Five furostanol saponins from fruits of Tribulus terrestris and their cytotoxic activities

Article abstract of DOI:10.1080/14786410902940990

Two new furostanol saponins, terrestroside A, 3-O-{β-D-xylopyranosyl- (1 → 3)-[ β-D-xylopyranosyl(1 → 2)] β - D-glucopyranosyl(1 → 4)-[α-L-rhamnopyranosyl(1 → 2)] β- D-galactopyranosy}-26- O -β D-glucopyranosyl-L-5a-furost-20(22)-en-(25R)-3,26-diol (1) an

Full text of DOI:10.1080/14786410902940990

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Five furostanol saponins from fruits of
Tribulus terrestris and their cytotoxic
activities
Jue Wang a , Xuyu Zu a & Yuyang Jiang a
a The Key Laboratory of Chemical Biology, Guangdong Province,
Graduate School at Shenzhen, Tsinghua University , Shenzhen,
Guangdong 518055, P.R. China
Published online: 22 Sep 2010.
To cite this article: Jue Wang , Xuyu Zu & Yuyang Jiang (2009) Five furostanol saponins from fruits
of Tribulus terrestris and their cytotoxic activities, Natural Product Research: Formerly Natural
Product Letters, 23:15, 1436-1444, DOI: 10.1080/14786410902940990
To link to this article: http://dx.doi.org/10.1080/14786410902940990
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Natural Product Research
Vol. 23, No. 15, 15 October 2009, 1436–1444
Five furostanol saponins from fruits of Tribulus terrestris and their
cytotoxic activities
*
Jue Wang, Xuyu Zu and Yuyang Jiang
The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen,
Tsinghua University, Shenzhen, Guangdong 518055, P.R. China
(Received 6 April 2009; final version received 21 April 2009)
Two new furostanol saponins, terrestroside A, 3-O-{ꢀ-D-xylopyranosyl-(1 ! 3)-
[ꢀ-D-xylopyranosyl(1 ! 2)]-ꢀ-D-glucopyranosyl(1 ! 4)-[ꢁ-L-rhamnopyranosyl
(1 ! 2)]-ꢀ-D-galactopyranosy}-26-O-ꢀ-D-glucopyranosyl-L-5a-furost-20(22)-en-
(25R)-3ꢀ,26-diol (1) and terrestroside B, 3-O-{ꢀ-D-xylopyran-osyl(1 ! 3)-[ꢀ-D-
xylopyranosyl(1 ! 2)]-ꢀ-D-glucopyranosyl(1! 4)-[ꢁ-L-rhamnopyranosyl-(1! 2)]-
ꢀ-D-galactopyranosy}-26-O-ꢀ-D-glucopyranosyl-5a-furostan-12-one-(25R)-22-
methoxy-3ꢀ,26-diol (2), together with three known compounds, chloromaloside E
(3), terrestrinin B (4) and terrestroneoside A (5) were isolated from the dry fruits
of Tribulus terrestris. Furthermore, the inhibitory effects of the compounds on
tumour cells were evaluated, and compounds 1–5 showed potential anti-tumour
activity.
Keywords: Tribulus terrestris; fruits; furostanol saponins
1. Introduction
Tribulus terrestris L. is an annual creeping herb used in traditional Chinese medicine, and
the fruit of this plant, which is known as ‘Ci Ji Li’, has long been used for the treatment of
eye trouble, oedema, abdominal distention, emission and morbid leucorrhoeas (Jiang,
1977). The steroidal saponins in the fruits of Tribulus terrestris include spirostanol
saponins and furostanol saponins. This article deals with the isolation and structure
elucidation of five furostanol saponins. Terrestrosides A (1) and B (2) were new
compounds.
The crude fractions of T. terrestris were subjected to normal-phase and reversed-phase
silica gel column chromatography and repeated HPLC to afford compounds 1–5
(Figure 1). They can be easily deduced to be furostanol saponins by the colour reaction
with Herlich’s spray reagent on TLC (Kiyosawa et al., 1968). After acid hydrolysis,
compounds 1–5 yielded glucose, xylose, rhamnose and galactose as sugar residues in a
2 : 2 : 1 : 1 ratio. They were analysed by GC-MS using standard aldononitrile peracetates as
authentic samples.
*Corresponding author. Email: jiangyy@sz.tsinghua.edu.cn
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2009 Taylor & Francis
DOI: 10.1080/14786410902940990
http://www.informaworld.com

