Triterpenoid saponins from Gynostemma pentaphyllum

Article abstract of DOI:10.1080/10286020.2010.547029

Four new dammarane-type triterpene saponins, 1-4, were isolated from the aerial parts of Gynostemma pentaphyllum (Thunb.) Makino. Their structural elucidations were accomplished mainly on the basis of spectroscopic methods, such as IR, HR-TOF-MS, and NMR. Compounds 1-4 showed moderate cytotoxic activities against cancer cell lines HL-60, Colon205, and Du145 in vitro.

Full text of DOI:10.1080/10286020.2010.547029

Journal of Asian Natural Products Research
Vol. 13, No. 2, February 2011, 168–177
Triterpenoid saponins from Gynostemma pentaphyllum
Lin Shia, Jia-Qing Caob, Sheng-Ming Shic and Yu-Qing Zhaobd*
b
aCollege of Food Science, Shenyang Agriculture University, Shenyang 110866, China; School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016,
d
China; cTianjin Institute of Pharmaceutical Research, Tianjin 300193, China; Key Laboratory of
Structure-based Drug Design and Discovery of Ministry of Education, Shenyang Pharmaceutical
University, Shenyang 110016, China
(Received 24 June 2010; final version received 7 December 2010)
Four new dammarane-type triterpene saponins, 1–4, were isolated from the aerial parts
of Gynostemma pentaphyllum (Thunb.) Makino. Their structural elucidations were
accomplished mainly on the basis of spectroscopic methods, such as IR, HR-TOF-MS,
and NMR. Compounds 1–4 showed moderate cytotoxic activities against cancer cell
lines HL-60, Colon205, and Du145 in vitro.
Keywords: Gynostemma pentaphyllum (Thunb.) Makino; dammarane-type; triter-
penoid saponin; cytotoxic activities
1. Introduction
dammar-24-ene 3-O-[a-L-rhamnopyranos
yl(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]-
b-D-glucopyranoside (2), (23S)-21R-O-
n-butyl-19-oxo-3b,20j,21-trihydroxy-21,
23-epoxydammar-24-ene 3-O-[a-L-rham-
nopyranosyl(1 ! 2)][b-D-xylopyranosyl
(1 ! 3)]-a-L-arabinopyranoside (3), and
(23S)-21S-O-n-butyl-19-oxo-3b,20j,21-
trihydroxy-21,23-epoxydammar-24-ene 3-
O-[a-L-rhamnopyranosyl(1 ! 2)][b-D-xy
lopyranosyl(1 ! 3)]-a-L-arabinopyrano-
side (4). In this paper, we report the
structural elucidation of the new dammar-
ane-type saponins (Figure 1).
Gynostemma pentaphyllum (Thunb.)
Makino is a herbal medicine with anticancer
activity [1], widely distributed in China,
Korea, and Japan. The biologically active
constituents are dammarane-type glyco-
sides, called gypenosides, which are struc-
turally correlated to the ginseng saponins
[2–5]. In our previous studies on the
anticancer natural medicines, Panax gin-
seng and Panax notoginseng, we have found
some active compounds [6,7]. As a continu-
ation of our work for discovering more
effective components, G. pentaphyllum was
investigated.
2. Results and discussion
From the extract of the aerial parts of
this plant, four new dammarane saponins
were characterized as (23S)-21R-O-n-
butyl-3b,20j,21-trihydroxy-21,23-epoxy-
dammar-24-ene 3-O-[a-L-rhamnopyranos
yl(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]-
b-D-glucopyranoside (1), (23S)-21S-O-n-
butyl-3b,20j,21-trihydroxy-21,23-epoxy-
Compound 1 was obtained as white
amorphous powder with mp 208–2108C
20
and ½aꢀD þ11.9 (c ¼ 0.14, MeOH). Its
molecular formula, C51H86O17, was
deduced from the HR-TOF-MS at m/z
993.5742 [M þ Na]þ. The IR spectrum
(KBr) showed absorption at 3418 cm21
*Corresponding author. Email: zyq4885@126.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2010.547029
http://www.informaworld.com

Journal of Asian Natural Products Research
169
26
27
25
2''''
4''''
O
O
21
23
O
O
24
3''''
19
1''''
18
20
HO17
13
HO
22
16
1
14
OH
5'
OH
O
6'
4'
5
3
30
7
2' O
O
O
O
HO
O
HO
OH
O
O
6
O
4'''
2'''
5'''
O
O
1'''
OH
28
29
3'
1'
1''
HO
HO
5''
HO
HO
3'''
6''
HO
3'' O
HO
2''
4''
HO
HO
OH
OH
1
2
O
O
HO
O
O
HO
OHC
OHC
OH
OH
O
O
O
O
O
O
O
O
O
O
OH
OH
HO
HO
HO
HO
O
O
HO
HO
HO
HO
OH
OH
3
4
Figure 1. Structures of compounds 1–4.
