Phytochemistry 157 (2019) 21–27

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Cucurbitane triterpenoids from the fruit of Momordica charantia L. and their T

anti-hepatic fibrosis and anti-hepatoma activities
Jiayin Yuea, Yuanyuan Sunc, Jing Xua, Jiaqing Caoa, Gang Chend, Huixing Zhanga,
Xiaoshu Zhanga,∗∗, Yuqing Zhaoa,b,∗
a
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
b
Key Laboratory of Structure-Based Drug Design & Discovery Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
c
School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China
d
School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China

ARTICLE INFO ABSTRACT

Keywords: Momordica charantia L. (Cucurbitaceae) is a popular vegetable and traditional folk medicine, that has been used
Momordica charantia L. for hundreds of years. In this study, three undescribed cucurbitane-type triterpene glycosides furpyr-
Cucurbitaceae onecucurbitane A, goyaglycoside I and charantagenin F along with nine known compounds were isolated from
Cucurbitane the immature fruit of Momordica charantia L. Their structures were identified on the basis of extensive 1D, 2D
Anti-hepatic fibrosis
NMR and HRESIMS spectroscopy analysis. All isolated compounds were examined for their anti-hepatic fibrosis
Anti-hepatic cancer
activity against murine hepatic stellate cells (t-HSC/Cl-6) and anti-hepatoma activity against two kinds of liver
cancer cell lines (HepG2 and Hep3B). Among them, karaviloside III exhibited excellent inhibitory activity
against activated t-HSC/Cl-6 cells and cytotoxic activity against Hep3B and HepG2 cell lines with IC50 values of
3.74 ± 0.13, 16.68 ± 2.07 and 4.12 ± 0.36 μM, respectively, which may potential to be developed as a
chemotherapy agent for treatment hepatic fibrosis or carcinoma and protection against both diseases.

1. Introduction
Momordica charantia L. (Cucurbitaceae) is an annual climbing vine,
that is also known as bitter gourd, bitter melon, kugua, balsam pear or
karela in Asia, South America, India, and East Africa. It is a popular
vegetable and traditional folk medicine, that has been used for hun-
dreds of years (Grover and Yadav, 2004; Anilakumar et al., 2015).
During the past two decades, there has been increasing evidence that M.
charantia possesses various beneficial antagonistic effects against oxi-
dation (Ghous et al., 2015; Panda et al., 2015), inflammation (Manabe
et al., 2003; Liaw et al., 2015), cancer (Singh et al., 2016; Raina et al.,
2016), diabetes (Joseph and Jini, 2013), obesity (Chan et al., 2005) and
antimicrobials (Saeed and Tariq, 2005; Puri et al., 2009; Mada et al.,
2013). Different beneficial effects have been partly attributed to the
various bioactive components of M. charantia. including triterpenoids,
polypeptides, flavonoids, polysaccharides, alkaloids and sterols (Jia
et al., 2017). Cucurbitane compounds are the main components of the
triterpenoids from the fruit, stem, vines, roots and leaves of M. charantia
(Okabe et al., 1982; Pan and Zhao, 2007; Guan and Zhao, 2007; Cao
et al., 2011; Li et al., 2015) and have been demonstrated to have an-
ticancer (Wang et al., 2012), antidiabetic (Chou et al., 2015; Jiang
et al., 2016; Yue et al., 2017), antioxidant (Liu et al., 2010) and other
biological activities.
In recent years, the potential for M. charantia. to protect the liver
has received more and more attention. Several studies have in-
vestigated these effects using M. charantia. extract in different experi-
mental animal models (Yu et al., 2013; Hussain et al., 2014; Xu et al.,
2014; Sagor et al., 2015; Bai et al., 2017; Deng et al., 2017). Following
on from this, the current study was designed to investigate the ther-
apeutic hepatic effects of cucurbitane-type triterpenoids, most of which
have been already isolated from M. charantia. As a result, three un-
described cucurbitane-type triterpene glycosides, furpyronecucurbitane
A (1), goyaglycoside I (2) and charantagenin F (3), as well as nine
known cucurbitane triterpenoids (4–12) were isolated from the im-
mature fruits of M. charantia. collected in Guangxi, China. The isolation,
identification, and activities against liver fibrosis and tumors are de-
scribed in this paper.

