Dammarane-type saponins from Gynostemma pentaphyllum and their cytotoxicities

Article abstract of DOI:10.1080/14786419.2020.1723093

Heat-processed Gynostemma pentaphyllum has shown strong activity against human lung carcinoma A549 cells. In this study, two dammarane-type saponins together with two known compounds were isolated from the ethanol extract of the heat-processed leaves of G. pentaphyllum. They were identified as 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-β-D-glucopyranoside (1, namely damulin E), 2α,3β,12β-trihydroxydammar-20,24-diene-3-O-β-D-glucopyranoside (2, namely damulin F), damulin A (3) and damulin B (4), respectively, using NMR and mass spectra. Damulin E and damulin F showed moderate activity against A549, H1299, T24, SH-SY5Y and K562 cell lines in?vitro using CCK-8 assay.

Full text of DOI:10.1080/14786419.2020.1723093

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Dammarane-type saponins from Gynostemma
pentaphyllum and their cytotoxicities
Wei-Ye Cui, Yulian Jin, Hui Liu, Ma-Li Zu, Xin-Fang Zhai, Ce Yang, Yu-Long Gu,
Yong Cheng & Xiang-Lan Piao
To cite this article: Wei-Ye Cui, Yulian Jin, Hui Liu, Ma-Li Zu, Xin-Fang Zhai, Ce Yang,
Yu-Long Gu, Yong Cheng & Xiang-Lan Piao (2020): Dammarane-type saponins from
Gynostemmaꢀpentaphyllum and their cytotoxicities, Natural Product Research, DOI:
10.1080/14786419.2020.1723093
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2020.1723093
Dammarane-type saponins from Gynostemma
pentaphyllum and their cytotoxicities
Wei-Ye Cuia, Yulian Jinb, Hui Liua, Ma-Li Zua, Xin-Fang Zhaia, Ce Yanga,
Yu-Long Gua, Yong Chenga and Xiang-Lan Piaoa
aCenter on Translational Neuroscience and School of Pharmacy, Minzu University of China, Beijing,
b
China; School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin, China
ABSTRACT
ARTICLE HISTORY
Received 6 December 2019
Accepted 19 January 2020
Heat-processed Gynostemma pentaphyllum has shown strong
activity against human lung carcinoma A549 cells. In this study,
two dammarane-type saponins together with two known
compounds were isolated from the ethanol extract of the heat-
processed leaves of G. pentaphyllum. They were identified as
2a,3b,12b-trihydroxydammar-20(22),24-diene-3-O-b-D-glucopyrano-
side (1, namely damulin E), 2a,3b,12b-trihydroxydammar-20,
24-diene-3-O-b-D-glucopyranoside (2, namely damulin F), damulin
A (3) and damulin B (4), respectively, using NMR and mass spectra.
Damulin E and damulin F showed moderate activity against
A549, H1299, T24, SH-SY5Y and K562 cell lines in vitro using
CCK-8 assay.
KEYWORDS
Gynostemma pentaphyllum;
saponin; damulin E;
damulin F; cytotoxicity
1. Introduction
Gynostemma pentaphyllum belongs to cucurbitaceae, which is widely grown in Asia.
As one of the well-known traditional Chinese medicines, G. pentaphyllum, called
Jiaogulan in China, is a perennial creeping medicinal plant widely distributed in
CONTACT Xiang-Lan Piao
xlpiao@muc.edu.cn; xlpiao@163.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1723093.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
W.-Y. CUI ET AL.
Shaanxi, Fujian, Gansu, Hubei, and Guangxi provinces in China as well as Japan, Korea
and Southeast Asian countries (Yin et al. 2004). According to published data, G. penta-
phyllum exhibit a variety of biological activities, including antioxidant and neuropro-
tective effects (Jia et al. 2015; Shang et al. 2016; Yu et al. 2016; Alhasani et al. 2018;
Xing et al. 2018; Zhang et al. 2020), immunomodulatory and anti-inflammatory activ-
ities (Liou et al. 2010; Shang et al. 2016; Wang et al. 2017; Shen et al. 2018), anti-
cancer (Lu et al. 2008; Liu et al. 2015; Zhang et al. 2018), anti-hyperlipidemic,
hypoglycemic, and atherosclerotic effects (Gao et al. 2016; Yang et al. 2017).