(OH). On acid hydrolysis, it yielded
glucose, xylose, and rhamnose, which
were identified by TLC comparison. The
1H NMR spectrum (Table 1) showed three
anomeric protons at d 4.90 (1H, d,
J ¼ 7.8 Hz, H-10), 5.00 (1H, m, H-1000)],
and 6.46 (1H, br s, H-100), a trisubstituted
olefin signal at d 5.63 (1H, d, J ¼ 8.4 Hz),
and seven methyl proton signals at d 0.75
(3H, s), 0.92 (3H, s), 0.94 (3H, s), 1.17
(3H, s), 1.23 (3H, s), 1.66 (3H, s), and
1.68 (3H, s). The carbon signals assignable
to the sugar moiety and the sapogenin part
in the 13C NMR spectrum (Table 1) were
almost superimposable on those of (23S)-
3b,20j,21j-trihydroxy-21,23-epoxydam-
mar-24-ene 3-O-[a-L-rhamnopyranosyl(1
! 2)][b-D-xylopyranosyl(1 ! 3)]-b-D-
glucopyranoside [8], except for the pre-
sence of the n-butyl group. In HMBC
spectrum, long-range correlations between
H-10000 and C-21, C-20000, and C-30000 could
testify the presentation of n-butoxy group
located at C-21 (Figure 2). The absolute
configuration for C-23 of 1 was deduced to
be S on the basis of the literature [8]. In the
NOESY spectrum, NOESY cross-peaks
were observed between the olefenic proton
signal at d 5.08 (H-21) and the signal at d
5.01 (H-23) (Figure 3). Up to this, the
aglycon part of 1 was determined as (23S)-
21R-O-n-butyl-3b,20j,21-trihydroxy-
21,23-epoxydammar-24-ene.
By gas chromatography (GC) analysis
of the trimethylsilyl ether derivatives of the
component monosaccharides (tR: 8.84,
9.76, and 26.59 min, respectively) and
comparing with the standard monosacchar-
ides under the same condition, together with
the analysis of the coupling constants in 1H
NMR spectrum, it was clear that 1 contained
one unit of b-D-glucose, one of b-D-xylose,
and one of a-L-rhamnose. The linkage sites
and sequences of the three saccharides and
of the aglycon were confirmed by the 2D
NMR experiments. In the HMBC spectrum
(Figure 2), the cross-peaks between H-10 of
the glucose and C-3 of the aglycon, H-100 of

170
L. Shi et al.
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 1 in C5D5N.
