Cucurbitane-type triterpene glycosides from the fruits of Momordica charantia

Article abstract of DOI:10.1002/mrc.2582

The chemical study of Momordica charantia fruits led to the isolation of three new cucurbitane triterpene glycosides, momordicosides U, V, and W (1-3). The structures of these compounds were determined to be (19R,23R)-5β, 19-epoxy-19-methoxycucurbita-6,24-diene-3β, 23-diol 3-O-β-D- allopyranoside (1), (23R)-5β, 19-epoxycucurbita-6,24-diene-3β, 23-diol 3-O-β-D-allopyranoside (2), and (19R)-5β, 19-epoxy-19,25- dihydroxycucurbita-6,23(E)-diene-3β-ol 3-O-β-D-glucopyranoside (3), by chemical and spectroscopic methods.

Full text of DOI:10.1002/mrc.2582

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Spectral Assignments and Reference Data
Received: 3 November 2009
Revised: 27 January 2010
Accepted: 27 January 2010
Published online in Wiley Interscience: 11 March 2010
(www.interscience.com) DOI 10.1002/mrc.2582
Cucurbitane-type triterpene glycosides
from the fruits of Momordica charantia
Nguyen Xuan Nhiem,a,b Phan Van Kiem,a Chau Van Minh,a Ninh Khac Ban,c
Nguyen Xuan Cuong,a Le Minh Ha,a Bui Huu Tai,a,b Tran Hong Quang,a,b
Nguyen Huu Tunga,b and Young Ho Kimb∗
The chemical study of Momordica charantia fruits led to the isolation of three new cucurbitane triterpene glycosides,
momordicosides U, V, and W (1–3). The structures of these compounds were determined to be (19R, 23R)-5β, 19-epoxy-
19-methoxycucurbita-6,24-diene-3β, 23-diol 3-O-β-D-allopyranoside (1), (23R)-5β, 19-epoxycucurbita-6,24-diene-3β, 23-diol
3-O-β-D-allopyranoside (2), and (19R)-5β, 19-epoxy-19,25-dihydroxycucurbita-6,23(E)-diene-3β-ol 3-O-β-D-glucopyranoside
c
(3), by chemical and spectroscopic methods. Copyright ꢀ 2010 John Wiley & Sons, Ltd.
Keywords: 1H NMR; 13C NMR; Momordica charantia; Cucurbitaceae; cucurbitane triterpene glycoside; momordicosides U-W
Introduction
32.7), C-22 (δC 44.4), C-24 (δC 129.0), and with C-25 (δC 133.6). This
permitted the positioning of the double bond at C-24/C-25 and
the hydroxy group at C-23 in the side chain. The stereochemistry
at C-23 of 1 was deduced to be R by comparing the 13C-NMR
chemical shifts of side chain [δC: 32.7 (C-20), 44.4 (C-22), 65.9
(C-23), 129.0 (C24), and 133.6 (C-25)] with those of (23R)-cycloart-
24-ene-3β,23-diol [δC 33.0 (C-20), 44.5 (C-22), 66.1 (C-23), 129.1
(C24), and 133.8 (C-25)] and (23S)-cycloart-24-ene-3β,23-diol [δC
33.5 (C-20), 44.5 (C-22), 67.3 (C-23), 128.4 (C24), and 135.6 (C-
25)].[17] On the other hand, the HMBC correlations (Fig. 1) were
observed between oxygenated methine H-19 at δH (4.59, 1H, s)
and methoxyl (δC 58.4), C-5 (δC 85.6), C-8 (δC 41.7), C-9 (δC 47.4), and
C-11(δC 23.0). Thisevidenceconfirmedthatmethoxylgroupwasat
C-19, and epoxy bridge was at C-5 and C-19. Compound 1 showed
a diagnostic NOE correlation (Fig. 2) between H-1β and H-19 in the
NOESY experiment, which supported the 19R stereochemistry.[16]
The 19R-stereochemistry allows very close spatial orientation of
H-19 to H-1β, which is consistent with the appearance of the NOE
correlation between H-1β and H-19 in the NOESY spectrum. Acid
hydrolysis of 1 (see Experimental) provided the allose (identified
as TMS derivatives). Moreover, the HMBC correlation between
allose H-1ꢁ at δH 4.66 (1H, d, 4.66) and C-3 (δC 82.9) confirmed
sugar moiety was attached to C-3. Based on the above evidence,
1 was identified as (19R,23R)-5β,19-epoxy-19-methoxycucurbita-
6,24-diene-3β,23-diol 3-O-β-D-allopyranoside, a new compound
named momordicoside U ( 1).