∗
Corresponding author. School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang, 110016, People's Republic of China.
∗∗
Corresponding author.
E-mail addresses: yuejiayin1990@163.com (J. Yue), xiaoshu2397@163.com (X. Zhang), zyq4885@126.com (Y. Zhao).

https://doi.org/10.1016/j.phytochem.2018.10.009
Received 10 April 2018; Received in revised form 24 September 2018; Accepted 8 October 2018
Available online 22 October 2018
0031-9422/ © 2018 Elsevier Ltd. All rights reserved.
J. Yue et al. Phytochemistry 157 (2019) 21–27

1
Table 1 H-NMR spectra (Table 1) showed proton resonances corresponding to
The NMR spectral data of compounds 1 in pyridine-d5 and DMSO‑d6. a β-D-allopyranoside moiety [δH 5.37 (1H, d, J = 7.8 Hz, 1′-H)], five
No. pyridine-d5 DMSO‑d6 tertiary methyl groups (δH 0.77, 0.80, 0.86, 1.86, 1.55, all s, 18, 30, 28,
27, 29, each 3H), one secondary methyl group [δH 1.03 (d, J = 6.4 Hz),
δC δH δC δH 21-H3], and four olefinic protons [δH 6.16 (dd, J = 9.7, 1.2 Hz,6-H),
5.51 (dd, J = 9.7, 3.6 Hz, 7-H), 5.08, (s, 26-Ha), 5.09 (s, 26-Hb)]. The
1 19.3 1.35 m 18.0 1.28 m 13
1.76 m 1.35 m C-NMR (Table 1), DEPT and HSQC spectra showed resonances of 42
2 27.9 1.75 m 26.5 1.63 m carbon signals composed of the six methyls (δC 15.4, 18.7, 20.5, 21.5,
2.39 m 1.92 m 24.9 and 25.9), eight methylenes (δC 19.3, 24.2, 27.9, 28.9, 31.5, 33.7,
3 85.6 3.62 br s 84.3 3.65 br s 39.6 and 41.4), three oxygenated methylenes (δC 63.7, 64.8 and 80.5),
4 39.3 38.2
four methines (δC 40.5, 52.6, 51.8 and 52.8), seven oxygenated me-
5 86.3 84.8
6 134.4 6.16 dd (9.7, 1.6) 133.7 6.04 dd (9.7, 1.6) thines (δC 69.6, 72.8, 73.2, 73.4, 76.5, 80.5 and 85.6), four olefinic
7 130.4 5.51 dd (9.7, 3.6) 129.3 5.47 dd (9.7, 3.6) carbons (δC 116.7, 130.4, 134.4 and 139.5), four quaternary carbons
8 52.6 2.27 s 51.4 2.29 br s (δC 39.3, 45.6, 45.9 and 49.3), and two oxygenated quaternary carbons
9 45.6 44.4
(δC 86.3 and 87.3), along with ketone (δC 208.1) and two anomeric
10 40.5 2.26 m 39.2 2.13 m
11 24.2 1.32 m 23.2 1.39 m carbons (δC 104.3 and 109.9). Analysis of the13C and 1H NMR spec-
1.59 m 1.61 m troscopic data and the molecular formula, suggested that compound 1 is
12 31.5 1.51 m 30.5 1.55 m a cucurbitane-type triterpene glycoside with an aglycone the same as
1.58 m 1.60 m those of (23E)-5β,19-epoxycucurbita-6,25-dien-3β-ol 3-O-β-D -allopyr-
13 45.9 44.9
anoside (charantoside VI) (Akihisa et al., 2007.) except for the signals
14 49.3 48.3
15 33.7 1.19 m 32.6 1.23 m due to the missing carbon atom at C-23-OCH3 and the six extra carbon
16 28.9 1.53 m 26.5 1.63 m signals (δC 41.4, 64.8, 73.2, 87.3, 109.9 and 208.1). HMBC correlations
2.00 m 1.91 m provided additional support for the assigned structure (Fig. 2). The
17 51.8 1.48 m 50.7 1.35 m
HMBC correlations of H-3 at 3.62 with δC 104.3(C-1′) as well as those
18 15.4 0.77 s 14.6 0.81s
19 80.5 3.58 d (8.0) 79.1 3.35 overlap
between the H-1′ at 5.39 with δC 85.6 (C-3), which indicated that the
3.77 d (8.0) 3.44 d (8.0) sugar unit was linked to C-3. Correlations were performed between the
20 33.4 2.04 m 32.1 1.63 m signals of Ha-26 at 5.08 and Hb-26 at 5.59 with 24.9(C-27), 52.8(C-24),
21 18.7 1.03 d (6.4) 18.0 0.87 d (6.4) 139.5(C-25); H-20 at 2.04 with 45.9(C-13), 51.8(C-17); Ha-22 at 1.13
22 39.6 1.13 m 38.3 0.74 m
with 18.7(C-21), 33.4(C-20); Hb-22 at 2.49 with 80.5 (C-23), led to the
2.49 m 1.94 m
23 80.5 4.88 m 78.7 4.46 m identification of the partial chain sides. Furthermore, the HMBC cor-
24 52.8 3.90 d (8.2) 50.6 3.35 overlap relations of H-23 at 4.88 with 87.3(C-3″); H-24 at δH 3.90 with δC
25 139.5 138.0 39.6(C-22), 80.5(C-23), 87.3(C-3″), 116.7(C-26), 139.5(C-25), 208.1(C-
