In Vitro Investigation of the Anticancer Activity of Peucedanum praeruptorum Dunn Extract on HepG2 Human Hepatoma Cells

Abstract

The anticancer activity of Peucedanum praeruptorum Dunn extract (PPDE) was investigated in vitro in the HepG2 human hepatoma cell line and compared to normal human liver cells (L02 cells). The effect of the PPDE on the proliferation of the cells was measured by MTT assays, and the levels of enzymes and small molecules implicated in oxidative stress regulation were measured using specific reagent kits. The expression levels of genes implicated in apoptosis (Bax, Bcl-2, caspase-3, caspase-8, and caspase-9) and oxidative stress (SOD1 and SOD2) were quantified by RT-qPCR. Lastly, HPLC was employed to analyze the composition of the PPDE. PPDE was found to significantly inhibit the proliferation of HepG2 cells but had little effect on the proliferation of normal liver cells. PPDE increased the levels of reactive oxygen species and malonaldehyde, a lipid peroxidation product, in HepG2 cells, and it reduced the activities of antioxidant enzymes, as well as the levels of γ-GCS and reduced glutathione (GSH), suggesting that it inhibited the ability of cancer cells to regulate intracellular oxidative stress. PPDE also increased the expression of the genes encoding Bax, caspase-3, caspase-8, and caspase-9 and decreased the expression of Bcl-2, SOD1, and SOD2 in HepG2 cells, suggesting that PPDE induced the apoptosis of the liver cancer cells. HPLC analysis identified that the components of PPDE included caffeic acid, isochlorogenic acid C, myricetin, baicalin, luteolin, and kaempferol, all of which have demonstrated antioxidant properties.

Full text

Research Article
In Vitro Investigation of the Anticancer Activity of Peucedanum
praeruptorum Dunn Extract on HepG2 Human Hepatoma Cells
Shiwen Hu,
1
,
2
Pan Wang,
3
Yongchun Chen,
4
Gang Kuang,
2
Cun Wang,
2
Jing Luo,
2
and Shaocheng Chen
1
,
2
1
Chongqing Field Scientic Observation and Research Station for Authentic Traditional Chinese Medicine in the ree Gorges
Reservoir Area, Chongqing University of Education, Chongqing 400067, China
2
College of Biological and Chemical Engineering, Chongqing University of Education, Chongqing 400067, China
3
Department of Traumatology, Chongqing University Central Hospital, Chongqing Emergency Medical Center,
Chongqing 400013, China
4
Wuxi County Agricultural Products Quality and Safety Supervision Station, Chongqing 405899, China
Correspondence should be addressed to Shaocheng Chen; cque@foxmail.com
Received 22 December 2022; Revised 23 August 2023; Accepted 1 September 2023; Published 15 September 2023
Academic Editor: Mohamed Addi
Copyright ©2023 Shiwen Hu et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
e anticancer activity of Peucedanum praeruptorum Dunn extract (PPDE) was investigated in vitro in the HepG2 human
hepatoma cell line and compared to normal human liver cells (L02 cells). e eect of the PPDE on the proliferation of the cells
was measured by MTT assays, and the levels of enzymes and small molecules implicated in oxidative stress regulation were
measured using specic reagent kits. e expression levels of genes implicated in apoptosis (Bax, Bcl-2, caspase-3, caspase-8, and
caspase-9) and oxidative stress (SOD1 and SOD2) were quantied by RT-qPCR. Lastly, HPLC was employed to analyze the
composition of the PPDE. PPDE was found to signicantly inhibit the proliferation of HepG2 cells but had little eect on the
proliferation of normal liver cells. PPDE increased the levels of reactive oxygen species and malonaldehyde, a lipid peroxidation
product, in HepG2 cells, and it reduced the activities of antioxidant enzymes, as well as the levels of c-GCS and reduced
glutathione (GSH), suggesting that it inhibited the ability of cancer cells to regulate intracellular oxidative stress. PPDE also
increased the expression of the genes encoding Bax, caspase-3, caspase-8, and caspase-9 and decreased the expression of Bcl-2,
SOD1, and SOD2 in HepG2 cells, suggesting that PPDE induced the apoptosis of the liver cancer cells. HPLC analysis identied
that the components of PPDE included caeic acid, isochlorogenic acid C, myricetin, baicalin, luteolin, and kaempferol, all of
which have demonstrated antioxidant properties.
