Two new triterpene saponins from Gynostemma pentaphyllum

Article abstract of DOI:10.1080/10286020.2012.700925

Two new dammarane-type triterpene saponins, gypenbiosides A (1) and B (2), were isolated from the aerial parts of Gynostemma pentaphyllum (Thunb.) Makino. Their structural elucidations were accomplished mainly on the basis of the interrelation of spectroscopic methods, such as IR, HR-TOF-MS, and NMR. The cytotoxic activity was evaluated against one human cancer cell line HL-60 using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Full text of DOI:10.1080/10286020.2012.700925

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Two new triterpene saponins from
Gynostemma pentaphyllum
Lin Shi a b , Fei Lu a , Hong Zhao c & Yu-Qing Zhao b
a College of Food Science, Shenyang Agriculture University,
Shenyang, 110866, China
b School of Traditional Chinese Materia Medica, Shenyang
Pharmaceutical University, Shenyang, 110016, China
c Dalian University, Dalian, 116622, China
Published online: 28 Aug 2012.
To cite this article: Lin Shi , Fei Lu , Hong Zhao & Yu-Qing Zhao (2012): Two new triterpene saponins
from Gynostemma pentaphyllum , Journal of Asian Natural Products Research, 14:9, 856-861
To link to this article: http://dx.doi.org/10.1080/10286020.2012.700925
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Journal of Asian Natural Products Research
Vol. 14, No. 9, September 2012, 856–861
Two new triterpene saponins from Gynostemma pentaphyllum
Lin Shia,b, Fei Lua, Hong Zhaoc and Yu-Qing Zhaob*
aCollege of Food Science, Shenyang Agriculture University, Shenyang 110866, China;
bSchool of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China; cDalian University, Dalian 116622, China
(Received 11 March 2012; final version received 5 June 2012)
Two new dammarane-type triterpene saponins, gypenbiosides A (1) and B (2), were
isolated from the aerial parts of Gynostemma pentaphyllum (Thunb.) Makino. Their
structural elucidations were accomplished mainly on the basis of the interrelation of
spectroscopic methods, such as IR, HR-TOF-MS, and NMR. The cytotoxic activity
was evaluated against one human cancer cell line HL-60 using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide assay.
Keywords: Gynostemma pentaphyllum; dammarane type; gypenbioside A;
gypenbioside B; cytotoxic activity
1. Introduction
2. Results and discussion
‘Jiao-Gu-Lan,’ the dried aerial parts of
Gynostemma pentaphyllum (Thunb.)
Makino (Cucurbitaceae), is a herbal
medicine with anticancer activity [1],
widely distributed in China, Korea, and
Japan, and has been used as a famous
folk medicine in China. The biological
active constituents are dammarane-type
glycosides, called gypenosides, which
are structurally correlated to the ginseng
saponins [2–6]. In our series of studies
on this anticancer natural medicine, we
have found some active compounds [7–
8]. As a continuation of our work for
discovering more effective components
of G. pentaphyllum, two new dammar-
ane saponins, gypenbiosides A (1) and B
(2), were isolated from the extract of the
aerial parts of this plant (Figure 1). In
this paper, we report the structural
elucidation of the two new dammarane-
type saponins as well as their antitumor
activities.
Compound 1, obtained as white amor-
phous powder, showed a pseudo-molecu-
lar ion peak at m/z 789.4410 [M þ Na]þ in
the HR-TOF-MS, pointing to the molecu-
lar formula C41H66O13. The IR spectrum
(KBr) showed peaks at 3427 cm21 (OH)
and 1635 cm21 (CvC). On acid hydroly-
sis, it yielded glucose and xylose which
were identified by thin layer chromatog-
1
raphy (TLC) comparison. The H NMR
spectrum (Table 1) showed two anomeric
protons at d 4.92 (1H, d, J ¼ 7.9 Hz, H-10),
5.28 (1H, d, J ¼ 7.5 Hz, H-100)], an olefinic
proton signal at d 5.58 (1H, d, J ¼ 8.0 Hz),
and seven methyl proton signals at d 0.74
(3H, s), 0.89 (3H, s), 0.92 (3H, s), 0.98
(3H, s), 1.30 (3H, s), 1.60 (3H, s), and 1.68
1
(3H, s). Analysis of the H and 13C NMR
spectra (Table 1) established that 1 was a
triterpene saponin with a 21,23-lactone
skeleton. In the previously published
literature [9], the carbon signals in the
13C NMR spectral data closely resembled
*Corresponding author. Email: linnashi@foxmail.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.700925
http://www.tandfonline.com

Journal of Asian Natural Products Research
857
26
27
25
O
O
O
21
O
24
23
22
HO
13
HO
12
20
17
16
15
11
9
19
10
18
1
2
3
14
30
OH
5'
OH
8
7
6'
4'
O
O
5
O
HO
HO
O
O
6
4
O
O
O
2'
28
29
1'
3'
5''
3''
OH
OH
4''
1''
OH
2''
OH
HO
HO
HO
HO
1
2
Figure 1. Structure of compounds 1 and 2.
