (23:77) to yield 12 (9.0 mg). Tubes 10–12 were
purified by semi-preparative HPLC with MeCN–H2O (26:74) to
yield 7 (7.7 mg). Fr. B4 was separated chromatographically on a sil-
ica-gel column with CHCl3–MeOH–H2O (3:1:0.01) to give 150
3.7. Compound 4
White amorphous power; ½a D20
ꢂ
+ 30.0 (c 0.04, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
Table 3
The effect of compounds 1, 2, and 4–16 on rat platelet activities.
Compounds Dose
Max. inhibition rate (%) Max. induction rate (%)
PGE1
50
l
g/mL 70.4
–
83
–
–
–
–
–
–
–
–
–
–
–
73
38
72
74
U46619
1
2
4
5
6
7
8
1.25
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
l
M
–
lM
16.8
7.2
9.6
18.1
7.2
9.6
14.5
10.8
13.2
15.6
-2.9
–
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
lM
9
10
11
12
13
14
15
16
–
–
–
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
7
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 609.4
lated with a one-ninth volume of 200 U (v/v) heparin. The blood
samples were immediately centrifuged at 2300g for 1 min at room
temperature, and the resulting supernatant platelet-rich plasma
(PRP) fraction was obtained. The remaining blood was further cen-
trifuged at 1500g for 10 min, and the resulting supernatant used as
the platelet-poor plasma (PPP) fraction. Platelet aggregation was
measured using a Lumi aggregometer (Chrono-Log, Havertown,
PA). The PRP was equilibrated at 37 °C without stirring for 3 min
prior to the initiation of each experiment, and compounds 1, 2,
[MꢀH]ꢀ, 591.4 [MꢀHꢀH2O]ꢀ, and 429.3 [MꢀHꢀH2OꢀGlc]ꢀ; HRE-
SIMS (negative) m/z 609.3638 [MꢀH]ꢀ (calc. for C33H53O10
,
609.3639).
3.8. Compound 5
White amorphous power; ½a D20
ꢂ
+ 25.7 (c 0.04, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 607.4
[MꢀH]ꢀ, 445.3 [MꢀHꢀGlc]ꢀ, and 427.3 [MꢀHꢀGlcꢀH2O]ꢀ; HRE-
and 4–12 (250
(DMSO) was added 2 min prior to the addition of U46619
(1.25
mol Lꢀ1) with continuous stirring at 37 °C. Compounds
13–16 were added without the addition of U46619 (1.25 )
mol Lꢀ1
l lg/mL) or dimethyl sulfoxide
mol Lꢀ1) or PGE1 (50
SIMS (negative) m/z 607.3497 [MꢀH]ꢀ (calc. for C33H11O10
,
l
607.3482).
l
with continuous stirring at 37 °C. The baseline was determined
from the PRP, and the maximum possible increase in light trans-
mission (platelet aggregation rate: 100%) was established with
PPP. H1 was the height of maximum aggregation by given each
compound with U46619 gathered away from PPP baseline, and
H0 was the height of maximum aggregation by giving U46619
without other compounds gathered away from the PPP baseline.
Then platelet max inhibition rate (MIR1) was obtained by the fol-
lowing formula System rate: MIR1 = (1ꢀH1/H0) ꢁ 100% and the
maximum induction rate (MIR2) was calculated based on the fol-
lowing formula System rate: MIR2 = H1/100 ꢁ 100%. U46619-
inducing rat platelet aggregation was used as positive control in
the biological test.
3.9. Compound 6
White amorphous power; ½a D20
ꢂ
+ 32.4 (c 0.09, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 767.4
[MꢀH]ꢀ, 749.4 [MꢀHꢀH2O]ꢀ, 587.3 [MꢀHꢀH2OꢀGlc]ꢀ, and
425.3 [MꢀHꢀH2Oꢀ2 ꢁ Glc]ꢀ; HRESIMS (negative) m/z 767.3849
[MꢀH]ꢀ (calc. for C39H59O15, 767.3854).
3.10. Compound 7
White amorphous power; ½a D20
ꢂ
ꢀ22.5 (c 0.05, pyridine); For 1H-
NMR (pyridine-d5, 600 MHz) and 13C-NMR (pyridine-d5, 150 MHz)
spectroscopic data, see Tables 1 and 2; ESIMS (negative) 931.5
[MꢀH]ꢀ, 769.4 [MꢀHꢀGlc]ꢀ, and 607.4 [MꢀHꢀ2 ꢁ Glc]ꢀ; HRE-
Acknowledgements
SIMS (negative) m/z 931.4531 [MꢀH]ꢀ (calc. for C45H71O20
,
This work was supported by the Major Program of Municipal
Natural Science Foundation of Beijing (7090001) and the National
Natural Science Foundation of China Union Foundation of Yunnan
Province (U10132601). The authors are grateful to Mei-feng Xu for
the determination of the NMR spectra.
