Gynostemosides A-E, megastigmane glycosides from Gynostemma pentaphyllum

Article abstract of DOI:10.1016/j.phytochem.2009.12.017

Megastigmane glycosides (1-5) together with seven (6-12) related known compounds were isolated from the whole plants of Gynostemma pentaphyllum. The structures were elucidated by means of spectroscopic methods, including 2D NMR, HR-ESIMS, and circular dichroism (CD), as well as chemical transformations to be (3R, 4R, 5S, 6S, 7E)-3,4,6-trihydroxymegastigmane-7-en-9-one-3-O-β-d-glucopyranoside (gynostemoside A, 1), (3S, 4S, 5R, 6R, 7E, 9R)-3,4,6,9-tetrahydroxymegastigmane-7-en-3-O-β-d-glucopyranoside (gynostemoside B, 2), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-9-O-β-d-glucopyranoside (gynostemoside C, 3), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-3-O-β-d-glucopyranoside (gynostemoside D, 4), and (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-4-O-β-d-glucopyranoside (gynostemoside E, 5), respectively.

Full text of DOI:10.1016/j.phytochem.2009.12.017

Phytochemistry 71 (2010) 693–700
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Gynostemosides A–E, megastigmane glycosides from Gynostemma pentaphyllum
Zhen Zhang a, Wei Zhang b, Yan-Ping Ji a, Yun Zhao a, Chuan-Gui Wang b, Jin-Feng Hu a,
*
a Department of Natural Products for Chemical Genetic Research, Key Laboratory of Brain Functional Genomics, Ministry of Education and Shanghai Key
Laboratory of Brain Functional Genomics (MOE&SBFG), East China Normal University, Shanghai 200062, PR China
b Institute of Biomedical Sciences, School of Life Science, East China Normal University, Shanghai 200241, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2009
Received in revised form 12 October 2009
Available online 25 January 2010
Megastigmane glycosides (1–5) together with seven (6–12) related known compounds were isolated from
the whole plants of Gynostemma pentaphyllum. The structures were elucidated by means of spectroscopic
methods, including 2D NMR, HR-ESIMS, and circular dichroism (CD), as well as chemical transformations
to be (3R, 4R, 5S, 6S, 7E)-3,4,6-trihydroxymegastigmane-7-en-9-one-3-O-b-
D-glucopyranoside (gynost-
emoside A, 1), (3S, 4S, 5R, 6R, 7E, 9R)-3,4,6,9-tetrahydroxymegastigmane-7-en-3-O-b-
(gynostemoside B, 2), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-9-O-b-
(gynostemoside C, 3), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-3-O-b-
(gynostemoside D, 4), and (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-4-O-b-
D
-glucopyranoside
-glucopyranoside
-glucopyranoside
Keywords:
Gynostemma pentaphyllum
Cucurbitaceae
Megastigmane glycosides
Absolute configuration
D
D
D-glucopyran-
oside (gynostemoside E, 5), respectively.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The whole plant of Gynostemma pentaphyllum (Thurb.) Makino
(Cucurbitaceae), named ‘‘Jiao-Gu-Lan” in Chinese, is used for a pop-
ular traditional Chinese medicine (TCM) as a cough suppressant,
diuretic, antipyretic and tonic (Jiangsu New Medicine College,
1977). The plant is widely distributed in Southern China in warm
and humid environments. Earlier phytochemical investigations
established that it contains dammarane-type glycosides, including
the protopanaxadiol-type ginsenosides (i.e., ginsenosides Rb1, Rb3,
Rc, Rd, F2, Rg3) previously isolated from Panax ginseng (Araliaceae)
(Razmovski-Naumovski et al., 2005; Hu et al., 1996). Modern phar-
macological studies also showed that G. pentaphyllum has a variety
of bioactivities similar to P. ginseng (Rujjanawate et al., 2004; Nor-
berg et al., 2004). Therefore, ‘‘Jiao-Gu-Lan” earned its favorable
name of ‘‘Southern Ginseng” (Liu et al., 2004a). During a reinvesti-
gation focusing on the constituents rather than dammarane-type
saponins from the ethanol extract of G. pentaphyllum, and in a con-
tinuation of our work with the discovery of novel anticancer agents
from plants (Wu et al., 2009, 2010), five new (1–5) and two known
(6, 7) megastigmane glycosides, together with five known meg-
astigmanes (8–12) were obtained (Fig. 1). The cytotoxicity of the
isolated compounds against a small panel of human tumor cell
lines (A549, H460, U251, U2OS, and MCF-7) was investigated. In
this paper, we report the isolation and absolute structure elucida-
tion of the new compounds and the cytotoxicity assay results.
2.1. Extraction and separation of megastigmane metabolites
The dried whole plants of G. pentaphyllum were extracted with
95% EtOH at room temperature. After evaporation of the solvent,
the aqueous residue was diluted with H2O and exhaustively ex-
tracted with n-BuOH. Evaporation of the solvent from the n-BuOH
layer under reduced pressure yielded a brown residue (77.8 g),
which was successively subjected to column chromatography
(CC) over silica gel, MCI gel and Sephadex LH-20, respectively. Fi-
nally, semi-preparative HPLC afforded compounds 1–12 (Fig. 1).
Based on the spectroscopic analysis and comparison with the liter-
ature, the known compounds were identified as citroside A (6)
(Umehara et al., 1988; Osorio et al., 1999), citroside B (7) (Umehara
et al., 1988), (3S, 4S, 5S, 6S, 9R)-3,4-dihydroxy-5,6-dihydro-b-ionol
(8) (Rodríguez et al., 1992; Pérez et al., 1996a), (3S, 5R, 6R,
7E, 9S)-3,5,6,9-tetrahydroxy-7-en-megastigmane
(9)
(Takeda
et al., 2000), (3S, 4S, 5R, 6R)*-3,4,6-trihydroxy-5,6-dihydro-b-ionol
(10) (Pérez et al., 1996b), (E)-4-(r-10,t-20,c-40-trihydroxy-20,60,60-
trimethylcyclohexyl)but-3-en-2-one (11) (Tan et al., 1989), and
40-dihydrophaseic acid (12) (Walton et al., 1973; Zaharia et al.,
2005).
2.2. Structure elucidation of compounds 1–5
The molecular formula of compound 1 was determined to be
C19H32O9 based on a pseudo-molecular ion peak at m/z 427.1974
[M+Na]+ in its positive mode HR-ESIMS. A strong absorption
* Corresponding author. Tel.: +86 21 62237510; fax: +86 21 62606791.
E-mail address: jfhu@brain.ecnu.edu.cn (J.-F. Hu).
(
mmax)
at 1641 cmꢀ1 in its IR spectrum together with the
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.12.017

694
Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
OR4
12
11
O
7
1
9
2
3
6
5
8
R3
10
OH
4
R1O
GluO
13
OR2
OH
1
R1
2: R1 = Glu, R2 = R4 = H, R3 = OH
3: R1 = R2 = R3 = H, R4 = Glu
4: R1 = Glu, R2 = R3 = R4 = H
5: R1 = R3 = R4 = H, R2 = Glu
8: R1 = R2 = R3 = R4 = H
R2
10: R1 = R2 = R4 = H, R3 = OH
6: R1 = H, R2 = Ac
7: R1 = Ac, R2 = H
HO
15
OGlu
13
12
6
R
7
9
1
10
2
3
8
O
OH
OH
COOH
4
5
11
HO
HO
14
OH
12
9: R = β-OH, α-H
11: R = O
Fig. 1. Isolated compounds 1–12.
maximum absorption (kmax) at 232 nm in its UV (MeOH) spec-
trum indicated a conjugated enone moiety in the structure of 1.
The 13C and DEPT NMR spectra (Table 1) of 1 showed, in addition
to the six signals attributed to a glucopyranosyl moiety, 13 car-
bon signals classified as four methyls, one methylene, five
methines, and three quaternary carbons. Comparison of these
data with those of the known compounds 6–11 suggested that
1 was a megastigmane glucoside.
