全 文 ： 2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 61

Chinese Journal of Natural Medicines 2011, 9(1): 0061−0064
doi: 10.3724/SP.J.1009.2011.00061
Chinese
Journal of
Natural
Medicines







Antitumor Activity of Leaves from Potentilla discolor on
Human Hepatocellular Carcinoma Cell Line HepG-2
JIN Quan, NAN Ji-Xing, LIAN Li-Hua*
College of Pharmacy, Yanbian University, Yanji 133000, China
Available online 20 Jan. 2011
[ABSTRACT] AIM: Potentilla discolor Bunge (PD), Chinese traditional medicinal herb, was investigated for its antitumor activity
and induction of apoptosis in vitro, using human HepG-2 hepatocellular carcinoma cell line. METHODS: The viability of HepG-2
cells was evaluated by MTT, after incubation with ethanolic extract of PD (5-200 mg⋅L−1). And HepG-2 cells were manipulated by
fluorescent microscopic analysis stained with DNA-specific fluorochrome 4, 6-diamidino-2-phenylindole dihydrochloride and per-
formed DNA fragmentation analysis. And caspase-3 activity and poly (ADP-ribose) polymerase (PARP) cleavage were detected by
western blot. RESULTS: PD decreased the cell viability in a concentration-dependent manner and exhibited typical characteristics of
apoptosis. Active form of caspase-3 and cleaved PARP were found in concentration-dependent manner. CONCLUSION: These results
indicated that PD may represent a potential new source of anticancer candidate.
[KEY WORDS] Potentilla discolor Bunge; HepG-2 cell; Antitumor; Apoptosis
[CLC Number] R965 [Document code] A [Article ID] 1672-3651(2011)01-0061-04

1 Introduction
The screening of promising extracts and subsequent iso-
lation of active compounds have been based on ethnophar-
macological assessment. Biological activity and the use of
traditional herb have a close correlation in the antitumor field.
In most developing countries, the traditional medicine and
herbs were used to help enhance their primary health care
needs.
Potentilla discolor Bunge (PD), a traditional Chinese
medicinal plant, is widely applied in diarrhoea, hepatitis,
functional uterine hemorrhage and traumatic hemorrhage [1].
In recent years, whole herbs of PD have also been used to
treat type 2 diabetes in clinical research [2]. Previous phyto-
chemical studies revealed the presence of triterpenes, poly-
phenols, such as flavonoids, ellagic acid derivatives, tannins
and coumarin, as well as some simple organic acids [3-4].
However, many of these studies have been focused on treat-

[Received on] 30-Aug-2010
[Research Funding] This project was partly supported by the Na-
tional Natural Science Foundation of China (No. 30660225 to Nan
JX).
[*Corresponding author] LIAN Li-Hua: Lecturer, Tel/Fax:
86-433-2660631, E-mail: lihualian@gmail.com
These authors have no any conflict of interest to declare.
ing diabetes, there is no scientific evidence for the effective-
ness of this plant as an anticancer agent yet. Although the
chemical constituents and pharmacological properties of PD
have been reported, investigation on PD effects had not been
carried out before our studies so as to demonstrate that fla-
vonoids-containing ethanol extract from PD presented re-
markable antitumor activity. Flavonoids are naturally occur-
ring polyphenolic compounds, which were ubiquitously
found in plants [5]. And flavonoids have been recognized to
possess antioxidants, enzyme inhibitors, precursors of toxic
substances, with anti-inflammatory, hepatoprotective, anti-
thrombotic, antiviral and anticarcinogenic activities [6-7].
In the present study, we used human HepG-2 hepatocel-
lular carcinoma cell line to examine the apoptotic effects
induced by PD.
2 Materials and Methods
2.1 Chemicals
RPMI-1640 medium and fetal bovine serum (FBS) was
purchased from Gibco Invitrogen Corporation (Grand Island,
NY, USA). Dimethyl sulfoxide (DMSO) in cells was main-
tained at 0.5%, which had no effect on cell growth or apop-
tosis. 3[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium
(MTT) and 4, 6-diamidino-2-phenylindole dihydrochloride
(DAPI) were purchased from Sigma (St. Louis, MO, USA).
JIN Quan, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 61−64
62 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期