to those of (20S,23S)-3b,20-dihydroxy-
dammar-24-en-21-oic acid 21,23-lactone
3-O-[a-L-rhamnopyranosyl(1 ! 2)][b-D-
xylopyranosyl(1 ! 3)]-b-D-glucopyrano-
side, but losing six signals due to L-
rhamnose of the side chain. And the
absolute configuration at C-20 of 1 was
deduced to be S on the basis of the
chemical shifts C-20 at d 79.0 and C-17 at
d 45.8 [4]. 1H–1H NOESY interactions
(Figure 2) and NMR analysis showed that
the chemical environment of Hb-22 and
Ha-22 is similar to each other except for
the impact of OH-C-20, which shifted the
signal of Hb-22 toward upfield. NOESY
cross peaks were observed between H-23
and Hb-22, H-24 and Ha-22, Me-26 and H-
24, Me-27 and H-23, suggesting that H-23
should be located on the b-side and the 2-
methylpropenyl group on the a-side,
which was also supported by the chemical
shifts of C-23 at d 74.1 and C-24 at d 125.4
[9]. Thus, the absolute configuration at C-
23 of 1 was deduced to be S. So, the
aglycon part of 1 was determined as
(20S,23S)-3b,20-dihydroxydammar-24-
en-21-oic acid 21,23-lactone. By GC
analysis of the trimethylsilyl (TMS) ether
derivatives of the component monosac-
charides and comparing with the standard
monosaccharides under the same con-
dition, together with the analysis of the
the linkage sites and sequences of the two
saccharides and the aglycon were con-
firmed by the 2D NMR experiments. In the
HMBC spectrum (Figure 3), the cross
peaks between H-10 of the glucose at d
4.92 and C-3 at d 89.1 of the aglycon, H-100
of the xylose at d 5.28, and C-30 at d 87.8
of the glucose were observed. Thus,
the structure of 1 was deduced as
(20S,23S)-3b,20-dihydroxydammar-24-
en-21-oic acid 21,23-lactone 3-O-[b-D-
xylopyranosyl(1 ! 3)]-b-D-glucopyrano-
side and named gypenbioside A.
Compound 2, a white amorphous
powder, showed a peak at m/z 789.4386
[M þ Na]þ in the HR-TOF-MS, pointing
to the molecular formula C41H66O13. The
IR spectrum (KBr) showed peaks at
3426 cm21 (OH) and 1637 cm21 (CvC).
On acid hydrolysis, it yielded glucose and
xylose which were identified by TLC
comparison. The 1H NMR spectrum
(Table 2) showed two anomeric protons
at d 4.90 (1H, d, J ¼ 7.8 Hz, H-10), 5.26
(1H, d, J ¼ 7.8 Hz, H-100)], an olefinic
proton signal at d 5.41 (1H, d, J ¼ 7.2 Hz),
and seven methyl proton signals at d 0.81
(3H, s), 0.90 (3H, s), 0.99 (3H, s), 1.01
(3H, s), 1.31 (3H, s), 1.63 (3H, s), and 1.68
(3H, s). Analysis of 1H and 13C NMR
spectra established that 2 was a triterpene
saponin with a 21,23-lactone skeleton.
1
1
coupling constants in H NMR spectrum,
it was clear that 1 contained one unit of b-
D-glucose and one unit of b-D-xylose. And
Comparison of H and 13C NMR spectra
(Table 2) of 2 with those of 1 indicated that
they had the same aglycon and sugar

858
L. Shi et al.
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 1 (in C5D5N, J in
Hz).