Position
dC
dH
1.41 (m), 0.82 (m)
2.27 (m), 1.83 (m)
3.38 (dd, J ¼ 11.8, 3.7 Hz)
HMBC correlations
1
2
3
4
5
6
7
8
39.4
26.6
88.6
39.3
56.3
18.1
35.9
40.3
50.5
36.5
22.1
25.3
41.5
50.5
30.9
27.3
45.7
15.4
16.2
82.7
104.1
43.6
C-10, C-4, C-28, C-29
C-3, C-4, C-9, C-10
0.73 (m)
1.41 (m), 1.39 (m)
1.49 (m), 1.21 (m)
9
1.26 (m)
C-8, C-10
10
11
12
13
14
15
16
17
18
19
20
21
22
1.38 (m), 1.29 (m)
2.10 (m), 1.97 (m)
2.26 (m)
1.72 (m), 1.12 (m)
2.05 (m), 1.38 (m)
2.07 (m)
0.94 (s)
0.75 (s)
C-14
C-7, C-8, C-9, C-14
C-1, C-5, C-9, C-10
5.08 (br s)
C-20, C-23, n-butyl-C-10000
C-20, C-21, C-24
2.43 (dd, J ¼ 7.2, 12.6 Hz),
2.13 (dd, J ¼ 7.4, 12.6 Hz)
5.01 (m)
5.63 (d, J ¼ 8.4 Hz)
23
24
25
26
27
28
29
30
73.4
129.0
133.5
25.4
17.6
27.5
16.5
16.5
104.6
76.6
87.9
69.3
77.8
62.2
101.5
72.2
72.2
73.7
69.5
18.4
104.6
74.5
78.0
70.3
67.0
67.3
32.4
20.0
14.1
C-26, C-27
1.66 (s)
1.68 (s)
1.23 (s)
1.17 (s)
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-13, C-14, C-15
C-300
0.92 (s)
4.90 (d, J ¼ 7.8 Hz)
3-O-glc-10
20
4.24 (m)
4.22 (m)
C-1
C-1000
30
40
4.76 (dd, J ¼ 7.0, 3.0 Hz)
50
3.91 (m)
4.51 (dd, J ¼ 11.7, 1.8 Hz), 4.29 (m)
6.46 (br s)
60
rha(1 ! 2)-100
C-20
C-30
200
4.60 (dd, J ¼ 9.6, 3.6 Hz)
4.81 (dd, J ¼ 9.6, 3.6 Hz)
4.30 (m)
4.13 (s)
1.71 (s)
5.00 (m)
3.95 (m)
4.19 (m)
4.11 (m)
4.24 (m), 3.69 (t, J ¼ 10.2 Hz)
3.96 (m), 3.57 (m)
1.61 (m), 1.58 (m)
1.48 (m), 1.34 (m)
0.81 (t, J ¼ 7.5 Hz)
300
400
500
600
xyl(1 ! 3)-1000
2000
3000
4000
5000
210000-O-n-butyl-10000
C-21, n-butyl-C-20000, 30000
n-butyl-C-10000, 30000, 40000
n-butyl-C-10000, 20000, 40000
n-butyl-C-20000, 30000
20000
3
40000

Journal of Asian Natural Products Research
171
O
O
21
HO
19
OH
3
O
O
HO
O
O
O 1'
OH
HO
HO
O
HO
HO
OH
Figure 2. The structure and key HMBC correlations of compound 1.
the rhamnose and C-20 of the glucose, H-1000
of the xylose and C-30 of the glucose were
displayed.
between them was the absolute configur-
ation of C-21. In the NOESY spectrum, no
cross-peak was observed between the
olefenic proton signal at d 5.26 (H-21)
and the signal at d 5.12 (H-23). Moreover,
comparing with 1, the downfield shifts at
C-20 (Dd þ 1.5), C-21 (Dd þ 4.1), and C-
22 (Dd þ 1.8), and the upfield shift at C-17
(Dd 2 0.8) could testify the difference. On
the basis of the above description, the
absolute configuration at C-21 of 2 was
deduced to be S. Thus, 2 was elucidated as
(23S)-21S-O-n-butyl-3b,20j,21-trihydro
xy-21,23-epoxydammar-24-ene 3-O-[a-L-
rhamnopyranosyl(1 ! 2)][b-D-xylopyra
nosyl(1 ! 3)]-b-D-glucopyranoside.
Thus, the structure of 1 was elucidated
as (23S)-21R-O-n-butyl-3b,20j,21-trihy-
droxy-21,23-epoxydammar-24-ene 3-O-
[a-L-rhamnopyranosyl(1 ! 2)][b-D-xylo
pyranosyl(1 ! 3)]-b-D-glucopyranoside.
Compound 2, a white amorphous
20
powder with mp 205–2078C and ½aꢀD
214.3 (c ¼ 0.49, MeOH), showed an ion
peak at m/z 993.5748 [M þ Na]þ in the
HR-TOF-MS, pointing to the molecular
formula C51H86O17. The IR spectrum
(KBr) showed absorption at 3439 cm21
(OH). On acid hydrolysis, it yielded
glucose, xylose, and rhamnose, which
were identified by TLC comparison.