2.2 Plant materials
The leaves of Potentilla discolor Bunge (PD) were col-
lected in Dongfoshi, Yanbian, Jilin province, China and the
identification was carried out by Prof. LV Hui-Zi (College of
Pharmacy of Yanbian University, China). The ethanolic ex-
tract was obtained from the dried leaves (200 g) of PD by
reflux extraction using 2.4 L 70% ethanol for two times at
80 °C. Then, the crude extracts were filtered and concen-
trated to yield a brown powder. The total flavonoids content
in the extract was measured using a colorimetric assay ac-
cording to the method of Jia et al [8]. The total flavonoids
yield (W/W) of PD were 18.89% and the total flavonoids
contained potengriffioside A, quercetin and kaempferol as
reported by Zhong et al [9].
2.3 Cell line and culture
HepG-2 cell line derived from a human hepatocellular
carcinoma was purchased from the Cell Bank of Shanghai
Institute of Biochemistry and Cell Biology, Chinese Academy
of Sciences (Shanghai, China). Cells were cultured in
RPMI-1640 medium supplemented with 10% FBS, 100
U⋅mL−1 penicillin and l00 µg⋅mL−1 streptomycin, being
maintained at 37 °C with 5% CO2 in a humidified atmos-
phere.
2.4 Cell viability assay
The viability of the cells was assessed by MTT assay.
MTT was added and incubated for an additional 3 h. Amount
of formazan was measured in an enzyme-linked immunosor-
bent assay (ELISA) plate reader at 540 nm.
2.5 DNA-specific fluorochrome 4,6-diamidino-2-pheny-
lindole dihydrochloride (DAPI) staining
HepG-2 cells, treated with PD, were centrifuged at 1 200
r·min-1 for 2 min and 3.7% neutral buffered formalin was
added to the cell pellet. The fixed cells were washed in
phosphate buffered saline (PBS), air dried and stained with
DAPI for 10 min. The adhered cells were rinsed with PBS,
air dried and mounted with 90% glycerol. The slides were
observed under Zeiss Axiovert 200M Fluorescence/Live cell
imaging microscope.
2.6 DNA fragmentation analysis
HepG-2 cells were treated with 100 mg⋅L−1 PD. Ge-
nomic DNA was prepared with the Wizard Genomic DNA
Purification Kit (Promega, Madison, WI, USA) in accordance
with the manufacturer’s guidance.
2.7 Caspase-3 activity assay
The activity of caspases-3 was determined by a caspase
colorimetric assay kit (R&D Systems, Inc., Minneapolis, MN,
USA) according to the manufacturers instructions. All of the
samples were assayed in three independent experiments.
2.8 Western blot analysis
The cleaved and non-cleaved forms of the enzymes poly
(ADP-ribose) polymerase (PARP) and cleaved caspase-3
were observed using western blot analyses. Briefly, proteins
(50 µg) were subjected to electrophoresis on SDS-polyacry-
lamide gels, followed by transfer to a nitrocellulose mem-
brane. The membrane was probed with the detected against
caspase-3 antibody (Cell Signaling Technology, Beverly, MA,
USA; 1/1000); PARP antibody (BD Biosciences Pharmingen,
San Diego, CA, USA; 1/1000). The membrane was incubated
with horseradish peroxidase-conjugated secondary antibody
(Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA;
1/2000). The detected protein signals were visualized by
Amersham ECL Plus™ Western Blotting Detection Reagents
(Amersham Biosciences, Piscataway, NJ, USA) according to
manufacturer’s recommendation.
2.9 Statistical analysis
Results are expressed as x ± s. Statistical differences
were determined between groups by one-way analysis of
variance (ANOVA) and Tukey’s multiple comparison test.
Statistically significant differences between groups were
defined as P < 0.05. Calculations were performed with the
GraphPad Prism program (GraphPad Software Inc., USA).
3 Results
3.1 Effect of PD on growth inhibition of HepG-2 cells
In this study, the inhibition of cell viability by PD was
evaluated on HepG-2 cells over various concentrations for 24
h. PD caused a concentration-dependent reduction in HepG-2
cell viability (Fig. 1A). After 100 mg⋅L−1 PD treatment, cell
viability was reduced to (49.94 ± 1.82) % at 24 h, relative to
normal cell. Therefore, the concentration of 100 mg⋅L−1 was

