5β,19-Epoxycucurbitane triterpenoids from momordica charantia and their anti-inflammatory and cytotoxic activity

Article abstract of DOI:10.1055/s-0034-1383307

Five new 5β,19-epoxycucurbitane triterpenoids, taikugausins A-E (1 -5), together with 5β,19-epoxy-25-methoxycucurbita-6,23-diene-3β,19-diol (6), have been isolated and characterized from the 70% EtOH extract of the fresh fruits of Momordica charantia. The

Full text of DOI:10.1055/s-0034-1383307

6
2
Original Papers
5
β,19‑Epoxycucurbitane Triterpenoids from
Momordica charantia and Their Anti‑Inflammatory
and Cytotoxic Activity
1
2
3,4
3
3
4
5
Authors
Chia‑Ching Liaw *, Hui‑Chi Huang *, Ping‑Chun Hsiao , Li‑Jie Zhang , Zhi‑Hu Lin , Syh‑Yuan Hwang , Feng‑Lin Hsu ,
Yao‑Haur Kuo3
,6,7
Affiliations
The affiliations are listed at the end of the article
Key words
Abstract
chemical shifts and the configuration of C‑19 with
an OMe group in 5β,19‑epoxycucurbitane are de-
scribed. Among them, compounds 3 and 4 exhib-
ited remarkable anti‑inflammatory activities by
the inhibition of nitric oxide production at the
concentration of 10 µg/mL. In addition, 3 and 4 al-
so showed moderate cytotoxicity against WiDr,
Hep G2, MCF‑7, and HEp‑2 human tumor cell
lines.
"
l Momordica charantia
!
"
l Cucurbitaceae
Five new 5β,19‑epoxycucurbitane triterpenoids,
taikugausins A–E (1–5), together with 5β,19‑ep-
oxy‑25‑methoxycucurbita‑6,23‑diene‑3β,19‑diol
(6), have been isolated and characterized from the
"
l 5β,19‑epoxycucurbitane
"
l taikuguasins
"
l anti‑inflammatory activity
"
l cytotoxicity
7
0% EtOH extract of the fresh fruits of Momordica
charantia. The chemical structures of compounds
–6 were elucidated on the basis of extensive
spectroscopic analyses, especially 2D NMR
HMQC, HMBC, and NOESY) experiments and
1
received
revised
accepted
May 22, 2014
Sept. 17, 2014
October 19, 2014
(
Supporting information available online at
http://www.thieme‑connect.de/products
HRESIMS data. The relationship between NMR
Bibliography
DOI http://dx.doi.org/
0.1055/s-0034-1383307
Published online December 3,
014
Planta Med 2015; 81: 62–70
Georg Thieme Verlag KG
1
Introduction
viously isolated over seventeen cucurbitane‑type
triterpenoids from the fruits of M. charantia [15–
16]. Our continuing study on the triterpenoid
constituents of the MeOH extract of M. charantia
fruits has now led to the isolation of 5 new cucur-
bitane triterpenoids [taikuguasins A–E (1–5)], and
a known one, 5β,19‑epoxy‑25‑methoxycucurbi-
2
!
The Cucurbitaceae plant Momordica charantia,
which is a climbing annual vine and widely culti-
vated as a vegetable crop in the tropical and sub-
tropical regions of world, is commonly called bit-
ter melon, goya in Japanese, and kugua in China
and Taiwan. The fruit of the plant has been used
in the folk medicine as stomachic and as a remedy
for diabetes in Asia, India, and South America [1–
©
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Dr. Yao‑Haur Kuo
Division of Chinese Materia
Medica Development
National Research Institute of
Chinese Medicine
No. 155‑1, Sec. 2, Linong St.
Taipei 112
Graduate Institute of Integrated
Medicine, College of Chinese
Medicine
China Medical University
No. 91 Hsueh‑Shih Road
Taichung 404
Taiwan
Phone: + 886228201999
ext. 7061
"
ta‑6,23‑diene‑3β,19‑diol (6) [17] (l Fig. 1). The
chemical structures of the isolates were eluci-
dated through detailed spectroscopic analyses,
1
1
2]. Pharmacological studies have demonstrated
including 2D NMR ( H‑ H COSY, HMQC, and
HMBC) and HRMS experiments, as well as in com-
parisons with those of related compounds. The
stereochemistries of the isolated cucurbitanes
(1–5) were determined by 1D NOE and NOESY
analyses. Compounds 1–6 were also evaluated
for cytotoxicity, as well as anti‑inflammatory ac-
tivities by lipopolysaccharide (LPS)‑induced nitric
oxide (NO) production on RAW264.7 macrophage
cell.
the extracts of M. charantia to possess wide me-
dicinal properties, such as antiviral [3], antitumor
[4,5], anti‑diabetic [6,7], antiulcer [8], anti‑in-
flammatory [3,9], and immunomodulatory [10]
activities. Various cucurbitane‑type, oleanane-
type, and ursane‑type triterpenoids, together
with polypeptide and sterols, have been isolated
from the fruits, leaves, and stems of this plant
[11–13]. So far, more than 110 cucurbitane‑type
triterpenoids or their saponins have been isolated
from M. charantia showing anti‑diabetic, anti-
cancer, anti‑obesity, and hypoglycemia activity
Fax: + 886228206150
kuoyh@nricm.edu.tw
Correspondence
Prof. Dr. Feng‑Lin Hsu
Graduate Institute of
Pharmacognosy
Taipei Medical University
No. 250 Wuxing Street
Taipei 110
Taiwan
Phone: + 886227361661
hsu320@tmu.edu.tw
Results and Discussion
!