26 116.7 5.09 br s 115.6 4.83 br s
2″); H-2″ at δH 5.55 with δC 52.8(C-24), 80.5(C-23), 87.3(C-3″), Ha-5″
5.60 br s 5.15 br s
27 24.9 1.86 s 24.1 1.68 s
at 2.80 with 87.3(C-3″), Hb-5″ at 3.28 with 73.2(C-6″), 64.8(C-7″), H-6″
28 25.9 0.86 s 25.2 0.80 s at 4.07 with 41.4(C-5″), 73.2(C-6″). Due to the lack of an active hy-
29 21.4 1.55 s 20.3 1.07 s drogen signal, it was not possible to determine whether the fused-ring
30 20.5 0.80 s 19.8 0.81 s or bridge-ring was in the side chain, so we measured the spectral data of
Sugar moiety
the sample dissolved in DMSO‑d6 and the 1H-NMR and 13C-NMR
1′ 104.3 5.37 d (7.8) 102.5 4.48 d (7.8)
2′ 73.4 3.97 m 71.4 3.12 m (DMSO‑d6) spectral data are shown in Table 1. Comparing with the data
3′ 72.8 4.69 m 70.9 3.81 m of the 1 dissolved in pyridine-d5, we were surprised to find a signal of
4′ 69.6 4.16 dd (9.3, 2.8) 67.6 3.27 m hydroxyl (δH 5.41) from the 1H-NMR spectrum data of the DMSO‑d6
5′ 76.5 4.46 m 74.4 3.47 overlap
dissolved 1. Furthermore, in the HMBC spectrum of 1, the important
6′ 63.7 4.38 dd (11.4, 5.1) 61.5 3.41 m
4.52 dd (11.4, 2.0) 3.61 m
correlation form OH to C-2″ (δC 107.8), C-3″ (δC 85.5), C-4″ (δC 206.7)
2″ 109.9 5.55 s 107.8 4.92 s and C-24 (δC 50.6), from H-2″ (δH 4.92) to C-23 (δC 78.7), C-24 and C-
3″ 87.3 85.5 3″, from Hb-5″ (δH 2.68) to C-4″, C-6″ (δC 71.2) and H-7″ (δC 63.2)
4″ 208.1 206.7 indicated that the bridge-ring was combined on the side chain.
5″ 41.4 2.80 dd (15.6, 2.5) 40.5 2.31 d (15.3, 2.5)
In the ROESY experiments of 1 dissolved in DMSO‑d6 (Fig. 3), cor-
3.28 m 2.67 m
6″ 73.2 4.06 m 71.2 3.63 m relations between H-3 and H-10/H3-28, H-10 and H-17/H3-30 were
7″ 64.8 3.98 m 63.2 3.47 overlap found, indicating the α-orientations of H-3, H-10 and H-17. However,
4.07 m the correlations of H-8/H2-19, H2-19/H3-18 and H3-18/H-20 suggested
OH 5.41 s β-orientations of H-8, H-19 and H-20. Furthermore, the observed long-
range correlations from H3-21 to H-23; from H-23 to H-24; from H-24 to
Hb-5″ (δH 2.67) respectively, indicated that H-23, H-24 and Hb-5″ were
α-orientations whereas the correlations from Ha-5″ (δH 2.31) to H-6″,
2. Results and discussion from H-6″ to H-2″ and from H-2″ to OH-3″ respectively, suggested that
Ha-5″, H-6″, H-2″and OH-3″ were β-orientations. The CD spectrum of 1
Ethanol 70% extracts of dried immature fruits slices of M. charantia. showed a first negative Cotton effect band at 201 nm and a second
were partitioned into saponin-rich and water-soluble extracts with Cotton effect band at 288 nm. A systematic analysis of conformational
water. Three undescribed cucurbitane-type glycosides (1, 2 and 3) and searches were carried out by means of the Spartan's 10 software using
nine known cucurbitane triterpenoid saponins were isolated and pur- Merck Molecular Force Field (MMFF). The conformers with Boltzmann-
ified from saponin-rich extract using various types of silica gel column, population of over 5% were chosen for ECD calculations, and then the
MCI, ODS and RP-HPLC with UV detector. conformers were initially optimized at B3LYP/6–31 + g (d, p) level in
Furpyronecucurbitane A (1) was obtained as a white amorphous MeOH using the CPCM polarizable conductor calculation model. The
powder with negative optical rotation. The HRESIMS spectrum of 1 theoretical calculation of ECD was conducted in MeOH using Time-
showed the molecular ion peak at m/z 783.4289 [M+Na]+ (calcd dependent Density functional theory (TDDFT) at the B3LYP/6–311 + g
783.4290) and indicated a molecular formula of 1 as C42H64O12. The (d, p) level for all conformers of compounds 1a and 1b. Rotatory

