Cucurbitane-type triterpenoids from the leaves of Momordica charantia

Article abstract of DOI:10.1080/10286020.2014.881801

Phytochemical investigation of the ethanol extract from the leaves of Momordica charantia L. led to the isolation of two new (1, 2) and four known (3-6) cucurbitane-type triterpenoids. Their structures were elucidated on the basis of extensive analyses of

Full text of DOI:10.1080/10286020.2014.881801

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Cucurbitane-type triterpenoids from
the leaves of Momordica charantia
Yu-Bo Zhanga, Huan Liub, Cun-Ya Zhub, Mao-Xin Zhangb, Yao-Lan
Lia, Bing Lingb & Guo-Cai Wanga
a Institute of Traditional Chinese Medicine and Natural Products,
College of Pharmacy, Jinan University, Guangzhou 510632, China
b Laboratory of Insect Ecology, South China Agricultural University,
Guangzhou 510642, China
Published online: 05 Feb 2014.
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To cite this article: Yu-Bo Zhang, Huan Liu, Cun-Ya Zhu, Mao-Xin Zhang, Yao-Lan Li, Bing Ling &
Guo-Cai Wang (2014) Cucurbitane-type triterpenoids from the leaves of Momordica charantia,
Journal of Asian Natural Products Research, 16:4, 358-363, DOI: 10.1080/10286020.2014.881801
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 4, 358–363, http://dx.doi.org/10.1080/10286020.2014.881801
Cucurbitane-type triterpenoids from the leaves of Momordica
charantia
Yu-Bo Zhanga, Huan Liub, Cun-Ya Zhub, Mao-Xin Zhangb, Yao-Lan Lia,
Bing Lingb* and Guo-Cai Wanga
*
aInstitute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan
University, Guangzhou 510632, China; Laboratory of Insect Ecology, South China Agricultural
University, Guangzhou 510642, China
b
(Received 21 October 2013; final version received 7 January 2014)
Phytochemical investigation of the ethanol extract from the leaves of Momordica
charantia L. led to the isolation of two new (1, 2) and four known (3–6) cucurbitane-
type triterpenoids. Their structures were elucidated on the basis of extensive analyses
of spectroscopic data including IR, UV, MS, 1D, and 2D NMR. Also the absolute
configurations of momordicines I (3) and II (4) were determined for the first time by
application of the modified Mosher’s method, acid hydrolysis, and GC analysis.
Keywords: Momordica charantia; Cucurbitaceae; cucurbitane-type triterpenoids;
absolute configuration
1. Introduction
were first isolated in 1984[4] and, to the best
of our knowledge, the absolute configur-
ation at C-23 remains undetermined. We
determined that the absolute configurations
of momordicines I (3) and II (4) at C-23
were R by the modified Mosher’s method
and acid hydrolysis.
Momordica charantia L. (Cucurbitaceae),
called bitter melon or ‘kugua,’ is a tropical
or subtropical vine widely distributed in
Asian, African, and Caribbean. It has been
extensively used as a folk remedy in various
Asian and African traditional medicine
systems for a long time [1]. In China, it
has been used to treat toothache, diarrhea,
furuncle, and diabetes [2]. In recent
decades, M. charantia has been widely
investigated for its antidiabetic properties.
There have been more than 70 cucurbitacins
and cucurbitane glycosides isolated from
the roots, stems, fruits, seeds, leaves, and
vines of the plant [3]. As part of our ongoing
search of chemical constituents for func-
tional foods or traditional medicine, we
carried out phytochemical investigations on
M. charantia. As a result, we have obtained
two new (1, 2) and four known (3–6)
cucurbitane-type triterpenoids from the
ethanol extract of M. charantia leaves
(Figure 1). Momordicines I (3) and II (4)
2. Results and discussion
Compound 1 was isolated as a white
powder. The molecular formula was
determined as C31H50O4 based on its
HR-ESI-MS at m/z 509.3615 [M þ Na]þ.
The IR spectrum exhibited absorption
bands at 3444 and 1644 cm21, indicating
the presence of hydroxyl and double bond.
1
The H NMR spectrum of 1 showed the
presence of seven methyl groups [dH 0.85
(3H, s), 0.95 (3H, s), 1.01 (3H, d,
J ¼ 6.4 Hz), 1.09 (3H, s), 1.25 (3H, s),
1.67 (3H, d, J ¼ 1.0 Hz), 1.70 (3H, d,
J ¼ 0.8 Hz)], a methoxyl [dH 3.26 (3H, s)],
and two olefinic protons [dH 5.17 (1H, dt,
J ¼ 8.4, 1.4 Hz), 5.99 (1H, d, J ¼ 4.6 Hz)].
*Corresponding authors. Email: gzhbling@scau.edu.cn; twangguocai@jnu.edu.cn
q 2014 Taylor & Francis