[14]. In the course of our search for potential anti-
tumor agents from natural sources, we have pre-
The EtOH extract yielded from the fresh fruits of
M. charantia was suspended in H O and succes-
2
sively subjected to Diaion HP‑20 column chroma-
tography to yield five fractions. A preliminary
*
Equal contribution as first authors.
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

Original Papers 63
3
.58, s) and acetal carbon (δ 113.1, C‑19) indicated that the me-
C
Fig. 1 Structures of
compounds 1–6 from
the fruits of Momordica
charantia.
thoxy group was located at C‑19. The structure of the sugar moi-
ety was determined by H‑ H COSY (H‑1′/H‑2′; H‑3′/H‑4′/H ‑6′)
1
1
2
and HMBC (H‑2′/C‑3; H‑4′/C‑3) correlations and further con-
firmed to be D‑ribo‑hexos‑3‑ulose (3‑keto‑Glu) by comparison
of the NMR data with the reported literature [22]. The D-form
was determined by specific rotation value of its sugar from acid
hydrolysis of compound 1 [23]. According to the coupling con-
stant (J = 7.6, H‑1′), the configuration of the sugar moiety was de-
termined as β‑D‑3‑keto‑glucose. Furthermore, the HMBC spec-
trum showed a long‑range correlation between H‑1′ and C‑3,
suggesting that the sugar moiety was attached at C‑3 in 1. The
1
planar structure of 1 was fully established and assigned by H-
1
H COSY, HMQC, and HMBC experiments. The stereochemistry
"
of 1 was determined by NOESY correlations (l Fig. 3), indicating
that H‑3, H‑10, H‑17, Me‑21, Me‑28 and Me‑30 had α‑orienta-
tion, while H‑8 and Me‑18 had β. The NOESY correlation between
H‑19 and H‑8 unambiguously evidenced that acetal carbon
(
C‑19) should have S conformation. On the basis of the above
observation, compound 1 was undoubtedly established to be
9S‑methoxy‑5β,19‑epoxycucurbita‑6,23‑dien-3β,25(E)‑diol 3-
1
O‑β‑D‑3‑ketoglycopyranoside and was named taikuguasin A.
The positive‑ion HRESIMS of taikuguasin B (2) displayed a quasi-
+
molecular ion at m/z 685.4304 ([M + Na] , calcd. 685.4294), indi-
cating a molecular formula of C38H62O . The IR spectrum re-
9
−
1
vealed the presence of hydroxyl (3408 cm ) and olefinic
1463 cm ) moieties. The 1D and 2D NMR spectroscopic data
l Tables 1 and 2; l Fig. 2) revealed that the aglycone of com-
−
1
(
(
"
"
pound 2 possessed a 5,19‑epoxycucurbitane triterpene structure,
similar to that of compound 1, except for the presence of an
ethoxy group (δC 65.9, C‑1′′; 15.6, C‑2′′) at C‑19 (δC 111.1) in 2,
rather than a methoxy group in 1. This was confirmed through
1
1
screening assay showed that the 70% EtOH fraction could inhibit
LPS‑induced NO production (IC50 = 16.5 µg/mL) and was cytotoxic
against human tumor cell lines (IC50 = ca. 30.0 µg/mL). Therefore,
the active fraction was further fractionated by sequential chro-
matography methods, by using silica gel column, solid‑phase ex-
traction (SPE) column, and reverse phase HPLC, to afford six
H‑ H COSY correlation of H ‑1′′ [δ 3.53 (m); 3.95 (m)]/Me‑2′′
2 H
[δH 1.10 (t, J = 7.2 Hz)] and HMBC correlations of H‑19 (δH 4.98)/
C‑1′′. The sugar moiety of compound 2 was determined to be a
D‑alloside on the basis of the C NMR data and comparison with
the reported literature of the sugar in acid hydrolysis [24]. The
relative configuration of 2 was determined by NOESY experiment
1
3
"
5
β,19‑epoxy cucurbitane‑type triterpenes, compounds 1–6
(l Fig. 3) and comparison with the published NMR spectroscopic
"
(
l Fig. 1).
data of cucurbitane‑type triterpenoids. The NOESY spectrum
showed correlations of Me‑18/H‑8, H‑20, which required that
H‑8, Me‑18, and H‑20 had β‑orientation, while H‑10, Me‑28,
and Me‑30 were α‑oriented due to the correlations of Me‑28/
H‑3, H‑10, and Me‑30/H‑10, H‑17. The configuration of C‑19
was determined as R based on the lack of correlations between
H‑19/H‑8 in NOESY spectrum of 2. Hence, the structure of com-
pound 2 was established as 19R‑ethoxy‑5β,19‑epoxycucurbita-
6,23‑dien‑3β,25(E)‑diol 3‑O‑β‑D‑allopyranoside, and the com-
pound was named taikuguasin B.
Compound 1 was isolated as a white amorphous powder, having
a molecular formula of C37
tion ESIMS data ([M + Na] m/z 669.4006). The IR spectrum
H
O calculated from the high‑resolu-
58
+
9
−
1
showed absorption bands at 3385, 1733, and 1447 cm , indicat-
ing hydroxyl, ketone, and olefinic functional groups, respectively.