The 1H NMR spectrum (Table 2) of 1 possessed signals of three
methyl groups singlet at d 2.26 (3H, s, Me-10), 1.37 (3H, s, Me-11),
and 1.04 (3H, s, Me-12), one methyl group doublet at d 1.21 (3H, d,
J = 7.0 Hz, Me-13), two oxymethines at d 4.57 (1H, br d, J = 2.2 Hz,
H-3) and 4.45 (1H, br s, H-4), an anomeric proton at d 4.96 (1H,
d, J = 7.8 Hz, H-10), and a pair of protons at d 6.87 (1H, d,
J = 15.7 Hz, H-7) and 6.82 (1H, d, J = 15.7 Hz, H-8) in a trans-disub-
stituted double bond conjugated with a ketone in the side-chain.
Table 1
13C (125 MHz) NMR spectroscopic data of 1–5.a
1 (DEPT)b
2 (DEPT)b
3 (DEPT)b
3 (DEPT)c
4 (DEPT)b
5 (DEPT)b
1
38.8 (C)
38.5 (C)
33.5 (C)
34.1 (C)
33.3 (C)
33.1 (C)
2
3
4
5
6
7
8
9
10
11
12
13
10
20
30
40
50
60
36.5 (CH2)
77.5 (CH)
72.2 (CH)
31.6 (CH)
80.0 (C)
36.8 (CH2)
78.1 (CH)
72.9 (CH)
32.1 (CH)
79.7 (C)
41.9 (CH2)
71.6 (CH)
74.7 (CH)
31.0 (CH)
50.4 (CH)
132.6 (CH)
134.8 (CH)
76.1 (CH)
21.1 (CH3)
32.6 (CH3)
23.9 (CH3)
17.6 (CH3)
102.2 (CH)
75.2 (CH)
78.3 (CH)
71.2 (CH)
78.0 (CH)
62.3 (CH2)
42.0 (CH2)
71.4 (CH)
72.4 (CH)
31.6 (CH)
51.2 (CH)
133.7 (CH)
135.7 (CH)
77.9 (CH)
21.4 (CH3)
32.9 (CH3)
24.0 (CH3)
17.4 (CH3)
102.2 (CH)
75.4 (CH)
78.1 (CH)
71.3 (CH)
78.0 (CH)
62.5 (CH2)
40.5 (CH2)
78.3 (CH)
70.8 (CH)
31.1 (CH)
50.1 (CH)
129.7 (CH)
138.1 (CH)
67.8 (CH)
24.5 (CH3)
32.1 (CH3)
23.3 (CH3)
17.5 (CH3)
103.3 (CH)
75.1 (CH)
78.5 (CH)
71.4 (CH)
78.1 (CH)
62.5 (CH2)
41.9 (CH2)
67.3 (CH)
81.3 (CH)
30.1 (CH)
50.8 (CH)
129.6 (CH)
138.2 (CH)
67.9 (CH)
24.5 (CH3)
31.9 (CH3)
23.5 (CH3)
17.3 (CH3)
102.9 (CH)
74.8 (CH)
78.4 (CH)
71.7 (CH)
78.0 (CH)
62.7 (CH2)
150.8 (CH)
130.6 (CH)
197.3 (C)
27.1 (CH3)
26.6 (CH3)
26.3 (CH3)
13.2 (CH3)
103.3 (CH)
75.0 (CH)
78.4 (CH)
71.3 (CH)
78.3 (CH)
62.5 (CH2)
131.3 (CH)
135.5 (CH)
67.7 (CH)
24.6 (CH3)
26.4 (CH3)
26.7 (CH3)
13.3 (CH3)
103.4 (CH)
75.0 (CH)
78.5 (CH)
71.4 (CH)
78.2 (CH)
62.5 (CH2)
a
b
c
Assignments were made by a combination of 1D and 2D NMR techniques (COSY, HSQC and HMBC).
Recorded in C5D5N.
Recorded in CD3OD.

Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
695
Table 2
1H (500 MHz) NMR spectroscopic data of 1–5 (in C5D5N).a
1
2
3
4
5
d (mult, J, Hz)
d (mult, J, Hz)
d (mult, J, Hz)
d (mult, J, Hz)
d (mult, J, Hz)
H-2ax
H-2eq
H-3eq
H-4eq
H-5ax
H-6
H-7
H-8
H-9
H-10
H-11
H-12
H-13
10
2.41 (1H, dd, 14.0, 2.2)
1.76 (1H, br d, 14.0)
4.57 (1H, br d, 2.2)
4.45 (1H, br s)
2.49 (1H, dd, 14.1. 2.0)
1.80 (1H, br d, 14.1)
4.59 (1H, br d, 2.0)
4.45 (1H, br s)
2.20 (1H, dd, 14.0, 2.5)
1.80 (1H, br d, 14.0)
4.48 (1H, br d, 2.5)
4.18 (1H, br s)
2.16 (1H, dd, 14.0, 2.5)
1.87 (1H, br d, 14.0)
4.53 (1H, br d, 2.5)
4.36 (1H, br s)
2.24 (1H, br d, 13.8)
1.78 (1H, br d, 13.8)
4.68 (1H, br s)
4.32 (1H, br s)
2.51 (1H, m)
2.27 (1H, dd, 11.5, 9.1)
5.64 (1H, dd, 15.5, 9.1)
5.71 (1H, dd, 15.5, 6.0)
4.58 (1H, dq, 6.5, 6.0)
1.45 (3H, d, 6.5)
0.82 (3H, s)
1.35 (3H, s)
1.24 (3H, d, 6.5)
4.92 (1H, d, 7.5)
4.04 (1H, dd, 8.9, 7.5)
4.19 (1H, dd, 9.1, 8.9)
4.22 (1H, dd, 9.1, 9.0)
3.84 (1H, m)
4.52 (1H, br d, 11.2)
4.38 (1H, dd, 11.2, 5.5)
2.73 (1H, dq, 7.0, 2.0)
2.69 (1H, br q, 6.8)
2.45 (1H, m)
2.42 (1H, m)
2.43 (1H, dd, 11.3, 9.3)
5.63 (1H, dd, 15.7, 9.3)
5.83 (1H, dd, 15.7, 6.7)
4.71 (1H, dq, 6.7, 6.5)
1.44 (3H, d, 6.5)
1.05 (3H, s)
1.39 (3H, s)
1.21 (3H, d, 5.5)
5.01 (1H, d, 7.5)
4.04 (1H, dd, 9.1, 7.5)
4.23 (1H, dd, 9.1, 8.9)
4.26 (1H, dd, 9.2, 8.9)
3.93 (1H, m)
4.50 (1H, dd, 11.5, 1.8)
4.38 (1H, dd, 11.5, 5.8)
2.39 (1H, dd, 11.5, 9.0)
5.56 (1H, dd, 15.6, 9.0)
5.80 (1H, dd, 15.6, 6.2)
4.59 (1H, dq, 6.5, 6.2)
1.46 (3H, d, 6.5)
0.94 (3H, s)
1.24 (3H, s)
1.19 (3H, d, 6.2)
4.98 (1H, d, 7.8)
4.04 (1H, dd, 8.9, 7.8)
4.21 (1H, dd, 9.2, 8.9)
4.23 (1H, dd, 9.2, 9.0)
3.86 (1H, m)
4.50 (1H, dd, 11.3, 1.8)
4.39 (1H, dd, 11.3, 5.8)
6.87 (1H, d, 15.7)
6.82 (1H, d, 15.7)
5.80 (1H, d, 15.5)
6.36 (1H, dd, 15.5, 6.1)
4.69 (1H, dq, 7.0, 6.1)
1.48 (3H, d, 7.0)
1.14 (3H, s)
2.26 (3H, s)
1.37 (3H, s)
1.04 (3H, s)
1.38 (3H, s)
1.21 (3H, d, 7.0)
4.96 (1H, d, 7.8)
4.07 (1H, dd, 8.9, 7.8)
4.21 (1H, dd, 9.2, 8.9)
4.26 (1H, dd, 9.2, 9.0)
3.89 (1H, m)
4.54 (1H, br d, 11.4)
4.40 (1H, dd, 11.4, 5.5)
5.61 (s)
1.31 (3H, d, 6.8)
4.97 (1H, d, 7.8)
4.03 (1H, dd, 9.0, 7.8)
4.21 (1H, dd, 9.0, 8.8)
4.25 (1H, dd, 9.1, 8.8)
3.88 (1H, m)
4.52 (1H, br d, 11.5)
4.39 (1H, dd, 11.5, 5.6)
5.60 (s)
20
30
40
50
60a
60b
OH-6
a
Assignments were made by a combination of 1D and 2D NMR techniques (COSY, NOESY, HSQC and HMBC).