Natural Product Research
1437
Figure 1. Five compounds isolated from the extract of T. terrestris, in which terrestrosides A (1) and
B (2) are new compounds.
2. Results and discussion
Terrestroside A (1) was obtained as a white amorphous power with negative optical
25
rotation (½ꢁꢀD ꢁ38.2ꢂ) (H2O, c 0.68), and was deduced to be a furostanol saponin by the
red colour produced with Ehrlich’s reagent. In the ESI-MS of 1, its negative and positive
molecular ion peaks were observed at m/z 1311.4 [M ꢁ H]ꢁ and 1335.3 [M þ Na]þ. The
molecular formula C61H100O30 was deduced from HR-TOF-MS and 13C-NMR (Table 1)
data analysis.
The 1H-NMR (pyridine-d5) spectrum of compound 1 displayed six signals of anomeric
protons at ꢂH 4.84 (1H, d, J ¼ 8.1 Hz), 4.82 (1H, d, J ¼ 7.8 Hz), 4.98 (1H, d, J ¼ 7.5 Hz),
5.22 (1H, d, J ¼ 7.8 Hz), 5.41 (1H, d, J ¼ 7.7 Hz), 6.16 (1H, brs), which were characteristic
of saponins with six sugar units. Detailed analysis of the 13C-NMR spectrum revealed that
34 signals were assignable to the sugar moiety, 27 to the aglycone, and two signals in the
olefinic carbon region at ꢂC (103.6, 152.3) could be assigned to C-20 and C-22, respectively.

1438
J. Wang et al.
Table 1. 13C-NMR data for the compounds 1–5 (in pyridine-d5, 100 MHz).
Position
1
2
3
4
5
Position
1
2
3
4
5
3-o-Glc
1
2
3
4
5
6
7
8
9
37.2
29.9
76.9
34.4
44.6
28.9
32.5
35.0
54.4
35.9
21.4
36.6
29.9
76.5
34.1
44.3
28.6
31.6
34.2
55.5
36.3
37.9
36.6
29.9
76.5
34.1
44.4
28.6
31.6
34.3
55.5
36.3
37.9
37.2
29.9
77.0
34.3
44.6
29.0
32.4
35.2
54.4
35.9
21.2
40.2
41.1
56.4
32.4
81.1
63.9
16.7
12.4
40.6
16.4
37.2
29.9
77.0
34.3
44.6
28.6
100
200
300
400
500
600
100.1 100.1 100.1 100.1 100.1
76.5
76.6
81.3
75.0
60.4
76.6
76.6
81.2
75.0
60.3
76.6
76.6
81.2
75.0
60.4
76.6
76.6
81.3
75.0
60.4
76.6
76.6
81.3
75.0
60.4
32.4 Rha
35.2
54.4
35.9
21.1
39.9
41.0
1000
101.9 101.9 101.9 101.9 101.9
2000
3000
4000
5000
6000
72.4
72.6
73.9
69.3
18.4
72.4
72.7
73.9
69.3
18.4
72.4
72.6
73.9
69.3
18.4
72.4
72.6
73.9
69.3
18.4
72.4
72.6
73.9
69.3
18.4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
26-o-Glc
10
39.8 212.7 212.9
43.7
54.7
34.4
84.5
64.6
14.3
12.4
103.6
11.7
55.6
55.7
31.3
79.8
55.5
15.9
11.7
41.0
14.9
55.7
55.8
31.6
79.6
55.0
16.1
11.8
41.2
15.2
56.3 Glc0000
32.0
81.3
64.3
16.4
12.3
40.4
10000
105.3 105.3 105.3 105.3 105.3
2
81.4
87.6
70.4
77.7
62.8
81.4
87.5
70.3
77.7
62.8
81.4
87.6
70.3
77.7
62.8
81.4
87.6
70.4
77.7
62.8
81.4
87.6
70.3
77.7
62.8
30000
40000
50000
60000
16.2 Xyl00000
152.3 112.6 110.7 110.6 112.6
1
105.0 104.9 105.0 105.0 105.0
23.6
31.4
33.4
74.9
17.3
30.7
28.1
34.1
75.1
17.0
37.0
28.3
34.2
75.2
17.4
37.2
28.3
34.2
75.2
17.4
32.4
28.1
34.1
75.1
17.1 Xyl000000
200000
300000
400000
500000
75.0
78.7
70.7
67.3
75.0
78.7
70.6
67.3
75.0
78.7
70.7
67.3
75.0
78.7
70.7
67.3
75.0
78.7
70.7
67.3
1
105.7 105.7 105.7 105.7 105.7
104.8 104.9 104.9 104.9 104.9
2000000
3000000
4000000
5000000
75.7
79.0
70.8
67.6
75.8
79.0
70.8
67.6
75.7
79.0
70.8
67.6
75.7
79.0
70.8
67.6
75.8
79.0
70.8
67.6
20
75.1
78.6
71.7
78.4
62.8
75.1
78.6
71.7
78.5
62.8
75.1
78.5
71.7
78.4
62.8
75.1
78.5
71.6
78.4
62.9
75.1
78.6
71.7
78.5
62.8
30
40
50
60
These assignments can be confirmed through long-range correlations in the hetero-
nuclear multiple bond correlation (HMBC) experiment. Namely, long-range correlations
were observed between the following protons and carbons: the methyl protons at ꢂH 0.69
(3H, s, 18-CH3) showed long-range correlations with the carbons at ꢂC 43.7 (C-13), 54.7
(C-14), 39.8 (C-12) and 64.6 (C-17); the methyl protons at ꢂH 0.84 (3H, s, 19-CH3) showed
long-range correlations with the carbons at ꢂC 35.9 (C-10), 37.2 (C-1), 44.6 (C-5) and 54.4
(C-9); the methyl protons at ꢂH 1.62 (3H, s, 21-CH3) showed long-range correlations with
the carbons at ꢂC 64.6 (C-17), 103.6 (C-20) and 152.3 (C-22); the methyl protons at ꢂ1.01
(3H, d, J ¼ 6.7 Hz, 27-CH3) showed long-range correlations with the carbons at ꢂC 31.4 (C-
24), 33.4 (C-25) and 74.9 (C-26); the methyl protons at ꢂH 1.69 (3H, d, J ¼ 6.1 Hz) were
assigned to 6-CH3 of the ꢁ-L-rhamnose (Figure 2). Thus, its aglycone moiety was deduced
to be a 5a-furost-20(22)-en-3ꢀ-2,6-diol structure.