1
"
The H NMR spectrum (l Table 1) of compound 1 revealed the
presence of six tertiary methyls (δH 0.80, 0.83, 0.89, and 1.53 × 3,
Me‑18, Me‑30, Me‑28, Me‑26, Me‑27, Me‑29), one secondary
methyl [δH 0.93 (br d, J = 4.4 Hz, Me‑21)], one methoxy [δH 3.58
(
s)], one acetal proton [δH 4.65 (s, H‑19)], and four olefinic pro-
tons [δH 6.30 (br d, J = 9.6 Hz, H‑6), 5.48 (dd, J = 9.6, 3.2 Hz, H‑7),
.89 (m, H‑24), and 5.93 (m, H‑23)], as well as a sugar moiety in-
Compound 3 was isolated as a white amorphous powder, pos-
sessing a molecular formula of C37H60O9 (m/z 671.4142, [M +
+
5
Na] ), calculated from the high‑resolution ESIMS data. The IR
−1
cluding one anomeric proton [δ 4.88 (d, J = 7.6 Hz, H‑1′)] and five
oxygenated protons [δH 4.98 (m, H‑4′), 4.68 (br d, J = 7.6 Hz, H‑2′),
4
(
spectrum displayed absorption bands of hydroxyl (3403 cm )
H
−
1
1
13
and olefinic (1447 cm ) functional groups. The H and C NMR
"
.55 (br d, J = 11.6 Hz, H‑6′), 4.46 (br d, J = 11.6, 4.8 Hz, H‑6′), 3.84
spectroscopic data (l Tables 1 and 2) showed that compound 3
m)]. The 13C NMR and DEPT spectra (l Table 2) clearly showed
"
possesses six singlet methyls [δC 15.0 (C‑18); 18.2 (C‑26); 25.8
(C‑27); 24.4 (C‑28); 21.0 (C‑29); 20.1 (C‑30)], one doublet methyl
[δH 1.17 (d, J = 6.0 Hz); δC 19.3 (C‑21)], one methoxy group [δH
3.32; δC 57.2 (C‑1′′)], one trisubstituted olefin [δH 5.62 (br d,
3
7 carbon signals, including eight methyls, eight methylenes,
fourteen methines, and seven quaternary carbons. These spectro-
scopic data, together with the reported constituents from
M. charantia [12,18–21] suggested that compound 1 was a
J = 9.0 Hz, H‑24); δ 129.0 (C‑24); 132.3 (C‑25)], a cis double band
C
5
β,19‑epoxycucurbitane triterpene with a sugar moiety and a
[δH 6.19 (dd, J = 9.6, 1.8 Hz, H‑6); 5.51 (dd, J = 9.6, 3.6 Hz, H‑7); δC
133.5 (C‑6); 130.5 (C‑7)], six methines (including two oxymeth-
"
methoxy group. HMBC correlations (l Fig. 2) between OMe (δ
H
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

6
4
Original Papers
Table 1 1H‑NMR spectroscopic data of compounds 1–5 (pyridine‑d5, 400 MHz).
Position
1
2
3
4
5
1
2
1.32 m; 2.55 m
1.78 m; 2.32 m
3.62 br s
1.41 m; 1.87 m
1.72 m; 2.15 m
3.69 br s
1.32 m; 2.55 m
1.79 m; 1.82 m
3.55 br d (11.4)
6.19 dd (9.6, 1.8)
5.51 dd (9.6, 3.6)
2.25 br s
1.42 m;1.65 m
1.79 m; 1.82 m
3.54 br d (8.4)
6.07 dd (9.6, 1.8)
5.63 dd (9.6, 3.6)
3.04 t (J = 3.0)
2.45 dd (12.0, 6.0)
1.60 m; 1.69 m
1.59 m (2H)
1.21 m (2H)
1.42 m; 1.87 m
1.48 m
1.51 m; 1.82 m
1.80 m; 1.95 m
3.56 br d (9.2)
6.18 br d (9.6)
5.72 dd (9.6, 3.2)
2.31 br s
3
6
6.30 br d (9.6)
5.48 dd (9.6, 3.2)
2.31 br s
6.13 dd (9.6, 1.6)
5.60 dd (J = 9.6, 3.6)
3.15 br s
7
8
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
3
0
1
2
5
6
7
8
9
0
1
2
3
4
6
7
8
9
0
1
2
3
4
5
6
2.29 m
2.41 dd (12.4, 5.6)
1.62 m; 1.72 m
1.51 m (2H)
1.26 m (2H)
1.28 m; 1.92 m
1.48 m
2.36 dd (12.6, 6.0)
1.65 m; 1.75 m
1.60 m (2H)
1.14 m; 1.22 m
1.45 m; 1.90 m
1.51 m
2.23 br d (5.6)
1.65 m; 1.82 m
1.55 m (2H)
1.30 m (2H)
1.32 m; 1.97 m
1.47 m
1.55 m; 1.90 m
1.50 m (2H)
1.25 m (2H)
1.30 m; 1.92 m
1.49 m
0.80 s
0.90 s
0.82 s
0.94 s
0.93 s
4.65 s
4.98 s
4.52 s
4.74 s
5.33 s
1.49 m
1.49 m
2.02 m
2.12 m
1.51 m
0.93 br d (4.4)
1.87 m; 2.54 m
5.93 m
0.96 br d (4.8)
1.82 m; 2.24 m
5.93 m
1.17 d (6.0)
1.18 m; 2.08 m
4.93 dt (9.0, 5.4)
5.62 br d (9.0)
1.75 s
1.16 d (6.6)
1.95 m; 2.08 m
4.92 dt (9.0, 5.4)
5.61 br d (8.4)
1.75 s
0.96 br d (4.8)
1.86 m; 2.44 m
5.59 m
5.89 m
5.89 m
5.56 m
1.53 s
1.52 s
1.32 s
1.53 s
1.53 s
1.67 s
1.66 s
1.32 s
0.89 s
0.79 s
0.88 s
0.84 s
0.88 s
1.53 s
1.42 s
1.42 s
1.36 s
1.41 s
0.83 s
0.85 s
0.86 s
0.88 s
0.84 s
′
′
′
′
′
′
4.88 d (7.6)
4.68 br d (7.6)
–
5.48 d (8.0)
3.92 m
4.96 d (7.8)
4.03 m
4.95 d (8.4)
4.03 t (7.8)
4.17 m
4.73 m
4.21 m
4.98 m
4.20 br d (J = 7.6)
3.47 m
4.21 m
4.21 m
3.84 m
3.89 m
3.8 dd (12.0, 3.0)
4.46 dd (12.0, 3.0)
4.32 dd (12.0, 6.3)
3.34 s
4.46 dd (11.6, 4.8)
4.39 dd (11.6, 4.8)
4.54 br (11.6)
3.53 m; 3.95 m
1.10 t (7.2)
4.47 dd (11.4, 2.4)
4.33 dd (12.0, 5.4)
3.32 s
4
.55 br d (11.6)
1
2
′′
′′
3.58 s
3.20 s
ines), an acetal group (δH 4.52; δ 114.8, C‑19), seven methylenes,
5β,19‑epoxycucurbita‑6,24‑dien‑3β,23R‑diol
23‑O‑β‑D‑gluco-
C
six quaternary carbons (including two oxygenated), and a sugar
pyranoside.
