Protective effects of dammarane-type triterpenes from hydrolyzate of Gynostemma pentaphyllum against H2O2-induced injury and anti-hepatic fibrosis activities

Article abstract of DOI:10.1016/j.phytol.2018.03.010

Gynostemma pentaphyllum (Thunb.) Makino is a kind of health food, and it is also used as a traditional medicine in Asia. In this study, phytochemical investigation of hydrolyzate of total G. pentaphyllum saponins led to the isolation of three previously undescribed triterpenes, Gypensapogenin R (1), Gypensapogenin S (2), and Gypensapogenin T (3). Their structures were mainly identified by 1D, 2D-NMR and HR-ESI–MS evidences. Anti-hepatic fibrosis activity was determined and the data showed that 2 and 3 exhibited more potent inhibitory effects (IC₅₀ values 14.93 and 29.19, respectively) on the growth of t-HSC/Cl-6 cells than Silymarin (positive control), while having much weaker effect in the growth of normal cell. Additionally, all the compounds exhibited excellent increased the ratio of viability of H9c2 induced by H₂O₂. Here in we report isolation, structure elucidation, as well as the evaluation of the anti-hepatic fibrosis and cardiomyocytes oxidative injury activities of these three compounds.

Full text of DOI:10.1016/j.phytol.2018.03.010

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
PhytochemistryLetters25(2018)33–36
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Protective effects of dammarane-type triterpenes from hydrolyzate of
Gynostemma pentaphyllum against H2O2-induced injury and anti-hepatic
fibrosis activities
⁎
Xiaoshu Zhanga,b, Guohui Shia,b, Mingbing Liua, Rong Chena, Xiaojun Wuc, Yuqing Zhaoa,b,
a
School of Functional Food and Wine, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China
Shenyang Pharmaceutical University, Shenyang, 110016, People’s Republic of China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Gynostemma pentaphyllum (Thunb.) Makino is a kind of health food, and it is also used as a traditional medicine
in Asia. In this study, phytochemical investigation of hydrolyzate of total G. pentaphyllum saponins led to the
isolation of three previously undescribed triterpenes, Gypensapogenin R (1), Gypensapogenin S (2), and
Gypensapogenin T (3). Their structures were mainly identified by 1D, 2D-NMR and HR-ESI–MS evidences. Anti-
hepatic fibrosis activity was determined and the data showed that 2 and 3 exhibited more potent inhibitory
effects (IC50 values 14.93 and 29.19, respectively) on the growth of t-HSC/Cl-6 cells than Silymarin (positive
control), while having much weaker effect in the growth of normal cell. Additionally, all the compounds ex-
hibited excellent increased the ratio of viability of H9c2 induced by H2O2. Here in we report isolation, structure
elucidation, as well as the evaluation of the anti-hepatic fibrosis and cardiomyocytes oxidative injury activities of
these three compounds.
Dammarane triterpenes
Gynostemma pentaphyllum
Anti-hepatic fibrosis
Cardiomyocytes oxidative injury
1. Introduction
diversity of new bioactive from Jiaogulan, a continuation work on
phytochemical investigation of the hydrolyzed saponins of G. penta-
Recently, Gynostemma pentaphyllum, which is an edible functional
food have been attracted extensive research and public attention be-
cause of their potential beneficial effects on human health. In China, G.
pentaphyllum were also used as a traditional Chinese medicine for al-
leviating various diseases and conditions, including liver protection,
enhance immunity, and lower cholesterol levels (Circosta et al., 2005;
Megalli et al., 2006; Suntararuks et al., 2008; Yeo et al., 2008). Previous
phytochemical investigations of G. pentaphyllum, various structure si-
milar dammarane gypenosides were discovered. Moreover, the struc-
ture of these gypenosides was similar to the ginseng saponins. Because
of this similarity structure, people claimed that drinking a tea or eat
some food made of G. pentaphyllum could longevity, more energy and
fewer illnesses (Lv et al., 2009). Pharmacology has demonstrated that
gypenosides isolated from G. pentaphyllum have various bioactivities,
including hepatoprotective, anti-inflammatory, cardiovascular and an-
tioxidant effects (Xie et al., 2010). Since 1990s, an extensive range of
health-food and beverages based on G. pentaphyllum have been devel-
oped and sold in China markets, such as total Jiaogulan saponin tablets,
Jiaogulan tea and Jiaogulan oral liquid (Lu et al., 2013). To search for
phyllum, three previously undescribed triterpenes namely gypensapo-
genin R (1), gypensapogenin S (2), and gypensapogenin T (3) were
obtained and identified by HR-ESI–MS, 1D and 2D NMR (Fig. 1).