The plant Momordica charantia L. (Cucurbitaceae) is widely
cultivated in many tropical regions of the world and its
fruit has been used as a bitter stomachic, a laxative, an
antidiabetic, and an anthelmintic agents for children in Chinese,
Indian, Vietnamese, and Indonesian traditional medicines.[1]
Cucurbitane-type triterpennoids are major components of this
plant, and more than 50 exemplars have been isolated from
the roots,[2] fruits,[3–11] seeds,[12] leaves, and vines[13–15] of this
plant. This paper deals with isolation and structure identification
of three new saponins from M. charantia fruits. Their structures
were elucidated as (19R,23R)-5β,19-epoxy-19-methoxycucurbita-
6,24-diene-3β,23-diol 3-O-β-D-allopyranoside (1), (23R)-5β,19-
epoxycucurbita-6,24-diene-3β,23-diol 3-O-β-D-allopyranoside (2),
and (19R)-5β,19-epoxycucurbita-6,23(E)-diene-3β,19,25-triol 3-O-
β-D-glucopyranoside (3) by physical, chemical, and spectroscopic
techniques, including 1D- and 2D-NMR as well as in comparison
with the reported corresponding data.
Results and Discussion
Compound 1 was obtained as a white powder. Its molecular
formula was determined as C37H60O9 due to the 37 carbon signals
in the 13C-NMR spectrum and an [M+H]+ ion peak in the positive
HRESIMS at m/z 649.4322 (calcd. for C37H61O9: 649.4316). A 13C-
NMR and DEPT experiments resolved the 37 carbon resonances
into eight methyl, 8 methylene, 15 methine, and 6 quaternary
carbons. The 1H- and 13C-NMR data (Table 1) were very similar to
those of charantosides I, II[16] except for the signals due to the side
chain. Compound 1 exhibited 1H-NMR signals for the side chain
protons at δH 0.91 (3H, d, 6.6); a secondary methyl, 1.61 (3H, s) and
1.63 (3H, s); two vinylic methyls, 4.40 (1H, dt, 3.0; 9.0), and 5.13
(1H, d, 9.0); an olefinic methine. In the HMBC spectrum (Fig. 1),
both methyl groups at δH 1.63 (3H, s, H-26) and 1.61 (3H, s, H-27)
correlated with carbons C-24 (δC 129.0) and C-25 (δC 133.6); proton
signal at δH 4.40 (1H, dt, 3.0, 9.0, H-23) correlated with C-20 (δC
∗
Correspondence to: Young Ho Kim, College of Pharmacy, Chungnam National
University, Daejeon 305-764, Korea. E-mail: yhk@cnu.ac.kr
a
Institute of Natural Products Chemistry, Vietnam Academy of Science and
Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
b
c
College of Pharmacy, Chungnam National University, Daejeon 305-764, Korea
Institute of Ecology and Biological Resources, VAST, 18 Hoang Quoc Viet,
Caugiay, Hanoi, Vietnam
c
Magn. Reson. Chem. 2010, 48, 392–396
Copyright ꢀ 2010 John Wiley & Sons, Ltd.