moiety [δ 104.0 (C‑1′); 75.6 (C‑2′); 78.9 (C‑3′); 71.8 (C‑4′); 78.3
Compound 4 showed a quasi‑molecular ion at m/z 671.4135 ([M
C
1
13
+
(C‑5′); 62.9 (C‑6′)]. Based on the similar H and C NMR spectro-
+ Na] ) in the HRESIMS spectrum and had the same molecular
1
13
scopic data as compounds 1 and 2, together with the HMBC cor-
formula C37H60O as 3. Their IR, H‑, and C NMR spectroscopic
9
"
"
relations (l Fig. 2) between H‑19 (δ 4.52)/C‑5 (δ 85.3) and OMe
data (l Tables 1 and 2) indicated that both compounds 3 and 4
H
C
(
δH 3.32)/C‑19 (δ 114.8), compound 3 was suggested as a 5β,19-
possessed the 5β,19‑epoxycucurbitane‑type triterpene with a
C
epoxycucurbitane‑type triterpene with an OMe group at C‑19
and a fused furan. The HMBC correlations between Me‑26, Me-
sugar moiety and methoxy group. Detailed comparisons of its
H‑ and C‑ NMR data with those of compound 3 revealed strong
resemblance in all signals except that both the C‑8 and H‑8 sig-
1
13
1
1
27 and C‑25, C‑24, together with H‑ H COSY correlations of Me-
2
1/H‑19/H ‑22/H‑23/H‑24 suggested that the side chain was –
nals in the spectra of 4 were shifted upfield to δ 42.1 and δH
2
C
1
1
CH(CH )CH CH(OH)CH=C(CH ) . This type of the side chain has
3.04 (t, J = 3.0 Hz), respectively. The H‑ H COSY and HMBC corre-
lations were similar as well, suggesting that both compounds 3
and 4 have a similar planar chemical structure. Moreover, their
3
2
3 2
been described previously in momordicoside U and charantoside
II isolated from the same title plant, M. charantia [19,21]. The
sugar moiety of compound 3 was determined to be a D‑glucose
on the basis of the 13C NMR data and comparison with the re-
ported literature of the sugar in acid hydrolysis. Moreover, the
HMBC correlations of H‑1′/C‑23 unambiguously assigned the sug-
ar moiety at C‑23. The relative stereochemistry of 3 was deter-
mined by NOESYexperiments and biogenetic consideration, indi-
cating that compound 3 has similar configurations with the fused
"
NOE spectra (l Fig. 4) showed the different correlations between
H‑19 and H‑8, confirming the R‑configuration of C‑19 on com-
pound 4. Thus, compound 4 was established as a C‑19 epimer de-
rivative of compound 3 and named taikuguasin D.
Compound 5 was assigned the molecular formula C31H50O , on
4
the basis of a quasimolecular ion peak at m/z 509.3619 [M + Na]+
in HRESIMS. Similar to compounds 1 and 2, compound 5 showed
−
1
ring moiety as compounds 1 and 2. Moreover, NOESY correlations
absorptions of IR spectrum at 3305 and 1638 cm , indicating hy-
droxyl and olefinic group functionalities, respectively. Detailed
"
(
l Fig. 3) of H‑21/Me‑18, H ‑22, Me‑21/H ‑22, H‑24, H ‑22/
β
α
β
1
13
"
H‑21, H‑23, and H‑23/H ‑22, as well as correlation between H‑8
inspection of H and C‑NMR spectroscopic data (l Tables 1
and 2) indicated the presence of the key structural features of a
5β,19‑epoxycucurbitane‑type triterpene skeleton with a me-
thoxy group (δH 3.20). Furthermore, compound 5 had very close
β
and H‑19, suggested that the stereochemistry of C‑23 and C‑19
adopted R‑ and S‑configuration, respectively. Based on the above
evidences, taikugausin C (3) was identified as 19S‑methoxy-
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

Original Papers 65
Position
1
2
3
4
5
Table 2 13C‑NMR spectroscopic
1
2
3
4
5
6
7
8
9
18.2 t
27.3 t
85.9 d
38.9 s
83.7 s
18.7 t
17.0 t
17.7 t
19.9 t
data of compounds 1–5 (pyridine-
d5, 100 MHz).