Comparison of the 1H and 13C NMR
spectra (Table 2) of 1 with those of 2
indicated that they had the same aglycon
and sugar moieties, and the only difference
Compound 3, a white amorphous
20
powder with mp 201–2038C and ½aꢀD
þ21.7 (c ¼ 0.83, MeOH), showed an ion
peak at m/z 977.5448 [M þ Na]þ in the
HR-TOF-MS, pointing to the molecular
formula C50H82O17. The IR spectrum
(KBr) showed absorption at 3438 (OH)
and 1702 (CvO) cm21. On acid hydroly-
sis, it yielded arabinose, xylose, and
rhamnose, which were identified by TLC
comparison. The 1H NMR spectrum
(Table 3) showed three anomeric protons
at d 4.88 (1H, d, J ¼ 5.4 Hz, H-10), 5.00
(1H, m, H-1000)], and 6.15 (1H, br s, H-100),
a trisubstituted olefin signal at d 5.61
O
O
23
21
H
H
H
HO
H
H
Figure 3. Key NOE correlations in
compound 1.

172
L. Shi et al.
Table 2. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 2 in C5D5N.
Position
dC
dH
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
39.7
26.9
88.9
39.7
56.7
18.5
35.8
40.7
51.0
37.1
21.8
24.9
41.4
50.2
31.7
27.0
44.9
15.9
16.6
84.2
108.2
45.4
73.6
129.5
133.1
25.6
18.0
27.9
16.9
16.7
105.1
77.0
88.3
69.9
78.1
62.6
101.8
72.6
72.5
73.9
69.8
18.7
104.9
74.9
78.3
70.7
67.3
66.4
32.4
20.0
14.1
1.44 (m), 0.85 (m)
2.28 (m), 1.85 (m)
3.38 (dd, J ¼ 11.4, 3.5 Hz)
C-10, C-4, C-28, C-29
C-4, C-9, C-10
0.74 (m)
1.49 (m), 1.38 (m)
1.53 (m), 1.22 (m)
1.30 (m)
C-8, C-10
1.51 (m), 1.43 (m)
2.02 (m), 1.89 (m)
2.27 (m)
1.74 (m), 1.13 (m)
1.47–1.50 (m)
2.46 (m)
0.92 (s)
0.74 (s)
C-7, C-8, C-14
C-1, C-5, C-9, C-10
5.26 (br s)
C-22, C-23, n-butyl-C-10000
C-20, C-21, C-24
2.51 (m), 1.96 (m)
5.12 (dt, J ¼ 7.2, 3.6, 11.8 Hz)
5.84 (d, J ¼ 7.2 Hz)
C-26
25
26
27
28
29
30
1.58 (s)
1.65 (s)
1.23 (s)
1.17 (s)
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-13, C-14, C-15
C-3, C-50
1.01 (s)
4.89 (d, J ¼ 7.8 Hz)
3-O-glc-10
20
4.25 (m)
4.18 (m)
C-100, C-30
C-1000, C-20
30
40
4.01 (m)
3.91 (m)
4.51 (m), 4.30 (m)
6.48 (br s)
4.61 (m)
4.82 (m)
4.31 (m)
4.78 (m)
1.68 (d, J ¼ 6.0 Hz)
5.01 (d, J ¼ 7.8 Hz)
3.96 (m)
50
60
rha(1 ! 2)-100
C-20
C-30
200
300
400
500
600
xyl(1 ! 3)-1000
2000
3000
4.08 (m)
4.10 (m)
4000
5000
4.24 (m), 3.71 (m)
3.90 (m), 3.45 (m)
1.63–1.65 (m)
1.48 (m), 1.45 (m)
0.90 (t, J ¼ 7.5 Hz)
210000-O-n-butyl-10000
C-21, n-butyl-C-20000, 30000
n-butyl-C-10000, 30000, 40000
n-butyl-C-10000, 20000, 40000
n-butyl-C-20000, 30000
20000
3
40000

Journal of Asian Natural Products Research
173
Table 3. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 3 in C5D5N.