Position
dC
dH (J in Hz)
HMBC correlations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
39.7
26.7
89.1
39.7
56.3
18.4
35.7
40.8
51.1
37.0
21.8
25.8
43.2
50.7
31.7
27.3
45.8
15.6
16.5
79.0
179.5
40.7
1.44 (1H, m), 0.73 (1H, m)
2.19 (1H, m), 1.80 (1H, m)
3.35 (1H, dd, 12.0, 4.6)
C-10, C-4, C-28, C-29
C-10
0.72 (1H, m)
1.51 (1H, m), 1.37 (1H, m)
1.48 (1H, m), 1.22 (1H, m)
1.24 (1H, m)
1.48 (1H, m), 1.25 (1H, m)
2.01 (1H, m), 1.67 (1H, m)
2.08 (1H, m)
1.60-1.68 (1H, m), 1.06-1.13 (1H, m)
2.41 (1H, m), 1.34 (1H, m)
2.53 (1H, m)
0.92 (3H, s)
0.74 (3H, s)
C-7, C-8, C-9, C-14
C-1, C-5, C-9, C-10
2.70 (1H, dd, 13.1, 6.0), 2.27
(1H, dd, 13.1, 5.9)
5.49 (1H, m)
C-17, C-20, C-21, C-23, C-24
23
24
25
26
27
28
29
30
74.1
125.4
138.5
25.6
18.1
28.0
17.0
16.5
106.6
75.4
87.8
69.6
78.2
62.7
106.4
74.7
78.3
71.0
67.5
C-24, C-25
C-22, C-23, C-26, C-27
5.58 (1H, d, 8.0)
1.60 (3H, s)
1.68 (3H, s)
1.30 (3H, s)
0.98 (3H, s)
0.89 (3H, s)
4.92 (1H, d, 7.8)
4.02 (1H, t, 7.8)
4.21 (1H, m)
4.11 (1H, m)
3.95 (m)
4.55 (1H, d, 10.8), 4.32 (1H, m)
5.28 (1H, d, 7.5)
4.06 (1H, m)
4.17 (1H, m)
4.18 (1H, m)
4.29 (1H, m), 3.69 (1H, t, 10.3)
C-24, C-25, C-27
C-24, C-25, C-26
C-4, C-29
C-4, C-28
C-13, C-14, C-15
C-3, C-50
3-O-glc-10
20
C-100
30
C-100, C-200, C-40
40
50
60
xyl(1 ! 3)-100
C-30
C-100
200
300
400
500
moieties, and the only difference between
them was in the absolute configurations of
C-20 and C-23. Moreover, comparing with
1, the downfield shifts at C-20 (Dd þ 2.2),
C-23 (Dd þ 1.1), and C-25 (Dd þ 0.9) and
the upfield shift at C-21 (Dd 21.1) and C-
22 (Dd 21.6) could also testify the
difference. The carbon signals of the
aglycon part in the 13C NMR spectrum
(Table 2) closely resembled to those
of (20R,23R)-3b,20-dihydroxydammar-
24-en-21-oic acid 21,23-lactone 3-O-[a-
L-rhamnopyranosyl-(1 ! 2)][b-D-xylo-
pyranosyl(1 ! 3)]-b-D-glucopyranoside

Journal of Asian Natural Products Research
859
13C NMR spectra were recorded on Bruker
AV-600 and ARX-300 spectrometer (Zur-
ich, Switzerland). HR-TOF-MS were
measured on BIC micro TOF-Q mass
spectrometer (New York City, NY, USA).
The GC was carried out on Agilent
technologies 6890N apparatus, with OV-
17 (30 m £ 0.32 mm) column (Santa Clara,
CA, USA). Preparative HPLC was carried
out on Beijing CXTH3000 system, P3000
pump, UV3000 spectrophotometric detec-
tor at 203 nm, and Daisogel C18 reversed-
phase column (10 mm, 30 £ 250 mm)
(Beijing Chuangxintongheng Company,
Beijing, China). Column chromatographies
were carried out with silica gel (SiO2: 200–
300 mesh, Qingdao Marine Chemical
Group, Co., Qingdao, China) and macro-
porous resin D101 (Cangzhou Company,
Hebei, China).
O
O
HO
H
H
H
H
Figure 2. Key NOE correlations of
compound 1.