27.3 t
83.4 d
39.0 s
85.4 s
133.2 d
131.5 d
42.2 d
47.9 s
41.5 d
23.1 t
30.8 t
45.2 s
48.3 s
33.8 t
28.1 t
50.3 d
14.7 q
111.1 d
36.6 d
18.8 q
39.5 t
124.2 d
141.7 d
69.7 s
30.8 q
30.8 q
24.8 q
21.3 q
19.9 q
102.4 d
73.7 d
71.8 d
69.2 d
76.5 d
63.2 t
65.9 t
15.6 q
27.8 t
76.3 d
37.4 s
85.3 s
133.5 d
130.5 d
49.8 d
49.3 s
38.2 d
21.9 t
27.8 t
76.1 d
37.7 s
86.9 s
131.6 d
132.7 d
42.1 d
48.2 s
41.0 d
23.4 t
28.2 t
76.5 d
37.5 s
84.5 s
134.0 d
130.5 d
50.0 d
48.9 s
38.7 d
22.6 t
31.0 t
45.4 s
48.3 s
36.4 t
28.1 t
50.4 d
14.9 q
107.8 d
36.4 d
18.9 q
39.7 t
128.4 d
137.6 d
74.8 s
26.5 q
26.0 q
24.5 q
21.3 q
17.9 q
135.0 d
129.2 d
49.5 d
49.1 s
39.1 d
21.8 t
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1
2
30.8 t
30.8 t
30.9 t
45.3 s
48.2 s
33.7 t
45.4 s
48.2 s
33.6 t
45.4 s
48.2 s
33.7 t
28.0 t
28.1 t
28.1 t
50.3 d
15.1 q
113.1 d
36.6 d
18.8 q
39.5 t
51.4 d
15.0 q
114.8 d
32.7 d
19.3 q
43.7 t
51.2 d
14.8 q
112.1 d
32.6 d
19.3 q
43.7 t
124.2 d
141.7 d
69.7 s
30.8 q
30.8 q
25.1 q
20.8 q
20.0 q
108.1 d
79.1 d
208.7 s
73.8 d
78.3 d
62.4 t
75.2 d
129.0 d
132.3 s
18.2 q
25.8 q
24.4 q
21.0 q
20.1 q
104.0 d
75.6 d
78.9 d
71.8 d
78.3 d
62.9 t
75.2 d
129.1 d
132.2 s
18.2 q
25.8 q
24.1 q
20.8 q
19.9 q
104.2 d
75.6 d
78.9 d
71.7 d
78.2 d
62.9 t
′
′
′
′
′
′
′′
′′
54.9 q
57.2 q
57.5 q
50.1 q
1
1
Fig. 2 H‑ H COSY and key HMBC correlations for
compounds 1–4.
1H‑NMR spectroscopic data to those of 5β,19‑epoxy‑25‑me-
thoxycucurbita‑6,23‑diene‑3β,19‑diol (6) [15]. However, in the
comparison of 13C NMR spectra with 5 and 6, the different chem-
ical shifts (C D N) of C‑19 (δ 107.8, 5; 105.1, 6), C‑8 (δ 50.0, 5;
31.6, 6), and C‑10 (δC 38.7, 5; 41.2, 6), together with the HMBC
correlation (l Fig. 2) of OMe/C‑25 in 5, assigned the OMe group
"
at C‑25 in 5 and C‑19 in 6, respectively. The NOESY correlation
"
(l Fig. 3) between H‑19/H‑8 in 5 confirmed the S configuration
6
5
C
C
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

6
6
Original Papers
H‑8 in the NOESY spectrum, the C‑19 was determined as having S
1
3
configuration, and the C NMR chemical shift of C‑8 should be
shifted downfield to δ 49–50 ppm. Moreover, the chemical shift
C
of H‑8 (δH 2.13–2.32) in 19S‑cucurbitane was more upfield
(
Δ = 0.5–1.0 ppm) than those of 19R‑cucurbitane. Based on the
above findings, the C‑19 stereochemistry of 5β,19‑epoxy‑19‑me-
thoxycucurbita‑6,23‑diene‑3β,25‑diol and 5β,19‑epoxy‑19,25-
dimethoxycucurbita‑6,23‑diene‑3β‑ol isolated from the leaves
of M. foetida may be considered as having 19S configuration [17].
The isolated compounds 1–4 were evaluated for anti‑inflamma-
tory activity by their inhibition of LPS‑induced NO production in
RAW264.7 macrophages, whereas the inhibition value for quer-
cetin, used as a positive control, was 2.86 ± 0.41 µM. As shown in
"
l Fig. 6, the isolated cucurbitanes 3 and 4 significantly decreased
NO production in a dose‑dependent manner and exhibited po-
tent inhibition activities with IC50 values of 8.61 and 6.01 µM, re-
spectively. In addition, the 70% EtOH extract exhibited potent in-
hibitory activity against several human tumor cell lines. There-
fore, compounds 1–4 and 6 were further examined against MCF-
7
(breast adenocarcinoma), Hep G2 (hepatocellular carcinoma),
HEp‑2 (laryngeal carcinoma), and WiDr (colon adenocarcinoma)
"
human tumor cell lines. Cytotoxicity results (l Table 4) showed
that compounds 3 and 4 had moderate cytotoxicity, while com-
pounds 1 and 2 were invalid for the above tumors. Interestingly,
the tumor cytotoxicity results also indicate cytotoxic effects at a
concentration of 20 µg/mL in RAW264.7 cell line. The bioassay re-
sults suggest that the side chain of 3 and 4 could play a crucial
role, compared with compounds 1‑2.
