Two new cucurbitane triterpenoids from the seeds of Momordica charantia

Article abstract of DOI:10.1080/10286020.2014.914502

Two new cucurbitane triterpenoids 1 and 2 were isolated, together with six known compounds, from the seeds of Momordica charantia L. The structures of new compounds were determined to be 3-O-{[β-d-galactopyranosyl(1 → 6)]-O-β-d-galactopyranosyl}-23(R), 24

Full text of DOI:10.1080/10286020.2014.914502

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Two new cucurbitane triterpenoids
from the seeds of Momordica charantia
Lin Maa, Ai-Hua Yuab, Li-Li Suna, Wan Gaoa, Meng-Meng Zhanga, Ya-
Lun Sua, Hua Liub, Teng-Fei Jia & Di-Zao Lic
a State Key Laboratory of Bioactive Substance and Function of
Natural Medicines, Institute of Materia Medica, Chinese Academy
of Medical Sciences & Peking Union Medical College, Beijing
100050, China
b Department of Medicinal Chemistry, Jiangxi University of
Traditional Chinese Medicine, Nanchang 330004, China
c State Key Laboratory of Medicinal Chemical Biology, State
Key Laboratory of Elemento-Organic Chemistry and Tianjin Key
Laboratory of Molecular Drug Research, College of Pharmacy,
Nankai University, Tianjin 300071, China
Published online: 29 Apr 2014.
To cite this article: Lin Ma, Ai-Hua Yu, Li-Li Sun, Wan Gao, Meng-Meng Zhang, Ya-Lun Su,
Hua Liu, Teng-Fei Ji & Di-Zao Li (2014) Two new cucurbitane triterpenoids from the seeds
of Momordica charantia, Journal of Asian Natural Products Research, 16:5, 476-482, DOI:
10.1080/10286020.2014.914502
To link to this article: http://dx.doi.org/10.1080/10286020.2014.914502
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Journal of Asian Natural Products Research, 2014
Vol. 16, No. 5, 476–482, http://dx.doi.org/10.1080/10286020.2014.914502
Two new cucurbitane triterpenoids from the seeds of
Momordica charantia
Lin Maa, Ai-Hua Yuab, Li-Li Suna, Wan Gaoa, Meng-Meng Zhanga, Ya-Lun Sua,
Hua Liub, Teng-Fei Jia* and Di-Zao Lic*
aState Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing
100050, China; bDepartment of Medicinal Chemistry, Jiangxi University of Traditional Chinese
c
Medicine, Nanchang 330004, China; State Key Laboratory of Medicinal Chemical Biology, State
Key Laboratory of Elemento-Organic Chemistry and Tianjin Key Laboratory of Molecular Drug
Research, College of Pharmacy, Nankai University, Tianjin 300071, China
(Received 22 January 2014; final version received 8 April 2014)
Two new cucurbitane triterpenoids 1 and 2 were isolated, together with six known
compounds, from the seeds of Momordica charantia L. The structures of new
compounds were determined to be 3-O-{[b-D-galactopyranosyl(1 ! 6)]-O-b-D-
galactopyranosyl}-23(R), 24(R), 25-trihydroxycucur-bit-5-ene (1), 3-O-[b-D-galacto-
pyranosyl]-25-O-b-D-galactopyranosyl-7(R), 22(S), 23(R), 24(R), 25-pentahydroxy-
cucurbit-5-ene (2), respectively. Their structures were elucidated by the combination of
mass spectrometry, one- and two-dimensional NMR experiments and chemical
reactions.
Keywords: Momordica charantia; seeds; cucurbitane triterpenoids; Cucurbitaceae
1. Introduction
hypoglycemic principles, we have exam-
ined the ethanolic extracts of M. charantia
purchased from Anguo of Hebei Province,
China. We report the isolation and
structural elucidation of two new cucurbi-
tane triterpenoids from the seeds of M.
charantia (Figure 1).
The fruit, seeds, aerial parts, and roots of
Momordica charantia L. (Cucurbitaceae)
have been used to treat diabetes. Over 100
compounds have been isolated from the
fruits, seeds, leaves, canes, and roots of
this genus, mainly cucurbitane- and
oleanene-type triterpenes, Recent studies
have discovered many new cucurbitane
triterpenoids from the fruits and the roots
of M. charantia L. [1–3], and cucurbitane
triterpenoids from the fruits of this genus
showed a significant enhancement of
glucose disposal and increases in fatty
acid oxidation. The cucurbitane triter-
penoids from M. charantia may provide
novel leads for the development of a new
class of AMPK-activating agents [4]. But a
little research on the seeds of M. charantia
was reported [5,6]. To search for the
2. Results and discussion
We have examined the ethanol extract of
the seed of M. charantia and have isolated
two new cucurbitane triterpenoids 1 and 2,
together with six known compounds
momocharaside A (3), momocharaside B
(4) [5], goyasaponin II (5), goyasaponin I
(6) [7], momordicoside C (7), and
momordicoside E (8) [6]. Compounds 3–
6 were isolated for the first time from the
seeds of M. charantia L., whose structures
were determined by comparing their
*Corresponding authors. Email: jitf@imm.ac.cn; zao78@163.com
q 2014 Taylor & Francis