Position
dC
dH
HMBC correlations
1
2
3
4
33.6 2.61 (m), 0.70 (m)
27.4 2.08 (m), 1.68 (m)
87.2 3.31 (dd, J ¼ 11.8, 3.9 Hz)
40.1
C-10, C-29
5
54.9 1.16 (m)
C-4, C-19, C-28, C-29
6
7
8
17.7 1.86 (m), 1.55 (m)
34.7 1.62 (m), 1.34 (m)
40.4
9
52.8 1.67 (m)
52.8
22.1 1.71 (m), 1.17 (m)
25.6 1.95 (m), 1.97 (m)
41.7 2.16 (m)
C-10, C-19
10
11
12
13
14
15
16
17
18
19
20
21
22
50.6
31.7 1.64 (m), 1.17 (m)
27.3 1.98 (m), 1.31 (m)
46.1 2.05 (m)
17.3 0.84 (s)
205.5 10.26 (s)
82.9
104.4 5.00 (br s)
43.6 2.40 (dd, J ¼ 6.9, 11.7 Hz),
2.12 (dd, J ¼ 7.8, 11.7 Hz)
73.7 4.99 (m)
C-7, C-8, C-9, C-14
C-1, C-9, C-10
C-17, C-20, C-22, C-23, n-butyl-C-10000
C-20, C-21, C-24
23
24
25
26
27
28
29
30
C-17, C-20, C-21, C-22, C-24
C-26, C-27
129.4 5.61 (d, J ¼ 8.3 Hz)
133.9
25.7 1.65 (s)
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-13, C-14, C-15
C-300
17.9 1.67 (s)
26.4 1.28 (s)
16.0 1.07 (s)
16.7 0.96 (s)
104.8 4.88 (d, J ¼ 5.4 Hz)
74.5 4.64 (dd, J ¼ 7.2, 6.0 Hz)
81.4 4.28 (m)
3-O-ara-10
20
C-1
C-1000
30
40
68.4 4.46 (m)
50
65.2 4.27 (m), 3.79 (d, J ¼ 9.6 Hz)
102.1 6.15 (br s)
72.6 4.56 (m)
72.5 4.74 (m)
73.9 4.27 (m)
rha(1 ! 2)-100
C-20
C-30
200
300
400
500
70.1 4.57 (m)
18.6 1.58 (d, J ¼ 6.0 Hz)
600
xyl(1 ! 3)-1000
105.2 5.00 (m)
2000
3000
4000
5000
74.7 3.90 (t, J ¼ 7.8 Hz)
77.8 4.07 (m)
70.9 4.10 (m)
67.0 4.30 (m), 3.64 (t, J ¼ 11.1 Hz)
201000-O-n-butyl-10000 67.7 3.97 (m), 3.56 (m)
C-21, n-butyl-C-20000, 30000
n-butyl-C-10000, 30000, 40000
n-butyl-C-10000, 20000, 40000
n-butyl-C-20000, 30000
20000
3
40000
32.2 1.57 (m), 1.34 (m)
19.7 1.33 (m), 1.36 (m)
14.0 0.80 (t, J ¼ 7.2 Hz)

174
L. Shi et al.
(1H, d, J ¼ 8.3 Hz), and six methyl proton
signals at d 0.84 (3H, s), 0.96 (3H, s), 1.07
(3H, s), 1.28 (3H, s), 1.65 (3H, s), and
1.67 (3H, s). The carbon signals assignable
to the sugar moiety and the sapogenin
part in the 13C NMR spectrum (Table 3)
were superimposable on those of (23S)-
3b,20j,21j-trihydroxy-19-oxo-21,23-epo
xydammar-24-ene 3-O-[a-L-rhamnopyra
nosyl(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]
-b-D-arabinopyranoside [8], except for the
presence of the n-butyl group. The HMBC
correlations between the H-10000 and C-21,
C-20000, and C-30000 could testify the presence
of n-butoxyl group located at C-21 (Figure
4). The absolute configuration at C-23 of 3
was deduced to be S on the basis of the
literature [8]. In addition, comparable to 1,
the absolute configuration at C-21 of 3 was
deduced to be R. Up to this, the aglycon
part of 3 was determined as (23S)-21R-O-
n-butyl-19-oxo-3b,20j,21-trihydroxy-21,
23-epoxydammar-24-ene.
and the sequence of three saccharides and
of the aglycon were confirmed by the 2D
NMR experiments. In the HMBC spectrum
(Figure 4), the cross-peaks between H-10 of
the arabinose and C-3 of the aglycon, H-100
of the rhamnose and C-20 of the arabincose,
H-1000 of the xyloseand C-30 of the arabinose
were displayed. Thus, the structure of 3
was elucidated as (23S)-21R-O-n-butyl-
19-oxo-3b,20j,21-trihydroxy-21,23-epox-
ydammar-24-ene 3-O-[a-L-rhamnopyrano
syl(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]-
a-L-arabinopyranoside.