Materials and Methods
!
General
Optical rotations were recorded on a JASCO P‑2000 polarimeter.
Infrared (IR) spectra were measured on a Mattson Genesis II
1
13
spectrophotometer. The 1D ( H, C, and NOE) and 2D (COSY,
HMBC, HMQC, and NOESY) spectra were recorded on Bruker Ad-
vance 400 MHz using C D N as solvent for measurement. Elec-
5
5
trospray ionization mass spectrometry (ESIMS) data were ob-
tained on a Finnigan ion trap tandem mass spectrometer. High
resolution electronic ionization mass spectrometry (HREIMS) da-
ta were measured on a Finnigan MAT‑95XL mass spectrometer.
Diaion HP‑20 (Mitsubishi Chemical Co.), Sephadex LH‑20 (GE),
and silica gel 60 (Merck, 70–230 and 230–400 mesh) were used
for column chromatography, and precoated silica gel (Merck, Kie-
selgel 60 F‑254, 1 mm) plates were used for TLC. The spots on TLC
were detected by spraying with an anisaldehyde‑sulphuric acid
solution and then heating at 100°C. HPLC separations were per-
formed on a Shimadzu LC‑6AD series apparatus with a SPD‑10A
UV detector and Varian 380‑LC ELSD detector, equipped with a
250 × 20 mm or 250 × 4.6 i.d. preparative Cosmosil 5C18 AR‑II col-
umn (Nacalai Tesque, Inc.).
Fig. 3 Key NOESY correlations for compounds 1–5.
of C‑19; whereas in 6, the OH group at C‑19 showed R‑configura-
tion. Based on the above observation, taikuguasin E (5), 19‑epi-
mer of 6, was determined as 19S‑5β,19‑epoxy‑25‑methoxycu-
curbita‑6,23‑diene‑3β,19‑diol.
In previous studies, the configurations of C‑19 in the known 5,19-
epoxycucurbitane compounds was mostly assigned as R. From
the characteristics of H and C NMR chemical shifts, the config-
urations of C‑19 in 5,19‑epoxycucurbitane derivatives were
known. In an attempt to understand the relationship between
NMR data and the configurations of C‑19, the chemical shifts for
Plant material
1
13
The fruits of Taiwanese M. charantia were provided from Starsci
Biotech Co. Ltd. and purchased in Nantou County Taiwan in Au-
gust 2009. They were identified by Dr. Syh‑Yuan Hwang working
in the Endemic Species Research Institute, Council of Agriculture,
Taiwan. A voucher specimen (NRICM, No. 20090901) has been
deposited in the National Research Institute of Chinese Medicine,
Taipei, Taiwan.
H‑8, C‑8, C‑9, C‑10, C‑11, and C‑19 are summarized as shown
"
(
l Table 3 and Fig. 5). The chemical shift of C‑8 and C‑11 clearly
appear at δ 41–43 ppm and 23–24 ppm, respectively, when C‑19
C
showed R form. Furthermore, due to a correlation between H‑19/
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

Original Papers 67
Fig. 4 NOE difference spectra of compounds 3
and 4.
Table 3 NMR chemical shifts and C‑19 configurations for 5β,19‑epoxycucurbitane.
Compound
C‑19 Con-
R group
NMR Data (ppm)
figuration
H‑8
C‑8
C‑9
C‑10
C‑11
C‑19
Taikaguasin A (1)a
Taikaguasin B (2)a
Taikaguasin C (3)a
Taikaguasin D (4)a
Taikaguasin E (5)a
S
R
S
R
S
S
R
R
R
R
R
R
R
R
OMe
OEt
2.31
3.15
2.25
3.04
2.31
2.31
3.12
3.13
3.13
3.14
3.12
3.13
3.13
2.82
49.5
42.2
49.8
42.1
50.0
50.1
42.3
42.3
42.2
42.2
42.3
42.2
42.2
41.4
49.1
47.9
49.3
48.2
48.9
50.0
48.2
48.3
48.1
48.1
48.2
48.2
48.2
48.5
39.1
41.5
38.2
41.0
38.7
38.7
41.7
41.7
41.7
41.6
41.6
41.7
41.6
40.6
21.8
23.1
21.9
23.4
22.6
28.2
23.3
23.4
23.2
23.3
23.3
23.3
23.4
30.5
113.1
111.1
114.8
112.1
107.8
107.8
112.4
112.5
112.3
112.2
112.5
112.4
112.4
105.4
OMe
OMe
OH
a
Kuguacin R [20]
OH
a
Goyaglycoside‑a [12]
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
a
Goyaglycoside‑b [12]
a
Goyaglycoside‑c [12]
a
Goyaglycoside‑d [12]
a
Goyaglycoside‑g [12]
a
Charantoside I [19]
a
Charantoside II [19]
5
β,19‑Epoxy‑25‑methoxycucurbita‑6,23‑diene‑3β,19‑diolb
[
17]
b
5
5
β,19‑Epoxycucurbita‑6, 23‑diene‑3β,19,25‑triol [17]
β,19‑Epoxy‑19‑methoxycucurbita‑6,23‑diene‑3β,25‑diolb
R
S
OH
2.82
2.13
41.4
49.8
48.5
48.9
40.6
37.9
30.5
30.4
105.4
114.7
OMe
[
17]
β,19‑Epoxy‑19,25‑dimethoxycucurbita‑6,23‑diene‑3β‑olb
17]
5
S
OMe
2.32
49.8
48.9
36.1
30.4
114.7
[
b
Momordicoside U [21]
R
R
R
R
OMe
OH
2.86
3.37
2.89
2.89
41.7
41.8
42.7
41.7
47.4
45.6
46.2
48.0
41.0
41.8
42.6
40.5
23.0
23.3
24.0
23.2
112.6
105.1
106.0
112.1
a
Momordicoside O [18]
c
Momordicoside W [21]
OH
(
19R,23E)‑5β,19‑Epoxy‑19‑methoxycucurbita‑6,23,25‑trien-
OMe
a
3
β‑ol [17]
a
Spectra recoded in pyridine‑d5; b spectra recoded in CDCl3; c spectra recoded in CD3OD
Extraction and isolation
plying SPE column and RP‑HPLC eluting chromatography with
60% MeOH, compounds 5 (1.5 mg, purity > 97% by HPLC) and 6
(7.6 mg, purity > 98% by HPLC) were obtained from Fr. C3 (1.4 g).