Gypenosides, dammarane-type saponins, are known to be the principal bioactive con-
stituents of G. pentaphyllum. The structures of gypenosides are closely similar to ginse-
nosides from Panax ginseng, G. pentaphyllum is also called “southern ginseng” or
“cheaper ginseng” (Shi et al. 2012).
By 2018, there were an estimated 18.1 million new cancer cases and 9.6 million
deaths of cancer patients (Bray et al. 2018). Blocked apoptosis caused the drug resist-
ance of cancer cells (Denicourt and Dowdy 2004). Therefore, inducing apoptosis is one
of the main mechanisms of anti-cancer therapy. It is known that caspases are activated
through two major apoptotic pathways, including the extrinsic or death receptor path-
way and the intrinsic or mitochondrial pathway (Kim et al. 2013). Several synthetic
anticancer agents have been suggested for the prevention and treatment of cancer,
various side effects and toxicities have been an issue (Lee et al. 2010). Therefore, phy-
tochemicals have attracted much attention and great effort has been made to search
for safe and effective therapeutic agents for cancer. In previous research, we isolated
gypenoside L, gypenoside LI, damulin A, damulin B, damulin C and damulin D from
heat-processed G. pentaphyllum and they were showed anti-cancer activities against
human lung carcinoma A549 cells and human hepatocellular carcinoma HepG2 cells
(Piao et al. 2013; Piao et al. 2014).
In this investigation, two compounds, damulin E (1) and damulin F (2), were newly
isolated with two known compounds, damulin A (3) and damulin B (4) (Figure 1), by
silica gel, HP-20 and further purification with HPLC from the leaves of heat processed
G. pentaphyllum. Although the structure of damulin F (2) was predicted in metabolite
analysis by LC-MS in our laboratory before (Chen et al. 2015), it was not obtained.
Therefore, the monomer was isolated and identified for the first time in this
experiment, and further activity was verified.
2. Results and discussion
Damulin E (1) was obtained as a white powder. In the negative mode ESI-MS of 1
(Figure 1), the HR-ESI-MS analysis indicated a protonated ion peak at m/z 619.4224
1
[M-H]ꢀ which corresponded to the molecular formula C36H60O8. The H NMR (C5D5N,
600 MHz) spectrum of 1 indicated the presence of 8 methyl signals at d 0.92 (3 H,s,
H-19), 0.97 (3 H, s, H-30), 1.03 (3 H, s, H-18), 1.08 (3 H, s, H-29), 1.43 (3 H, s, H-28), 1.59
(3 H, s, H-27), 1.64 (3 H, s, H-26) and 1.84 (3 H, s, H-21), two olefinic proton signals at
d 5.51 (1 H, m, H-22) and d 5.24 (1 H, m, H-24), and one anomeric proton signal at
d 5.00 (1 H, d, J ¼ 7.9 Hz, H-1’). According to the anomeric proton coupling constant
value of 7.9 Hz, the configuration of the sugar could be identified as b-type. The 13C

NATURAL PRODUCT RESEARCH
3
Figure 1. Chemical structures of isolated saponins from G. pentaphyllum.
NMR and DEPT spectra (C5D5N, 150 MHz) showed 36 carbon signals, of which 30 were
assigned to the aglycone, four olefinic carbon signals at d 140.7 (C-20), 124.0 (C-22),
124.3 (C-24) and 131.8 (C-25), three oxygenated carbons at d 67.4 (C-2), 96.0 (C-3) and
73.0 (C-12). The DEPT90 spectrum showed two CH carbons (C-22 and C-24) from four
olefinic carbons. The 1H-1H COSY spectrum revealed the correlations between H-1
(d 2.38, 1.15)/H-2 (d 4.04)/H-3 (d 3.33), H-5 (d 0.89)/H-6 (d 1.52, 1.41)/H-7 (d 1.50, 1.26),
H-9 (d 1.54)/H-11 (d 2.11, 1.51)/H-12 (d 3.93)/H-13 (d 2.01)/H-17 (d 2.82)/H-16 (d 1.95,
1.55)/H-15 (d 1.68, 1.13) and H-22 (d 5.51)/H-23 (d 2.81, 2.42)/H-24 (d 5.24) (Table S1).