22
J. Yue et al.
Fig. 1. Chemical structures of the isolated compounds 1–12 from the fruits of M. charantia L.
strengths for a total of 50 excited states were calculated. ECD spectra
were generated using the program SpecDis 1.6 (University of Würzburg,
Würzburg, Germany) and GraphPad Prism 5 (University of California
San Diego, USA) from dipole-length rotational strengths by applying
Gaussian band shapes with sigma = 0.3 eV. The obtained ECD spectrum
of 1 (Fig. 4) matched the experimental results closely, which suggested
an absolute configuration of 23S, 24S, 2″S, 3″R, and 6″R for compound
1. On the basis of the above evidence, the structure of 1 was established
as shown in Fig. 1 and named furpyronecucurbitane A. This is the first
example of this type of cucurbitane-type triterpene glycoside from M.
charantia.
Goyaglycoside I (2) was obtained as a white amorphous powder
23
Phytochemistry 157 (2019) 21–27
with negative optical rotation. The HRESIMS spectrum of 2 showed a
pseudo-molecular ion peak at m/z 685.4291 [M+Na]+ and indicated a
molecular formula of C38H62O9 (calcd 685.4286) in conjunction with
NMR data analysis. The 1H-NMR spectra (Table 1) showed proton re-
sonances corresponding to a β-D-allopyranoside moiety [δH 5.39 (1H, d,
J = 7.8 Hz, 1′-H)], six tertiary methyl groups (δH 0.85, 0.89, 0.92, 1.35,
1.35, 1.48, all s, 18, 30, 28, 26, 27, 29, each 3H)), one secondary methyl
group [δH 0.96 (d, J = 5.4 Hz), 21-H3], two methoxyl proton (δH 3.23,
3.59, 3H, both s, 25-OMe,19-OMe), four olefinic protons [δH 6.30 (dd,
J = 9.8, 1.2 Hz,6-H), 5.52 (m, 7-H), 5.57 (m, 23-H), 5.67 (m, 24-H)].
The 13C-NMR (Table 1) spectra showed resonances of 38 carbons sig-
nals, which were closely similar to those of 19(R), 25-dimethoxy-5β,19-
J. Yue et al. Phytochemistry 157 (2019) 21–27

Fig. 2. Key HMBC correlations of compounds 1–3.