[9]. And the absolute configuration at C-20
of 2 was deduced to be R on the basis of the
chemical shifts C-20 at d 81.2, and C-17 at
d 45.4 [4]. Therefore, the structure of 2
was elucidated as (20R,23R)-3b,20-dihy-
droxydammar-24-en-21-oic acid 21,23-
lactone 3-O-[b-D-xylopyranosyl(1 ! 3)]-
b-D-glucopyranoside and named gypen-
bioside B.
Compounds 1 and 2 were tested for
in vitro cytotoxicity against a human
promyelocytic leukemia cell line (HL-60)
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT)
method [10]. Both of them showed
cytotoxicity against HL-60 cells and their
IC50 values (mg ml21) were 47.12 and
45.50, respectively.
3.2 Plant material
The aerial parts of G. pentaphyllum were
collected from Shaanxi province of China
by Xi’an Tianyi Co. Ltd. A voucher
specimen of the plant (No. 2007016) at
our laboratory was identified by Prof.
Qishi Sun of Shenyang Pharmaceutical
University.
3. Experimental
3.1 General experimental procedures
3.3 Extraction and isolation
IR spectra were recorded on Bruker IFS-55
spectrophotometer (Karlsruhe, Germany).
Optical rotations were measured on Perkin-
Elmer polarimeter (Beijing, China). 1H and
Dried aerial parts of G. pentaphyllum
(8.0 kg) were extracted with 75% EtOH
( £ 3), and the water soluble extract of the
O
O
O
O
23
HO
20
HO
19
OH
OH
O
3
O
HO
HO
O
O
O
O
O
O
3'
1'
OH
OH
1''
OH
OH
HO
HO
HO
HO
1
2
Figure 3. Key HMBC correlations of compounds 1 and 2.

860
L. Shi et al.
Table 2. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 2 in C5D5N (J in Hz).
Position
dC
dH (J in Hz)
HMBC correlations
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
39.8
26.8
89.1
39.8
56.4
18.4
35.7
40.7
51.1
37.1
21.8
26.3
45.0
50.2
31.7
28.0
45.4
15.7
16.5
81.2
178.4
39.1
75.2
124.0
139.4
25.6
18.2
28.0
16.8
16.3
106.5
75.4
87.8
69.9
78.2
62.7
106.4
74.7
78.3
71.0
67.5
1.61 (1H, m), 0.98 (1H, m)
2.23 (1H, m), 1.86 (1H, m)
3.38 (1H, m)
C-10, C-28, C-29
C-4
0.74 (1H, m)
1.50 (1H, m), 1.39 (1H, m)
1.49 (1H, m), 1.23 (1H, m)
1.33 (1H, m)
1.58 (1H, m), 1.30 (1H, m)
2.01 (1H, m), 1.67 (1H, m)
1.85 (1H, m)
1.60 (1H, m), 1.11 (1H, m)
2.53 (1H, d, 11.5), 1.50 (1H, m)
2.70 (1H, m)
0.90 (3H, s)
0.81 (3H, s)
C-8, C-13, C-14
C-1, C-9, C-10
2.60 (1H, m), 2.12 (1H, m)
5.70 (1H, m)
5.41 (1H, d, 7.2)
C-20, C-21
25
26
27
28
29
30
1.68 (3H, s)
1.63 (3H, s)
1.31 (3H, s)
1.01 (3H, s)
0.99 (3H, s)
4.90 (1H, d, 7.8)
4.03 (1H, t, 7.8)
4.22 (1H, m)
4.12 (1H, m)
3.96 (1H, m)
4.54 (1H, dd, 11.9, 2.4), 4.32 (1H, m)
5.26 (1H, d, 7.8)
4.08 (1H, m)
4.17 (1H, m)
4.19 (1H, m)
4.29 (1H, m), 3.70 (1H, t, 9.6)
C-24, C-25, C-27
C-24, C-25, C-26
C-3, C-4, C-5, C-29
C-3, C-4, C-5, C-28
C-8, C-14
C-3
C-10
C-100, C-20
3-O-glc-10
20
30
40
50
60
xyl(1 ! 3)-100
C-30
C-100
200
300
400
500
plant was separated by a macroporous
resin column to get the 70% EtOH eluates
which upon drying afforded the total
saponins (80 g). The total saponins were
chromatographed repeatedly over silica
gel with CHCl3/MeOH/H2O (7:2:1–
7:3:1–7:4:1) to provide five fractions A–
E. Fraction B (0.5 g) was separated into
five fractions, frs CaZCe, by HPLC (80%
MeOH). Fraction Cc (0.07 g) was then
subjected to preparative RP-HPLC (85%
MeOH), with the spectrophotometric
detector at 203 nm, C18 reversed-phase
column (10 mm, 30 £ 250 mm; flow rate
14.0 ml min21), to yield 1 (15 mg, tR
15 min) and 2 (18 mg, tR 20 min).