Journal of Asian Natural Products Research
359
21
18
26
20
23
25
27
12
H
H
H
OR2
OMe
H
19
OR2
OR
OHC
OHC
17
15
30
OR1
H
H
H
H
H
1
2
3
9
10
5
8
7
O
HO
HO
HO
OR1
6
28
29
1 R1 = Me, R2 =H
2 R = Glc
5 R1 = H, R2 = H
6 R1 = Glc, R2 = Me
3 R1 = H, R2 = H
4 R1 = H, R2 =Glc
Figure 1. Structures of compounds 1–6.
The 13C NMR spectrum displayed an
aldehyde group (dC 209.7), three oxy-
methine carbons (dC 66.7, 76.8, 77.2), and
two double bonds (dC 122.2, 130.6, 133.6,
148.6). Comparison of the 1H and 13C
NMR data of 1 (Table 1) with those of
momordicine I [4] showed that they were
similar, except for the presence of an
additional methoxyl (dC 56.3) in 1, and an
oxy-methine at dC 65.6 (C-7) in momordi-
cine I shifted to dC 76.8 in 1, indicating
that C-7 was substituted by a methoxyl.
And this was confirmed by the HMBC
correlation (Figure 2) between the meth-
oxyl at dH 3.26 and C-7 at dC 76.8.
According to the absolute configuration
of momordicine I (3) determined by the
modified Mosher’s method as follows,
C-23 in 1 was also assigned as R.
Therefore, compound 1 was deduced and
named as charantin A.
the presence of hydroxyl and double bond.
The 1H spectrum of 2 showed the presence
of seven methyls [dH 0.88 (3H, s), 0.89
(3H, s), 0.90 (3H, s), 0.99 (3H, d,
J ¼ 6.4 Hz), 1.20 (3H, s), 1.70 (6H, s)], a
methoxyl [dH 3.40 (3H, s)], and three
olefinic protons [dH 5.24 (1H, d,
J ¼ 8.8 Hz), 5.58 (1H, dd, J ¼ 9.8,
3.6 Hz), 5.96 (1H, dd, J ¼ 9.8, 2.1 Hz)].
The 13C NMR spectrum showed two oxy-
methine carbons at dC 76.9 and 77.8, and
two C ¼ C double bonds at dC 129.0,
132.1, 134.0, and 134.3 (Table 1). The
NMR data were in good agreement with
those of (19R,23E)-5b,19-epoxy-19-meth-
oxycucurbita-6,23,25-trien-3b-ol [5],
except for the signals of the side-chain
moiety, suggesting that 2 had a 19-
methoxy-5b,19-epoxy-3b-hydroxy-10a-
cucurbitan-6-ene ring system [5]. The
NMR data of 2 also showed the presence
of a b-D-glucopyranosyl moiety [dH 4.26
(d, J ¼ 7.8 Hz, H-10)], and a long chain of
C-atoms [dC 18.5 (C-26), 19.9 (C-21), 26.2
(C-27), 33.8 (C-20), 44.3 (C-22), 76.9
(C-23), 129.0 (C-24), 134.3 (C-25)], which
Compound 2 was also isolated as a
white powder. Its molecular formula was
determined as C37H60O9 according to the
HR-ESI-MS at m/z 671.4106 [M þ Na]þ.
The IR spectrum exhibited absorption
bands at 3415 and 1648 cm21, indicating
OCH3
O
H
O
OH
O
OH
O
HO
HO
HO
OCH3
HO
OH
1
2
HMBC
Figure 2. Key HMBC correlations of 1 and 2.