epoxycucurbita-6,23-dien-3β -ol 3-O-β-D-allopyranoside (goyaglycoside

Fig. 3. Key ROESY correlations of compounds 1.
Fig. 4. Experimental and calculated ECD spectra of compound 1.
d) (Murakami et al., 2001) except for the signals due to the stereo-
chemistry at C-19. Thus, the ΔδC values [δC (goyaglycoside d)- δC(2)]
for the related signals were calculated as 1.1(C-5), −7.6(C-8), −0.5(C-
9), 2.2(C-10), 1.0(C-11) and 2.0(C-19) from the 13C-NMR data of
goyaglycoside d and 2 (Table 1), which were almost consistent with the
ΔδC values [δC (19R) − δC (19S)] 1.8(C-5), −7.8(C-8), −0.7(C-9),
2.6(C-10), 1.8(C-11) and 2.6(C-19) calculated from the 13C-NMR data
of 5β,(19R)- and 5β,(19S)-epoxy-19,23-dimethoxycucurbita-6,24-dien-
3β-ol (Zeng et al., 2014). The actual linkage positions were further
confirmed on the basis of HMBC correlations between H-3 (δH 3.65)
and the C-1' (δC 104.3) of the allose moiety and between H-1′ of the
allose moiety (δH 5.39) and C-3 of the aglycon (δC 85.1) group. (Fig. 2).
Furthermore, long-range correlations were also observed between the
25-methoxyl protons (δH 3.23) and the C-25 (δC 75.2). Consequently,
the 1D and 2D spectroscopic data of 2, along with the molecular for-
mula suggested that compound 2 is a cucurbitane-type triterpene gly-
coside and the structure of compound 2 could be assigned as 19(S), 25-
dimethoxy-5β,19-epoxycucurbita-6,23-dien-3β-ol 3-O-β-D-allopyrano-
side (goyaglycoside I).
Charantagenin F (3) was obtained as a white amorphous powder
with negative optical rotation. The HRESIMS spectrum of 3 showed a
pseudo-molecular ion peak at m/z 685.4285 [M+Na]+ and indicated a
molecular formula of C38H62O9 (calcd 685.4286) in conjunction with
NMR data analysis. The 1H-NMR spectra (Table 1) showed proton re-
sonances corresponding to a β-D- glucopyranosyl moiety [δH 4.93 (1H,
d, J = 7.7 Hz, 1′-H)], six tertiary methyl groups (δH 0.87, 0.85, 0.91,
1.43, 1.70, 1.71, all s, 18, 28, 30, 29,27, 26 each 3H)), one secondary
methyl group [δH 1.05 (d, J = 4.8 Hz), 21-H3], two methoxyl proton
(δH 3.44, 3.29, 3H, both s, 19-OMe, 23-OMe), three olefinic protons [δH
6.15 (dd, J = 9.7, 1.7 Hz,6-H), 5.60 (dd, J = 9.7, 3.5 Hz, 7-H), 5.13 (d,
9.3, 24-H),]. The 13C-NMR (Table 1) spectra showed resonances of 38
carbons signals, which were closely similar to those of (19R, 23S)-5
β,19-epoxy-19,23-dimethoxycucurbita-6,24-dien-3β-ol-3-O-β-D-allo-
pyranoside (charantagenin E, 4) (Wang et al., 2012) except for an
anomeric carbon atom (δC 106.3) and a series of oxygenated carbon
signals (δC 63.4, 72.3, 76.6, 78.3 and 79.1) assigned to a β-D- gluco-
pyranosyl residue (Akihisa et al., 2007). The actual linkage positions
were further confirmed on the basis of HMBC correlations between H-3
(δH 3.71) and the C-1' (δC 106.3) of the glucose moiety and between H-
1′ of the glucose moiety (δH 4.93) and C-3 of the aglycon (δC 84.2)