The fresh fruits of M. charantia (wet, 3.6 kg) were sliced and ex-
tracted three times with 70% EtOH (7.0 L) at 50°C for 24 h and
concentrated under reduced pressure. The EtOH extract (75 g)
was subjected to Diaion HP‑20 column chromatography (9 ×
25
D
Taikuguasin A (1): white amorphous powder. [α] − 68 (c 0.40,
MeOH); IR (KBr) νmax 3385, 2967, 2872, 1733, 1616, 1447, 1372,
−
1
1
5
0 cm) eluting with H O, 40% EtOH, 70% EtOH, 95% EtOH, and
1155, 1111, 1081, and 1047 cm ; H‑NMR (400 MHz, pyridine-
2
"
13
100% EtOAc (each 2.5 L), respectively, to obtain five fractions (Fr.
d ) spectroscopic data, see l Table 1; C‑NMR (100 MHz, pyri-
5
"
A–E). Fr. C (9.8 g) was chromatographed by silica gel column
12 × 36 cm) eluting with a gradient of CHCl /MeOH solvent sys-
dine‑d ) spectroscopic data, see l Table 2; ESIMS m/z 669 [M +
5
+
+
(
Na] ; HRESIMS m/z 669.4006 [M + Na] (calcd. for C37H58O Na,
3
9
tem to afford four subfractions (Fr. C1 to Fr. C4). Fr. C2 (3.3 g) was
669.3981).
25
subjected by a C18 SPE column with MeOH/H O solvent system
Taikuguasin B (2): white amorphous powder. [α]
D
− 88 (c 0.68,
2
and further purified by reverse phase (RP) HPLC with ELSD detec-
tor eluting with 60% MeOH to yield compounds 1 (3.4 mg, purity
MeOH). IR (KBr) νmax 3408, 2968, 2875, 1639, 1463, 1375, 1260,
−
1
1
1151, 1114, 1085, and 1029 cm ; H‑NMR (400 MHz, pyridine-
"
13
>
98% by HPLC), 2 (9.4 mg, purity > 98% by HPLC), 3 (50.8 mg, pu-
d ) spectroscopic data, see l Table 1; C‑NMR (100 MHz, pyri-
5
"
rity > 99% by HPLC), and 4 (27.6 mg, purity > 97% by HPLC). By ap-
dine‑d ) spectroscopic data, see l Table 2; ESIMS m/z 685 [M +
5
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

6
8
Original Papers
"
13
spectroscopic data, see l Table 1; C‑NMR (100 MHz, pyridine-
"
+
d ) spectroscopic data, see l Table 2; ESIMS m/z 671 [M + Na] ,
HRESIMS m/z 671.4135 [M + Na]+ (calcd. for C37H60O Na,
5
9
6
71.4135).
Taikuguasin E (5): white amorphous powder; [α]
MeOH); IR (KBr) ν 3305, 2987, 1638, 1372, 1224, 1164, and
2
5
D
− 38 (c 0.20,
max
−
1
1
1089 cm ; H‑NMR (400 MHz, pyridine‑d ) spectroscopic data,
5
"
13
see l Table 1; C‑NMR (100 MHz, pyridine‑d ) spectroscopic da-
ta, see l Table 2; ESIMS m/z 509 [M + Na] ; HRESIMS m/z
5
"
+
+
509.3619 [M + Na] (calcd. for C31H50O Na, 509.3601).
4
Fig. 5 Difference between several carbon chemical shifts of 5β,19-
epoxycucurbitanes. (Color figure available online only.)
Acid hydrolysis of compounds 1–4
Compounds 1–4 (each 1.0 mg) were refluxed with 1 N HCl (diox-
ane‑H O, 1:1, 2 mL) at 90°C for 1 h, respectively. The residue was
2
partitioned with CH Cl and H O. The aqueous layer was neutral-
2
2
2
ized with aqueous 1 N Na CO3 and then evaporated under re-
2
+
+
Na] ; HRESIMS m/z 685.4304 [M + Na] (calcd. for C38H62O Na,
duced pressure. The evaporated filtrate was dissolved in a solu-
tion of 1‑(trimethylsilyl)imidazole in pyridine (0.2 mL), and
stirred at 60°C for 5 min. After the reaction mixture was dried
9
6
85.4294).