1. Introduction
Peucedanum praeruptorum Dunn is a member of the
Apiaceae family of aromatic owering plants and is native to
China [1]. It is a perennial herb whose roots and stems are
prepared into traditional Chinese medicines (TCMs) [2]
because it can relieve the symptoms of fever, cold, cough,
and bronchitis and act as an expectorant, mainly due to the
presence of coumarin compounds [3–5]. In particular, an-
gular dihydropyrano coumarins, such as praeruptorin A and
ulopterol, that are extracted from P.praeruptorum Dunn
have been used as raw materials for various health products
[6]. Similarly, ulopterol extracted from P.praeruptorum
Dunn is formulated into health drinks [7]. P.praeruptorum
Dunn can also be mixed with plants such as Platycodon
grandiorus and green tea to prepare herbal teas for ther-
apeutic applications. Furthermore, P.praeruptorum Dunn
plants are commonly cooked and mixed with honey for
direct consumption [6, 7].
Previous studies have demonstrated the anticancer ef-
fects of P.praeruptorum Dunn extracts (PPDE) in in vitro
rectal and cervical cancer models [8]. ese anticancer
properties might involve the ability of the chemical com-
ponents of the extract to degrade reactive oxygen species
Hindawi
Journal of Food Quality
Volume 2023, Article ID 9010344, 10 pages
https://doi.org/10.1155/2023/9010344

(ROS), including superoxide anions (O
2
−
), peroxide ions
(O
22−
), hydroxyl radicals (
•
OH), hydrogen peroxide (H
2
O
2
),
peroxynitrite (ONOO
−
), organic peroxy free radicals
(ROO
•
), lipid peroxy free radicals (LOO
•
), and other by-
products of cellular respiration. It is commonly accepted that
excessive ROS buildup in cells induces oxidative stress,
which can harm or even kill cells. However, despite being
naturally resistant to oxidative stress, cancer cells might
succumb to ROS-induced death if this resistance is lost [9].
Excessive ROS production can damage cancer cells by in-
creasing the peroxidation of polyunsaturated fatty acids in
cell membranes and degrading other biological macro-
molecules such as proteins and nucleic acids [10]. Antiox-
idant compounds may therefore aid in the prevention of
cancer by reducing the capacity of cells to defend themselves
against oxidative stress.
Inducing apoptosis has been shown to be an eective
method for preventing cancer cells from proliferating.
Apoptosis can be triggered by external agents that regulate
the expression of specic genes, including those that encode
members of the Bcl-2 family of cell death-regulating pro-
teins. In addition, activating specic caspases or increasing
the expression of the genes that encode these caspases can
also result in apoptosis in cancer cells [11]. erefore, the
bioactive ingredients found in health products or edible
plants may have the ability to kill cancer cells by modulating
the expression of genes from the Bcl-2 and caspase families,
which are essential for apoptosis induction [12, 13].
e objective of this research was to explore the possible
anticancer eect of PPDE on liver cancer through in vitro
experiments. By doing so, we aim to establish a theoretical
foundation for the utilization and advancement of PPDE.
More specically, our study will concentrate on assessing the
application of PPDE in in vitro cancer models, as liver cancer
is a prevalent malignancy in Asia and poses a substantial
threat to individuals’ well-being [14].
2. Materials and Methods
2.1. PPDE Extraction
P.praeruptorum Dunn (Wenyaotang Trading Co., Ltd.,
Yulin, Guangxi, China) was freeze-dried and ground into
a ne powder. A 100 g sample of the powder was heated in
1000 mL of an 80% (v/v) aqueous ethanol solution at 50°C
for 1 hour. e suspension was ltered, and the extraction
process was repeated on the remaining solid residue. e
soluble ltrates were collected and combined, and the
ethanol was removed by rotary evaporation to obtain the
PPDE. e ground powder was extracted three times in
multiple repetitions to obtain variations in the composition
of the extract.