24
J. Yue et al. Phytochemistry 157 (2019) 21–27

group. Furthermore, long-range correlations were also observed be- Table 3

tween the 23-methoxyl protons (δH 3.29) and the C-23 (δC 76.8) Antiproliferative activity of the isolated compounds 1–12 on the t-HSC/Cl-6,
(Fig. 2). Consequently, the 1D and 2D spectroscopic data of 3, along Hep3B and HepG2 cells.
with the molecular formula suggested that compound 3 is a cucurbi- Compounds IC50(μM)a,b
tane-type triterpene glycoside and the structure of compound 3 could
be assigned as (19R, 23S)-5β,19-epoxy-23-methoxycucurbita-6,24-dien- t-HSC/Cl-6 Hep3B HepG2
3β-ol 3-O-β-D-glucopyranoside (charantagenin F).
1 71.00 ± 1.06 24.14 ± 3.07 23.06 ± 6.68
By analysis of their 13C and 1H NMR data and comparison to the 2 24.72 ± 1.10 50.75 ± 0.56 58.13 ± 7.4
literature data, the remaining known cucurbitane were identified as 3 41.20 ± 7.89 55.93 ± 4.37 25.8 ± 2.57
charantagenin E (4) (Wang et al., 2012), (19R, 23S)-5β,19-epoxy-19,23- 4 58.78 ± 3.76 > 100 86.67 ± 5.35
5 > 200 > 100 > 100
dimethoxycucurbita-6,24-dien-3β-ol (5) (Zeng et al., 2014), charantoside
6 63.934 ± 8.29 > 100 68.89 ± 4.37
D (6) (Yen et al., 2014), charantoside E (7) (Yen et al., 2014), 19(R)- 7 > 200 > 100 > 100
5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol (8a) (Mulholland et al., 8a and 8b 28.13 ± 3.21 32.55 ± 3.68 84.72 ± 6.76
1997), 19(S)-5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol (8b) (Chen 9 59.50 ± 7.58 44.46 ± 1.11 89.89 ± 10.3
et al., 2009), 7β,25-dimethoxycucurbita-5(6),23(E)-dien-19-al 3-O-β-D- 10 29.64 ± 2.22 48.36 ± 5.41 14.59 ± 3.7
11 3.74 ± 0.13 16.68 ± 2.07 4.12 ± 0.27
allopyranoside (9) (Liu et al., 2008), 7β,25-dimethoxycucurbita-
12 23.81 ± 0.63 68.42 ± 4.97 24.76 ± 2.9
5(6),23(E)-dien-19-al (10) (Kimura et al., 2005), karaviloside III (11) Silymarinc 201.44 ± 34.22
(Nakamura et al., 2006) and karavilagenin B (12) (Nakamura et al., 5-FUc 15.49 ± 0.62 33.58 ± 6.07
2006). Compared the chemical structures of the twelve isolated com-
a
pounds with previous reported from the fruits of Momordica chanratia, IC50:Half inhibitory concentrations measured by the MTT assay.
b
IC50values represent the means ± SD of three parallel measurements.
their structure is basically the same except for the changes of chain sides c
Positive control.
and whether there is some groups in C-19 including aldehyde group or
methyl. Remarkably, another unique type, there is one bridge-ring be-
tween C-5 and C-19 in some compounds which derived from aldehyde
group of C-19. In our current work, twelve compounds were obtained moderate inhibitory activity against HepG2 cells with IC50 values
from bitter gourd and all of them belonged to the above types. However, of < 30 μM. It's worth mentioning that karaviloside III (11) the most
it is notable that compound 1 has a unique side chain structure, and it is potent antiproliferative activity against the t-HSC/Cl-6, Hep3B and
also the latest discovery in M. charantia. HepG2 cells line with IC50 values of 3.74 ± 0.13, 16.68 ± 2.07 and
All isolated compounds were assayed for their activity against ac- 4.12 ± 0.27 μM, respectively. This study suggests some cucurbitane
tivated t-HSC/Cl-6 cells and for cytotoxicity against Hep3B and HepG2 compounds in M. charantia have certain anti-hepatic fibrosis and anti-
cells line (Table 3). Compounds 2, 8 and 10–12 had moderate in- hepatoma activities, especially karaviloside IIIFurther research is
hibitory activity against t-HSC/Cl-6 cells with IC50 values of < 30 μM. needed to fully investigate the detailed molecular mechanisms under-
Compounds 1 and 11 had moderate inhibitory activity against Hep3B lying the growth inhibitory effects of karaviloside III.
cells with IC50 values of < 30 μM. Compounds 1, 3, 10–12 had

Table 2
The NMR spectral data of compounds 2 and 3 in pyridine-d5.
No. Compound 2 Compound 3 No. Compound 2 Compound 3

δC δH δC δH δC δH δC δH

1 18.6 1.33 m 19.1 1.41 m 21 19.3 0.96 d (5.4) 20.3 1.05 d (4.8)
2.52 m 1.70 m
2 28.0 1.76 m 27.7 2.26 m 22 40.0 1.85 m 43.4 1.58 m
2.28 m 2.25 m 1.70 m
3 85.1 3.65 br s 84.2 3.71 br s 23 128.8 5.57 m 76.8 4.09 m
4 39.3 39.5 24 138.0 5.67 m 127.7 5.13 d (9.3)
5 84.3 85.9 25 75.2 136.1
6 135.8 6.30 dd (9.8, 1.2) 133.5 6.15 dd (9.7, 1.7) 26 26.4 1.35 s 26.2 1.71 s
7 129.7 5.52 m 132.0 5.60 dd (9.7, 3.5) 27 26.9 1.35 s 18.8 1.70 m
8 49.8 2.34 br s 42.6 3.08 br s 28 20.5 0.92 s 25.3 0.85 s
9 48.6 48.5 29 21.4 1.48 s 21.6 1.43 s
10 39.4 2.35 m 42.0 2.45 dd (12.4, 5.4) 30 25.6 0.89 s 20.4 0.91 s
11 22.3 1.80 m 23.7 1.65 m OMe-19 56.1 3.59 s 58.0 3.44 s
1.87 m 1.72 m
12 31.2 1.58 m 31.4 1.52 m OMe-23 55.7 3.29 s
1.60 m 1.58 m
13 45.7 45.7 OMe-25 50.5 3.23 s
14 48.6 48.7 Sugar moiety
15 34.1 1.27 m 34.2 1.28 m 1′ 104.2 5.39 d (7.8) 106.3 4.93 d (7.8)
1.33 m 1.52 m
16 28.4 1.51 m 29.0 1.32 m 2′ 73.7 3.96 m 76.6 4.09 m
1.96 m 1.95 m
17 50.8 1.52 m 51.7 1.55 m 3′ 72.8 4.72 m 78.3 4.25 m
18 15.5 0.85 s 15.0 0.87 s 4′ 69.5 4.26 dd (9.4, 2.7) 72.3 4.20 m
19 114.2 4.65 s 112.8 4.83 s 5′ 76.3 4.47 m 79.1 3.99 m
20 36.8 1.53 m 34.2 1.54 m 6′ 63.6 4.42 m 63.4 4.39 dd (11.8, 5.5)
4.55 dd (11.3, 1.7) 4.58 dd (11.8, 2.1)