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Spectral Assignments and Reference Data
Table 1. The NMR spectral data of compounds 1–3
1
2
3
a
a
b
Positions
1
δC
δHa (J in Hz)
δC
δHa (J in Hz)
δC
δHb (J in Hz)
1.41 (1H, α)c
18.2
1.35 (1H, α)c
1.55 (1H, β)c
1.80 (2H, dd, 4.2; 13.2)
3.47 (1H, br s)
–
18.3
1.30 (1H, α)c
1.56 (1H, β)c
19.3
1.55 (1H, dd, 4.8; 16.5) (β)
2.12 (2H, dd, 4.2; 13.2)
3.36 (1H, br s)
–
2
27.2
82.9
38.7
85.6
132.1
132.0
41.7
47.4
41.0
23.0
30.7
27.3
84.2
38.5
86.3
132.7
130.6
51.9
44.8
39.5
23.3
30.8
1.89 (2H, dd, 4.2; 13.2)
3.35 (1H, br s)
–
27.9
87.0
39.7
86.3
134.1
133.6
42.7
46.2
42.6
24.0
31.8
3
4
5
–
–
–
6
5.97 (1H, d, 9.6)
5.53 (1H, d, 9.6)
2.86 (1H, br s)
–
5.97 (1H, d, 9.6)
5.51 (1H, d, 9.6)
2.28 (1H, br s)
–
6.05 (1H, dd, 1.8; 9.6)
5.54 (1H)c
7
8
2.89 (1H, br s)
–
9
10
11
12
2.31 (1H, dd, 6.0; 13.2)
1.55 (2H)c
2.18 (1H, dd, 4.2; 10.8)
1.59 (2H)c
2.43 (1H, dd, 5.4; 13.2)
1.60 (1H, dt, 2.4; 7.8)
1.58(1H)c
1.29 (2H)c
1.54 (2H)c
1.67(1H, d, 2.4)
–
13
14
15
16
17
18
19
45.1
48.1
33.5
28.2
50.9
14.6
112.6
–
38.5
48.6
33.1
28.1
50.8
14.8
79.7
–
49.4
39.7
34.7
29.0
51.3
15.2
106.0
–
–
–
1.30 (2H)c
1.33 (2H, m)
1.39 (1H, m)
0.84 (3H, s)
4.59 (1H, s)
1.23 (2H)c
1.36 (2H)c
1.33 (2H, m)
1.37 (1H, m)
0.81 (3H, s)
3.41 (1H, d, 7.8)
3.56 (1H, d, 7.8)
1.62 (1H, m)
0.90 (3H, d, 6.6)
1.59 (2H, m)
1.98 (2H, m)
1.52 (1H, m)
0.90 (3H, s)
5.05 (3H, s)
20
21
22
32.7
18.6
44.4
1.62 (1H, m)
0.91 (3H, d, 6.6)
1.60 (2H, m)
32.5
18.6
44.3
37.6
19.1
40.3
1.51 (1H, m)
0.91 (3H, d, 6.6)
1.77 (1H, m)
2.16 (1H, br s)
5.54 (1H)c
23
24
25
26
27
28
29
30
19-OMe
1ꢁ
65.9
129.0
133.6
25.7
18.1
21.0
24.9
19.7
58.4
100.6
72.3
70.1
68.2
74.2
63.0
4.40 (1H, dt, 3.0; 9.0)
5.13 (1H, d, 9.0)
–
65.8
129.0
133.5
25.7
4.39 (1H, dt, 3.0; 9.0)
5.12 (1H, d, 9.0)
–
125.9
140.8
71.2
30.1
30.1
20.5
25.7
20.2
5.54 (1H)c
–
1.63 (3H, s)
1.63 (3H, s)
1.61 (3H, s)
1.08 (3H, s)
0.80 (3H, s)
0.78 (3H, s)
1.23 (3H, s)
1.23 (3H, s)
1.12 (3H, s)
0.85 (3H, s)
0.90 (3H, s)
1.61 (3H, s)
18.1
1.13 (3H, s)
20.5
0.79 (3H, s)
25.5
0.78 (3H, s)
19.9
3.38 (3H, s)
4.66 (1H, d, 7.8)
3.37 (1H, dd, 3.0; 7.8)
4.18 (1H, d, 3.0)
3.54 (1H, dd, 3.0; 10.2)
3.63 (1H, m)
102.1
71.4
70.5
68.3
73.8
63.0
4.66 (1H, d, 7.8)
3.36 (1H, dd, 3.0; 7.8)
4.17 (1H, d, 3.0)
107.5
75.4
77.8
71.8
77.6
62.8
4.21 (1H, d, 7.8)
3.19 (1H, t, 7.8)
2ꢁ
3ꢁ
3.20 (1H, t, 8.4)
4ꢁ
3.52 (1H, dd, 3.0; 10.2)
3.62 (1H, m)
3.25 (1H, t, 8.4)
5ꢁ
3.33 (1H, m)
6ꢁ
3.70 (1H, dd, 5.4; 10.8)
3.83 (1H, dd, 4.2; 10.8)
3.70 (1H, dd, 5.4; 10.8)
3.83 (1H, dd, 4.2; 10.8)
3.62 (1H, dd, 5.4; 11.4)
3.80 (1H, dd, 2.4; 11.4)
Assignments were done by HMQC, HMBC, COSY, and NOESY experiments.