Fig. 1 Effects of PD-induced apoptosis in HepG-2 cells. (A)
HepG-2 cells were incubated with PD for 24 h. Cell viability
was measured with MTT assay. Results are expressed as a
percentage of normal cell viability at 24 h, which was set to
100%. Data are x ± s (n = 6) for each concentration. ***P <
0.001 vs normal control values. (B) DNA fragments were
isolated from HepG-2 cells treated with PD for 24 h. M
represents 123 base-pair marker. (C) Treated and untreated
cells were stained with DAPI and examined for condensation
and fragmentation. Stained nuclei were then observed under
fluorescent microscope using blue filter (× 400). Normal cells
(lower left panel) have a round or an ovoid shape. Cells
treated with 100mg·L−1 of PD (lower right panel) are showing
condensation and fragmentation of the nuclei, suggesting
they are undergoing apoptosis.
JIN Quan, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 61−64
2011 年 1 月 第 9 卷 第 1 期 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 63

chosen for further experiments.
3.2 Effect of PD-induced apoptosis on HepG-2 cells
As a biochemical hallmark of apoptotic cell death, DNA
fragmentation was determined. To evaluate whether the cell
death induced by PD was associated with apoptosis, HepG-2
cells were treated with 100 mg⋅L−1 of PD for 24 h. DNA ex-
tract from the cells treated with PD for 24 h displayed ladder
patterns of discontinuous DNA fragments, suggesting that
cell death induced by the PD treatment was mainly caused by
apoptosis (Fig. 1B). No DNA ladder was detected in the DNA
sample isolated from normal cells. To confirm whether the
induction of cell death is related to apoptosis, we manipulated
the cells by fluorescent microscopic analysis of apoptotic
cells stained with DAPI. Normal cells have a round or an
ovoid shape, while the number of apoptotic cells was in-
creased by PD treatment and the characteristic condensed
nuclei of apoptotic cells were clearly visible (Fig. 1C).
3.3 Caspase-3 activation and PARP cleavage in
PD-induced apoptosis
Caspase-3 plays a pivotal role in the execution phase of
apoptosis induced by diverse stimuli. To monitor the potential
involvement of caspase-3 in HepG-2 cell apoptosis, we per-
formed the enzymatic activity of caspase-3, a downstream
caspase. PD-induced activation of caspase-3 occurred
dose-dependently (Fig. 2). Significant activation of caspase-3
was detected after treatment with 50, 100 or 200 mg⋅L−1 of
PD, at a level 6.51, 8.94 or 11.32-fold higher than normal
value (P < 0.001). Additional evidence of caspase-3 activa-
tion is the proteolysis of procaspase-3 into small active frag-