25
J. Yue et al.
3. Experimental
3.1. General experimental procedures
UV spectra were recorded using a Shimadzu UV-2201 spectrometer
(Kyoto, Japan). Optical rotation was measured on a JASCO P-2000
polarimeter (Jasco Co., Tokyo, Japan). NMR spectra were recorded on
Bruker AV-600 spectrometer with TMS as internal standard, J in Hz; IR
spectra on a Bruker IFS-55 infrared spectrometer; High-resolution
electrospray ion-ization mass spectra (HR-ESI-MS) were recorded on an
Agilent 1100 LC-MSD TOF (time-of-flight) system; Column chromato-
graphy (cc): silica gel (SiO2 300–400 mesh, Qingdao Marine Chemical
Group, Co.); MCI gel (75–150 μm, Mitsubishi, Japan); ODS (300–400
mesh, Agela Technologies Co.); Sephadex LH-20 (Pharmacia, Co.).
HPLC were performed on a HPLC system (LC3000, Beijing
ChuangXinTongHeng Science & Technology Co., Ltd., Beijing, China)
consisting of a P3000 pump and a UV3000 spectrophotometric detector
at 203 nm (Beijing ChuangXinTongHeng Science & Technology Co.,
Ltd.) using a YMC- Pack-ODS-A column (250 × 10 mm, 5 μm and
250 × 4.6 mm, 5 μm, respectively).
3.2. Reagents
All extraction solvents were purchased from Qingdao Haiyang
Chemical Co., Ltd. Deionized water for HPLC analysis and for sample
preparation was obtained from Wahaha Group Co., Ltd (Hangzhou,
China). Methanol and acetonitrile (HPLC-grade) as well as other sol-
vents (analytical grade) were purchased from Tianjin Kangkede
Technology Co. Ltd (Tianjin, China). 3-(4, 5 dimethylthiazol-2-yl)-2,5-
diphenyltetra zolium (MTT) and DMSO were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). The cell culture reagents were from
Gibco/Invitrogen (Grand Island, NY, USA); KBr pellets.
3.3. Plant material
The dried immature fruits (moisture < 13%) of Momordica charantia
L. (Cucurbitaceae) slices were purchased from the farmer's market in
Guangxi province, China (25.03°S, 109.17°E) in November 2013. They
were harvested in Wutang town, Nanning city, Guangxi province, China
(108.555489°E, 22.945332°S) in May 2013, when the air humidity was
about 70%. They were identified by Professor Jincai Lu (School of
Traditional Chinese Materia Medica, Shenyang Pharmaceutical
University, China), and voucher specimen (NO. 2013122) was de-
posited at the same site.
3.4. Extraction and isolation
Slices of the dried fruits of M. charantia (10.0 kg) were cut into
smaller pieces and then extracted three times with 70% ethanol
(3 × 10 L) for 2 h under reflux to yield 3000 g of extract on solvent
evaporation. The extract was then dispersed in deionized water and the
mixture was stored overnight (Zeng et al., 2014). Upon filtration of the
mixture, 120.0 g of the saponin-rich extract was obtained. The extract
was applied to chromatographic purification on a silica gel column
(80 × 1200 mm, 850 g) eluting with petroleum ether-acetone (from
15:1 to 1:1 gradient system, v/v). Five fractions (A-E) were obtained
based on TLC analysis.
Fraction B (2.0 g) was further separated with a step gradient elution
of PE-acetone (from 9:1 to 1:1, v/v) to afford Fr. B-1, Fr. B-2. and Fr. B-
3. Compounds 5 (10 mg, 43 min) were obtained from Fr. B-1 using a
semi-preparative RP-HPLC column and eluted with MeOH-H2O (93:7,
v/v, 3.0 mL/min). Further purification of Fr. B-2 using preparative RP-
HPLC (MeOH-H2O, 89:11, 3.0 mL/min) yielded compounds 8a and 8b
(nearly 1:2 mixtures of inseparable isomers, 30 mg, 25 min). Fr. B-3 was
subjected to Sephadex LH-20 column with MeOH as the eluent to re-
move the pigment and further purified by RP-HPLC (MeOH-H2O, 87:13,
26
Phytochemistry 157 (2019) 21–27
v/v) to yield compounds 10 (13 mg, 19 min) and 12 (17 mg, 21 min).