These data demonstrated that 1 has features very similar to
The molecular weight of compound 2 and its chemical formula
of C19H34O9 were deduced from its positive mode HR-ESIMS, which
resulted in a [M+Na]+ ion peak at m/z 429.2064. Its 1H and 13C NMR
spectroscopic data (Tables 1 and 2) showed that 2 is an analogue of
1, with an additional oxymethine at d 4.69 (1H, dq, J = 7.0, 6.1 Hz,
H-9, dC: 67.7) in place of the ketone carbonyl at C-9 (dC: 197.3) in
the side-chain of 1. This was supported by the absence of the IR
those of lasianthionoside A, a megastigmane glucoside previously
isolated from the leaves of Lasianthus fordii (Rubiaceae) (Takeda
et al., 2004). Its aglycone as 3,4,6-trihydroxy-megastigmane-7-
en-9-one was determined by detailed 1D and 2D NMR (COSY,
HSQC and HMBC) spectroscopic analyses. In the COSY NMR spec-
trum of 1, a spin system of –CH2–CH(O)–CH(O)–CH(CH3)– was
found between: H2-2 and H-3; H-3 and H-4; H-4 and H-5; and
H-5 and Me-13. The chemical shifts of the corresponding carbons
were subsequently deduced through an HSQC NMR experiment
to be d 36.5 (C-2), 77.5 (C-3), 72.2 (C-4), 31.6 (C-5), and 13.2 (C-
13), respectively. In the HMBC spectrum, the anomeric proton at
d 4.96 was found to have a clear 3J correlation with the oxymethine
carbon at d 77.5, which enabled us to assign C-3 as the glycosidic
linkage position, implying a free secondary hydroxyl group located
at C-4 and a free tertiary hydroxyl group bonded to C-6 (d 80.0).
The b-glycosidic linkage was determined based on the observed
coupling constant (7.8 Hz) of the anomeric proton. Notably, the
proton chemical shift of the tertiary hydroxyl group at C-6 can
be unambiguously assigned at d 5.61 (1H, s) through the HMBC
correlations (2J, 3J) between this exchangeable proton (OH-6) and
C-5 (d 31.6), C-6 (d 80.0), and C-7 (d 150.8).
The relative stereochemistry at C-3, C-4, C-5, and C-6 was
characterized through extensive analyses of the coupling patterns
of the protons bonded to the cyclohexane ring and the NOE cor-
relations in the NOESY NMR experiment. The small coupling con-
stants (Table 2) found for H-3 and H-4 indicated both protons
have an equatorial orientation. In the NOESY NMR spectrum, clear
NOE correlations (Fig. 2) were observed between: H-2ax at d 2.41
and Me-12 at d 1.04; H-2eq at d 1.76 and Me-11 at d 1.37; Me-11
and H-5 at d 2.73; H-2ax and OH-6 at d 5.61; as well as Me-13 at d
1.21 and OH-6. Similar to the known compound (3S, 5R, 6S, 7E)-
3,5,6-trihydroxy-7-megastigmane-9-one (Sun et al., 2007), the S
configuration at C-6 in 1 was determined from a negative Cotton
absorption band of the a,b-unsaturated ketone moiety in 2. There-
fore, 2 was the C-9 ketone reduction product of 1. As for 1, the gly-
cosidic linkage position at C-3 and the proton chemical shift [d 5.60
(1H, s)] of the exchangeable tertiary hydroxyl group at C-6 were
determined by analysis of the HMBC correlations. In the HMBC
NMR spectrum, the anomeric proton at d 4.97 was found to have
a clear 3J correlation with C-3 at d 78.1 (CH), while the exchange-
able proton at d 5.60 exhibited 2J or 3J correlations with C-5 (d
32.1, CH), C-6 (d 79.7, C) and C-7 (d 131.3, CH). Similar to com-
pound 1, the coupling constant (7.8 Hz) of the anomeric proton
in 2 also indicated a b-glycosidic linkage.
The relative stereochemistry at the chiral centers in the cyclo-
hexane of 2 was judged from the NOE correlations (Fig. 2) in its
NOESY NMR spectrum and the observed coupling patterns (Table 2)
of the protons linked to this ring. To further determine the absolute
configuration, compound 2 was subjected to enzymatic hydrolysis
with b-glucosidase to yield D-glucose and 3,4,6,9-tetrahydroxy-7-
en-megastigmane (2a) as its aglycone. Compound 2a was then
subjected to a modified Mosher’s method (Takeda et al., 2000;
Ohtani et al., 1991) by treatment with (S)- and (R)-a-methoxy-a-
trifluoromethylphenyl acetic acid (MTPA) in the presence of 1-
ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride
(EDCꢁHCl) and 4-dimethylaminopyridine (4-DMAP) to give its
3,9-di-(S)-MTPA ester (2b) and 3,9-di-(R)-MTPA ester (2c). In their
1H NMR spectra, although the signals of H-7, H-8 and H-9 of 2c
overlapped around d 5.56–5.63, the chemical shifts of H-7 and H-
8 in 2b could be observed at higher fields (
at least), while its Me-10 resonated at a lower field (D
D
d: negative, <ꢀ0.03
effect (
D
e238nm ꢀ8.69) in its CD spectrum. In addition, enzymatic
d: positive)
hydrolysis of 1 with b-glucosidase gave only
D
-glucose as the su-
(Fig. 3) when compared with those of 2c, indicating an R configu-
ration at C-9 in 2a. The S configuration at C-3 was confirmed by
the observed difference of the chemical shifts of the protons
bonded to C-2, C-4, C-5 and C-13. In 2b, H-4, H-5, and Me-13 were
gar moiety, which was identified by direct comparison with an
authentic sample. Therefore, compound 1 was elucidated to be
(3R, 4R, 5S, 6S, 7E)-3,4,6-trihydroxymegastigmane-7-en-9-one-3-O
-b-D-glucopyranoside.
at lower fields (Dd: positive) and the two protons at C-2 were

696
Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
(3S, 4S, 5R, 6R, 7E, 9R)-3,4,6,9-tetrahydroxy-megastigmane-7-en-
3-O-b- -glucopyranoside. Meanwhile, 2a was found to be identical
5.61
OH
D
2.41
H
to 10 [whose absolute configuration at C-9 was previously un-
known (Pérez et al., 1996b)] by comparing their spectroscopic data
and physical properties. Thus, the structure of compound 10 can be
now updated as (3S, 4S, 5R, 6R, 9R)-3,4,6-trihydroxyl-5,6-dihydro-
b-ionol.