931.4539).
3.11. Acid hydrolysis
Compounds 1–7 (1.5 mg each) were individually treated in 2 N
CF3COOH–H2O (2 mL) at 95 °C for 4 h. Each reaction mixture was
extracted with CH2Cl2 (2 mL) three times, with the aqueous layer
repeatedly evaporated to dryness until neutral. Then, in the mono-
saccharide mixture, glucose and galactose were detected by TLC
analysis on a cellulose plate using n-BuOH–EtOAc–C5H5N–H2O
(6:1:5:4) as the developing solution and aniline-o-phthalic acid
as the detection solution and were then compared with the control
samples: glucose (Rf 0.46) and galactose (Rf 0.69). The sugar resi-
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.phytochem.
2014.08.003.
References
due in pyridine (1 mL) was added to
chloride (3.0 mg), and the mixture was kept at 60 °C for 1 h. Then,
HMDS-TMCS (hexamethyldisilazane–trimethylchlorosilane)
(0.6 mL) was added to the reaction mixture, which was then kept
at 60 °C for 0.5 h. The supernatant (1.0 mL) was analyzed by GC
under the following conditions: Agilent Technologies 6890 gas
chromatograph was the equipment, carrying a 5973 mass spectro-
L-cysteine methyl ester hydro-
Agrawal, P.K., 1992. NMR spectroscopy in the structural elucidation of
oligosaccharides and glycosides. Phytochemistry 31, 3307–3330.
Agrawal, P.K., Jain, D.C., Pathak, A.K., 1995. NMR spectroscopy of steroidal
sapogenins and steroidal saponins: an update. Magn. Reson. Chem. 33, 923–
953.
Agrawal, P.K., 2005. Assigning stereodiversity of the 27-Me group of furostane-type
steroidal via NMR chemical shifts. Steroids 70, 715–724.
Bedir, E., Khan, I.A., Walker, L.A., 2002. Biologically active steroidal glycosides from
Tribulus terrestris. Pharmazie 57, 491–493.
Bhutani, S.P., Chibber, S.S., Seshadri, T.R., 1969. Flavonoids of the fruits and leaves of
Tribulus pentandrus: constitution of tribuloside. Phytochemistry 8, 299–303.
Cai, L.F., Wu, Y.J., Zhang, J.G., Pei, F.Q., Xu, Y.J., Xie, S.X., Xu, D.M., 2001. Steroidal
saponins from Tribulus terrestris. Planta Med. 67, 196–198.
Chen, G., Liu, T., Lu, X., Wang, H.F., Hua, M.H., Pei, Y.H., 2012. New steroidal
glycosides from Tribulus terrestris L.. J. Asian Nat. Prod. Res. 14, 780–784.
Chen, G., Su, L., Feng, S.G., Lu, X., Wang, H.F., Pei, Y.H., 2013. Furostanol saponins
from the fruits of Tribulus terrestris. Nat. Prod. Res. 27, 1186–1190.
Cong, Y., Liu, X.L., Kang, L.P., Yu, Z.Y., Zhao, Z.H., Li, J., Ma, B.P., Cong, Y.W., 2012.
Pennogenin tetraglycoside stimulates secretion-dependent activation of rat
platelets: evidence for critical roles of adenosine diphosphate receptor signal
pathways. Thromb. Res. 129, 209–216.
Cong, Y., Liu, X.L., Yu, Z.Y., Kang, L.P., Ma, B.P., Cong, Y.W., 2010. Screening of active
ingredients of timosaponin for anti platelet aggregation, and analysis of
structure activity relationship. Med. J. Chin. PLA 35, 1370–1373.
Fattorusso, E., Iorizzi, M., Lanzotti, V., Targlialatela-Scafati, O., 2002. Chemical
composition of shallot (Allium ascalonicum Hort.). J. Agric. Food Chem. 50, 5686–
5690.
graph
detector,
and
a
HP-5
capillary
column
(30 m ꢁ 0.25 mm ꢁ 0.25
l
m) was used. The conditions were as fol-
low: column temperature: 180 °C/250 °C; programmed increase,
15 °C/min; carrier gas: N2 (1 mL/min); injection and detector tem-
perature: 250 °C; injection volume: 4.0 lL; and split ratio: 1/50.