360
Y.-B. Zhang et al.
Table 1. 1H and 13C NMR spectral data of 1 and 2 (in CD3OD, d in ppm, J in Hz).
1
2
No.
dH
dC
dH
dC
1
2
3
4
5
6
7
8
1.49 (m), 1.56 (m)
1.72 (m), 1.97 (m)
3.54 (br s)
–
22.2
30.0
77.2
42.6
148.6
122.2
76.8
46.9
51.3
37.7
23.4
30.4
47.1
48.9
36.0
28.7
52.2
15.4
209.7
33.9
19.4
45.7
66.7
130.6
133.6
18.3
26.0
27.9
26.1
19.0
56.3
1.51 (m), 1.61 (m)
1.66 (m), 1.83 (m)
3.37 (m)
18.5
28.3
77.8
38.5
88.1
132.1
134.0
43.3
49.3
42.1
24.4
32.1
46.5
49.7
34.8
29.2
52.3
15.3
113.4
33.8
19.9
44.3
76.9
129.0
134.3
18.5
26.2
20.9
24.6
20.5
–
–
–
5.99 (d, 4. 6)
3.52 (br d, 5.8)
2.02 (br s)
–
5.58 (dd, 9.8, 3.6)
5.96 (dd, 9.8, 2.1)
2.90 (br s)
–
2.46 (t, 9.8)
1.65 (m), 1.80 (m)
1.65 (m), 1.65 (m)
–
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7-OMe
19-OMe
10
2.59 (m)
1.45 (m), 2.37 (m)
1.26 (m), 1.72 (m)
–
–
–
1.38 (m), 1.38 (m)
1.41 (m), 1.94 (m)
1.53 (m)
1.35 (m), 1.35 (m)
1.43 (m), 1.96 (m)
1.46 (m)
0.95 (s)
9.77 (s)
1.73 (m)
0.90 (s)
4.70 (s)
1.81 (m)
1.01 (d, 6.4)
0.97 (m), 1.63 (m)
4.42 (td, 9.5, 3.3)
5.17 (dt, 8.4, 1.4)
–
0.99 (d, 6.4)
1.00 (m), 1.80 (m)
4.53 (m)
5.24 (d, 8.8)
–
1.70 (s)
1.70 (s)
1.20 (s)
1.67 (d, 1.0)
1.70 (d, 0.8)
1.09 (s)
1.25 (s)
0.85 (s)
3.26 (s)
0.88 (s)
0.89 (s)
3.40 (s)
4.26 (d, 7.8)
3.12 (m)
3.29 (m)
3.29 (m)
58.2
103.9
75.7
78.6
71.8
78.0
63.0
20
30
40
50
3.14 (m)
3.65 (dd, 11.8, 5.3)
3.80 (dd, 11.8, 2.5)
60
Notes: Overlapped signals were reported without designating multiplicity. Spectra were measured at 400 MHz.
were almost superimposable on those of
momordicine II (4) [4].
nected to C-23. Consequently, the struc-
ture of 2 was determined and named as
charantin B.
The HMBC correlation (Figure 2)
between H-21 at dH 0.99 and C-17 at dC
52.3 suggested that the long chain was
connected to C-17. Besides, the HMBC
correlation between H-10 at dH 4.26
and C-23 at dC 76.9 indicated that the
b-D-glucopyranosyl moiety was con-
The known compounds were identified
as momordicines I (3) and II (4) [4],
3b,7b,25-trihydroxycucurbita-5,(23E)-
dien-19-al (5) [6], and momordicoside K
(6) [7], by comparing their physical and
spectroscopic data with the literature.