a Measured in CDCl3.
b Measured in MeOD.
c Overlapped signals.
Compound 2 possesses the molecular formula of C36H58O8
as determined from HRESIMS ([M+H]+) m/z 619.4216 (calcd. for
C36H59O8: 619.4210). The 13C-NMR and DEPT spectra (Table 1) of
2 showed 36 carbon signals including 7 methyl, 9 methylene, 14
methine, and 6 quaternary carbons. Comparing 1H- and 13C-NMR
data of 2 with those of 1 showed that the side chain portion of 2
was the same as that of 1 (Fig. 3). This confirmed the positioning
of the double bond at C-24/C-25, the hydroxy group at C-23 in the
side chain and the stereochemistry at C-23 of 2 was deduced to be
R. The exception remarkable between 1 and 2 was on the C-19 (δC
112.6 and 79.7 in 1 and 2, respectively. On the other hand, the 13C-
NMR data of 2 were very similar to those of charantosides IV, VI[16]
except for the signals of the side chain. Moreover, the HMBC cor-
relations (Fig. 1) between H-19 [δH 3.41 (1H, d, 7.8) and 3.56 (1H, d,
7.8)] and C-5 (δC 86.3), C-8 (δC 51.9), C-9 (δC 44.8), and C-10 (δC 39.5)
confirmed that epoxy bridge was at C-5 and C-19. The above data
indicated that the aglycone of 2 was (23R)-5β,19-epoxycucurbita-
6,24-diene-3β,23-diol.[7] TheHMBCcorrelationbetweenalloseH-1ꢁ
c
Magn. Reson. Chem. 2010, 48, 392–396
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
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N. X. Nhiem et al.
OMe
OH
HO
O
O
HO
O
OH
1
OH
OH
HO
O
O
HO
O
OH
OH
2
OH
OH
HO
O
O
HO
O
HO
OH
3
Figure 1. Key HMBC correlations of 1–3.
Me
H
H
H
H
Me
Me
OMe
Me
H
H
H
H
HO
H
O
H
H
H
H
RO
Me
H
H
H
H
Me
H
H
H
H
Me
H
H
Figure 2. Major NOE correlations (↔) for compound 1.
(δH 4.66) and C-3 (δC 84.2) confirmed the sugar moiety attached to
C-3 and identified as D-allose, similar to 1. Accordingly, 2 was iden-
tified as (23R)-5β,19-epoxycucurbita-6,24-diene-3β,23-diol 3-O-β-
D-allopyranoside, a new compound named momordicoside V ( 2).