The absolute configurations of the monosaccharides were con-
firmed by comparison of the retention times of the monosaccha-
ride derivatives with those of standard samples:
(17.95 min) and -galactose (18.57 min). The -glucose was
-galactose were
D-glucose
D
D
detected for compounds 1–6, and
detected for compound 7.
D-glucose and D
3.12. Platelet aggregation assay
Blood samples were obtained by cardiac puncture from male
Wistar rats weighing 200–250 g, and the blood was anti-coagu-
Fu, Y.L., Yu, Z.Y., Tang, X.M., Zhao, Y., Yuan, X.L., Wang, S.Q., Ma, B.P., Cong, Y.W.,
2008. Pennogenin glycosides with spirostanol structure are strong platelet
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003

8
L.-P. Kang et al. / Phytochemistry xxx (2014) xxx–xxx
agonists: structural requirement for activity and mode of platelet agonist
synergism. J. Thromb. Haemost. 6, 524–533.
Tomova, M.P., Panova, D., Wulfson, N.S., 1974. Steroid saponins and sapogenins IV:
saponins from Tribulus terrestris. Planta Med. 25, 231–237.
Hamed, A.I., Oleszek, W., Stochmal, A., Pizza, C., Piacente, S., 2004. Steroidal
saponins from the aerial parts of Tribulus pentandrus Forssk. Phytochemistry 65,
2935–2945.
Huang, J.W., Tan, C.H., Jiang, S.H., Zhu, D.Y., 2003. Terrestrinins A and B, two new
steroid saponins from Tribulus terrestris. J. Asian Nat. Prod. Res. 5, 285–290.
Inoue, T., Mimaki, Y., Sashida, Y., Nikaido, T., Ohmoto, T., 1995. Steroidal saponins
from the tubers of Dichelostemma multiflorum and their inhibitory activity on
cyclic-AMP phosphodiesterase. Phytochemistry 39, 1103–1110.
Jin, J.M., Zhang, Y.J., Yang, C.R., 2004. Spirostanol and furostanol glycosides from the
fresh tubers of Polianthes tuberosa. J. Nat. Prod. 67, 5–9.
Tschesche, R., Javellana, A.M., Wulff, G., 1974. Steroidsaponine wit mehr als einer
Zuckerkette, IX Purpureagitosid, ein bisdesmosidicsches 22-hydroxyfurostanol-
glycoside aus den blattern von Digitalis purepure L.. Chem. Ber. 107, 2828–2834.
Wang, J., Zu, X.Y., Jiang, Y.Y., 2009. Five furostanol saponins from fruits of Tribulus
terrestris and their cytotoxic activities. Nat. Prod. Res. 2009, 1436–1444.
Wang, Y., Ohtani, K., Kasal, R., Yamasaki, K., 1996. Steroidal saponins from fruits of
Tribulus terrestris. Phytochemistry 42, 1417–1422.
Wang, Y., Ohtani, K., Kasal, R., Yamasaki, K., 1997. Steroida sapoinins from fruits of
Tribulus terrstris. Phytochemistry 45, 811–817.
Wo, T.S., Shi, L.S., Kuo, S.C., 1999. Alkaloids and other constituents from Tribulus
terrestris. Phytochemistry 50, 1411–1415.
Kang, L.P., Zhang, J., Cong, Y., Li, B., Xiong, C.Q., Zhao, Y., Tan, D.W., Yu, H.S., Yu, Z.Y.,
Cong, Y.W., Liu, C., Ma, B.P., 2012. Steroidal glycosides from the rhizomes of
Anemarrhena asphodeloides and their anti-platelet aggregation activities. Planta
Med. 78, 611–616.
Wu, K.L., Kang, L.P., Xiong, C.Q., Zhao, Y., Yu, H.S., Zhang, J., Ma, B.P., 2012. Study on
chemical components of steroidal saponins from Tribulus terrestris L.. J. Tianjin
Univ. Trad. Chin. Med. 31, 225–228.
Kang, L.P., Ma, B.P., Shi, T.J., Zhang, J., Xiong, C.Q., 2006. Two new furostanol
saponins from the rhizomes of Anemarrhena asphodeloides. Acta Pharm. Sin. 41,
527–532.