Journal of Asian Natural Products Research
861
3.3.1 Gypenbioside A ((20S,23S)-3b,20-
dihydroxydammar-24-en-21-oic acid 21,23-
lactone 3-O-[b-D-xylopyranosyl(1 ! 3)]-b-
D-glucopyranoside; 1)
trimethylchlorosilane (0.2 ml) were added
to the solution to obtain the TMS ethers.
The mixture was refluxed at 208C for
15 min, the solvent was dried, then
dissolved in ethyl acetate (1 ml), and
extracted with H2O (1 ml). The
aqueous layer was examined by GC (H2
flame ionization detector, column tem-
perature: 100–2808C, programmed
increase: 108C min21, carrier gas: N2
(1.5 ml min21), injector and detector tem-
perature: 2808C, injection volume: 1 ml,
split ratio: 10:1). The derivatives of D-
xylose and D-glucose were detected at tR
(min): 8.84 and 26.59 min, respectively.
The standard monosaccharides were sub-
jected to the same operation and GC
analysis under the same condition.
White amorphous powder. Libermann–
Burchard and Molish reactions were
28
positive. ½aꢀD 217.5 (c ¼ 0.54, MeOH).
IR (KBr) nmax: 3427, 2943, 1756, 1635,
1388, 1043, and 618 cm21 1H and 13C
.
NMR spectral data (Table 1). HR-TOF-
MS: m/z 789.4410 [M þ Na]þ (calcd for
C41H66O13Na, 789.4396).
3.3.2 Gypenbioside B ((20R,23R)-3b,20-
dihydroxydammar-24-en-21-oic acid 21,23-
lactone 3-O-[b-D-xylopyranosyl(1 ! 3)]-b-
D-glucopyranoside; 2)
White amorphous powder. Libermann–
Burchard and Molish reactions were
28
positive. ½aꢀD þ 31.5 (c ¼ 0.52, MeOH).
3.6 Antitumor bioassay
IR (KBr) nmax: 3427, 2943, 1756, 1636,
1451, 1386, 1080, 1042, 981, and
618 cm21. H and 13C NMR spectral data
Antitumor activity was evaluated by MTT
assay as reported in the literature [10],
with Rg3 as the positive control.
1
(Table 2). HR-TOF-MS: m/z 789.4386
[M þ Na]þ (calcd for C41H66O13Na,
789.4396).
References
[1] W.L. Hsiao, W.C. Shing Tai, Z. Mo, and
P.K. Wu, J. Biotechnol. 1365, 522 (2008).
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K. Hino, T. Nakajima, and M. Okuhira,
Yakugaku Zasshi 104, 1155 (1984).
[4] S. Piacente and C. Pizza, J. Nat. Prod. 58,
512 (1995).
3.4 Acid hydrolysis of 1 and 2
Each compound (2 mg) was heated in
2.5 ml of 1 M HCl/MeOH (4:1) at 908C for
6 h in a water bath. After cooling, the
reaction mixture was diluted to 10 ml with
water and then extracted with CHCl3
(10 ml £ 3). After concentration, the aqu-
eous layer was examined by TLC (CHCl3/-
MeOH/H2O 55:45:10) and compared with
the authentic samples.
[5] F. Yin, L.H. Hu, F.C. Lou, and R.X. Pan,
J. Nat. Prod. 67, 942 (2004).
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1126 (2005).
[10] M.J. Tang, D.D. Shen, Y.C. Hu, S. Gao,
and S.S. Yu, J. Nat. Prod. 67, 1969
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3.5 Determination of sugar components
The monosaccharide subunits were
obtained by HCl hydrolysis as described
above. The aqueous layer was concen-
trated to dryness to give a residue which
was dissolved in pyridine (1 ml), and then
hexamethyl disilazane (0.4 ml) and