The HMBC spectrum revealed the correlations of H-18 (d 1.03) to C-7 (d 35.8), C-8 (d
40.8) and C-9 (d 51.3), H-19 (d 0.92) to C-1 (d 48.4), C5 (d 56.8), C-9 (d 51.3) and C-10
(d 38.5), H-21 (d 1.84) to C17 (d 50.9), C-20 (d 140.7) and C-22 (d 124.0), H-26 (d 1.64)
to C-C24 (d 124.3), C-25 (d 131.8) and C-27 (d 18.2), H-27 (d 1.59) to C-24 (d 124.3),
C-25 (d 131.8) and C-26 (d 26.2), H-28 (d 1.43) to C-3 (d 96.0), C-4 (d 41.4), C-5 (d 56.8)
and C-29 (d 18.5), H-29 (d 1.08) to C-3 (d 96.0), C-4 (d 41.4), C-5 (d 56.8) and C-28
(d 29.0), H-30 (d 0.97) to C-8 (d 40.8), C-14 (d 51.4) and C-15 (d 33.1). The anomeric
proton of glucose H-10 (d 5.00) correlated with C-3 (d 96.0) of the aglycon was
revealed by the HMBC (Table S1). These data supported the fact that 1 has a dammar-
ane skeleton, which were similar to those of damulin A (Nguyen et al 2011; Piao et al.
2013). The COSY spectrum allowed the sequential assignments of all resonances
for the monosaccharide, starting from the anomeric proton (Table S1). The absolute
configuration of the sugar in the compound (1) was confirmed as D-glucose by
comparing the retention time of the hydrolyzed sugar in 1 with that of D-glucose and
D-galactose, two six-carbohydrate sugar standards, in the ion chromatographic column

4
W.-Y. CUI ET AL.
(D-galactose, 5.892 min; D-glucose, 6.502 min) (Figure S9). On the basis of the obtained
data, the structure of compound 1 was assigned as 2a,3b,12b-trihydroxydammar-
20(22),24-diene-3-O-b-D-glucopyranoside (1, namely damulin E).
Compound 2 was isolated as white powder. HR-ESI-MS analysis indicated a proto-
nated ion peak at m/z 619.4216 [M-H]ꢀ which corresponded to the molecular formula
1
1
C36H60O8.The H NMR, 13C NMR, H-1H COSY and HMBC spectroscopic properties for
compound 2 were similar to those of 1, except for the peaks related to the side chain
(Figure 1). The chemical shifts of C-20 (d 156.0) and C-21 (108.6) revealed that there
1
is a double bond in C-20(21). The H NMR (C5D5N, 600 MHz) spectrum of 2 indicated
the presence of 7 methyl signals at d 0.92 (3 H, s, H-19), 0.97 (3 H,s, H-30), 1.03 (3 H, s,
H-18), 1.08 (3 H, s, H-29), 1.44 (3 H, s, H-28), 1.61 (3 H, s, H-27) and 1.68 (3 H, s, H-26),
three olefinic proton signals at d 5.24 (1 H, bs, H-21), 4.93 (1 H, bs, H-21) and 5.31 (1 H,
m, H-24), and one anomeric proton signal at d 5.02 (1 H, d, J ¼ 7.9 Hz, H-10). According
to the anomeric proton coupling constant value of 7.9 Hz, the configuration of the
sugar could be identified as b-type. The 13C NMR and DEPT spectra (C5D5N, 150 MHz)
showed 36 carbon signals, of which 30 were assigned to the aglycone, four olefinic
carbon signals at d 156.0 (C-20), 108.6 (C-21), 125.93 (C-24) and 131.7 (C-25), three