Herein, the purification procedure and structure elucidation of these
compounds were described. Meanwhile, all the obtained compounds
were tested protective effects against cardiomyocytes injury induced by
H2O2, and their cytotoxic activity against t-HSC/Cl-6 cells and normal
cell lines.
2. Results and discussion
2.1. Structure determination
Acid hydrolysis of the crude saponins of G. pentaphyllumwith a
MeOH solution of HCl provided crude hydrolysates. The hydrolysates
were subjected to silica gel, Sephadex LH-20, and prep-HPLC chroma-
tograpic methods to afford three new compounds 1-3.
Gypensapogenin R (1) was isolated as a white amorphous solid from
the hydrolyzed saponins of G. pentaphyllum. The HR-ESI mass spectrum
⁎
Corresponding author at: School of Functional Food and Wine, Shenyang Pharmaceutical University, No.103, Wenhua Road, Shenhe District, Shenyang, 110016, Liaoning, People’s
Republic of China.
E-mail address: zyq4885@126.com (Y. Zhao).
https://doi.org/10.1016/j.phytol.2018.03.010
Received 15 January 2018; Received in revised form 24 February 2018; Accepted 1 March 2018
1874-3900/©2018PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
X. Zhang et al.
PhytochemistryLetters25(2018)33–36
Fig. 1. Structures of Compounds 1-3.
of compound 1 showed [M + Na]+ at m/z 807.4817 (calcd. for
42H72O13Na, 807.4865). The 1H NMR spectrum included signals due
fibrosis activity by examining the inhibitory activity against t-HSC/Cl-6
cells (Table 2). In particular, our results showed that all the tested
compounds exhibited inhibitory activity against t-HSC/Cl-6 cells. Of
them, compounds 2 and 3 displayed potent inhibitory activity with IC50
C
to seven methyl groups (δ 0.76, 0.90, 0.98, 1.00, 1.32, 1.40 and 1.41),
an olefinic proton at δ 5.70 (1H, t, J = 7.4 Hz), three oxygen-bonded
methine and methylene groups (δ 3.38, 4.52 and 4.72) and two
anomeric proton at δ 4.91 (1H, d, J = 7.7 Hz) and δ 4.96 (1H, d,
J = 7.8 Hz) (Table 1). Meanwhile, from the 13C NMR spectrum, 42
carbon were observed including that for two olefinic carbon at δ 131.6
and 138.8, two glucopyranosyl subunits at δ 103.7 (C-1′), 75.6 (C-2′),
79.0 (C-3′), 72.4 (C-4′), 78.7 (C-5′), 63.5 (C-6′) and δ 107.3 (C-1′), 76.2
(C-2′), 79.1 (C-3′), 72.3 (C-4′), 78.7 (C-5′), 63.3 (C-6′). The skeleton of
compound 1 suggested by the 1H and 13C NMR spectrum data that the
basic mother nucleus of 1 was similar to that of 3-O-β-D-glucopyranosyl-
gypensapogenin D (Li et al., 2012a,b), including a similar side chain of
gypensapogenin L (Zhang et al., 2015). Meanwhile, the observed long-
range correlations from δ 4.96 (H-1′) to δ 89.2 (C-3); from δ 4.91 to δ
66.2 (C-21), indicated that glycosidation of the alcoholic function at C-3
and C21 was informed. Acid hydrolysis of 1 gave a D-glucose, which
was clarified by GC analysis (Li et al., 2012b). Its β-glycosidic linkages
were evident from the J values in the 1H NMR spectrum (J1′2′ = 7.8 Hz
and J1"2" = 7.7). Based on HRESIMS, 1H NMR, 13C NMR, HSQC, HMBC
and NOESY spectra, the structure of compound 1 was identified to be
gypensapogenin R (Fig. 1).
values ranging from 14.93
compounds 1 showed lower inhibitory activity. Impressively, all the
tested compounds exhibited more significant that the positive control
1.69 and 29.19
2.33 μM, whereas
Silymarin with IC50 values of 225.27
to normal cells (Fig. 2).