H
1.04
H3C
OH
CH3
1.76
H
6.87
CH3
The 1H and 13C NMR spectra (Tables 1 and 2) of compound 3
showed general features similar to those of 2. The most obvious
difference between these two compounds was that the oxygen-
bearing quaternary carbon signal at d 79.7 (C-6) in 2 was replaced
by a methine carbon signal at d 50.4 in 3, and hence an additional
proton was observed at d 2.40 (1H, dd, J = 11.3, 9.3 Hz, H-6) in 3. In
the COSY spectrum of 3, a spin system of –CH2–CH(O)–CH(O)–
CH(CH3)–CH–CH@CH–CH(O)–CH3 was found, indicating that 3 is
a 6-deoxy analogue of 2. This was further supported by its chemi-
cal formula C19H34O8 deduced from a pseudo-molecular ion peak at
m/z = 413.2143 [M+Na]+ in its positive mode HR-ESIMS. In the
HMBC NMR spectrum of 3, a 3J correlation was observed between
the anomeric proton at d 5.01 (1H, d, J = 7.5 Hz) and C-9 at d 76.1,
demonstrating C-9 as the glycosidic linkage position. Compound 3
has a structure identical to the known compound C-1 (Ibraheim
and Abdallah, 1994) previously isolated from the Egyptian plant
Maerua crassifolia Forssk. However, their NMR spectroscopic data
recorded in C5D5N are quite different, indicating that the two com-
pounds are diastereomers. Enzymatic hydrolysis of 3 with b-gluco-
H
H
O
1.21
1.37
CH3
H
OGlu
H
2.73
1
1.38
OGlu
CH3
2.69
1.80
H
H
H
5.80
OH
1.14
CH3
H
H
CH3
H
CH3
2.49
OH
1.31
H
OH
5.60
2
sidase gave
D-glucose and an aglycone, which was identical to
compound 8 (Pérez et al., 1996a; Rodríguez et al., 1992) based on
the analyses of their HPLC chromatograms and 1H NMR spectra.
In addition, a 13C NMR spectrum in CD3OD (Table 1) of 3 showed
that the chemical shift (d 77.9) of C-9 was almost consistent with
1.39
CH3
OH
1.80
2.45
H
H
H
that of in (9R)- rather than in (9S)-3-oxo-a-ionol-9-O-b-D-glucopy-
5.63
OGlu
1.05
CH3
ranoside [9R: C-9 (d 77.0), 9S: C-9 (d 74.7)] (Pabst et al., 1992) pre-
viously isolated from raspberry fruits. Thus, 3 was deduced to be
(3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-9-O-b-
H
H
CH3
H
D-glucopyranoside, which was in full agreement with the NOE cor-
CH3
1.21
2.20
OH
relations (Fig. 2).
H
Based on their HR-ESIMS, the chemical formulae of com-
pounds 4 and 5 were both determined to be the same as com-
pound 3. The NMR spectroscopic features (Tables 1 and 2) of
compounds 3–5 were also very similar. Like compounds 1–3,
the homonuclear proton connectivities in compounds 4 and 5
were determined by analysis of their COSY spectra, and their het-
eronuclear proton and carbon connectivities were deduced by
HSQC and HMBC NMR experiments. In their HMBC spectra, clear
3J correlations were observed between the anomeric proton at d
4.98 (1H, d, J = 7.8 Hz) and C-3 at d 78.3 in 4, and between the
anomeric proton at d 4.92 (1H, d, J = 7.5 Hz) and C-4 at d 81.3
in 5, indicating the only difference between 4 and 5 was the gly-
cosidic linkage position. The enzymatic hydrolysis of 4 and 5 with
H
2.43
3
Fig. 2. Key NOE correlations in NOESY NMR spectra of compounds 1–3.
- 0.22
- 0.07
OR
< - 0.03
- 0.03
- 0.07
b-glucosidase both gave D-glucose and the same aglycone, whose
< - 0.03
+ 0.05
structure was also identical to compound 8 (Pérez et al., 1996a;
Rodríguez et al., 1992). Consequently, the structures of 4 and 5
were elucidated to be (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymeg-
OH
+ 0.26
astigmane-7-en-3-O-b-D-glucopyranoside, and (3S, 4S, 5S, 6S, 7E,
9R)-3,4,9-trihydroxymegastigmane-7-en-4-O-b-D-glucopyranoside,
RO
+ 0.10
+ 0.09
respectively.
OH
2.3. Other known megastigmane metabolites
2b: R = (S)-MTPA
2c: R = (R)-MTPA
All the previously known compounds (6–12) from other plant
sources were isolated from G. pentaphyllum for the first time. Their
1H NMR spectroscopic data recorded either in CD3OD (7, 11, 12) or
in C5D5N (8, 9) (Table 3), and their 13C NMR data recorded either in
CD3OD (6, 7, 12) or in C5D5N (9) (Table 4) were reported for the
first time. Among them, the chemical shifts of 40-dihydrophaseic
Fig. 3. Results with the modified Mosher’s method (
D
dH = dS ꢀ dR).
shifted to higher fields (
those of 2c. Therefore, structure
D
d: negative) (Fig. 3) when compared with
was elucidated as
2

Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
697
Table 3
1H (500 MHz) NMR spectroscopic data of 7–9, 11 and 12.
7a
8b
9b
11a
12a,c
d (mult, J, Hz)
dH (mult, J, Hz)
d (mult, J, Hz)
d (mult, J, Hz)
d (mult, J, Hz)
2
1.90 (1H, overlapped)
1.25 (1H, m)
4.36 (1H, m)
2.48 (1H, br d, 13.2)
1.90 (1H, overlapped)
2.24 (1H, dd, 14.2, 2.4)
1.82 (1H, br d, 14.2)
4.50 (1H, br d, 2.4)
4.22 (1H, br s)
2.59 (1H, t, 12.2, 11.8)
2.05 (1H, dd, 12.2, 2.3)
4.91 (1H, m)
2.51 (1H, overlapped)
2.47 (1H, overlapped)
1.42 (2H, m)
1.85 (1H, dd, 12.2, 7.0)
1.66 (1H, dd, 12.2, 2.0)
4.11 (1H, m)
2.03 (1H, dd, 13.5, 7.5)
1.74 (1H, dd, 13.5, 3.0)
3
4
4.12 (1H, m)
1.46 (2H, m)
5
6
7
8
2.49 (1H, overlapped)
2.48 (1H, overlapped)
5.74 (1H, dd, 15.5, 9.0)
5.85 (1H, dd, 15.5, 6.5)
4.63 (1H, dq, 6.5, 6.2)
1.49 (1H, d, 6.2)
6.83 (1H, d, 15.9)
6.51 (1H, dd, 15.9, 6.1)
4.78 (1H, qd, 6.3, 6.1)
1.51 (3H, d, 6.3)
1.66 (3H, s)
6.36 (1H, d, 15.2)
7.40 (1H, d, 15.2)
6.50 (1H, d, 15.8)
7.96 (1H, d, 15.8)
5.92 (1H, s)
9
10
11
12
2.28 (3H, s)
1.13 (3H, s)
1.45 (3H, s)
2.31 (3H, s)
1.11 (3H, s)
0.85 (3H, s)
5.77 (1H, br s)
1.02 (3H, s)
1.42 (3H, s)
1.27 (3H, s)
3.80 (1H, d, 7.5)
3.71 (1H, d, 7.5)
0.93 (3H, s)
13
14
15
10
1.45 (3H, s)
1.26 (3H, d, 6.5)
1.64 (3H, s)
1.25 (3H, s)
1.15 (3H, s)
2.08 (3H, br s)
4.55 (1H, d, 7.9)
20
3.16 (1H, dd, 9.0, 7.9)
3.35 (1H, dd, 9.0, 8.9)
3.24 (1H, dd, 8.9, 8.8)
3.20 (1H, m)
3.80 (1H, br d, 11.8)
3.62 (1H, dd, 11.8, 4.8)
30
40
50
60
a
b
c
Recorded in CD3OD.
Recorded in C5D5N.
Assignments were made by a combination of 1D and 2D NMR techniques (COSY, HSQC and HMBC).