oxygenated carbons at d 67.4 (C-2), 96.0 (C-3) and 72.8 (C-12). The DEPT135 showed
one methylidene carbon at d 108.6 (C-21) and the DEPT90 appeared one CH carbon at
d 125.9 (C-24), which were similar to those of damulin B (Nguyen et al 2011; Piao
1
et al. 2013). The H-1H COSY spectrum revealed the correlations between H-1 (d 2.34,
1.18)/H-2 (d 4.05)/H-3 (d 3.33), H-5 (d 0.89)/H-6 (d 1.57, 1.41)/H-7 (d 1.52, 1.28), H-9
(d 1.59)/H-11 (d 2.13, 1.54)/H-12 (d 3.92)/H-13 (d 2.11)/H-17 (d 2.84)/H-16 (d 2.11, 1.59)/
H-15 (d 1.73, 1.16) and H-22 (d 2.50)/H-23 (d 2.39, 2.33)/H-24 (d 5.31) (Table S1). The
HMBC spectrum revealed the correlations of H-18 (d 1.03) to C-7 (d 35.8), C-8 (d 40.7)
and C-9 (d 51.4), H-19 (d 0.92) to C-1 (d 48.4), C5 (d 56.8), C-9 (d 51.4) and C-10
(d 38.5), H-21 (d 5.17, 4.93) to C17 (d 48.8) and C-22 (d 34.4), H-26 (d 1.68) to C-C24
(d 125.9), C-25 (d 131.7) and C-27 (d 18.3), H-27 (d 1.61) to C-24 (d 125.9), C-25 (d
131.7) and C-26 (d 26.3), H-28 (d 1.44) to C-3 (d 96.0), C-4 (d 41.4), C-5 (d 56.8) and
C-29 (d 18.5), H-29 (d 1.08) to C-3 (d 96.0), C-4 (d 41.4), C-5 (d 56.8) and C-28 (d 29.0),
H-30 (d 0.97) to C-8 (d 40.7), C-14 (d 51.7) and C-15 (d 33.1). The anomeric proton of
glucose H-1’ (d 5.02) correlated with C-3 (d 96.0) of the aglycon was revealed by the
HMBC (Table S1). These data supported the fact that 2 has a dammarane skeleton,
which were similar to those of damulin B (Nguyen et al. 2011; Piao et al. 2013). The
absolute configuration of the sugar in 2 was confirmed as D-glucose by comparing
the retention time of the hydrolyzed sugar in 2. Consequently, on the basis of the
obtained data, the structure of compound 2 was assigned as 2a,3b,12b-trihydroxy-
dammar-20(21),24-diene-3-O-b-D-glucopyranoside, namely damulin F (Figure 1). The
1H and 13C NMR assignments for compounds 1 and 2 are listed in (Table S1).
In the previous study, damulin B showed strong activity against A549 cells (Kim
et al. 2012). Compared with control (ginsenoside Rg3), damulin F showed stronger
cytotoxic activity against A549 cells with the IC50 value of 19.8 0.4 lM (Table S2),
whereas damulin E appeared weaker activity with the IC50 value of 38.9 0.6 lM.
In addition, we compared the effects of damulin E and damulin F with that of
damulin A and damulin B, and found that damulin E and damulin F showed stronger

NATURAL PRODUCT RESEARCH
5
cytotoxicity against A549 cells than that of damulin A (IC50 value of 59.2 1.2 lM) and
damulin B (IC50 value of 29.6 0.8 lM), respectively (Figure S2). Therefore, 3-O-b-D-
mono-glucosyl saponins could increase the cytotoxic activity compared with 3-O-b-D-
di-glucosyl saponins and double bond in C20(21) position showed stronger cytotoxic
activity than that of double bond in C20(22) position in saponins (Xing et al. 2016).