Taikuguasin C (3): white amorphous powder; [α]
MeOH); IR (KBr) νmax 3403, 2943, 1447, 1380, 1304, 1274, 1184,
151, 1082, and 132 cm ; H‑NMR (400 MHz, pyridine‑d ) spec-
troscopic data, see l Table 1; C‑NMR (100 MHz, pyridine‑d )
spectroscopic data, see l Table 2; ESIMS m/z 671 [M + Na] ; HRE-
SIMS m/z 671.4142 [M + Na] (calcd. for C37H60O Na, 671.4135).
Taikuguasin D (4): white amorphous powder; [α]
2
5
D
− 47 (c 0.10,
under a stream of N , each residue was partitioned between
2
−1 1
1
CH Cl and H O (1:1 v/v). Each CH Cl fraction was subjected to
5
2
2
2
2
2
"
13
GC by using a CP‑Chirasil‑L‑Val column (25 m × 0.25 mm,
0.12 mm; column temperature, 50–150°C, 30°C/min, 150–180°
5
"
+
+
C, 0.8°C/min; injector temperature, 200°C; He carrier gas,
9
2
5
3
D
− 59 (c 0.10,
2.0 kg/cm ; mass detector, Thermo, DSQ2; electron energy,
MeOH); IR (KBr) νmax 3396, 2943, 2875, 1449, 1414, 1380, 1157,
70 eV). The peaks of each hydrolysate from compounds 2–4 were
identified by comparison with authentic samples (D‑glucose,
−
1
1
1113, 1081, and 1049 cm ; H‑NMR (400 MHz, pyridine‑d5)
Fig. 6 NO production inhibition effect of compounds 1–4 on RAW 264.7 macrophage cells induced by LPS. (Color figure available online only.)
Liaw C-C et al. 5β,19-Epoxycucurbitane Triterpenoids from… Planta Med 2015; 81: 62–70

Original Papers 69
Cpd/cell linea
ED50 (µM)
Table 4 Cytotoxic activities of the
isolated compounds from the fruits
of M. charantia.
WiDr
(−)b
Hep G2
MCF‑7
HEp‑2
1
59.92 ± 0.52
(−)
(−)
(−)
2
57.28 ± 1.06
27.39 ± 0.55
33.88 ± 1.03
(−)
(−)
(−)
3
28.76 ± 0.26
29.35 ± 1.04
(−)
42.60 ± 0.91
42.66 ± 0.60
(−)
28.82 ± 0.40
46.26 ± 0.66
(−)
4
6
Mitomycin c
0.29 ± 0.02
0.44 ± 0.02
0.29 ± 0.02
0.32 ± 0.02
a
Cell lines: WiDr (human colon adenocarcinoma), Hep G2 (human hepatocellular carcinoma), MCF‑7 (human breast adenocarcinoma),
and HEp‑2 (human laryngeal carcinoma) tumor cell lines; b (−): ED50 > 60 µM. Mitomycin c was used as positive control. All values are pre-
sented as mean ± SD (n = 3)
t 30.60 min; D‑allose, t 36.54 min). The D‑ribo‑hexos‑3‑ulose
Acknowledgments
!
R
R
(3‑Keto‑Glc) of 1 was confirmed by comparing the NMR data
and the optical rotation value with the reported literature.
The grants from the National Research Institute of Chinese Medi-
cine and Starsci Biotech Co. Ltd. are gratefully acknowledged. We
thank also the Instrument Center of National Taiwan University
for the HR‑ESI‑MS measurements and the National Center for
High‑Performance Computing for checking the database.
Measurement of nitric oxide production
The mouse macrophage cell line RAW 264.7 was obtained from
ATCC and cultured in DMEM containing 5% heat‑inactivated fetal
calf serum, 100 U/mL penicillin and streptomycin and grown at
3
7°C with 5% CO in fully humidified air. Cells were plated at a
2
4
density of 5 × 10 cells/well in 96‑well culture plate and stimu-
lated with LPS (1.0 µg/mL) in the presence or absence of different
concentrations of tested compounds (1 ~ 20 µg/mL) for 24 h
simultaneously. Compounds 1–4 were dissolved in DMSO and
further diluted with sterile PBS. NO2− accumulation in the me-
dium was used as an indicator of NO production which was mea-
sured by adding Griess reagent (1% sulfanilamide in 5% phos-
Conflict of Interest
!
There is no conflict of interest.
Affiliations
1
R&D Department, Starsci Biotech Co. Ltd., Taipei, Taiwan
Department of Chinese Pharmaceutical Sciences and Chinese Medicine
2
Resources, China Medical University, Taichung, Taiwan
Division of Chinese Materia Medica Development, National Research Institute
of Chinese Medicine, Taipei, Taiwan
Endemic Species Research Institute, Council of Agriculture, Nantou, Taiwan
Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei,
Taiwan
Graduate Institute of Integrated Medicine, College of Chinese Medicine,
China Medical University, Taichung, Taiwan
3
phoric acid and 0.1% naphthylenediamine in D.D. H O). NaNO2
2
was used to generate a standard curve, and azo production was
determined by measuring optical density at 540 nm. All experi-
ments were performed in triplicate. NO production by LPS stim-
ulation was designated as 100% for each experiment. Quercetin
4
5
6
(
Sigma‑Aldrich, purity > 95%) was used as a positive control.
Statistical significance is expressed as * p < 0.05, ** p < 0.01, and
** p < 0.001.
7
Ph.D Program for the Clinical Drug Discovery from Botanical Herbs, School of
Pharmacy, Taipei Medical University, Taipei, Taiwan
*
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