Compound 4, a white amorphous
20
powder with mp 203–2058C and ½aꢀD
211.0 (c ¼ 0.46, MeOH), showed a
peak at m/z 977.5443 [M þ Na]þ in the
HR-TOF-MS, pointing to the molecular
formula C50H82O17. The IR spectrum
(KBr) showed absorption at 3430 (OH),
and 1707 (CvO) cm21. On acid hydroly-
sis, it yielded arabinose, xylose, and
rhamnose, which were identified by TLC
By GC analysis of the trimethylsilyl
ether derivatives of the component mono-
saccharides (tR: 6.20, 8.84, and 9.76 min,
respectively) and the standard monosac-
charides under the same condition, together
comparison. Comparison of the 1H and 13
C
NMR spectra (Table 4) of 3 with those of 4
indicated that they had the same aglycon
and sugar moieties, and the only difference
between them was the absolute configur-
ation at C-21. Namely, comparable to 3,
the downfield shifts at C-20 (Dd þ 1.2),
C-21 (Dd þ 3.7), and C-22 (Dd þ 1.7),
and the upfield shift at C-17 (Dd 2 1.3)
1
with the coupling constants in H NMR
spectrum, it was clear that 3 contained one
unit of a-L-arabinose, one of b-D-xylose,
and one of a-L-rhamnose. The linkage sites
O
O
HO
CHO
OH
O
O
O
O
O
OH
HO
HO
O
HO
HO
OH
Figure 4. The structure and key HMBC correlations of compound 3.

Journal of Asian Natural Products Research
175
Table 4. 1H (600 MHz) and 13C (150 MHz) NMR spectral data for compound 4 in C5D5N.
Position
dC
dH
HMBC correlations
1
2
3
4
5
6
7
8
33.7
27.6
87.4
40.1
54.9
17.8
34.8
40.5
52.9
52.9
22.3
24.9
41.2
50.1
32.1
27.0
44.8
17.4
205.5
84.1
108.1
45.3
2.64 (m), 0.74 (m)
2.07 (m), 1.68 (m)
3.32 (dd, J ¼ 11.7, 3.9 Hz)
C-10, C-4, C-28, C-29
C-4, C-6, C-10, C-19
1.19 (m)
1.87–1.89 (m)
1.68 (m), 1.37 (m)
9
1.72 (m)
C-8, C-10, C-19
10
11
12
13
14
15
16
17
18
19
20
21
22
1.78 (m), 1.13 (m)
2.02 (m), 1.89 (m)
2.20 (t, J ¼ 11.4 Hz)
1.70 (m), 1.21 (m)
1.80 (m), 1.45 (m)
2.41 (m)
0.85 (s)
10.26 (s)
C-13
C-7, C-8, C-9, C-14
C-1, C-10
5.21 (br s)
C-22, C-23, n-butyl-C-10000
C-20, C-21, C-24
2.47 (dd, J ¼ 9.0, 12.6, 3.6 Hz),
1.94 (dd, J ¼ 9.0, 12.6, 3.6 Hz)
5.10 (m)
5.82 (d, J ¼ 8.4 Hz)
23
24
25
26
27
28
29
30
73.6
129.4
132.9
25.6
18.0
26.4
16.0
16.5
104.8
74.5
81.7
68.4
65.1
102.1
72.6
72.5
73.9
70.1
18.6
105.3
74.6
77.8
70.9
67.0
66.3
32.3
19.9
14.1
C-21
C-26, C-27
1.59 (s)
1.66 (s)
1.21 (s)
1.07 (s)
1.03 (s)
4.87 (d, J ¼ 6.0 Hz)
4.62 (dd, J ¼ 7.2, 6.0 Hz)
4.28 (m)
C-24, C-25, C-27
C-24, C-25, C-26
C-4, C-5, C-29
C-4, C-5, C-28
C-8, C-13, C-14, C-15
C-300
3-O-ara-10
20
C-1
C-1000
30
40
4.30 (m)
4.26 (m), 3.79 (d, J ¼ 9.6 Hz)
50
rha(1 ! 2)-100
6.13 (br s)
4.57 (m)
C-20
C-30
200
300
4.73 (m)
4.25 (m)
4.56 (m)
1.58 (s)
400
500
600
xyl(1 ! 3)-1000
5.00 (d, J ¼ 7.2 Hz)
2000
3.92 (m)
4.11 (m)
4.10 (m)
3000
4000
5000
4.30 (m), 3.64 (t, J ¼ 9.0 Hz)
3.88 (m), 3.42 (m)
1.63 (m), 1.43 (m)
1.41 (m), 1.43 (m)
0.90 (t, J ¼ 7.5 Hz)
210000-O-n-butyl-10000
C-21, n-butyl-C-20000, 30000
n-butyl-C-10000, 30000, 40000
n-butyl-C-10000, 20000, 40000
n-butyl-C-20000, 30000
20000
3
40000