2.2. Determination of Cell Viability. L02 and HepG2 cells
were maintained in Dulbecco’s modied Eagle medium
(DMEM, Invitrogen, New York, USA) containing 10% inac-
tivated calf serum in a humidied incubator at 37°C with a 5%
CO
2
atmosphere. After one week of culture, the medium was
discarded, and the cells were lifted from the plates with 0.05%
trypsin-EDTA (Invitrogen). e cells were collected by cen-
trifugation at 3000 rpm and then resuspended in each well at
a density of 1 ×10
4
cells/mL. is suspension was inoculated
into a 96-well culture plate at a volume of 200 L per well. After
the cells were adherent, the upper liquid medium was dis-
carded. en, aliquots (200 L) of PPDE of dierent con-
centrations (50, 100, and 200 g/mL) were added to each well,
and the cells were incubated for an additional 48 h. Meanwhile,
doxorubicin hydrochloride (1 M) was added to each well, and
the cells were incubated for another 48 h. After discarding the
upper liquid medium, 200 L of 5 mg/mL MTT (Invitrogen)
was added to each well, and the cells were incubated for 4 h.
Finally, 200 L of DMSO was added to each well to dissolve the
purple-colored formazan product, and the plates were allowed
to shake for 30 min. e absorbance of the solution in each well
was measured at 570 nm, and rates of cell survival were cal-
culated. e control group and PPDE treatments were repeated
in 8 parallel experiments. e survival rate was calculated as
a percentage with the formula OD
P
/OD
c
×100, wherein OD
P
is
the absorbance of the PPDE-treated well, and OD
c
is the
absorbance of the control well. e proliferation inhibition rate
was calculated as a percentage using the formula (OD
c
−OD
P
)/
OD
c
×100 [12].
2.3. Determination of Intracellular Malondialdehyde (MDA)
Production. After the cells were treated as described in
Section 2.2, a thiobarbituric acid colorimetric assay was
employed to measure MDA production in the cells [15].
After rinsing with phosphate buered saline (PBS), all cells
were collected with a cell scraper and added to a precooled
cell lysis solution. en, a sample (500 L) of the cell lysis
supernatant was mixed with 15% trichloroacetic acid and
0.67% thiobarbituric acid (400 L) and added to a 5 mL glass
test tube. e mixture was thoroughly mixed and incubated
for 20 min in a 95°C water bath. After cooling, 3 mL of
isopropyl alcohol was added to extract the colored com-
pound, and the absorbance of the solution was measured at
532 nm. e total protein content of the cells was quantied
using a commercially available kit (NanJing JianCheng
Bioengineering Institute, Nanjing, Jiangsu, China).
2.4. Determination of Intracellular ROS Content. After the
cells were treated in a 6-well cell culture plate as described in
Section 2.2, DMEM containing 20 mol/L 2′,7′-
dichlorodihydrouorescein diacetate was added, and the
mixture was incubated for 20 min at 37°C. e treated cells
were washed twice with cold PBS [15] and resuspended in x,
and a FLUOstar OPTIMA uorimeter was used to measure
the uorescence intensity (λ
ex
: 485 nm; λ
em
: 530 nm) to
determine the relative ROS content.
2.5. Determination of Intracellular Antioxidant Enzyme Ac-
tivity, GSH Content, and c-GCS Activity. Cells were seeded
into 6-well plates at a density of 2 ×10
5
cells/well and then
treated as described in Section 2.2 [16]. After the treatment,
the cell lysates were assayed for superoxide dismutase
2Journal of Food Quality

(SOD), catalase (CAT), glutathione peroxidase (GSH-Px),
glutathione (GSH), and gamma-glutamylcysteine synthetase
(c-GCS) activity using commercial kits (NanJing JianCheng
Bioengineering Institute, Nanjing, Jiangsu, China) accord-
ing to the instructions in each kit. Enzyme activities were
expressed as enzyme-specic activity units (U/mg protein),
while the GSH content and c-GCS activity were expressed as
units of mol/mg protein, and these values were corrected
for total cellular protein content.