Natural Product Research
1439
Figure 2. Key HMBC correlations of terrestroside A (1).
The C-25 configuration of the compound 1 was ‘R’, which was determined by
comparison of the 13C-NMR of 26-O-ꢀ-D-glucopyranosyl(25R)-5a-furost-20(22)-en12-
one-3ꢀ,26-diol-3-O-ꢀ-D-galactopyranosyl(1-2)-ꢀ-D-glucopyranosyl(1-4)-ꢀ-D-galacto-pyra-
noside (Tu, Ma, & Li, 2002; Wang & Ohtani, 1997). After acid hydrolysis, glucose, xylose,
rhamnose and galactose as sugar residues in a 2 : 2 : 1 : 1 ratio was identified by GC-MS
analysis with standard aldononitrile peracetates as authentic samples.
Complete assignments of each glycosidic proton system were achieved by analysis of
2D NMR experiments (COSY, TOCSY, HMQC, HMBC and NOESY). Evaluation of
spin–spin couplings and chemical shifts allowed the identification of two ꢀ-D-
glucopyranosyl units, two ꢀ-D-xylopyranosyl units, one ꢀ-D-galactopyranosy unit and
one ꢁ-L-rhamnopyranosyl unit. The linkage of sugar units was established by HMBC
experiment cross peaks between ꢀ-D-Glc (H-10)/C26–O; ꢀ-D-Gal (H-100)/C3–O; ꢁ-L-Rha (H-
1000)/ꢀ-D-Gal (C-200); ꢀ-D-Glc (H-10000)/ꢀ-D-Gal (C-400); ꢀ-D-Xyl (H-100000)/ꢀ-D-Glc (C-20000);
and ꢀ-D-Xyl (H-1000000)/ꢀ-D-Glc (C-30000) (Figure 1). Comparison of spectroscopic data with
that of the structure offered references (Q. Yang & C. Yang, 2000) indicated that they
have the same sugar sequence. Thus, the structure of compound 1 was established to be
3-O-{ꢀ-D-xylopyranosyl(1 ! 3)-[ꢀ-D-xylopyranosyl(1 ! 2)]-ꢀ-D-glucopyranosyl(1 ! 4)-[ꢁ-
L-rhamnopyranosyl(1 ! 2)]-ꢀ-D-galactopyranosy}-26-O-ꢀ-D-glucopyranosyl-5a-furost-20(22)-
en-(25R)-3ꢀ,26-diol.