3. Experimental
3.1. General experimental procedures
Silica gel (SiO2: 200–300 mesh) was purchased from Qingdao Marine Chemical Group,
Co. Ltd, China. Macroporous resin HP-20 was purchased from Mitsubishi Chemical Co.,
Ltd., Japan. Inertsil ODS-SP column (4.6 ꢁ 250 mm, 5 lm) was purchased from GL
Sciences, Inc., Japan. HPLC-grade acetonitrile was purchased from Fisher Chemical,
America. Water for HPLC was purified using Water Purification Systems (Heal Force
Bio-Meditech Holdings Ltd., China). All other reagents were of analytical reagent grade.
A549, H1299, T24, SH-SY5Y and K562 cells were purchased from China Infrastructure
of Cell Line Resources (National Infrastructureof Cell Resourse, China). DMEM and RPMI
1640 were purchased from Gibco, America. D-galactose (>99%), D-glucose (>99%)
were purchased from Chengdu Pufei De Biotech Co., Ltd, China. Sodium hydroxide
(50% mass fraction) (Thermo Fisher Scientific, America). Sugars were analysed using
Dionex Carbo PacTM PA20 column (150 mm ꢁ 3 mm, 6.5 lm) (Thermo Fisher Scientific,
America). The NMR spectra were recorded in C5D5N on a Bruker AV-600 NMR
spectrometer (Bruker, Switzerland, 600 MHz for 1 H) and the chemical shifts are
reported in ppm relative to the residual undeuterated solvent. Mass spectra and
accurate mass measurements were recorded on LCMS-IT-TOF spectrometer (Shimadzu,
Japan). Cytotoxic activity was detected on Molecular Devices FlexStation 3 microplate
reader (Molecular Devices, America). Ion chromatograms were recorded in ICS-3000
Ion Chromatography with ampere detector, Chromeleon 6.8 chromatographic
workstation (Thermo Fisher Scientific, America).
3.2. Plant material
G. pentaphyllum was collected from Zhangzhou, Fujian in China and identified
professionally. Voucher specimen (No.GP2018-01) was deposited at the Isolation and
Structure Identification Laboratory in School of Pharmacy, Minzu University of China.
3.3. Extraction and isolation
Dried leaves of G. pentaphyllum (20 kg) were steam treated at 130 ꢂC for 3 h and was
refluxed twice with 80% ethanol in H2O. The organic solvent was removed under a
vacuum to give 6.3 kg of ethanol extract. The extract was separated by resin HP-20 in
succession with water, 20% and 95% ethanol. The 95% ethanol fraction was chromato-
graphed over silica gel column (80 ꢁ 450 mm). Elution with CHCl2/MeOH gradient (20:1
to 2:1) yielded 13 fractions. Compound 1 (10 mg), 2 (9 mg), 3 (20 mg) and 4 (16 mg)

6
W.-Y. CUI ET AL.
were isolated from fraction 8 by HPLC (Inertsil ODS-SP column, 4.6 ꢁ 250 mm, 5 lm)
using acetonitrile/H2O (50%, v/v) at the flow rate of 1 mL/min.