2.6. Quantitative RT-PCR. Total RNA was extracted from
the cells using the TRIzol reagent (Invitrogen) according to
the manufacturer’s instructions, and the concentration of
total RNA in each sample group was adjusted to the same
level with xafter determination of purity by UV spectro-
photometry. Equivalent amounts of mRNA (2 g) from each
treatment group were added to sterilized PCR tubes con-
taining oligo-dT18, RNase, dNTP, and MLV enzyme (1 L
each) as well as 5x reaction buer (10 L). e cDNA was
synthesized in three steps: 37°C for 120 min, 99°C for 4 min,
and 4°C for 3 min [15]. en, the mRNA expression was
quantied by real-time uorescent quantitative PCR (RT-
qPCR). A sample of the cDNA (2 L), upstream and
downstream primers (10 mol/L, ermo Fisher Scientic,
Waltham, MA, USA), SYBR Premix Ex Taq II (10 L), ROX
Reference Dye (0.4 L), and sterilized double-distilled water
(5.6 L) were added to the mix solution to yield a total
reaction volume of 20 L. e reactions were performed in
a StepOne Plus PCR apparatus (ermo Fisher Scientic).
Reaction conditions included 40 cycles at 95°C for 35 s,
55−59°C for 30 s, 95°C for 15 s, and 60°C for 60 s, after which
a nal extension at 95°C for 15 s was performed. e cDNA
samples of each gene were amplied 3 times in parallel, and
the expression level of the target gene was reported as the
mean of the three C
t
values. e housekeeping gene glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH) was used
as an internal reference [17, 18].
2.7. Chromatographic Analysis Method (HPLC).
High-performance liquid chromatography (HPLC, UltiMate
3000, ermo Fisher Scientic) was utilized to analyze the
composition of sample compounds. Standard samples
(2 mg, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai,
China) were placed in separate centrifuge tubes each con-
taining 5 mL of x. en, 2 mL of methanol was added to each
tube, and the mixture was thoroughly mixed to prepare
standard stock solutions. Test solutions were prepared by
dissolving standard substances and extracts from the sam-
ples in methanol. e compositions of the PPDE samples
were determined using the following chromatographic pa-
rameters: diode array detector; 4.6 mm ×150 mm, 5 m
AcclaimTM120 C18 column; mobile phase consisting of
methanol and 0.5% acetic acid; detection wavelength set at
359 nm; column temperature maintained at 35°C; ow rate
set at 0.6 mL/min; and sample injection volume of 20 L. A
gradient elution using acetonitrile (mobile phase C) and
0.5% acetic acid (mobile phase B) was used, the conditions of
which are shown in Table 1.
2.8. Statistical Analysis. e SAS9.1 statistical software was
used to analyze the mean value of the results of three parallel
experiments. e Tukey post hoc test using one-way analysis
of variance (ANOVA) was used to analyze the statistical
dierences between groups. A P<0.05 was considered
statistically signicant.
3. Results
3.1. Compound Composition of PPDE. Analysis of the
composition of the PPDE by HPLC found that the PPDE
comprised six dierent components: caeic acid, iso-
chlorogenic acid, myricetin, baicalin, luteolin, and kaemp-
ferol (Figure 1).