1440
J. Wang et al.
Table 2. Results of inhibitory effects of compounds 1–5 on tumour cell bioassay
(MTT based) in vitro.
IC50 (ꢃM)
Samples
NCI-H46
SF-268
MCF-7
HepG2
1
2
3
4
5
9.26
4.31
3.11
5.68
7.21
7.53
3.47
3.03
6.14
5.81
6.18
3.98
2.75
4.77
8.31
*
6.66
*
3.59
*
Note: *The compounds showed no effects.
Terrestroside B (2) was obtained as a colourless crystal with negative optical rotation
25
(½ꢁꢀD ꢁ25.2ꢂ) (H2O, c 0.56), and was deduced to be a furostanol saponin by the red colour
produced with Ehrlich’s reagent. In the ESI-MS of 2, its negative and positive molecular
ion peaks were observed at m/z 1357.6 [M ꢁ H]ꢁ and 1381.6 [M þ Na]þ. The molecular
formula C62H102O32 was deduced from HR-TOF-MS and 13C-NMR (Table 1) data
analysis.
Comparing spectroscopic data with that of compound 1, it was found that they share
the same sugar sequence and a similar aglycone. However, there were no signals in the
olefinic carbon region. In the 13C-NMR spectrum of the compound 2, a remarkable
carbonyl signal at ꢂC 212.7 could be assigned to C-12, which was determined by HMBC
experiment.
Using the techniques of 2D NMR (COSY, TOCSY, HMQC, HMBC and NOESY)
and comparing with compound 1, the structure of compound 2 was established to be 3-O-
{ꢀ-D-xylopyranosyl (1 ! 3)-[ꢀ-D-xylopyranosyl (1 ! 2)]-ꢀ-D-glucopyranosyl (1 ! 4)-[ꢁ-L-
rhamnopyranosyl (1 ! 2)]-ꢀ-D-galactopyranosy}-26-O-ꢀ-D-glucopyranosyl-5a-furost-12-
one-(25R)-22-methoxy-3ꢀ, 26-diol.
Compounds 3 (Q. Yang & C. Yang, 2000), 4 (Bedir, Khan, & Walker, 2002; Huang,
Tan, Jiang, & Zhu, 2003) and 5 (Sun, Gao, Tu, Guo, & Zhang, 2002; Xu et al., 2000) were
known compounds, whose structures were elucidated by comparison of their spectral data
with those in the literature.
2.1. Anti-tumour activity
The inhibitory effects of compounds 1–5 were examined on NCI-H460, SF-268, MCF-7
and HepG2 tumour cells. Five triterpenoid saponins (compounds 1–5) all showed potent
anti-tumour activity, and the IC50 values for these compounds are shown in Table 2.
3. Experimental
The following instruments were used to obtain physical data: a YANACO digital
micromelting point apparatus, uncorrected; NMR (ppm, J in Hz): a Bruker AVANCE-
400 NMR; ESI-MS: a Bruker Esquire 2000; HPLC: [Detector: an RID-10A SHIMADZU
refractive index detector; Pump: an LC-8A SHIMADZU liquid chromatograph; Column:
a SHIM-PACK PREP-ODS column (20 ꢃ 250 mm)]. MPLC: an EYELA CERAMIC
[pump VSP-3050; Fraction collector: DC-1200]. CC: an RP-18 (Merck). Resin: D101.
Optical rotation: a Jasco P-1020.