Xu, T.H., Xu, Y.J., Liu, Y., Xie, S.X., Si, Y.S., Xu, D.M., 2009. Two new furostanol
saponins from Tribulus terrestris L.. Fitoterapia 80, 354–357.
Xu, Y.J., Huang, X.L., Xie, S.X., Xu, T.H., Xu, D.M., Zhang, Y.J., 2007. Isolation and
identification of a new furosteroidal saponin from fruits of Tribulus Terrestris L..
Chem. Res. Chin. Univ. 28, 484–486.
Kostova, I., Dinchev, D., 2005. Saponins in Tribulus terrestris
– chemistry and
bioactivity. Phytochem. Rev. 4, 111–137.
Liu, T., Chen, G., Yi, G.Q., Xu, J.K., Zhang, T.L., Hua, H.M., Pei, Y.H., 2010. New
pregnane and steroidal glycosides from Tribulus terrestris L.. J. Asian Nat. Prod.
Res. 12, 209–214.
Mimaki, Y., Kuroda, M., Kameyama, A., Yokosuka, A., Sashida, Y., 1998. Steroidal
saponins from the rhizomes of Hosta sieboldii and their cytostatic activity on
HL-60 cells. Phytochemistry 48, 1361–1369.
Mahato, S.B., Sahu, N.P., Ganguiy, A.N., Miyahara, K., Kawasaki, T., 1981. Steroidal
glycosides of Tribulus terrestris Linn. J. Chem. Soc., Perkin Trans. 1 (9), 2405–
2410.
Saleh, N.A.M., Ahmed, A.A., Abdalla, M.F., 1982. Flavonoid glycosides of Tribulus
pentandrus and Tribulus terrestris. Phytochemistry 21, 1995–2000.
Su, L., Chen, G., Feng, S.G., Wang, W., Li, Z.F., Chen, H., Liu, Y.X., Pei, Y.H., 2009.
Steroidal saponins from Tribulus terrestris. Steroids 74, 399–403.
Tomova, M.P., Gjulemetova, R., Zarkova, S., Peeva, S., Pangarova, T., Simova, M.,
1981. Steroidal spooning from Tribulus terrestris L. with a stimulating action on
the sexual functions. In: First International Conference on Chemical,
Biotechnological and Biologically Active Natural Products, Proceedings Varna,
September 3, 299–303.
Xu, Y.J., Xu, T.H., Yang, J.Y., Xie, S.X., Liu, Y., Si, Y.S., Xu, D.M., 2010a. Two new
furostanol saponins from Tribulus terrestris L.. Chin. Chem. Lett. 21, 580–583.
Xu, Y.J., Xu, T.H., Zhou, H.Q., Li, B., Xie, S.X., Si, Y.S., Liu, Y., Liu, T.H., Xu, D.M., 2010b.
Two new furostanol saponins from Tribulus terrestris. J. Asian Nat. Prod. Res. 12,
349–354.
Xu, Y.X., Chen, H.S., Liu, W.Y., Gu, Z.B., Liang, H.Q., 1998. Two sapogenins from
Tribulus terrestris. Phytochemistry 49, 199–201.
Yin, H.J., Zhou, D.Y., Jiang, Y.R., Luo, L., Shi, D.Z., 2005. Effect of total saponins of
Tribulus on platelet activation and carotid arterial thrombosis in hyperlidemia
rats. Chin. J. Integr. Med. Cardio-/Cerebrovasc. Dis. 3, 138–140.
Zhou, X.L., He, X.J., Zhou, G.X., Ye, W.C., Yao, X.S., 2006. Steroidal glycosides from
Solanum nigrum. Chin. Tradit. Herbal Drugs 37, 1618–1621.
Zhang, J.Y., Meng, Z.Y., Zhang, M.Y., Ma, D.S., Xu, S.X., Kodama, H., 1999. Effect of six
steroidal saponins isolated from anemarrhenae rhizoma on platelet aggregation
and hemolysis in human blood. Clin. Chim. Acta 289, 79–88.
Zhang, S., Yang, R.J., Li, H., Yin, Z.Y., Zhou, H.Y., Li, X.W., Jin, Y.R., Yang, S.J., 2010.
Separation and bio-activities of spirostanol saponin from Tibulus terrestris.
Chem. Res. Chin. Univ. 26, 915–921.
Please cite this article in press as: Kang, L.-P., et al. Steroidal saponins from Tribulus terrestris. Phytochemistry (2014), http://dx.doi.org/10.1016/
j.phytochem.2014.08.003