3.2. Eects of PPDE on the Proliferation of L02 Normal Liver
Cells and HepG2 Hepatoma Cells. As shown in Figure 2,
incubation of L02 cells with dierent concentrations of
PPDE (0–200 g/mL) did not have any detectable eect on
the proliferation of normal human liver cells, with the
growth rate of L02 cells remaining consistently close to
100%. In contrast, the proliferation of HepG2 hepatocellular
carcinoma cells was negatively correlated with the con-
centration of PPDE. Higher concentrations of PPDE
treatment resulted in lower rates of proliferation for HepG2
cells. Comparatively, doxorubicin, a chemotherapeutic drug,
demonstrated signicantly higher potency against HepG2
cells compared to PPDE; this strong inhibition of cell
proliferation was also observed in the L02 cells. ese
ndings suggested that PPDE inhibited the proliferation of
cancer cells without having an apparent impact on normal
cells at the concentrations tested. In contrast, while doxo-
rubicin potently inhibited the proliferation of liver cancer
cells, it also displayed high toxicity. ese results highlighted
the advantages of PPDE, as it exhibited minimal toxicity
toward normal cells and demonstrated in vitro anticancer
eects in live cells. erefore, 50, 100, and 200 g/mL PPDE
were selected for further study. Using MTT assays, treatment
of HepG2 hepatocellular carcinoma cells with PPDE at 50,
100, and 200 g/mL resulted in growth inhibition of
15.5 ±1.6%, 50.4 ±2.5%, and 78.4 ±2.4%, respectively, rel-
ative to untreated cells. PPDE, therefore, was found to in-
hibit the growth of HepG2 hepatocellular carcinoma cells
in vitro.
3.3. Eects of PPDE on MDA Content and ROS Level in
HepG2 Hepatoma Cells. As shown in Figure 3, the MDA
content in HepG2 HCC cells after PPDE treatment was
signicantly higher compared to that in normal HepG2
HCC cells (P<0.05), with the highest MDA content
Table 1: Flow phase gradient elution conditions.
t(min) V(mobile phase B) (%) V(mobile phase C) (%)
0 88 12
30 60 40
35 0 100
40 0 100
Journal of Food Quality 3

60.0
55.0
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
-5.0
-10.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0
Mixed standard chromatogram
PPDE chromatogram
180.0
175.0
162.5
150.0
137.5
125.0
112.5
100.0
87.5
75.0
62.5
50.0
37.5
25.0
12.5
0.0
-12.5
-20.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0 26.0 28.0 30.0 32.0 34.0 36.0 38.0 40.0
Figure 1: Compound composition of the Peucedanum praeruptorum Dunn extract (PPDE) detected by HPLC.
0
20
40
60
80
100
120
Survival rate (%)
50 100 150 200 250 3000
Concentration of PPDE (μg/mL)
(a)
50 100 150 200 250 3000
Concentration of PPDE (μg/mL)
0
20
40
60
80
100
120
Survival rate (%)
(b)
Figure 2: Continued.
4Journal of Food Quality

observed in the cells treated with 200 g/mL PPDE. Simi-
larly, after treatment with dierent concentrations of PPDE
for 24 h, the levels of ROS in HepG2 HCC cells increased as
the PPDE concentration increased in a dose-dependent
manner.
3.4. Eects of PPDE on the Activities of CAT, SOD, and GSH-Px
in HepG2 Hepatoma Cells. As shown in Figure 4, PPDE was
found to reduce the activities of CAT, SOD, and GSH-Px in
HepG2 hepatoma cells. As the PPDE concentration increased,
the activities of CAT, SOD, and GSH-Px in the lysates of the
treated cancer cells decreased signicantly (P<0.05).
3.5. Eects of PPDE on c-GCS Activity and GSH Content in
HepG2 Hepatoma Cells. As shown in Figure 5, the activity of
c-GCS and the levels of GSH were highest in the untreated
HepG2 cells compared to both the untreated L02 cells and
the HepG2 cells treated with PPDE. Specically, after
treatment of the HepG2 cells with PPDE (50, 100, and
200 g/mL) for 24 h, the activity of c-GCS and the levels of
GSH in the HepG2 cells decreased gradually.