3.4. Spectroscopic data of compounds 1 and 2
Damulin E (1): white amorphous powder; [a]2D5: -4 (c 0.1, MeOH); HR-ESI-MS m/z
1
619.4224 [M-H]ꢀ (calcd for C36H59O8, 619.4210). H NMR (C5D5N, 600 MHz) (Table S1):
2.38 (1 H, m, H-1), 1.15 (1 H, m, H-1), 4.04 (1 H, m, H-2), 3.33 (1 H, d, J ¼ 9.2 Hz, H-3),
0.89 (1 H, m, H-5), 1.52 (1 H, m, H-6), 1.41 (1 H, m, H-6), 1.50 (1 H, m, H-7), 1.26 (1 H, m,
H-7), 1.54 (1 H, m, H-9), 2.11 (1 H, m, H-11), 1.51 (1 H, m, H-11), 3.93 (1 H, m, H-12), 2.01
(1 H, m, H-13), 1.68 (1 H, m, H-15), 1.13 (1 H, m, H-15), 1.95 (1 H, m, H-16), 1.55 (1 H, m,
H-16), 2.82 (1 H, m, H-17), 1.03 (3 H, s, H-18), 0.92 (3 H, s, H-19), 1.84 (3 H, s, H-21), 5.51
(1 H, m, H-22), 2.81 (1 H, m, H-23), 2.42 (1 H, m, H-23), 5.24 (1 H, m, H-24), 1.64 (3 H, s,
H-26), 1.59 (3 H, s, H-27), 1.43 (3 H, s, H-28), 1.08 (3 H, s, H-29), 0.97 (3 H, s, H-30), 5.00
(1 H, d, J ¼ 7.9 Hz, H-1’), 4.14 (1 H, m, H-2’), 4.29 (1 H, m, H-3’), 4.25 (1 H, m, H-4’), 4.13
(1 H, m, H-5’), 4.65 (1 H, m, H-6’), 4.38 (1 H, m, H-60). 13C NMR (C5D5N, 150 MHz): 48.4
(C-1), 67.4 (C-2), 96.0 (C-3), 41.4 (C-4), 56.8 (C-5), 19.0 (C-6), 35.8 (C-7), 40.8 (C-8), 51.3
(C-9), 38.5 (C-10), 33.0 (C-11), 73.0 (C-12), 51.5 (C-13), 51.4 (C-14), 33.1 (C-15), 29.4
(C-16), 50.9 (C-17), 16.4 (C-18), 18.1 (C-19), 140.7 (C-20), 13.7 (C-21), 124.0 (C-22), 28.0
(C-23), 124.3 (C-24), 131.8 (C-25), 26.2 (C-26), 18.2 (C-27), 29.0 (C-28), 18.5 (C-29), 17.5
(C-30), 107.1 (C-10), 76.1 (C-2’), 79.3 (C-30), 72.2 (C-4’), 79.1 (C-50), 63.1 (C-6’).
Damulin F (2): white amorphous powder; [a]2D5: -8 (c 0.1, MeOH); HR-ESI-MS m/z
1
619.4216 [M-H]ꢀ (calcd for C36H59O8, 619.4210). H NMR (C5D5N, 600 MHz) (Table S1):
2.34 (1 H, m, H-1), 1.18 (1 H, m, H-1), 4.05 (1 H, m, H-2), 3.33 (1 H, d, J ¼ 9.3 Hz, H-3),
0.89 (1 H, m, H-5), 1.57 (1 H, m, H-6), 1.41 (1 H, m, H-6), 1.52 (1 H, m, H-7), 1.28 (1 H, m,
H-7), 1.59 (1 H, m, H-9), 2.13 (1 H, m, H-11), 1.54 (1 H, m, H-11), 3.92 (1 H, m, H-12), 2.11
(1 H, m, H-13), 1.73 (1 H, m, H-15), 1.16 (1 H, m, H-15), 2.11 (1 H, m, H-16), (1.59 (1 H, m,
H-16), 2.84 (1 H, m, H-17), 1.03 (3 H, s, H-18), 0.92 (3 H, s, H-19), 5.24 (1 H, bs, H-21),
4.93 (1 H, bs, H-21), 2.50 (1 H, m, H-22), 2.39 (1 H, m, H-23), 2.33 (1 H, m, H-23), 5.31
(1 H, m, H-24), 1.68 (3 H, s, H-26), 1.61 (3 H, s, H-27), 1.44 (3 H, s, H-28), 1.08 (3 H, s,
H-29), 0.97 (3 H, s, H-30), 5.02 (1 H, d, J ¼ 7.9 Hz, H-1’), 4.14 (1 H, m, H-2’), 4.30 (1 H, m,
H-3’), 4.28 (1 H, m, H-4’), 4.15 (1 H, m, H-5’), 4.65 (1 H, m, H-6’), 4.37 (1 H, m, H-6’).