Compound 3 possesses the molecular formula of C36H58O9
as determined from HRESIMS ([M+H]+) m/z 635.4144 (calcd. for
stereochemistry at C-19 of 3 was deduced to be R by comparing
the13C-NMRchemicalshiftsofC-5(δC 86.3)andC-19(δC 106.0)with
those of (19R)-5β,19-epoxycucurbita-6,23,25-triene-3β,19-diol[18]
[C-5 (δC 86.5) and C-19 (δC 105.3)] and (19S)-5β,19-epoxycucurbita-
6,23,25-triene-3β,19-diol[18] [C-5 (δC 85.0) and C-19 (δC 107.4)]. The
R configuration of C-19 was also supported by the NOE correlation
between H-1β and H-19 in the NOESY spectrum. On the other
hand, two methyl groups [δH 1.23 (3H, s, H-26) and δH 1.23 (3H,
d, H-27) correlated with carbons C-24 (δC 140.8) and C-25 (δC
71.2) in the HMBC spectrum, indicating the positioning of the
double bond at C-23/C-24 and the hydroxy group at C-25 in the
side chain. Acid hydrolysis of 3 (see Experimental) provided the
D-glucose (identified as TMS derivatives). Moreover, the HMBC
cross peaks from glucose H-1ꢁ at δH (4.21, 1H, d, 7.8) to C-3 (δH 87.0)
C
36H59O9: 635.4159). The 13C-NMR and DEPT spectra of 3 showed
36carbonsignalsincluding7methyl,8methylene,15methine,and
1
6 quaternary carbons. The H- and 13C-NMR data (Table 1) were
very similar to those of goyaglycoside-a[4] except for the signals of
C-19. The HMBC correlations were observed between oxygenated
methine H-19 at δH 5.05 (1H, s) and C-5 (δC 86.3), C-9 (δC 46.2), C-10
(δC 42.6), and C-11 (δC 24.0). This evidence confirmed that hydroxy
group was at C-19, and epoxy bridge was at C-5 and C-19. The
c
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Spectral Assignments and Reference Data
24
27
HPLC system (600 pump, 600 controller, and a 996 photodiode
array detector). Column chromatography was performed using a
silica-gel (Kieselgel 60, 70–230 mesh and 230–400 mesh, Merck,
Darmstadt, Germany) or YMC RP-18 resins (30–50 µm, Fujisilisa
Chemical Ltd., Kyoto, Japan), and TLC using a pre-coated silica-
gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm,
Merck).
21
18
23
20
17
R
26
OH
11
13
19
9
6′
HO
O
30
3
5
O
4
2′
O
4′
HO
1′
OH
1
2
R = OCH3
R = H
28
29
Plant material
OH
The fruits of M. charantia were collected in Vuthu, Thaibinh
province, Vietnam during June, 2009, and identified by one of
authors (Dr Ninh Khac Ban). A voucher specimen (INPC MC0609)
was deposited at the Herbarium of Institute of Natural Products
Chemistry, VAST, Vietnam.
OH
OH
HO
HO
O
O
O
Extraction and isolation
HO
3
OH
The dried fruits of M. charantia (2.0 kg) were extracted with MeOH
three times under reflux for 15 h to yield 300 g of a dark solid
extract, which was then suspended in water and successively
partitioned with chloroform (CHCl3) and ethyl acetate (EtOAc)
to obtain CHCl3 (150.0 g), EtOAc (50.0 g), and water (100.0 g)
extracts after removal of the solvents in vacuo. The CHCl3 extract
(150.0 g) was chromatoghraphed on a silica-gel column eluting
with CHCl3 –MeOH gradient (100 : 0–5 : 1, v/v) to obtain four sub-
fractions designated F1 (50.5 g), F2 (33.4 g), F3 (38.0 g), and
F4 (28.1). Sub-fraction F2 was chromatographed on a silica-
gel column eluting with CHCl3 –MeOH (10 : 1, v/v) to obtain
compounds 1 (21.5 mg) and 3 (15.2 mg). Sub-fraction F3 was
further chromatographed on a silica-gel column eluting with
CH2Cl2 –MeOH (90 : 10, v/v) to give five smaller fractions, F3A–F3E.
Fraction F3B was chromatographed on an YMC RP-18 column
eluting with acetone–water (1 : 1, v/v) to yield 2 (19.6 mg).
Figure 3. Structures of 1–3.
confirmed sugar moiety was attached to C-3. Based on the above
evidence, 3 was identified as (19R)-5β,19-epoxycucurbita-6,23(E)-
diene-3β,19,25-triol 3-O-β-D-glucopyranoside, a new compound
named momordicoside W ( 3).