176
L. Shi et al.
could also testify the difference. And the
absolute configuration at C-21 of 4 was
deduced to be S. Thus, 4 was elucidated as
(23S)-21S-O-n-butyl-19-oxo-3b,20j,21-
trihydroxy-21,23-epoxydammar-24-ene 3-
O-[a-L-rhamnopyranosyl(1 ! 2)][b-D-
xylopyranosyl(1 ! 3)]-b-D-arabinopyra-
noside.
3.3 Extraction and isolation
Dried aerial parts of G. pentaphyllum
(Thunb.) Makino (8.0 kg) were extracted
with 75% EtOH ( £ 3), and the water
soluble extract of the 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 repeat-
edly over silica gel with CHCl3–MeOH–
H2O (7:2:1–7:4:1) to provide five
fractions A–E. Fraction D (1.0 g) was
separated into five fractions, Da–De, by
HPLC (ODS, 80% MeOH). Fraction Da
(0.3 g) was then subjected to preparative
RP-HPLC (75% MeOH) to yield 3
(100 mg, tR ¼ 18 min) and 1 (25 mg,
tR ¼ 23 min), 2 (30 mg, tR ¼ 27 min), and
4 (40 mg, tR ¼ 32 min).
3. Experimental
3.1 General experimental procedures
Optical rotations were obtained on Perkin-
Elmer polarimeter. Melting points were
determined with the Electrothermal melt-
ing point apparatus (Beijing Taike X-4
spectrophotometer) and are uncorrected.
UV spectra were measured on Shimadzu
UV-2201 spectrophotometer; MeOH
soln.; in lmax (log e). IR spectra were
recorded on Bruker IFS-55 spectropho-
1
tometer; KBr pellets; n in cm21. H and
3.3.1 (23S)-21R-O-n-Butyl-3b,20j,21-tr
ihydroxy-21,23-epoxydammar-24-ene
3-O-[a-L-rhamnopyranosyl(1 ! 2)][b-D-
xylopyranosyl(1 ! 3)]-b-D-glucopyrano
side (1)
13C NMR spectra were recorded using
Bruker AV-600 and ARX-300 spec-
trometer; d in ppm relative to Me4Si as
internal standard, J in Hz. HR-TOF-MS
were recorded by BIC micro TOF-Q mass
spectrometer; in m/z (rel. %). Prep. GC
were recorded on Agilent technologies
6890N apparatus, OV-17 (30 m £ 0.32
mm) column. HPLC (Beijing CXTH3000
System) was performed through P3000
pump, UV3000 spectrophotometric detec-
tor at 203 nm, Daisogel C18 reversed-phase
column (10 mm, 30 £ 250 mm; flow rate
14.0 ml/min). Column chromatography
was performed on silica gel (SiO2: 200–
300 mesh, Qingdao Marine Chemical
Group, Co., Qingdao, China) and macro-
porous resin D101.
White amorphous powder, Libermann–
Burchard and Molish reactions were
20
positive. MP 208–2108C, ½aꢀD þ11.9
(c ¼ 0.14, MeOH). IR (KBr): nmax 3418,
2937, 1645, 1452, 1384, 1265, 1097, 1046,
811, 613 cm21. For 1H and 13C NMR
spectral data see Table 1. HR-TOF-MS:
m/z 993.5742 [M þ Na]þ (calcd for
C51H86O17Na, 993.5757).
3.3.2 (23S)-21S-O-n-Butyl-3b,20j,21-
trihydroxy-21,23-epoxydammar-24-ene
3-O-[a-L-rhamnopyranosyl(1 ! 2)][b-D-
xylopyranosyl(1 ! 3)]-b-D-glucopyrano
side (2)
3.2 Plant material
The aerial parts of G. pentaphyllum
(Thunb.) Makino were collected in July
2006 from Xi’an city, Shanxi province of
China by Xi’an Tianyi Co. Ltd. A voucher
specimen (No. 2007016) has been depos-
ited at the herbarium of the department.