13C NMR (C5D5N, 150 MHz): 48.4 (C-1), 67.4 (C-2), 96.0 (C-3), 41.4 (C-4), 56.8 (C-5), 19.0
(C-6), 35.8 (C-7), 40.7 (C-8), 51.4 (C-9), 38.5 (C-10), 33.4 (C-11), 72.8 (C-12), 53.0 (C-13),
51.7 (C-14), 33.1 (C-15), 31.3 (C-16), 48.8 (C-17), 16.3 (C-18), 18.1 (C-19), 156.0 (C-20),
108.6 (C-21), 34.4 (C-22), 27.6 (C-23), 125.9 (C-24), 131.7 (C-25), 26.3 (C-26), 18.3 (C-27),
29.0 (C-28), 18.5 (C-29), 17.5 (C-30), 107.1 (C-1’), 76.1 (C-2’), 79.3 (C-3’), 72.2 (C-4’),
79.1 (C-5’), 63.1 (C-6’).
3.5. Acid hydrolysis of compounds 1-2 and identification of sugar components
Each isolated compounds (1 mg) was dissolved in 1 M HCl (500 lL) and stirred for 6 h
at 80 ꢂC. Then neutralize with sodium hydroxide and filter into a clean bottle with
a 0.22 lm filter for later use. Standard references, D-galactose and D-glucose, were
prepared to 5 mg/mL of solution. Sugars were separated using Dionex CarboPacTM

NATURAL PRODUCT RESEARCH
7
PA20 column and Dionex CarboPac PG20 guard column at 30 ꢂC by ion chromatog-
raphy with pulse ampere detector. The flow rate was 0.5 mL/min and injection volume
was 20 lL. Mobile phase B, 13.125 mL of 50% sodium hydroxide solution dissolved
into 1 L of water, was isocratic eluted at 6% from 0 to 20 min.
3.6. Cell culture and cytotoxicity assay
A549 cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum
at 37 ꢂC in a humidified atmosphere of 5% CO2. Stock solutions of the test compounds
were prepared in dimethylsulfoxide (DMSO) at a concentration of 1 mg/mL and stored
at ꢀ20 ꢂC. The cytotoxicity was determined by Cell Counting Kit-8. Briefly, cells were
seeded in 96-well plates (5 ꢁ 103 cells/well) and grown overnight. Cells were then
incubated in DMEM medium containing different amounts of test compounds (0, 10,
20, 30, 40 and 50 lM) for 24 h. The solution was removed from the medium and
100 lL of 10% CCK-8 solution was added to each well. After incubation for 3 h at
37 ꢂC, the absorbance of each well was measured at 450 nm using a microplate reader.
Inhibition was calculated via the following equation: Inhibition ¼ [(AsampleꢀAblank)/
(AcontrolꢀAblank)] ꢁ 100%. Asample is the test sample absorbance, Ablank is the blank
absorbance, Acontrol is the negative control absorbance. IC50 (concentration in mM
required to inhibit cancer cells by 50%) was used to determine the inhibition.
3.7. Statistical analysis
The results of each group are expressed as mean SD values. Data were analysed by
the one-way ANOVA between control and sample treated groups by SPSS 22.0
(International Business Machines Corporation, Armonk, America). A value of p < 0.05
was considered to represent a statistically significant difference among groups.
4. Conclusion
Damulin E and damulin F were isolated and identified from the heat-processed
G. pentaphyllum using resin HP-20, silica gel and HPLC for the first time. They showed
stronger A549 cytotoxic activity than that of damulin A and damulin B, respectively.
3-O-b-D-mono-glucosyl saponins could increase the cytotoxic activity compared
with 3-O-b-D-di-glucosyl saponins and double bond in C20(21) position showed
stronger cytotoxic activity than that of double bond in C20(22) position in
saponins (Xing et al. 2016). Damulin F showed the strongest activity against A549
cells. In the further, the potential therapeutic effect of damulin F against cancer
need be verified in vitro and in vivo.
Disclosure statement
No potential conflict of interest was reported by the author(s).

8
W.-Y. CUI ET AL.
Funding
This work was supported by the National Science Fundation of China (No.81673692,
81511140297); National Key R&D Program of China (No.2017YFC1704000); and the Doctoral
Student’s Independent Research Project of Minzu University of China (No.BBZZKY-2019088).
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