2.73 and without any toxicity
2.3. Protective effects of the compounds against H2O2-induced myocardial
cell injury
Compounds 1-3 were tested for their protective effects against
H2O2-induced myocardial cell injury (Table 3). The activity was eval-
uated on various concentrations. Three compounds were thought to
increase the rate of viability of H9c2 induced by H2O2-induced myo-
cardial cell injury in a dose-dependent relationship. Notably, the effect
of low dose was also outstanding, which closed to positive control Vi-
tamin E, while no observable toxicity. Of them, compound 2 were even
more significant than Vitamin E. From the data, we speculated that this
result was related to the number of sugar and the site of methoxy group.
Three previously undescribed compounds were isolated and char-
acterized from hydrolyzate of total Gynostemma pentaphyllum saponins.
Results of bioassay showed that all the obtained compounds exhibited
protective effects against H2O2-induced myocardial cell injury and anti-
hepatic fibrosis activity. Further studies are warranted to clear their
potential mechanism and to reveal their other bio-activities.
Gypensapogenin S (2) was isolated as a white amorphous solid from
the hydrolyzed saponins of G. pentaphyllum. The HR-ESI mass spectrum
of compound 2 showed [M + Na]+ at m/z 659.4493 (calcd. for
C
37H64O8Na, 659.4493). The NMR data of 2 was similar to that of 1
except for inexistence of glucopyranosyl moiety and highfield of C-3
and presence of one methoxy group. On the basis of 1H NMR, 13C NMR,
HSQC and HMBC spectra the structure of 2 was identified and named
gypensapogenin S (Fig. 1).
3. Experimental
Gypensapogenin T (3) was isolated as a white amorphous solid from
the hydrolyzed saponins of G. pentaphyllum. The HR-ESI mass spectrum
of compound 1 showed [M + Na]+ at m/z 705.4481 (calcd. for
3.1. General experimental procedures
Saponins of G. pentaphyllum (> 80%) were purchased from Hu Nan
Province Jiuhui modern Chinese materia medica Co. Ltd. Optical ro-
tations: Perkin-Elm erpolarimeter. UV absorption spectra were mea-
sured on Unico UV-2800AH UV–vis Spectrophotometer (Unico
Instruments Ltd., Shanghai). IR absorption spectra were recorded on an
IR S-55 Infrared spectropho-tometer (Bruker, Germany) with KBr pel-
lets. HR-ESI–MS spectra were recorded using an Agilent 1100 LC–MSD
time-of-flight system. NMR spectra were recorded on Bruker AV-600
spectrometer with TMS as internal standard, J in Hz. Column chro-
matography (cc): silica gel (SiO2: 200–300 mesh, Qingdao Marine
Chemical Group, Co. Ltd., Shandong, China); Sephadex LH-20 (phar-
macia, Co.). Prep. HPLC (Beijing CXTH3000 system): P3000 pump,
UV3000 spectrophotometric detector at 210 nm, YMC C18 reversed-
phase column (5 μm, 10 × 250 nm; flow rate 3.0 mL/min). All chemi-
cals and solvents were of analytical or HPLC grade. FBS (fetal bovine
serum), RPMI 1640 medium and DMEM medium were bought from
Thermo Fisher Scientific Co., Ltd. MTT reagent was acquired from
Sigma Aldrich (St. Louis, MO, USA). Silimarin was purchased from
C
38H66O10Na, 705.4548). The 1H NMR spectrum included signals due
to seven methyl groups (δ 0.75, 0.89, 0.95, 0.98, 1.29, 1.31 and 1.40),
two methoxy group (δ 3.22 and 3.28), three oxygen protons (δ 3.37,
3.84, and 4.00) and an anomeric proton at δ 4.94 (1H, d, J = 7.8 Hz)
(Table 1). Meanwhile, from the 13C NMR spectrum, 36 carbon were
observed including that for one glucopyranosyl subunit at δ 107.3 (C-
1′), 76.1 (C-2′), 79.1 (C-3′), 72.2 (C-4′), 78.7 (C-5′), 63.4 (C-6′). The
observed long-range correlations from δ 4.94 (H-1′) to δ 89.2 (C-3),
indicated that glycosidation of the alcoholic function at C-3 was in-
formed. The HMBC correlations from the proton signals at δ 3.22
(-OCH3) to 76.1 (C-23), δ 3.28 (-OCH3) to 81.2 (C-21). On the basis of
1H NMR, 13C NMR, HSQC and HMBC spectra the structure of 3 was
identified and named gypensapogenin T (Fig. 1).