Natural Product Research
1441
3.1. Plant material
The fruit of T. terrestris L. was from the Ji-Lin province of China and identified
by a company in Nanjing. The voucher of this plant material is on file in our laboratory.
3.2. Extraction and isolation
The dried fruits of T. terrestris (1.8 kg) were thrice extracted with 70% EtOH. The
solutions were combined and evaporated to dryness to give a crude extract of 450.6 g. The
extract was then subjected to D101 resin column chromatography eluted gradiently with
H2O/EtOH solutions to give H2O fraction (244.7 g), 20% EtOH fraction (56.2 g), 70%
EtOH fraction (66.7 g) and 90% EtOH fraction (83.4 g), respectively. The 20% EtOH
fraction (56.2 g) was then subjected to silica gel column chromatography (Wako gel C-300,
Wako pure Chemical Industry Ltd., 700 g, 12 ꢃ 17 cm, eluted with CHCl3 : MeOH : H2O
(8 : 2 : 0.2; 7 : 3 : 0.5)) to give nine portions. The fractions were combined on the basis of
TLC with Ehrlich’s spray reagent. The fourth portion was then separated by silica gel
column chromatography and eluted with CHCl3 : MeOH 100 : 0–0 : 100) into fractions 1–7,
and fraction 4 was further purified by HPLC (Senshu pak PEGASIL ODS Al,
10 ꢃ 250 mm, MeOH : H2O (70 : 30), flow rate 4 mL minꢁ1; UV detector at 254 nm) to
give five furostanol saponins: compound 1 (504 mg), compound 2 (10 mg), compound 3
(15.4 mg), compound 4 (15.1 mg) and compound 5 (20.3 mg).
3.3. Structure and identification
25
Compound 1: White power, [m.p.] 215–217ꢂC, ½ꢁꢀD –38.2ꢂ(H2O, c 0.68), [HR-TOF-MS]
1335.6185 [M þ Na þ H]þ. ESI-MS: 1311.4 [M ꢁ H]ꢁ, 1335.3 [M þ Na]þ. For 1H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
25
Compound 2: Colourless crystal, [m.p.] 220–222ꢂC, ½ꢁꢀD –25.2ꢂ (H2O, c 0.56), [HR-TOF-
MS] 1381.6250 [M þ Na]þ, ESI-MS: 1357.6 [MꢁH]ꢁ, 1381.6 [M þ Na]þ. For 1H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
25
Compound 3: White power, [m.p.] 214–216ꢂC, ½ꢁꢀD ꢁ29.6ꢂ (H2O, c 0.28), [HR-TOF-MS]
1
1367.6095 [M þ Na]þ, ESI-MS: 1343.6 [M ꢁ H]ꢁ, 1367.5 [M þ Na]þ. For H and 13C-
NMR (pyridine-d5) data, see Tables 1 and 3, respectively.
25
Compound 4: White power, [m.p.] 230–232ꢂC, ½ꢁꢀD –39.3ꢂ (H2O, c 0.50), ESI-MS: 1329.6
[M ꢁ H]ꢁ, 1353.6 [M þ Na]þ. For 1H and 13C-NMR (pyridine-d5) data, see Tables 1 and 3,
respectively.
25
Compound 5: Colourless crystal, [m.p.] 225ꢁ227ꢂC, ½ꢁꢀD –32.5ꢂ (H2O, c 0.48), ESI-
MS: 1343.8 [MꢁH]ꢁ, 1367.7 [M þ Na]+. For H and 13C-NMR (pyridine-d5) data, see
1
Tables 1 and 3, respectively.
3.4. Acid hydrolysis and GC-MS
Each glycoside (5 mg) was heated in a sealed tube with 5mL of aqueous 2 M HCl at 100ꢂC
for 2 h. The aglycone was extracted with dichloromethane, and the aqueous residue was
evaporated under reduced pressure. Then, 1 mL pyridine and 2 mg NH2OH ꢄ HCl were
added to the dry residue, and the mixtures were heated at 100ꢂC for 1 h. After the reaction