Table 4
verted by either distinct transferase or synthase enzymes (Enzell,
1985; Baumes et al., 2002; Setha et al., 2004). In fact, various carote-
noids have been detected in G. pentaphyllum (Liu et al., 2004b). Nat-
urallyoccurringmegastigmane derivativeswere previouslyfoundto
have anti-proliferative (Zhou et al., 2009; Liu et al., 2008; Janakiram
et al., 2008; Chen et al., 2006; Liu et al., 2004c; Liu et al., 2004d; Dun-
can et al., 2004; Yu et al., 1995), anticancer (Ito et al., 2002; Jung et al.,
1998), and cytotoxiceffects (Kuang et al., 2008; Alija et al., 2006; Per-
rone et al., 2005; Kubo and Morimitsu, 1995). Our isolated com-
13C (125 MHz) NMR spectroscopic data of 6, 7, 9 and 12.
6 (DEPT)a
7 (DEPT)a
9 (DEPT)b
12 (DEPT)a,c
1
2
3
4
5
6
7
8
37.0 (C)
36.9 (C)
40.2 (C)
49.4 (C)
49.9 (CH2)
62.9 (CH)
48.1 (CH2)
77.8 (C)
49.9 (CH2)
62.8 (CH)
46.8 (CH2)
77.7 (C)
47.0 (CH2)
64.1 (CH)
46.7 (CH2)
76.9 (C)
44.5 (CH2)
66.1 (CH)
46.0 (CH2)
87.9 (C)
119.1 (C)
200.7 (C)
101.4 (CH)
212.9 (C)
26.6 (CH3)
26.7 (CH3)
30.1 (CH3)
32.5 (CH3)
119.5 (C)
202.1 (C)
101.5 (CH)
212.9 (C)
27.3 (CH3)
27.5 (CH3)
29.8 (CH3)
32.8 (CH3)
78.3 (C)
83.4 (C)
130.4 (CH)
136.2 (CH)
68.2 (CH)
24.8 (CH3)
27.6 (CH3)
26.2 (CH3)
27.6 (CH3)
135.1 (CH)
132.2 (CH)
151.0 (C)
120.3 (CH)
170.6 (C)
77.4 (CH2)
16.2 (CH3)
19.5 (CH3)
21.1 (CH3)
pounds,
however,
were
tested
against
human
lung
9
adenocarcinoma (A549, H460), human glioma (U251), human oste-
osarcoma (U2OS), and human breast (MCF-7) cancer cells using the
MTT method (Wu et al., 2009, 2010), but were inactive.
10
11
12
13
14
15
10
20
30
40
50
60
3. Concluding remarks
98.7 (CH)
75.3 (CH)
78.7 (CH)
71.8 (CH)
78.6 (CH)
63.8 (CH2)
98.5 (CH)
75.4 (CH)
79.4 (CH)
71.6 (CH)
78.8 (CH)
63.8 (CH2)
Through a combination of silica gel, MCI gel and Sephadex LH-
20 column chromatography, as well as semi-preparative HPLC, five
new megastigmane glycosides (1–5) and several related known
compounds (6–12) were isolated from the well-known traditional
Chinese medicine ‘‘Jiao-Gu-Lan” (G. pentaphyllum). Based on exten-
sive spectroscopic methods and a few chemical transformations,
the absolute configuration of the new megastigmane glycosides
was completely elucidated. Megastigmane derivatives have, to
date, never been reported from this plant. This class of naturally
occurring compounds could stimulate future phyochemical
genomics studies.
a
b
c
Recorded in CD3OD.
Recorded in C5D5N.
Assignments were made by a combination of 1D and 2D NMR techniques
(COSY, HSQC and HMBC).
acid (12) (Walton et al., 1973; Zaharia et al., 2005) were unambig-
uously assigned here for the first time by detailed 1D and 2D NMR
spectroscopic analyses, with the relative stereochemistry was fur-
ther investigated using a NOESY NMR experiment.
4. Experimental
4.1. General procedures
2.4. Biogenetic consideration and cytotoxic assay
Optical rotations were measured with a Perkin–Elmer 341
polarimeter. CD spectra were obtained on a JASCO 810 spectrome-
ter. IR and UV spectra were recorded on Thermo Nicolet NEXUS
670 FT-IR and Libra S35 spectrometer, respectively. NMR spectra
Based on a biogenetic considerations, the isolated megastigmane
derivatives 1–12 are degraded carotenoid-like compounds probably
formed by initial cleavage of cartenoids and subsequently intercon-

698
Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
were measured on Bruker AM 500 Avance DRX-500 spectrometer.
Chemical shifts (d in ppm) were referenced to the residual solvent
signals [CD3OD (dH/dC: 3.30/49.0), C5D5N (dH: 7.20, 7.57, 8.72; dC:
149.8, 135.5, 123.4)]. Low-resolution electrospray ionization mass
spectrometry (LR-ESIMS) was carried out on a Bruker Esquire
3000plus instrument. HR-ESIMS was measured on a Bruker Dalton-
ics micrOTOF mass spectrometer. Semi-preparative HPLC was per-
formed on a Beckman System consisting of a Beckman Coulter
System Gold 508 autosampler, Gold 126 gradient HPLC pumps
with a Beckman System Gold 166 single wavelength UV detector
(254 nm), a Sedex 80 (SEDERE, France) evaporative light-scattering
detector (ELSD), and a Beckman Coulter Ultrasphare ODS column
v) for 32 min, followed by CH3CN–H2O (95:5, v/v) for 5 min (flow
rate: 3 mL/min; 8: tR = 27.5 min).
Fr.4 (CH2Cl2–MeOH 8:1, 1.0 g) was subjected to MCI gel CC
(5.0 ꢂ 45 cm) using a stepwise gradient elution with MeOH/H2O
(from 3:7 to MeOH neat, v/v) to afford compound 9 (30.2 mg),
which was further purified by GPC on Sephadex LH-20 (column:
4.0 ꢂ 130 cm) in MeOH. Compounds 1 (3.0 mg), 6 (150.2 mg),
and 7 (2.0 mg) were obtained from Fr.5 (CH2Cl2–MeOH 6:1, 9.6 g)
through silica gel CC (column: 3.0 ꢂ 70 cm) with EtOAc–MeOH–
HAc–H2O (80:3.5:3:1, v/v)]. Compound 6 was further purified by
GPC on Sephadex LH-20 (column: 4.0 ꢂ 130 cm) in MeOH. Both
compounds 1 and 7 were then re-purified by using semi-prepara-
tive HPLC. The method employed an isocratic gradient of CH3CN–
H2O containing 0.1% (v/v) HAc (1:9, v/v) for 36 min, followed by
CH3CN–H2O (95:5, v/v) for 5 min (flow rate: 3 mL/min; 1:
tR = 24.3 min, 7: tR = 29.4 min).
(250 ꢂ 10 mm, 5
lm). The solvents for chromatography were ana-
lytical grade (Shanghai Chemical Reagents Co. Ltd., China) and
those for HPLC were HPLC grade (Jiangsu Hanbon Science and
Technology Co. Ltd., China). b-Glucosidase (emulsion), (R)- and
(S)-MTPAs, 4-DMAP and EDCꢁHCl were all purchased from Sig-
ma–Aldrich Chemical Co. (St. Louis, MO, USA). Column chromatog-
raphy (CC) was performed using silica gel (200–300 mesh, Qingdao
Fr.6 (CH2Cl2–MeOH 4:1, 15 g) was subjected to silica gel CC
(5.0 ꢂ 70 cm) with a CH2Cl2–MeOH gradient (9:1–1:1–MeOH neat,
v/v) to afford four subfractions (Fr.6A–Fr.6D). Compounds
3
Ji-Yi-Da Silysia Chemical Ltd., China), MCI gel CHP20P (75–120
l
,
(3.1 mg), 4 (13.0 mg) and 5 (5.6 mg) were isolated from Fr.6B
(CH2Cl2–MeOH 9:1, 2100 mg) via silica gel CC (4.0 ꢂ 70 cm) with
EtOAc–MeOH–HAc–H2O (80:3.5:3:1, v/v) and were further purified
by using semi-preparative HPLC. The method was developed in a
way that resulted in an isocratic gradient of MeOH in H2O contain-
ing 0.1% (v/v) HAc (24:76, v/v) for 36 min, and followed by MeOH–
H2O (95:5, v/v) for 5 min (flow rate: 3 mL/min; 3: tR = 29.6 min, 4:
tR = 23.9 min, 5: tR = 19.9 min). Fr.6C (CH2Cl2–MeOH 6:1, 1500 mg)
was subjected to silica gel CC (4.0 ꢂ 70 cm) with EtOAc–MeOH–
HAc–H2O (80:3.5:3:1, v/v) as the eluent to furnish compound 2
(14.1 mg), which was further purified by Sephadex LH-20 GPC (col-
umn: 3.0 ꢂ 98 cm) in MeOH.
Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (GE
Healthcare Bio-Sciences AB, Sweden). Silica gel-precoated plates
(GF254, 0.25 mm, Yantai Kang-Bi-Nuo Silysia Chemical Ltd., China)
were used for TLC. TLC was performed with EtOAc–AcOH–
MeOH–H2O, CH2Cl2–MeOH–H2O or CH2Cl2–MeOH as mobile
phases. Spots were visualized by spraying with 5% vanillin in EtOH
(containing 1% H2SO4) followed by heating to 150 °C.
4.2. Plant material
The dried whole plants of G. pentaphyllum were purchased from
Shanghai Kang-Qiao TCM Materials Co. Ltd., and were originally
collected from Anhui province of China in December 2004. The
plant was identified by Prof. Jian-Wei Chen (College of Pharmacy,
Nanjing University of Traditional Chinese Medicine). A voucher
specimen (No. 060416) was deposited at the Herbarium of the
Shanghai Key Laboratory of Brain Functional Genomics, East China
Normal University.
4.4. Gynostemoside A (1)
White amorphous power; ½a D22
ꢃ
ꢀ26.1 (c 0.13, MeOH); UV
e238nm ꢀ8.69,
(MeOH) kmax (log
e
) 232 (3.71); CD (MeOH)
D
De208nm
+7.77; IR (film, MeOH) mmax 3359 (br), 2922, 2850, 1641, 1560,
1465, 1426, 1360, 1274, 1168, 1138 cmꢀ1; for 13C and 1H NMR
spectroscopic data, see Tables 1 and 2; ESIMS m/z = 427 [M+Na]+,
831 [2M+Na]+, 449 [M+HCOOꢀ]ꢀ, 807 [2MꢀH]ꢀ; HR-ESIMS m/
z = 427.1974 [M+Na]+ (calcd for C19H32O9Na, 427.1944,
Dm = +7.0 ppm).
4.3. Extraction and isolation
The air-dried and ground material (10 kg) was extracted with
EtOH–H2O (10 L ꢂ 4, 95:5, v/v) at room temperature (each process
lasting 24 h). After removal of the solvent by evaporation, the res-
idue (520 g) was suspended in H2O (750 mL), and then extracted
with n-BuOH (5 ꢂ 750 mL). The combined n-BuOH fractions were
concentrated in vacuo to give a residue (77.8 g), which was sub-
jected to silica gel CC (8.0 ꢂ 82 cm) using a CH2Cl2–MeOH gradient
(10:1–1:1–MeOH neat, v/v) to yield 10 fractions (Fr.1–Fr.10). Fr.2
(CH2Cl2–MeOH 10:1, 3.0 g) was applied to an MCI gel (column:
5.0 ꢂ 45 cm), this being eluted using a stepwise gradient elution
with MeOH/H2O (from 1:4 to MeOH neat, v/v) to afford four sub-
fractions (Fr.2A–Fr.2D). Compounds 10 (8.2 mg) and 11 (2.1 mg)
were isolated from Fr.2B (MeOH/H2O 3:7, 510 mg) through silica
gel CC (column: 3.0 ꢂ 45 cm) with CH2Cl2–MeOH–H2O (20:
1:0.05) as eluant, with both further purified by gel permeation
chromatography (GPC) on Sephadex LH-20 (column: 3.0 ꢂ 98 cm)
in MeOH. Fr.2C (MeOH/H2O 2:3, 360 mg) was subjected to silica
gel CC (3.0 ꢂ 45 cm) with a CH2Cl2–MeOH gradient (15:1–1:1–
MeOH neat, v/v) to yield compound 12 (10.0 mg). Compound 8
(3.2 mg) was isolated from Fr.2D (MeOH/H2O 1:1, 74.0 mg) first
via silica gel CC (3.0 ꢂ 45 cm) with CH2Cl2–MeOH–H2O (15:1:
0.05, v/v), and then was further purified by using semi-preparative
HPLC. The method was developed in a way that resulted in an iso-
cratic gradient of CH3CN–H2O containing 0.1% (v/v) HAc (13:87, v/
4.5. Gynostemoside B (2)
White amorphous power; ½a D22
ꢀ17.9 (c 0.34, MeOH); IR (film,
ꢃ
MeOH) mmax 3392 (br), 2971, 2926, 2900, 1407, 1393, 1251, 1077,
1040, 1027, 973, 891 cmꢀ1; for 13C and 1H NMR spectroscopic data,
see Tables 1 and 2; ESIMS m/z = 429 [M+Na]+, 451 [M+HCOOꢀ]ꢀ,
811 [2MꢀH]ꢀ; HR-ESIMS m/z = 429.2064 [M+Na]+ (calcd for
C19H34O9Na, 429.2101, Dm = ꢀ8.5 ppm).
4.6. Gynostemoside C (3)
White amorphous power; ½a D22
ꢀ9.9 (c 0.15, MeOH); IR (film,
ꢃ
MeOH) mmax 3416 (br), 2987, 2971, 2901, 1407, 1394, 1382, 1250,
1230, 1076, 1066, 1056, 1028, 892 cmꢀ1; for 13C and 1H NMR spec-
troscopic data, see Tables 1 and 2; ESIMS m/z = 413 [M+Na]+, 803
[2M+Na]+, 435 [M+HCOOꢀ]ꢀ, 779 [2MꢀH]ꢀ; HR-ESIMS m/
z = 413.2143 [M+Na]+ (calcd for C19H34O8Na, 413.2151,
Dm = ꢀ2.0 ppm).
4.7. Gynostemoside D (4)
White amorphous powder; ½a D22
ꢀ27.3 (c 0.64, MeOH); IR (film,
ꢃ
MeOH) mmax 3379 (br), 2922, 2866, 1455, 1346, 1055, 1033,

Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
699
1014 cmꢀ1; for 13C and 1H NMR spectroscopic data, see Tables 1
Acknowledgments
and 2; ESIMS m/z = 413 [M+Na]+, 803 [2M+Na]+, 435 [M+HCOOꢀ]ꢀ,
779 [2MꢀH]ꢀ; HR-ESIMS m/z = 413.2182 [M+Na]+ (calcd for
C19H34O8Na, 413.2151, Dm = +7.4 ppm).
The authors gratefully acknowledge Prof. Jian-Wei Chen (Nan-
jing University of TCM) for the plant identification. This work
was supported by NSFC Grants (90713040, 30640068), NCET Grant
(NCET-06-0422), and STCSM Grants (07DZ22006, 06DZ19002,
06PJ14033).
4.8. Gynostemoside E (5)
White amorphous powder; ½a D22
ꢀ30.9 (c 0.23, MeOH); IR (film,
ꢃ
MeOH) mmax 3385 (br), 2962, 2924, 1367, 1167, 1074, 1039 cmꢀ1
;
Appendix A. Supplementary data
for 13C and 1H NMR spectroscopic data, see Tables 1 and 2; ESIMS:
m/z = 413 [M+Na]+, 435 [M+HCOOꢀ]ꢀ, 779 [2MꢀH]ꢀ; HR-ESIMS:
m/z = 413.2113 [M+Na]+ (calcd for C19H34O8Na, 413.2151,
Dm = ꢀ9.3 ppm).