2.2. Anti-hepatic fibrosis activity
The obtained compounds 1-3 were assessed for the anti-hepatic
34

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
X. Zhang et al.
PhytochemistryLetters25(2018)33–36
Table 2
IC50 values of the isolated compounds for inhibition t-HSC/Cl-6 cells and normal cells.
Table 1
1H (600 MHz) and 13C (150 MHz) NMR data of 1-3 in C5D5N.
No.
1
2
3
Compounds
IC50 μM
δC
δH, mult. (J
in Hz)
δC
δH, mult. (J
in Hz)
δC
δH, mult. (J in
Hz)
t-HSC/Cl-6
H9c2
gastric mucosa cells (GES-1)
1
2
3
108.40
3.48
1.69
2.33
2.73
> 300
> 300
> 300
> 300
> 300
> 300
> 300
> 300
1
2
3
40.0
28.5
89.2
0.78, m
1.47, m
1.29, m
1.83, m
3.38, dd,
11.8, 4.4
–
0.73, m
1.48, m
1.66, m
1.21, m
1.53, m
–
40.0
28.7
78.4
0.94, m
1.62, m
1.32, m
1.86, m
3.45, m
39.6
29.0
89.2
0.75, m
1.44, m
1.30, m
1. 85, m
3.37, dd,
11.8, 4.3
–
0.73, m
1.49, m
1.65, m
1.21, m
1.50, m
–
14.93
29.19
225.27
Silymarina
a
Silymarin was used as positive control.
4
5
6
40.1
56.8
18.8
40.0
56.8
19.1
–
40.1
56.8
18.8
(1500 mL) were treated with 10% HCl (1000 mL) and heated at 50 °C
for 8 h. After 3 days standing, washed with plenty of water, filtration,
and then vacuum evaporated to recover the solvent. A 70 g portion of
the residue was subjected to silica gel CC (CH2Cl2: MeOH
100:0 ∼ 0:100), which resulted in eight fractions (Fr.A-H). Fr. F (2.1 g)
chromatographed over silica gel eluting with a gradient of EtOAc-
MeOH (0:100 ∼ 100:0) to obtain five subfractions F1-5. Subfraction F3
(1.6 g) was further separated on a Sephadex LH-20 column (CH2Cl2:
MeOH, 1:1) to yield four portions (F3a-F3d). Portion F3c was purified
by semipreparative HPLC eluting with 87% and 75% MeOH-H2O to
yield 3 (28 mg). Similarly, F3e was purified using Sephadex LH-20 and
semipreparative HPLC eluting with 76% MeOH-H2O to obtain 2
(22 mg). Compound 1 (31 mg) was obtained from Fr. G (1.4 g) by
semipreparative HPLC using MeOH-H2O (77:23).