1442
J. Wang et al.
Table 3. 1H-NMR data for the compounds 1–5 (in pyridine-d5, 400 MHz).
Position
1
2
3
4
5
1
2
3
0.77, 1.54
1.72, 2.00
3.90
0.77, 1.33
1.74, 2.00
4.11
0.70, 1.33
1.71, 1.95
4.11
0.72, 1.54
1.74, 2.00
3.90
0.72, 1.54
1.74, 2.00
3.89
4
5
6
1.63, 1.87
0.89
1.15
1.63, 1.89
0.84
1.14
1.64, 1.89
0.84
1.14
1.64, 1.89
0.90
1.13
1.64, 1.89
0.89
1.14
7
8
9
0.79, 1.50
1.36
0.49
0.71, 1.52
1.74
0.86
0.71, 1.52
1.74
0.86
0.78, 1.50
1.40
0.50
0.79, 1.50
1.40
0.49
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
26-o-Glc
100
/
/
/
/
/
1.22, 1.42
1.04, 1.70
/
2.23, 2.37
/
/
1.33
1.53, 2.00
4.37
2.88
1.03
0.85
2.18
1.41
/
2.03
1.32, 1.77
1.91
3.58, 3.91
0.98
2.25, 2.40
/
/
1.36
1.53, 2.05
4.84
2.88
1.11
0.86
2.18
1.54
/
2.03
1.61, 2.03
1.91
3.60, 3.91
0.98
1.22, 1.39
1.68, 1.04
/
1.17, 1.38
1.00, 1.63
/
0.79
1.01
0.98
1.32, 1.94
4.78
2.42
0.69
0.84
/
1.62
/
2.21
1.80, 1.42
1.93
3.60, 3.92
1.01
1.39, 2.00
4.90
1.93
0.85
0.84
2.21
1.31
/
0.78
1.77, 2.01
1.91
3.60, 3.91
0.97
1.32, 1.94
4.43
1.72
0.78
0.84
2.19
1.18
/
0.78
1.32, 1.77
1.89
3.58, 3.91
0.99
4.82
3.99
4.20
4.18
3.92
4.35, 4.52
4.83
3.99
4.21
4.21
3.92
4.35, 4.52
4.80
3.99
4.20
4.20
3.92
4.35, 4.52
4.79
3.99
4.20
4.18
3.92
4.35, 4.52
4.84
3.99
4.20
4.18
3.92
4.35, 4.52
200
300
400
500
600
3-o-Glc
100
4.84
4.44
4.11
4.46
3.92
4.16, 4.66
4.82
4.44
4.11
4.46
3.92
4.16, 4.66
4.81
4.44
4.11
4.46
3.92
4.19, 4.68
4.83
4.44
4.11
4.46
3.92
4.19, 4.68
4.84
4.44
4.11
4.46
3.92
4.19, 4.68
200
300
400
500
600
Rha
10000
20000
30000
40000
50000
60000
Glc
10000
20000
6.16
4.71
4.50
4.18
4.89
1.69
6.18
4.72
4.52
4.20
4.87
1.69
6.17
4.73
4.51
4.20
4.87
1.69
6.16
4.73
4.53
4.20
4.87
1.69
6.18
4.71
4.50
4.18
4.87
1.71
4.98
4.23
4.98
4.23
4.98
4.23
4.98
4.23
4.99
4.23
(Continued )

Natural Product Research
1443
Table 3. Continued.
Position
1
2
3
4
5
30000
40000
4.04
3.77
3.80
3.99, 4.48
4.05
3.81
3.81
3.99, 4.48
4.05
3.80
3.80
4.05
3.80
3.80
4.04
3.80
3.80
50000
60000
3.99, 4.48
3.99, 4.48
3.99, 4.48
Xyl000000
1
5.22
3.92
4.04
4.07
5.23
3.92
4.04
4.07
5.22
3.92
4.04
4.07
5.22
3.92
4.04
4.07
5.23
3.92
4.04
4.07
2000000
3000000
4000000
5000000
Xyl000000
1
2000000
3000000
4000000
5000000
3.64, 4.19
3.64, 4.19
3.64, 4.19
3.64, 4.19
3.64, 4.19
5.41
3.39
4.03
4.45
5.42
3.39
4.03
4.45
5.42
3.39
4.03
4.45
5.40
3.39
4.03
4.45
5.42
3.39
4.03
4.45
3.46, 4.73
3.46, 4.73
3.46, 4.73
3.46, 4.73
3.46, 4.73
mixtures were cooled, 1.5 mL of Ac2O was added and the mixtures were heated at 100C for
1 h. The reaction mixtures were evaporated under reduced pressure, and the resulting
aldononitrile peracetates were analysed by GC-MS using standard aldononitrile peracetates
as authentic samples. Glucose, xylose, rhamnose and galactose were identified for
compounds 1 and 2 in a 2 : 2 : 1 : 1 ratio.
3.5. Inhibition on NCI-H460, SF-268, MCF-7 and HepG2 tumour cell bioassay
The inhibitory effects of compounds on tumour cells were measured using the
3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay method
(Mosmann, 1983).
Acknowledgements
The authors are grateful to the Shenzhen Traditional Chinese Medicine and Natural Product
Institute for the use of NMR and ESI-MS, and to the Shanghai Institute of Materia Medica, Chinese
Academy of Sciences for the use of HR-TOF-MS. The authors would also like to thank the financial
support from the Ministry of Science and Technology of China (2007AA02Z160), and the Chinese
National Natural Science Foundation (20872077, 90813013).
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