Journal of Asian Natural Products Research
361
In addition, the absolute configurations of
momordicines I (3) and II (4) at C-23 were
determined as R configuration by the
modified Mosher’s method and acid
hydrolysis.
£ 250 mm; Nacalai Tesque). All the
reagents were purchased from Tianjin
Damao Chemical Company (Damao,
Tianjin, China).
3.2 Plant material
3. Experimental
The leaves of M. charantia were collected
in an experimental field of College of
Resources and Environment, South China
Agricultural University in August, 2005
and authenticated by Prof. Mao-Xin Zhang.
A voucher specimen (2010110723) has
been deposited at the Laboratory of Insect
Ecology, College of Resources and
Environment, South China Agricultural
University.
3.1 General experimental procedures
Optical rotations were determined by a
Jasco P-1020 digital polarimeter (Jasco,
Shanghai, China). UV spectra were
recorded using a Jasco V-550 UV/VIS
spectrophotometer (Jasco). A Jasco FT/IR-
480 plus FT-IR spectrometer was used
to determine IR spectra (Jasco). ESI-MS
data were obtained with a Finnigan LCQ
Advantage Max mass spectrometer
(Thermo Electron, Massachusetts, USA).
HR-ESI-MS data were measured on an
Agilent 6210 LC/MSD TOF mass spec-
trometer (Waters, Massachusetts, USA).
1D and 2D NMR spectra were performed
on a Bruker AV-400 spectrometer (Bruker,
Faellanden, Switzerland) using TMS as the
internal standard, and chemical shifts (d)
were expressed in ppm with reference to
the solvent signals. Open column chroma-
tography (CC) was performed using
macroporous resin (Diaion HP-20, Shang-
hai, China), silica gel (200–300 mesh;
Qingdao Marine Chemical Plant, Qingdao,
China), ODS silica gel (50 mm; YMC,
Tokyo, Japan), and Sephadex LH-20 (25–
100 mm, Fluka, Buchs, Switzerland). Thin-
layer chromatography (TLC) was per-
formed using precoated silica gel plates
(GF254, Yantai Jiangyou Silica Gel
Technology Development Co., Ltd., Yan-
tai, China). Analytical HPLC was carried
out on a Waters chromatograph equipped
with an evaporative light-scattering detec-
tor, a P680 pump, and a C18 reversed-
phase column (Cosmosil, 5 mm, 4.6 mm
£ 250 mm; Nacalai Tesque, Kyoto, Japan).
Preparative HPLC was performed on a
Varian Prostar system equipped with UV
detectors (Varian, California, USA) and a
preparative Cosmosil C18 column (20 mm
3.3 Extraction and isolation
Air-dried leaves of M. charantia (1.0 kg)
were macerated with 90% EtOH under
room temperature. The combined extracts
were evaporated in vacuo to afford a crude
extract which was suspended in water and
partitioned successively with petroleum
ether (19 g), EtOAc (20 g), and n-BuOH
(60 g). The EtOAc fraction was chromato-
graphed on a silica gel column, using
mixtures of petroleum ether, EtOAc, and
MeOH with increasing polarity as eluents
to yield nine fractions. Fraction 7 (0.54 g)
was purified by Sephadex LH-20 (CHCl3–
MeOH, 1:1) and then by preparative HPLC
to yield compound 1 (MeOH/H2O, 70:30,
8 ml/min, 210 nm; tR 45.3 min, 8.7 mg) and
compound 2 (MeOH/H2O, 70:30, 8 ml/
min, 210 nm; tR 22.5 min, 6.8 mg), respect-
ively. Fraction 6 (2.5 g) was subjected to
repeated chromatograph on silica gel
(CHCl3/MeOH, 100:1 ! 85:15) to yield
3 (50 mg) and 5 (25 mg). Fraction 8 (4.6 g)
was applied to silica gel CC (CHCl3/
MeOH, 100:1 ! 80:20) to yield 4 (30 mg).
3.3.1 Compound 1
27:0
White powder; ½aꢀD þ 176.0 (c ¼ 0.67,
MeOH);UV(MeOH)lmax (nm)(log1):210