Fig. 2 Caspase activity in terms of PD dose. (A) The caspase
activity of normal cells was set to 100% and relative changes
in activity are shown in terms of dose. Values represent the
results of three separate experiments. ***P < 0.001 vs normal
control values. (B) HepG-2 cells were treated with various
concentrations of PD for 24 h, and then analyzed by western
blotting. The 17 kDa subunit of activated caspase-3 was de-
tected. PARP was cleaved from the intact proform of 116 kDa
to a fragment of 85 kDa. Actin was used as the loading con-
trol. The names of proteins are shown on the left and sizes on
the right in each panel.
ments and cleavage of PARP. Therefore, activation of cas-
pase-3 was also confirmed with western blot analysis. As
shown in Fig. 2B, procaspase-3 was degradated to 17 kDa
fragment. Furthermore, exposure of HepG-2 cells to PD
caused the degradation of 116 kDa PARP into an 85 kDa
fragment (Fig. 2B).
4 Discussion
This study has shown that the leaves of Potentilla dis-
color Bunge (PD) significantly inhibited cell proliferation
and induced apoptosis in HepG-2 cells at a low concentration.
These results prompted us to undertake a more thorough
study of these findings. Recently it has been reported that
flavonoids are polyphenolic compounds found in various
foods of plants, such as fruits, beverages and vegetables. It is
well known that flavonoids are involved in a wide range of
biological activities, including free radical scavenging, apop-
tosis induction, cancer chemoprevention, and protection from
vascular disease [10]. It was reported that total flavonoids in
the leaves of Ginkgo biloba induced apoptosis of eosinophil
[11]. We were intrigued to know whether the total flavonoids
from PD could induce the apoptosis of cancer cell.
In the present work, we firstly observed the cytotoxic
effect of PD on HepG-2 cells and then the apoptotic effect of
PD in tumor cells by measuring the morphological changes.
In the cytotoxicity experiment, MTT was used to measure the
cytotoxic effect with various concentrations of PD (5, 0.4, 10,
50, 100, 200 mg⋅L−1) on HepG-2 cell line. Our results
showed that 100 mg⋅L−1 PD treatment reduced cell viability
to (49.94 ± 1.82) % at 24 h. Therefore, HepG-2 cells evi-
dence the great sensitivity toward the cytotoxic effect of PD.
Based on the cell morphological changes of HepG-2
cells, DNA fragmentation and involvement of caspases, we
concluded that PD caused apoptosis in HepG-2 cells in a
dose-dependent manner. The morphologies of PD-treated
cells revealed the typical features of apoptosis. These apop-
totic phenomena were further confirmed by DNA laddering
and DAPI staining.
Previous reports have demonstrated that the caspase
family proteases play essential roles in the process of apop-
tosis [12]. Caspase-3 is an executioner caspase that can be
activated by the following mitochondrial pathway involving
the activation of caspase-9 due to the release of cytochrome c
to the cytosol, which leads to its binding to apoptosis prote-
ase activation factor-1, or a death receptor pathway involving
caspase-8 [13-15]. This study demonstrated that the activation
of caspase-3 was involved in PD-induced cell death. Activa-
tion of caspases leads to cleavage and inactivation of key
cellular proteins such as PARP. In our model, PARP cleav-
age was observed within 24 h of PD treatment, which was
accompanied by the appearance of 17 and 19 kDa procas-
pase-3 cleavage intermediates suggesting the activation of
caspase-3. Cleavage of the caspase-3 substrate PARP, an
indicator of caspase-3 activation, was significantly elevated
JIN Quan, et al. /Chinese Journal of Natural Medicines 2011, 9(1): 61−64
64 Chin J Nat Med Jan. 2011 Vol. 9 No. 1 2011 年 1 月 第 9 卷 第 1 期

after the treatment with PD.
Taking together, our findings demonstrated that HepG-2
cells are relatively sensitive to the cytotoxic effect of PD
which induces apoptosis in HepG-2 cells via the activation of
caspase-3. Potentilla discolor Bunge may be a potential
chemopreventive or therapeutic agent in hepatocellular car-
cinoma, and these possibilities needed further investigation.
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翻白草诱导人肝癌细胞 HepG-2凋亡的研究
金 泉, 南极星, 廉丽花*
延边大学药学院, 延吉 133000
【摘 要】 目的：研究翻白草乙醇提取物对人肝癌细胞 HepG-2 体外增殖的抑制作用及凋亡的诱导作用。方法：用不同浓
度的翻白草乙醇提取物对人肝癌细胞 HepG-2, MTT 法测定对细胞生长的抑制率, 用琼脂糖凝胶电泳观察 DNA 梯状条带和 DAPI
染色观察凋亡小体, 再用 Western blot 法检测对 caspase-3 和 PARP 蛋白表达的影响。结果：翻白草乙醇提取物对人肝癌细胞
HepG-2 有较强的浓度依赖性抑制作用, 诱导细胞出现明显的形态学改变, 琼脂糖凝胶电泳出现典型的 DNA 梯状条带, Western
blot 分析结果发现, 翻白草乙醇提取物促进 caspase-3 活化而引发下游 PARP 裂解, 进而诱导人肝癌细胞 HepG-2 细胞走向自体凋
亡的途径。结论：翻白草乙醇提取物可能通过抑制人肝癌细胞 HepG-2 的增殖及诱导其凋亡而产生抗肿瘤作用。
【关键词】 翻白草; HepG-2 细胞; 抗肿瘤; 细胞凋亡

【基金项目】 国家自然科学基金项目资助 (No. 30660225)