Journal of Asian Natural Products Research
477
OH
22
OH
24
OH
21
18
27
20
17
23
25
22
21
18
27
20
17
23
25
26
12
24
OH
O
O
26
OH
12
11
OH
HO
13
14
11
19
10
OH
16
13
14
19
1
4
16
1
4
2
9
6
OH
2
15
9
8
1''
15
10
8
3
30
OH
3
30
HO
HO
5
7
O
O
OH
7
5
O
O
OH
O
OH
O
OH
1'' OH
OH
6
28
29
28
OH
29
OH
1'
OH
1'
OH
1
2
Figure 1. Structures of compounds 1 and 2 from the seeds of M. charantia.
physical properties and the spectral data
with those reported in the literature.
were very similar to those of 23(R), 24(R),
25-trihydroxycucurbit-5-ene 3-O-{[b-glu-
copyranosyl(1 ! 6)]-O-b-glucopyrano-
syl}-25-O-b-gluco-pyranoside [1]. The
analysis of HMBC spectrum (Figure 2)
confirmed that 1 was 3-O-{[b-D-galacto-
pyranosyl(1 ! 6)]-O-b-D-galactopyrano-
syl}-23(R), 24(R), 25-trihydroxycucurbit-
5-ene.
Compound 1 was obtained as a white
powder. The positive-ion quasimolecular
ion peak was observed at m/z 823
[M þ Na]þ and the molecular formula
C42H72O14 was determined by positive-ion
HR-ESI-MS measurement (m/z 823.4814
[M þ Na]þ; calcd 823.4813). The 1H
NMR spectrum of 1 (Table 1) showed
signals for seven tertiary methyl groups at
d 0.73, 0.81, 0.82, 1.07, 1.45, 1.57, and
1.58 (each 3H, s), a secondary methyl at d
1.25 (3H, d, J ¼ 6.6 Hz), and one olefinic
proton at d 5.41 (1H, d, J ¼ 5.5 Hz). Acid
hydrolysis of 1 furnished galactose, which
was identified by HPLC comparison with
an authentic sample. In the 13C NMR
spectrum of 1 (Table 1), 30 aglycon carbon
signals and 12 sugar signals were found,
indicating that 1 was a triterpene saponin.
The 1H and 13C NMR (Table 1) spectra of
compound 1 exhibited two sugar anomeric
protons assignable to two b-D-galactopyr-
anosyl moiety (dH 5.25, 1H, d, J ¼ 8.0 Hz;
dH 4.86, 1H, d, J ¼ 8.0 Hz). The identities
of the sugar chain sequence were deter-
mined by a combination of DEPT and two-
dimensional NMR experiments (such as
HMQC and HMBC). The sequence of the
sugar chain was deduced from the HMBC
correlations of the anomeric proton signal
H-10 at dH 5.25 (1H, d, J ¼ 8.0 Hz) and C-
3 at dC 87.3, H-100 at dH 4.86 (1H, d,
J ¼ 8.0 Hz) and C-60 at dC 70.7. The 13C
NMR data of the aglycone of compound 1
Compound 2 was obtained as a white
powder. The positive-ion quasimolecular
ion peak was observed at m/z 855
[M þ Na]þ and the molecular formula
C42H72O16 was determined by positive-ion
HR-ESI-MS measurement (m/z 855.4713
[M þ Na]þ; calcd 855.4716). The 1H
NMR spectrum of 2 (Table 1) showed
signals for seven tertiary methyl groups at
d 0.78, 0.81, 0.83, 1.03, 1.44, 1.71, and
1.82 (each 3H, s), a secondary methyl at d
1.37 (3H, d, J ¼ 6.5 Hz), and one olefinic
proton at d 5.42 (1H, d, J ¼ 4.2 Hz). Acid
hydrolysis of 2 furnished galactose, which
was identified by HPLC comparison with
an authentic sample. In the 13C NMR
spectrum of 2 (Table 1), 30 aglycone
carbon signals and 12 sugar signals were
found, indicating that 2 was a triterpene
1
saponin. The H and 13C NMR (Table 1)
spectra of compound 2 exhibited two sugar
anomeric protons assignable to two b-D-
galactopyranosyl moiety (dH 5.21, 1H, d,
J ¼ 8.0 Hz; dH 4.91, 1H, d, J ¼ 7.5 Hz).
The identities of the sugar chain sequence
were determined by a combination of
DEPT and two-dimensional NMR exper-