Pivotal NMR, CD and LR-/HR-ESIMS spectra of isolated com-
pounds (1–9, 11, 12) and semisynthetics (2b, 2c) are available as
Supplementary data. Supplementary data associated with this arti-
cle can be found, in the online version, at doi:10.1016/j.phytochem.
2009.12.017.
4.9. Enzymatic hydrolysis of compounds 1–5
A solution of compounds 1–5 (1.4, 13.0, 1.6, 5.5, 1.5 mg, respec-
tively) in 0.2 M acetic acid and sodium acetate buffer (pH 3.8,
1.5 mL) was treated with lyophilized b-glucosidase (13.2, 41.2,
15.0, 36.3, 14.9 mg, respectively) from almonds, and the solution
was stirred at 40 °C for 48 h. After cooling, the reaction mixture
References
Alija, A.J., Bresgen, N., Sommerburg, O., Langhans, C.D., Siems, W., Eckl, P.M., 2006. b-
Carotene breakdown products enhance genotoxic effects of oxidation stress in
primary rat hepatocytes. Carcinogenesis 27, 1128–1133.
of compound 1 was extracted with EtOAc (2 mL ꢂ 3).
D
-glucose
Baumes, R., Wirth, J., Bureau, S., Gunata, Y., Razungles, A., 2002. Biogeneration of
C13-norisoprenoid compounds: experiments supportive for an apo-carotenoid
pathway in grapevines. Anal. Chim. Acta 458, 3–14.
Chen, W.L., Liu, Q.F., Wang, J., Zou, J., Meng, D.H., Zuo, J.P., Zhu, X.Z., Zhao, W.M., 2006.
New guaiane, megastigmane and eudesmane-type sesquiterpenoids and anti-
inflammatory constituents from Youngia japonica. Planta Med. 72, 143–150.
Duncan, R.E., Lau, D., El-Sohemy, A., Archer, M.C., 2004. Geraniol and b-ionone
in the aqueous residue was detected by comparison with an
authentic sample. For compound 2: after cooling, the reaction mix-
ture was extracted with EtOAc (2 mL ꢂ 3). The EtOAc extract was
then concentrated under reduced pressure, and the residue was
purified by semi-preparative HPLC [ACN–H2O–HAc (16:84:0.1, v/
v/v)] to give 2a (4.9 mg, 62.7%). Similarly, each EtOAc extract from
the enzymatic hydrolysis product of 3–5 was concentrated and
then subjected to silica gel CC [2.0 ꢂ 25 cm, EtOAc–MeOH–HAc–
H2O (36:3.5:3:1, v/v/v/v)] to afford 8 with a yield of 0.7 (74.8%),
2.0 (62.2%), 0.7 (79.8%) mg, respectively.
inhibit proliferation, cell cycle progression, and cyclin-dependent kinase
2
activity in MCF-7 breast cancer cells independent of effects on HMG-CoA
reductase activity. Biochem. Pharmacol. 68, 1739–1747.
Enzell, C., 1985. Biodegradation of carotenoids – an important route to aroma
compounds. Pure Appl. Chem. 57, 693–700.
Hu, L.H., Chen, Z.L., Xie, Y.Y., 1996. New triterpenoid saponins from Gynostemma
pentaphyllum. J. Nat. Prod. 59, 1143–1145.
Ibraheim, Z.Z., Abdallah, O.M., 1994. Miner constituents of Maerua crassifolia Forssk
growing in Egypt. Bull. Fac. Pharm. Cairo Univ. 32, 407–410.
Ito, H., Kobayashi, E., Li, S.H., Hatano, T., Sugita, D., Kubo, N., Shimura, S., Itoh, Y.,
Tokuda, H., Nishino, H., 2002. Antitumor activity of compounds isolated from
leaves of Eriobotrya japonica. J. Agric. Food Chem. 50, 2400–2403.
Janakiram, N.B., Cooma, I., Mohammed, A., Steele, V.E., Rao, C.V., 2008. b-Ionone
inhibits colonic aberrant crypt foci formation in rats, suppresses cell growth,
4.10. Preparation of 3,9-di-O-(S)-MTPA ester (2b) and 3,9-di-O-(R)-
MTPA ester (2c) of 2a
A solution of 2a (2.4 mg) in dry CH2Cl2 (1.5 mL) was treated
with (S)-MTPA (37.7 mg) in the presence of EDCꢁHCl (31.2 mg)
and 4-DMAP (19.7 mg), and the mixture was stirred at room tem-
perature for 90 min. The reaction mixture was added to CH2Cl2
(1.5 mL) and successively washed with H2O (1.5 mL), 2 M HCl
(1.5 mL), NaHCO3-saturated H2O (1.5 mL), and brine (1.5 mL),
respectively. The organic layer was combined and dried (Na2SO4)
and evaporated under reduced pressure. The residue was purified
by semi-preparative HPLC [ACN–H2O–HAc (72:28:0.1, v/v/v)] to
furnish 2b (2.6 mg, 39.1%). Through a similar procedure, 2c
(2.5 mg, 37.6%) was generated from 2.4 mg of 2a using (R)-MTPA
(35.6 mg) in a CH2Cl2 solution containing EDCꢁHCl (30.1 mg) and
4-DMAP (18.6 mg). (3S, 4S, 5R, 6R)-3,4,6-Trihydroxyl-5,6-dihydro-
b-ionol-3,9-di-O-(S)-MTPA ester (2b): 1H NMR (CDCl3, 500 MHz)
d 0.58 (3H, s, CH3-12), 0.71 (3H, s, CH3-11), 0.94 (3H, d, J = 7.0 Hz,
CH3-13), 1.41 (3H, d, J = 6.5 Hz, CH3-10), 1.42 (1H, overlapped, H-
2eq), 1.93 (1H, m, H-5), 2.13 (1H, dd, J = 16.0, 3.5 Hz, H-2ax), 3.54
(3H, s, OCH3), 3.55 (3H, s, OCH3), 3.68 (1H, br s, H-4), 5.26 (1H, d,
J = 3.5 Hz, H-3), 5.50 (1H, d, J = 15.6 Hz, H-7), 5.53 (1H, dd,
J = 15.6, 6.2 Hz, H-8), 5.59 (1H, m, H-9), 7.33–7.37 (3H, m), 7.41–
7.43 (3H, m), and 7.50–7.53 (4H, m). (3S, 4S, 5R, 6R)-3,4,6-Trihydr-
oxyl-5,6-dihydro-b-ionol-3,9-di-O-(R)-MTPA ester (2c): 1H NMR
(CDCl3, 500 MHz) d 0.78 (3H, s, CH3-11), 0.80 (3H, s, CH3-12),
0.85 (3H, d, J = 7.1 Hz, CH3-13), 1.36 (3H, d, J = 6.2 Hz, CH3-10),
1.45 (1H, d, J = 16.1 Hz, H-2eq), 1.67 (1H, m, H-5), 2.20 (1H, dd,
J = 16.1, 3.0 Hz, H-2ax), 3.51 (3H, s, OCH3), 3.54 (3H, s, OCH3), 3.58
(1H, br s, H-4), 5.27 (1H, d, J = 3.0 Hz, H-3), 5.56–5.63 (3H, H-7,
H-8 and H-9), 7.35–7.40 (6H, m), and 7.50–7.51 (4H, m).
and induces retinoid ꢂ receptor-
a in human colon cancer cells. Mol. Cancer
Ther. 7, 181–190.
Jiangsu New Medicine College, 1977. Dictionary of Chinese Medicinal Materials.