Fraction C (12.0 g) was subjected to a silica gel CC, using a gradient of
CH2Cl2-MeOH as the eluent to provide four fractions (Fr. C-1 to Fr. C-4).
Fr. C-2 was subjected to MCI eluted with MeOH-H2O and was purified
by preparative RP-HPLC (MeOH-H2O, 87:13, 3.0 mL/min) to yield
compounds 6 (7.0 mg, 25 min), 11 (12 mg, 31 min), 9 (20.4 mg,
45 min) and 3 (10.9 mg, 47 min). Similarly, Fr. C-3 was subjected to
MCI eluted with MeOH-H2O and was also purified by preparative RP-
HPLC (MeOH-H2O, 89:11, 3.0 mL/min) to yielded compounds 2
(7.0 mg, 20 min), 4 (5.0 mg, 23 min) and 7 (6.4 mg, 28 min). Fr. D-1-4
was separated on an ODS column (50 × 500 mm, 60 g) eluted with a
gradient of MeOH-H2O (30:70–90:10, v/v) to provide Fr. D-1-4-1 and
Fr. D-1-4-2. The Fr. D-1-4-1 was separated by preparative HPLC
(MeOH/H2O, 80:20, 3.0 mL/min) to obtain compounds 1 (8.0 mg,
16min).
3.4.1. Furpyronecucurbitane A (1)
White amorphous powder; [ ]25 D -37.7 (c 0.1, MeOH); IR (KBr) vmax
(cm−1): 3424, 2925, 1631, 1402, 1384, 1271, 1127, 1009, 832, 703; 1H
NMR (600 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5,
DMSO‑d6): see Table 1; HR-ESI-MS m/z 783.4289 [M+Na]+ (calcd. for
C42H64O12Na, 783.4289).
3.4.2. Goyaglycoside I (2)
White amorphous powder; [ ]25
D -108.5 (c 0.1, MeOH); IR (KBr) vmax
(cm-1): 3422, 2928, 1631, 1401, 1383, 1271, 1077, 1027, 832, 703; 1H
NMR (400 MHz, pyridine-d5) and 13C NMR (100 MHz, pyridine-d5):
see Table 2; HR-ESI-MS m/z 685.4291 [M+Na]+ (calcd. for
C42H64O12Na, 685.4308).
3.4.3. Charantagenin F (3)
White amorphous powder; [ ]25
D -72.2 (c 0.1, MeOH); IR (KBr) vmax
(cm−1): 3424, 2936, 1631, 1443, 1383, 1271, 1082, 917, 832, 702; 1H
NMR (400 MHz, pyridine-d5) and 13C NMR (150 MHz, pyridine-d5): see
Table 2; HRESIMS m/z 685.4285 [M+Na]+ (calcd. for C42H64O12Na,
685.4308).
3.5. Cell culture and viability assay
The hepatocellular carcinoma cell lines (HepG2 and Hep3B) and the
murine hepatic stellate cell line (t-HSC/Cl-6) were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum, L-glutamine 2 mM and antibiotics (100 U/mL pe-
nicillin and streptomycin) in a humidified atmosphere of 5% CO2 at
37 °C.
Cell viability was assessed via MTT assay (Mosmann, 1983). The
cells were plated overnight in 96-well plates with 1 × 104 cells per well,
and then treated with the compounds (N = 12) at various concentra-
tions for a further 48 h. After removing all of the liquid left in the wells,
100 μL of fresh medium with MTT reagent (5 μg/mL) was added to each
well for incubation at 37 °C for 4 h. The formazan crystal was then
dissolved with DMSO, and the reduction in cell viability determined at
490 nm using a microplate reader. For this experiment, the compounds
(N = 12) were dissolved in DMSO to give a 0.1 M stock solution.
3.6. Statistical analysis
All data represent the mean ± S.D. of three independent experi-
ments. Analysis of variance was performed by two-way ANOVA pro-
cedures, using SPSS 17.0 (Statistical Program for Social Sciences, SPSS
Inc., Chicago, IL, USA) program.
Conflicts of interest
The authors declare that there are no conflicts of interest.
J. Yue et al.
Acknowledgements
The research was supported by National Natural Science Foundation
of China (Grant No. 81703389), Foundation of Liaoning Province
Education Administration (201610163L35), Youth development sup-
port plan of Shenyang Pharmaceutical University (ZQN2016023). We
are glad to acknowledge the Analytical Center of Shenyang
Pharmaceutical University for NMR measurements.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.phytochem.2018.10.009.
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