362
Y.-B. Zhang et al.
(3.6); IR (KBr) nmax: 3444, 2954, 1697,
1
1.015 (3H, d, J ¼ 4.9 Hz, H-21), 6.114
(1H, m, H-23), 5.409 (1H, m, H-24), 1.868
(3H, br s, H-26), 1.741 (3H, br s, H-27),
1.205 (3H, s, H-28), 1.205 (3H, s, H-29),
0.722 (3H, s, H-30).
1644, 1454, 1378, 1081cm21; for H and
13C NMR spectral data, see Table 1; HR-
ESI-MS m/z: 509.3615 [M þ Na]þ (calcd
for C31H50O4Na, 509.3601).
Compound 3b: 1H NMR (300 MHz,
C5D5N): d 5.181 (1H, br s, H-3), 6.033
(1H, overlapped, H-6), 5.703 (1H, d,
J ¼ 5.5 Hz, H-7), 2.045 (1H, s, H-8),
0.886 (3H, s, H-18), 9.813 (1H, s, H-19),
1.067 (3H, d, J ¼ 6.2 Hz, H-21), 6.068
(1H, m, H-23), 5.245 (1H, m, H-24), 1.866
(3H, br s, H-26), 1.712 (3H, br s, H-27),
1.292 (3H, s, H-28), 1.212 (3H, s, H-29),
0.810 (3H, s, H-30).
3.3.2 Compound 2
27:0
White powder; ½aꢀD 252.2 (c ¼ 1.0,
MeOH); UV (MeOH) lmax (nm) (log 1):
208 (3.9); IR (KBr) nmax: 3415, 2941,
1648, 1449, 1378, 1077 cm21; for H and
1
13C NMR spectral data, see Table 1; HR-
ESI-MS m/z: 671.4106 [M þ Na]þ (calcd
for C37H60O9Na, 671.4130).
3.4 Preparation of (R)- and (S)-MTPA
esters (3a and 3b) of momordicine I (3)
3.5 Acid hydrolysis and GC analysis of 4
Solution of compound 4 (1.5 mg) was
hydrolyzed with 1.5 ml of 2 N HCl
(MeOH) for 3 h at 808C. The reaction
mixture was dissolved in H2O and
extracted with CH2Cl2. The aglycone
was elucidated as 3 by comparison of
The absolute configuration of momordi-
cine I (3) was determined for the first time
by the modified Mosher’s method [8].
Compound 3 (1.5 mg) was dissolved
in deuterated pyridine (0.5 ml) in a clean
NMR tube under a gentle nitrogen stream
25:4
their optical rotations (½aꢀD þ 83.9
25:4
1
and a H NMR spectrum was recorded as
(c ¼ 1.0, MeOH) for the aglycone,
a reference. Then (S)-a-methoxy-a-(tri-
fluoromethyl) phenylacetic chloride (5 ml)
was added into the NMR tube under the
N2 gas stream and immediately shaken
until uniformly mixed. After sealing with
parafilm, the reaction NMR tube was kept
for 8 h at room temperature. The 1H NMR
spectrum, recorded directly from the
reaction NMR tube, showed the pro-
duction of the corresponding (R)-MTPA
ester (3a). In an identical fashion, another
portion of compound 3 (1.5 mg) was
reacted in a second NMR tube with (R)-
a-methoxy-a-(trifluoromethyl)phenylace-
tic chloride (5 ml) at room temperature for
8 h using deuterated pyridine (0.5 ml) as
the solvent, to afford the (S)-MTPA ester
of 3 (3b).
½aꢀD þ 86.7 (c ¼ 1.0, MeOH) for com-
pound 3), TLC, HPLC, and NMR spectral
data. The aqueous layer was concentrated
and dried by N2. Then, 1 ml of dry
pyridine and 2 mg of L-cysteine methy-
lester hydrochloride were added to the
residue. The mixture was heated at 608C
for 2 h and concentrated to dryness with
N2. N-(trimethylsilyl) imidazole (0.2 ml)
was added into the mixture and then kept
at 608C for 1 h. At last, the solution was
diluted with H2O (1 ml) and extracted with
hexane (1 ml). The organic layer was
analyzed using GC under the following
conditions: AT-SE-30 (0.5 mm £ 0.32 mm
£ 30 m), detector: FID, column tempera-
ture: 2208C, detector temperature: 2708C,
injector temperature: 2708C, and carried
gas: N2. The standard D-glucose was
subjected to the same reaction and GC
analysis under the above conditions. As a
result, D-glucose was detected from the
hydrolysates of 4.
Compound 3a: 1H NMR (300 MHz,
C5D5N): d 5.262 (1H, br s, H-3), 6.056
(1H, overlapped, H-6), 5.676 (1H, d,
J ¼ 5.3 Hz, H-7), 2.040 (1H, s, H-8),
0.782 (3H, s, H-18), 9.939 (1H, s, H-19),

Journal of Asian Natural Products Research
363
[4] M. Yasuda, M. Iwamoto, H. Okabe, and
T. Yamauchi, Chem. Pharm. Bull. 32, 2044
(1984).
Acknowledgments
This study was carried out with the support
from the program of National Natural Science
Foundation of China (Project Nos: 81273390
and 81202429) and the Natural Science
Foundation of Guangdong Province (No.
S2013020012864).
[5] Y. Kimura, T. Akihisa, N. Yuasa, M.
Ukiya, T. Suzuki, M. Toriyama, S.
Motohashi, and H. Tokuda, J. Nat. Prod.
68, 807 (2005).
[6] M.O. Fatope, Y. Takeda, H. Yamashita,
H. Okabe, and T. Yamauchi, J. Nat. Prod.
53, 1491 (1990).
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