0.82, m
1.46, m
1.57, m
1.24, m
1.58, m
–
7
36.2
36.3
36.1
8
9
10
11
41.1
51.6
37.4
22.0
41.2
51.7
37.9
22.1
41.0
51.5
37.7
22.2
1.26, m
–
1.32, m
–
1.27, m
–
1.45, m
1.51, m
1.14, m
1.78, m
2.31, m
–
1.11, m
1.20, m
1.22, m
1.82, m
2.54, m
0.98, s
0.90, s
–
1.80, m
1.94, m
1.16, m
1.43, m
1.96, m
–
1.24, m
1.57, m
1.87, m
1.93, m
2.93, m
1.01, s
0.83, s
–
1.18, m
1.41, m
1.83, m
2.23, m
2.14, m
–
1.06, m
1.54, m
1.82, m
1.53, m
1.89, m
0.95, s
0.89, s
–
12
25.7
26.0
27.1
13
14
15
46.8
49.9
32.2
45.9
50.0
32.5
47.1
50.7
32.0
16
29.3
27.8
28.0
17
18
19
20
21
48.1
16.2
16.3
138.8
66.2
41.3
16.4
16.9
138.0
71.4
44.3
16.1
16.8
83.8
81.2
3.3. Spectroscopic data of the new compounds
Gypensapogenin R (1): amorphous powder, [α]2D5−10°(c 0.2, MeOH);
UV (MeOH) λmax (log ε) 238 (1.04) nm; IR (KBr) νmax 3417, 2925,
2857, 1687, 1587, 1319, 1032, 656; The data of 1H and 13C NMR were
shown in Table 1; HR-ESI–MS 807.4817 [M + Na]+ (C42H72O13Na,
Calcd. 807.4865).
4.52, m
4.72, m
4.01, m
4.39, m
3.84, m
22
23
24
131.6 5.70, t, 7,4
130.7 5.94, t, 7.4
37.4
76.1
62.2
2.16, m
2.20, m
4.00, m
Gypensapogenin
S
(2): amorphous powder, [α]2D5−22°(c 0.06,
24.0
45.3
2.66, m
22.9
41.0
2.28, m
2.30, m
1.56, m
1.56, m
–
1.15, s
1.15, s
1.23, s
1.04, s
0.92, s
MeOH); UV (MeOH) λmax (log ε) 230 (1.20) nm; IR (KBr) νmax 3424,
2943, 2798, 1645, 1569, 1354, 1044, 608; The data of 1H and 13C NMR
were shown in Table 1; HR-ESI–MS 659.4493 [M + Na]+
(C37H64O8Na, Calcd. 659.4493).
1.82, m
1.84, m
–
1.40, s
1.41, s
1.32, s
1.00, s
0.76, s
2.51, m
25
26
27
28
29
30
C3-1′
2′
3′
4′
5′
6′
70.0
30.1
30.4
29.0
17.2
16.8
74.6
25.5
25.4
29.0
16.7
16.2
72.1
23.6
23.6
28.5
17.2
16.8
–
1.40, s
1.31, s
1.29, s
0.98, s
0.75, s
Gypensapogenin
T
(3): amorphous powder, [α]2D5−17°(c 0.15,
MeOH); UV (MeOH) λmax (log ε) 226 (1.18) nm; IR (KBr) νmax 3427,
2941, 2835, 1623, 1567, 1366, 1039, 647; The data of 1H 13C NMR
were shown in Table 1; HR-ESI–MS 705.4481 [M + Na]+
(C38H66O10Na, Calcd. 705.4548).
107.3 4.96, d, 7.8
107.3 4.94,m
76.2
79.1
72.3
78.7
63.3
4.05, m
4.26, m
4.23, m
4.02, m
4.39, m
4.60, m
76.1
79.1
72.2
78.7
63.4
4.04, m
4.26, m
4.20, m
4.02, m
4.39, m
4.59, m
3.4. Acid hydrolysis of compounds 1–3
The acid hydrolysis was used to identify the type and configuration
of sugar moiety and aglycone (Li et al., 2012b). Each compound (2 mg)
in 2 M HCl/MeOH (4:1) (8 mL) was stirred at 90 °C for 2 h. After
cooling, the reaction mixture was diluted to 30 mL with water and then
extracted with CH2Cl2 (30 mL × 3). The water layer was neutralized
with 1 M aqueous KOH.