White amorphous powder, Libermann–
Burchard and Molish reactions were
20
positive. MP 205–2078C, ½aꢀD 214.3
(c ¼ 0.49, MeOH). IR (KBr): nmax 3439,
2933, 1645, 1384, 1044, 616 cm21. For 1H
and 13C NMR spectral data see Table 2.

Journal of Asian Natural Products Research
177
HR-TOF-MS: m/z 993.5748 [M þ Na]þ
above. The aqueous layer was concentrated
to dryness to give a residue which was
dissolved in pyridine (1 ml), and then
hexamethyldisilazane (0.4 ml) and tri-
methylchlorosilane (0.2ml) were added to
the solution to obtain the trimethylsilyl
(TMS) ethers. The mixture was kept at 208C
for 15min, and extracted with H2O (1 ml).
Each aqueous layer was examined by GC
(H2 flame ionization detector, column
temperature: 100–2808C, programmed
increase: 108C/min, carrier gas: N2
(1.5ml/min), injector and detector tempera-
ture: 2808C, injection volume: 1 ml, split
ratio: 10:1). The derivatives of L-arabinose,
D-xylose, L-rhamnose, and D-glucose were
detected. tR: 6.20, 8.84, 9.76, and 26.59 min,
respectively. The standard monosacchar-
ides were subjected to the same operation
and GC analysis under the same condition.
(calcd for C51H86O17Na, 993.5757).
3.3.3 (23S)-21R-O-n-Butyl-19-oxo-3b,
20j,21-trihydroxy-21,23-epoxydammar-
24-ene 3-O-[a-L-rhamnopyranosyl
(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]-a-L-
arabinopyranoside (3)
White amorphous powder, Libermann–
Burchard and Molish reactions were
20
positive. MP 201–2038C, ½aꢀD þ21.7
(c ¼ 0.83, MeOH). IR (KBr): nmax 3438,
2942, 1702, 1651, 1540, 1453, 1384, 1259,
C
1041, 817, 783, 610 cm21. For 1H and 13
NMR spectral data see Table 3. HR-TOF-
MS: m/z 977.5448 [M þ Na]þ (calcd for
C50H82O17Na, 977.5444).
3.3.4 (23S)-21S-O-n-Butyl-19-oxo-3b,
20j,21-trihydroxy-21,23-epoxydammar-
24-ene 3-O-[a-L-rhamnopyranosyl
(1 ! 2)][b-D-xylopyranosyl(1 ! 3)]-a-L-
arabinopyranoside (4)
3.6 Antitumor bioassay
Antitumor activities were evaluated by
MTT assay [9] with Rg3 as the positive
control. Compounds 1–4 showed moder-
ate antitumor activities against HL-60,
Colon205, and Du145.
White amorphous powder, Libermann–
Burchard and Molish reactions were
20
positive. MP 203–2058C, ½aꢀD 211.0
(c ¼ 0.46, MeOH). IR (KBr): nmax 3430,
2929, 1707, 1637, 1384, 1043, 612 cm21
.
For 1H and 13C NMR spectral data see
Table 4. HR-TOF-MS: m/z 977.5443
[M þ Na]þ (calcd for C50H82O17Na,
977.5444).
References
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[2] M. Nagai, K. Izawa, S. Nagumo, N.
Sakurai, and T. Tankino, Chem. Pharm.
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[3] T. Takemoto, S. Arihara, K. Yoshikawa, K.
Hino, T. Nakajima, and M. Okuhira,
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3.4 Acid hydrolysis of 1–4
Each compound (4 mg) was heated in 5 ml
of 2 M HCl–MeOH (4:1) at 908C for 6 h in
a water bath. After cooling, the reaction
mixture was diluted to 20 ml with water
and then extracted with CHCl3
(20 ml £ 3). After concentration, each
aqueous layer was examined by TLC
(CHCl3–MeOH–H2O 55:45:10) and com-
pared with the authentic samples.
[7] W. Wang, Y.Q. Zhao, E.R. Rayburn, D.L.
Hill, H. Wang, and R. Zhang, Cancer
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[9] C. Lammer, S. Wagerer, and R. Saffrich,
J. Cell Sci. 11, 2445 (1998).
3.5 Determination of sugar components
The monosaccharide subunits were
obtained by HCl hydrolysis as described