Shanghai Scientific and Technological Publisher, Shanghai. p. 16–17.
Jung, M., Mo, H.B., Elson, C.E., 1998. Synthesis and biological activity of b-ionone-
derived alcohols for cancer chemoprevention. Anticancer Res. 18, 189–192.
Kuang, H.X., Xia, Y.G., Yang, B.Y., Wang, Q.H., Lü, S.W., 2008. Sesquiterpene
glucosides from Chloranthus japonicus Sieb. Chem. Biodivers. 5, 1736–1742.
Kubo, I., Morimitsu, Y., 1995. Cytotoxicity of green tea flavor compounds against
two solid tumor cells. J. Agric. Food Chem. 43, 1626–1628.
Liu, X., Ye, W.C., Mo, Z.Y., Yu, B., Zhao, S.X., Wu, H.M., Che, C.T., Jiang, R.W., Mak,
T.C.W., Hsiao, W.L.W., 2004a. Five new ocotillone-type saponins from
Gynostemma pentaphyllum. J. Nat. Prod. 67, 1147–1151.
Liu, H.L., Kao, T.H., Chen, B.H., 2004b. Determination of carotenoids in the Chinese
medicine herb Jiao-Gu-Lan (Gynostemma pentaphyllum Makino) by liquid
chromatography. Chromatographia 60, 411–417.
Liu, J.R., Yang, B.F., Chen, B.Q., Yang, Y.M., Dong, H.W., Song, Y.Q., 2004c. Inhibition of
b-ionone
on
SGC-7901
cell
proliferation
and
upregulation
of
metalloproteinases-1 and -2 expression. World J. Gastroenterol. 10, 167–171.
Liu, J.R., Chen, B.Q., Yang, B.F., Dong, H.W., Sun, C.H., Wang, Q., Song, G., Song, Y.Q.,
2004d. Apoptosis of human gastric adenocarcinoma cells induced by b-ionone.
World J. Gastroenterol. 10, 348–351.
Liu, J.R., Sun, X.R., Dong, H.W., Sun, C.H., Sun, W.G., Chen, B.Q., Song, Y.Q., Yang, B.F.,
2008. b-Ionone suppresses mammary carcinogenesis, proliferative activity and
induces apoptosis in the mammary gland of the Sprague-Dawley rat. Int. J.
Cancer 122, 268–2698.
0
Norberg, ÅA., Hoa, N.K., Liepinsh, E., Phan, D.V., Thuan, N.D., Jörnvall, H., Sillard, R.,
Östenson, C.G., 2004. A novel insulin-releasing substance, phanoside, from the
plant Gynostemma pentaphyllum. J. Biol. Chem. 279, 41361–41367.
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR
application of Mosher’s method. The absolute configurations of marine
terpenoids. J. Am. Chem. Soc. 113, 4092–4096.
Osorio, C., Duque, C., Fujimoto, Y., 1999. C13-norisoprenoid glucoconjugates from
Lulo (Solanum quitoense L.) leaves. J. Agric. Food Chem. 47, 1641–1645.
Pabst, A., Barron, D., Sémon, E., Schreier, P., 1992. Two diastereomeric 3-oxo-
b- -glucosides from raspberry fruit. Phytochemistry 31, 1649–1652.
a-ionol
D

700
Z. Zhang et al. / Phytochemistry 71 (2010) 693–700
Pérez, C., Trujillo, J., Almonacid, L.N., Trujillo, J., Navarro, E., Alonso, S.J., 1996a.
Absolute structures of two new C13-norisoprenoids from Apollonias barbujana. J.
Nat. Prod. 59, 69–72.
Tan, S.T., Wilkins, A.L., Holland, P.T., 1989. Isolation and X-ray crystal structure of
(E)-4-(r-10,t-20,c-40-trihydroxy-20,60,60-trimethyl-cyclohexyl)but-3-en-2-one,
a
constituent of New Zealand thyme honey. Aust. J. Chem. 42, 1799–1804.
Pérez, C., Trujillo, J.M., Almonacid, L.N., Navarro, E., Alonso, S.J., 1996b. New C13
-
Umehara, K., Hattori, I., Miyase, T., Ueno, A., Hara, S., Kageyama, C., 1988. Studies on
the constituents of leaves of Citrus unshiu Marcov. Chem. Pharm. Bull. 36, 5004–
5008.
norisoprenoids from Apollonias barbujana. Nat. Prod. Lett. 8, 1–6.
Perrone, A., Plaza, A., Bloise, E., Nigro, P., Hamed, A.I., Belisario, M.A., Pizza, C.,
Piacente, S., 2005. Cytotoxic furostanol saponins and a megastigmane glucoside
from Tribulus parvispinus. J. Nat. Prod. 68, 1549–1553.
Razmovski-Naumovski, V., Huang, T.H.W., Tran, V.H., Li, G.Q., Duke, C.C., Roufogalis,
B.D., 2005. Chemistry and pharmacology of Gynostemma pentaphyllum.
Phytochem. Rev. 4, 197–219.
Rodríguez, M.L., Brito, B.I., González, A.G., Almonacid, L.N., Pérez, C., Trujillo, J.M.,
1992. Structure of a new C13-norisoprenoid. Acta Cryst. C48, 2192–2194.
Rujjanawate, C., Kanjanapothi, D., Amornlerdpison, D., 2004. The anti-gastric ulcer
effect of Gynostemma pentaphyllum Makino. Phytomedicine 11, 431–435.
Setha, S., Kondo, S., Hirai, N., Ohigashi, H., 2004. Xanthoxin, abscisic acid and its
metabolite levels associated with apple fruit development. Plant Sci. 166, 493–
499.
Walton, D.C., Dorn, B., Fey, J., 1973. The isolation of an abscisic-acid metabolite 40-
dihydrophaseic acid, from non-imbibed Phaseolus vulgaris seed. Planta (Berl.)
112, 87–90.
Wu, S.B., Ji, Y.P., Zhu, J.J., Zhao, Y., Xia, G., Hu, Y.H., Hu, J.-F., 2009. Steroids from the
leaves of Chinese Melia azedarach and their cytotoxic effects on human cancer
cell lines. Steroids 74, 761–765.
Wu, S.B., Pang, F., Wen, Y., Zhang, H.-F., Zhao, Z., Hu, J.-F., 2010. Antiproliferative and
apoptotic activities of linear furocoumarins from Notopterygium incisum on
cancer cell lines. Planta Med. 76, 82–85.
Yu, S.G., Anderson, P.J., Elson, C.E., 1995. Efficacy of b-ionone in the
chemoprevention of rat mammary carcinogenesis. J. Agric. Food Chem. 43,
2144–2147.
Sun, Y., Zhan, Y.C., Sha, Y., Pei, Y.H., 2007. Norisoprenoids from Ulva lactuca. J. Asian
Nat. Prod. Res. 9, 321–325.
Takeda, Y., Okada, Y., Masuda, T., Hirata, E., Shinzato, T., Takushi, A., Yu, Q., Otsuka,
H., 2000. New megastigmane and tetraketide from the leaves of Euscaphis
japonica. Chem. Pharm. Bull. 48, 752–754.
Takeda, Y., Shimizu, H., Masuda, T., Hirata, E., Shinzato, T., Bando, M., Otsuka, H.,
2004. Lasianthionosides A–C, megastigmane glucosides from leaves of
Lasianthus fordii. Phytochemistry 65, 485–489.
Zaharia, L.I., Galka, M.M., Ambrose, S.J., Abrams, S.R., 2005. Preparation of
deuterated abscisic acid metabolites for use in mass spectrometry and
feeding studies. J. Labelled Compd. Radiopharm. 48, 435–445.
Zhou, J.M., Geng, G.Y., Batist, G., Wu, J.H., 2009. Syntheses and potential anti-
prostate cancer activities of ionone-based chalcones. Bioorg. Med. Chem. Lett.
19, 1183–1186.