C21-1”
2”
3”
4”
5”
103.7 4.91, d, 7.7
104.0 5.02, d, 7.8
75.6
79.0
72.4
78.7
63.5
4.04, m
4.26, m
4.18, m
4.02, m
4.39, m
4.60, m
75.7
79.1
72.4
78.8
63.4
4.12, m
4.29, m
4.23, m
4.01, m
4.39, m
4.60, m
6”
C21-OCH3
C23-OCH3
C25-OCH3
56.5
49.5
3.28, s
3.22, s
3.5. Anti-hepatic fibrosis activity
49.4
3.15, s
Hepatic stellate cells (t-HSC/Cl-6) were obtained from American
Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in
a Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented
with 10% heat inactivated fetal bovine serum and antibiotics (100 U/
mL penicillin and streptomycin) in moist air containing 5% CO2 at
37 °C. First, the t-HSC/Cl-6 cells were plated in 96-well plates (4000
cells/well) for 12 h, and then cells were treated by compounds at dif-
ferent concentrations (10 μM, 30 μM, 60 μM, 100 μM) for 48 h. Four
replicate wells were used at each point in the experiment with four
parallel control wells. 100 μL fresh medium with MTT reagent (5 μg/
Furui Medical Technology Co., Ltd. (Nei Menggu, China). Vitamin E
was purchased from J&K Scientific Ltd. (Beijing, China). All the cell
lines were obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA).
3.2. Extraction and isolation
The total saponins (1000 g) of G. pentaphyllum dissolved in MeOH
35

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
X. Zhang et al.
PhytochemistryLetters25(2018)33–36
Fig. 2. The key HMBC correlations of 1-3.
Table 3
Protective effects of compounds 1-3 on H2O2-induced H9c2 cell injury.
Compounds
Viabilities of H9c2 cardiomyocyte (%)
0 μM
10 μM
30 μM
60 μM
100 μM
1
2
3
46.81
48.19
43.63
45.75
1.87
2.16
1.42
1.90
52.89
76.68
43.30
52.47
1.25
1.68
1.01
2.06
80.24
87.64
61.54
54.33
2.19
3.16
2.55
1.93
78.35
85.83
59.93
61.79
3.41
2.89
3.22
3.31
80.71
83.57
79.98
75.01
3.61
4.12
3.59
3.20
Vitamin Ea
a
Positive control.
mL) was added to each well for 4 h of incubation at 37 °C after re-
moving all the left liquid in the wells. After the incubation period, the
formazan crystal was dissolved with DMSO, and the reduction of cell
viability was determined at 490 nm using a microplate reader (Bio-Rad,
USA) (Mosmann, 1983).
training program for College Students of Shenyang Pharmaceutical
University.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.phytol.2018.03.010.
3.6. Assay for testing the protective effects against myocardial cell injury
induced by H2O2
References
The rat H9C2 cardiomyocytes were obtained from ATCC (American
Type Culture Collection, Manassas, VA, USA) and routinely maintained
in DMEM medium supplemented with 10% heat inactivated fetal bo-
vine serum and antibiotics (100 U/mL penicillin and streptomycin) in a
humidified atmosphere containing 5% CO2 at 37 °C, and allowed to
adhere for 24 h before the tested compounds (1-3). The tested com-
pounds with five different concentrations (10–100 μM) were added and
following 12 h of incubation, 200 μM H2O2 was added to each well for
24 h (Fearon and Faux, 2009; Yasuoka et al., 2004). The cells in the
control groups were treated with the same volume of PBS. Cell viability
was evaluated by MTT assay, based on the reduction of MTT by the
mitochondrial dehydrogenase of intact cells to a purple formazan pro-
duct. The reduction of cell viability was determined at 490 nm using a
microplate reader (Bio-Rad, USA). All experiments were repeated three
times.
Circosta, C., Pasquale, D.R., Occhinto, F., 2005. Cardiovascular effects of the aqueous
extract of Gynostemma pentaphyllum Makino. Phytomedicine 12, 638–643.