Experimental
General experimental procedures
All NMR spectra were recorded on a Jeol ECA 600 spectrom-
eter operated at 600 and 150 MHz for hydrogen and carbon
respectively. NMR measurements, including 1H, 13C, HSQC, gra-
dient heteronuclear multiple bond coherence (gHMBC) NOESY
and COSY, experiments, were carried out using 5-mm probes at
22.2 ◦C from CDCl3, or CD3OD solutions, with TMS as the internal
standard. Chemical shifts are reported in parts per million from
TMS. Signals are reported as follows: chemical shift (ppm), cou-
pling constants (Hz) and assignment. The pulse conditions were
as follows: for the 1H spectrum, spectrometer frequency (SF) =
600.17 MHz, acquisition time (AQ) = 1.4549 s, relaxation delay (RD)
= 5 s, pulse 90◦ width = 14.4 µs, spectral width (SW) = 11261 Hz,
and digital resolution (DR) = 0.6873 Hz; for the 13C spectrum, SF
= 150.91 MHz, AQ = 0.692 s, RD = 2 s, pulse 90◦ width = 11.1 µs,
SW = 47348 Hz, and DR = 1.45 Hz; for the COSY spectrum, AQ
= 0.158 s, RD = 1.5 s, SW = 6464 Hz, and number of points (NP)
= 2048; for the NOESY spectrum: RD=1.5 s, SW = 8080 Hz, NP =
2048, number of increments (NI) = 256, and mixing time 0.25 s;
for the HMQC experiment, AQ = 0.1289 s, RD = 1.5 s, SW = 7947
(1H) and 33200 (13C) Hz, NP = 1028, FT size = 1024 × 4096, and
140 Hz one-bond coupling constant; for the HMBC experiment,
AQ = 0.2577 s, RD = 1.5 s, SW = 7947 (1H) and 34200 (13C) Hz,
NP = 2048, FT size = 2048 × 4096, and 8-Hz long-range coupling
constant. Data processing was carried out with JEOL Delta NMR
v4.3.6 programs.
Acid hydrolysis
Each compound (1–3, 2 mg) was dissolved in HCl 1N (dioxane-
H2O, 1 : 1, 1 ml) and then heated to 80 ◦C in a water bath for
3 h. The acidic solution was neutralized with silver carbonate
and the solvent thoroughly driven out under N2 gas overnight.
After extraction with CHCl3, the aqueous layer was concentrated
to dryness using N2 gas. The residue was dissolved in 0.1 ml
of dry pyridine, and then L-cysteine methyl ester hydrochloride
in pyridine (0.06 M, 0.1 ml) was added to the solution. The
reaction mixture was heated at 60 ◦C for 2 h, and 0.1 ml of
trimethylsilylimidazolewasadded, followedbyheatingat60 ◦Cfor
1.5 h. The dried product was partitioned with n-hexane and H2O
(0.1 ml each), and the organic layer was analyzed by gas–liquid
chromatography (GC): column SPB-1 (0.25 mm × 30 m); detector
FID, column temp 210 ◦C, injector temp 270 ◦C, detector temp
300 ◦C, carrier gas He (2 ml/min). Under these conditions, standard
sugars gave peaks at tR (min) 5.17, 8.24, 11.11 and 14.26 for
D-allose, L-allose, D-glucose, and L-glucose, respectively. Peaks at
tR (min) 5.17 of D-allose was observed for both compounds 1
and 2, and peak at tR (min) 11.11 of D-glucose was observed for
compound 3.
The HRTOF mass spectra were obtained using a JEOL JMS-
T100LC spectrometer. The ESI-MS was obtained on an AGILENT
1200 SERIES LC-MSD Trap spectrometer. GC spectra were
recorded on a Shidmazu-2010 spectrometer. Optical rotations
were determined on a Jasco DIP-370 automatic polarimeter. The
FT-IR spectra were obtained from a Jasco Report-100 infrared
spectrometer. Preparative HPLC was carried out using a Waters
Momordicoside U (1): An amorphous white powder, [α]D25
:
−24.3◦ (c = 0.5 in CHCl3); IR (KBr) νmax cm−1: 3341, 1446, 1028;
positive ESI-MS m/z: 671 [M+Na]+, HRESIMS found m/z: 649.4322
[M+H]+; Calcd. C37H61O9 for 649.4316, 1H- and 13C-NMR (Table 1).
Momordicoside V (2): An amorphous white powder, [α]D25
:
−35.1◦ (c = 0.5 in CHCl3); IR (KBr) νmax cm−1: 3329, 1431, 1018;
c
Magn. Reson. Chem. 2010, 48, 392–396
Copyright ꢀ 2010 John Wiley & Sons, Ltd.
www.interscience.wiley.com/journal/mrc