478
L. Ma et al.
d

Journal of Asian Natural Products Research
479

480
L. Ma et al.
OH
24
21
18
27
20
17
23
22
25
OH
12
11
OH
26
13
14
19
16
1
4
9
6
2
15
10
8
3
30
HO
O
H
7
5
O
O
OH
OH
O
28
OH 1''
1'
29
OH
H
OH
OH
C
H
HMBC
Figure 2. Key HMBC correlations of compound 1.
iments (such as HMQC and HMBC). The
sequence of the sugar chain was deduced
from the HMBC correlations of the
anomeric proton signal H-10 at dH 5.21
(1H, d, J ¼ 8.0 Hz) and C-3 at dC 86.9, H-
100 at dH 4.91 (1H, d, J ¼ 7.5 Hz) and C-25
at dC 80.1 (Figure 3). The 13C NMR data of
the aglycone of compound 2 were very
similar to those of 3-O-[b-D-glucopyrano-
syl(1/6)-b-D-glucopyranosyl]-25-O-b-D-
glucopyranosyl-22(S), 23(R), 24(R), 25-
tetrahydroxycucurbit-5-ene [4]. The rela-
tive configuration of C-7 in compound 2
was assigned on the basis of NOESY
correlation of H-7 and CH3-30, so 7-OH
was b-configuration. The absolute con-
figuration of C-7 was R. So the structure of
compound 2 was determined as 3-O-b-D-
galactopyranosyl-25-O-b-D-galactopyra-
nosyl-7(R), 22(S), 23(R), 24(R), 25-penta-
hydroxyl cucurbit-5-ene.
The structures of compounds 3–8 were
identified by comparison of their spectral
data with those reported in the literature.
OH
22
OH
24
21
18
27
O
20
17
23
25
12
11
26
OH
HO
13
14
19
10
16
O
OH
OH
1
4
9
6
2
1''
15
8
H
30
OH
3
HO
OH
5
OH
7
O
H
O
28
29
1'
OH
H
C
OH
HMBC
Figure 3. Key HMBC correlations of compound 2.

Journal of Asian Natural Products Research
481
3. Experimental
concentrated under reduced pressure to
yield an extract (1.6 kg). The alcohol
extract was partitioned successively with
CHCl3, EtOAc, and n-BuOH. The n-
BuOH-soluble extract (175 g) was sub-
jected to silica gel column chromatog-
raphy with gradient elution [CHCl3–
MeOH 20:1 (2 l), CHCl3 –MeOH 9:1
(2 l), CHCl3–MeOH 4:1 (2 l), CHCl3–
MeOH–H2O 7:3:0.5 (2 l), CHCl3 –
MeOH–H2O 6:4:0.5 (2 l), MeOH (2 l)] to
give 11 fractions (Fr1–11). Compounds 1
(16 mg) and 2 (20 mg) from Fr8 (9.2 g)
were purified by repeated column chro-
matography over silica gel column chro-
matography with gradient elution
[CHCl3 –MeOH–H2O 7:3:0.5], ODS
(MeOH–H2O, 65:35), and Sephadex LH-
20 (MeOH). Further fractionation of Fr6
by silica gel column chromatography with
gradient elution [CHCl3 –MeOH–H2O
3.1 General experimental procedures
Optical rotations were measured with a
Perkin-Elmer 241 MC polarimeter (Perkin-
Elmer, Foster City, CA, USA). UV spectra
were recorded with a UV-2401 spec-
trometer (Shimadzu Corp., Kyoto, Japan).
IR spectra were obtained on a Nicolet 5700
IR spectrometer (Thermo corp., West Palm
Beach, FL, USA). NMR spectra were
recorded on a VARIAN INOVA 500 (1H,
500 MHz; 13C, 125 MHz) spectrometer
(Varian, Palo Alto, CA, USA). ESI-MS
was carried out with Angilent 1100 LC/
MSD (Angilent Technologies Ltd, Santa
Clara, CA, USA). For column chromatog-
raphy, silica gel (200–300 mesh; Qingdao
Marine Chemical Inc., Qingdao, China),
ODS (40–60 mm; YMC Co. Ltd, Kyoto,
Japan), and Sephadex LH-20 (Pharmacia
Biotech AB, Uppsala, Sweden) were used.
The analytical HPLC was carried out on
Angilent 1200 LC with DAD, and the
preparative HPLC was carried out on
Shimadzu LC-20A (Shimadzu Corp.,
Kyoto, Japan) with YMC-Pack ODS
column (20 mm £ 250 mm, 10 mm; YMC
Co. Ltd).
7:3:0.5
(4 l),
CHCl3 –MeOH–H2O
6:4:0.5 (4 l)] gave six subfractions (Fr10 –
60). Compounds 3 (15 mg), 4 (460 mg), 5
(24 mg), and 6 (17 mg) from Fr60 (11.4 g)
were isolated by repeated column chro-
matography over silica gel with CHCl3–
MeOH–H2O 6:4:0.5, ODS (MeOH–H2O,
60:40), and Sephadex LH-20 (MeOH).
Compound 7 (21 mg) from Fr50 (4.9 g) was
separated by repeated column chromatog-
raphy over silica gel with CHCl3 –
MeOH–H2O 7:3:0.5, Sephadex LH-20
(MeOH), and preparative HPLC
(MeOH–H2O, 65:35; flow rate: 7 ml/min;
tR: 20.03 min; detection wavelengths:
210 nm). Compound 8 (12 mg) from Fr5
(6.4 g) was isolated by repeated normal
phase silica gel with CHCl3–MeOH–H2O
6:4:0.5, Sephadex LH-20 (MeOH), and
preparative HPLC (MeOH–H2O, 60:40;
flow rate: 7 ml/min; tR: 26.80 min; detec-
tion wavelengths: 210 nm).
3.2 Plant material
The seeds of M. charantia were purchased
from Anguo of Hebei Province in 2011, and
identified by Associate Professor Lin Ma at
Institute of Materia Medica, Chinese Acad-
emy of Medical Sciences and Peking Union
Medical College. A voucher specimen
(No. ID-S-2547) has been deposited at our
laboratory in the Institute of Materia Medica,
Chinese Academy of Medical Sciences and
Peking Union Medical College.
3.3 Extraction and isolation
The seeds of M. charantia (15.0 kg) were
defatted three times by petroleum ether
(90 l each time), then extracted three times
under reflux in 95% ethanol (90 l each
time), and the combined solution was
3.3.1 Compound 1
25
White powder. ½aꢀD þ 100:0 (c ¼ 1.0,
MeOH); UV (MeOH) lmax nm (log 1):
233 (0.41), 327 (0.57); IR (KBr) nmax
:

482
L. Ma et al.
3402, 2954, 1695, 1603, 1281, 1170, 1020,
1
808C for 6 h. The aglycone was extracted
with chloroform, and the aqueous layer
was evaporated under reduced pressure
and taken as preparations of the normal
sugar derivatives. Then, compounds
1 and 2 only furnished D-galactose
(tR ¼ 13.20 min), which were identified
by HPLC analysis of the derivatives [8]
with standard D-galactose derivative, the
absolute configurations of the galactose
units in compounds 1 and 2 were
determined as D.
817 cm–1; for H and 13C NMR spectro-
scopic data, see Table 1; HR-ESI-MS: m/z
823.4814 [M þ Na]þ (calcd for
C42H72O14Na, 823.4813).
3.3.2 Compound 2
25
White powder. ½aꢀD þ 84:0 (c ¼ 1.0,
MeOH); UV (MeOH) lmax nm (log 1):
232 (0.39), 329 (0.53); IR (KBr) nmax
3413, 2948, 1696, 1601, 1283, 1168,
:
1
819 cm–1; for H and 13C NMR spectro-
scopic data, see Table 1; HR-ESI-MS: m/z
855.4713 [M þ Na]þ (calcd for
C42H72O16Na, 855.4716).
Acknowledgments
This work was supported financially by
National Science and Technology Project of
China (No. 2011ZX09307-002-01). The
authors acknowledge the Department of Instru-
mental Analysis, Institute of Materia Medica,
Chinese Academy of Medical Sciences and
Peking Union Medical College for all spectral
analysis.
3.4 Acidhydrolysisofcompounds1and2
About 80 ml of D-glucose, D-galactose, L-
rhamnose, D-xylose, and L-arabinose aqu-
eous solutions (each 2 mg/ml) was mixed
with 80 ml of 0.5-mol/l PMP CH3OH
solution and 80 ml of 0.3-mol/l NaOH
aqueous solution. The mixtures were
heated at 708C for 30 min and then cooled
to room temperature to which 80 ml of 0.3-
mol/l HCl aqueous solution was added.
The resulted mixture was extracted with
CHCl3 (0.5 ml, 3 times), and the water
fractions were identified by HPLC analysis
(Phenomenex C18, 250 mm £ 4.6 mm,
5 mm); flow phase: A: CH3CN–20 mmol/
l NH4OAc aqueous solution (15:85), B:
CH3CN–20 mmol/l NH4OAc aqueous sol-
ution (40:60); flow rate: 1.2 ml/min;
gradient elution, 0 ! 20 min, volume
fraction of B from 0% to 60%; detection
wavelengths: 245 nm; sample volume:
20 ml.
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aqueous 2-M HCl-1,4-dioxane (1:1) at
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