Fearon, I.M., Faux, S.P., 2009. Oxidative stress and cardiovascular disease: novel tools
give (free) radical insight. J. Mol. Cell. Cardiol. 47, 372–381.
Li, N., Wu, C.F., Xu, X.Y., Liu, Z.Y., Li, X., Zhao, Y.Q., 2012a. Triterpenes possessing an
unprecedented skeleton isolated from hydrolyzate of total saponins from
Gynostemma pentaphyllum. Eur. J. Med. Chem. 50, 173–178.
Li, W., Cao, J.Q., Tang, Y., Zhang, L.H., Xie, Q.M., Shen, H.J., Zhao, Y.Q., 2012b. Cyclic
bisdesmosides from Actinostemma lobatum MAXIM (Cucurbitaceae) and their in
vitro cytotoxicity. Fitoterapia 83, 147–152.
Lu, J.G., Zhu, L., Lo, K.Y.W., Leung, A.K.M., Ho, A.H.M., Zhang, H.Y., Zhao, Z.Z., Fong,
D.W.F., Jiang, Z.H., 2013. Chemical differentiation of two taste variants of gynos-
temma pentaphyllum by using UPLC?Q-TOF-MS and HPLC–ELSD. J. Agric. Food
Chem. 61, 90–97.
Lv, Y., Yang, X., Zhao, Y., Ruan, Y., Yang, Y., Wang, Z., 2009. Separation and quantifi-
cation of component monosaccharides of the tea polysaccharides from Gynostemma
pentaphyllum by HPLC with indirect UV detection. Food Chem. 112, 742–746.
Megalli, S., Davis, N.M., Roufogalis, B.D., 2006. Anti-hyperlipidemic and hypoglycemic
effects of Gynostemma pentaphyllum in the Zucker fatty rat. J. Pharm. Pharm. Sci. 9,
281–291.
Mosmann, T.J., 1983. Rapid colorimetric assay for cellular growth and survival: appli-
cation to proliferation and cytotoxicity assays. J. Immunol. Methods 65, 55–63.
Suntararuks, S., Yoopan, N., Rangkadilok, N., Worasuttayangkurn, L., 2008.
Immunomodulatory effects of cadmium and gynostemma pentaphyllum herbal tea on
rat splenocyte proliferation. J. Agric. Food Chem. 56, 9305–9311.
Xie, Z., Liu, W., Huang, H., Slavin, M., Zhao, Y., Whent, M., 2010. 2010 chemical com-
position of five commercial gynostemma pentaphyllum samples and their radical
scavenging, antiproliferative, and anti-inflammatory properties. J. Agric. Food Chem.
58, 11243–11249.
Yasuoka, C., Ihara, Y., Ikeda, S., Miyahara, Y., Kondo, T., Kohno, S., 2004. Antiapoptotic
activity of Akt is down-regulated by Ca2+ in myocardiac H9c2 cells Evidence of
Ca2+-dependent regulation of protein phosphatase 2Ac. J. Biol. Chem. 279,
51182–51192.
3.7. Statistical analysis
All the presented data and results were performed in at least three
independent experiments and expressed as the mean
comparisons were made by Student's t-test and One-way ANOVA
method. Statistical calculations were performed using the SPSS 16.0 for
Windows software package.
SD. Statistical
Acknowledgements
Yeo, J., Kang, Y.J., Jeon, S.M., Jung, U.J., Lee, M.K., Song, H., 2008. Potential hy-
poglycemic effect of an ethanol extract of Gynostemma pentaphyllum in C57BL/KsJ-
db/db mice. J. Med. Food. 11, 709–716.
Zhang, X.S., Cao, J.Q., Zhao, C., Wang, X.D., Wu, X.J., Zhao, Y.Q., 2015. Novel dam-
marane-type triterpenes isolated from hydrolyzate of total Gynostemma penta-
phyllum saponins. Bioorg. Med. Chem. Lett. 25, 3095–3099.
The research was supported by National Nature Science Foundation
of China (Grant No. 81703389), Education fund item of Liaoning
Province (201610163L35), Youth development support plan of
Shenyang Pharmaceutical University (ZQN2016023) and Innovative
36