3.6. Eects of PPDE on the mRNA Expression of Bax, Bcl-2,
Caspase-3, Caspase-8, Caspase-9, SOD1, and SOD2 in HepG2
Cells. e expression levels of several apoptosis-related
genes were quantied by RT-qPCR. As shown in Fig-
ure 6, the expression levels of the genes encoding Bax,
caspase-3, caspase-8, and caspase-9 in HepG2 cells were
higher after treatment with 50, 100, and 200 g/mL PPDE
than in the untreated cells. Furthermore, the mRNA ex-
pression levels of Bcl-2, SOD1, and SOD2 were lower in the
PPDE-treated HepG2 cells than in untreated cells. e ex-
pression levels of caspase-3, caspase-8, and caspase-9 genes
50 100 150 200 250 3000
Concentration of PPDE (μg/mL)
0
20
40
60
80
100
120
Survival rate (%)
(c)
50 100 150 200 250 3000
Concentration of PPDE (μg/mL)
0
20
40
60
80
100
120
Survival rate (%)
(d)
Figure 2: Eects of Peucedanum praeruptorum Dunn extract (PPDE) and positive control drug of doxorubicin on proliferation of the
human normal liver cell line L02 and HepG2 human hepatoma cells (n�8). (a) PPDE-treated L02 cells, (b) PPDE-treated HepG2 cells,
(c) doxorubicin-treated L02 cells, and (d) doxorubicin-treated HepG2 cells.
D
C
B
A
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
0.5
1.0
1.5
2.0
2.5
3.0
MDA (ng/mg pro)
(a)
D
C
B
A
Control
(untreated)
50 100
PPDE (μg/mL)
200
0
50
100
150
200
250
300
ROS (%)
(b)
Figure 3: Eects of Peucedanum praeruptorum Dunn extract (PPDE) on the levels of MDA (a) and ROS (b) in HepG2 human hepatoma
cells (n�8).
A–D
Dierent lowercase letters indicate signicant dierences between the mean values of each group (P<0.05).
Journal of Food Quality 5

A
B
C
D
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
0.5
1.0
1.5
2.0
2.5
CAT (U/mg pro)
(a)
A
B
C
D
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
1.0
2.0
3.0
4.0
5.0
6.0
SOD (U/mg pro)
(b)
A
B
C
D
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
0.5
1.0
1.5
2.0
2.5
GSH-Px (U/mg pro)
(c)
Figure 4: Eect of Peucedanum praeruptorum Dunn extract (PPDE) on the activities of CAT (a), SOD (b), and GSH-Px (c) in HepG2
human hepatoma cells (n�8).
A–D
Dierent lowercase letters indicate signicant dierences between the mean values of each group
(P<0.05).
A
B
C
D
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
0.2
0.4
0.6
0.8
1.0
1.2
GSH (μmol/mg pro)
(a)
A
B
C
D
Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
1.0
2.0
3.0
4.0
5.0
γ-GCS (μmol/mg pro)
(b)
Figure 5: Eects of Peucedanum praeruptorum Dunn extract (PPDE) on the levels of GSH (a) and c-GCS (b) in HepG2 human hepatoma
cells (n�8).
A–D
Dierent lowercase letters indicate signicant dierences between the mean values of each group (P<0.05).
6Journal of Food Quality

d
c
b
a
Bax
a
b
c
d
dc
b
a
d
c
b
a
d
c
b
a
a
b
c
d
a
b
c
d
Control
(untreated)
50 100
PPDE (μg/mL)
200 Control
(untreated)
50 100
PPDE (μg/mL)
200
Control
(untreated)
50 100
PPDE (μg/mL)
200
Bcl-2
Caspase-8Caspase-3
Control
(untreated)
50 100
PPDE (μg/mL)
200
Caspase-9
SOD1 (Cu/Zn-SOD) SOD2 (Mn-SOD)
0.0
1.0
2.0
3.0
4.0
5.0
mRNA relative expression intensity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mRNA relative expression intensity
0.0
1.0
2.0
3.0
4.0
mRNA relative expression intensity
0.0
1.0
2.0
3.0
4.0
5.0
mRNA relative expression intensity
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
mRNA relative expression intensity
Control
(untreated)
50 100
PPDE (μg/mL)
200
Control
(untreated)
50 100
PPDE (μg/mL)
200 Control
(untreated)
50 100
PPDE (μg/mL)
200
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mRNA relative expression intensity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
mRNA relative expression intensity
Figure 6: Eects of Peucedanum praeruptorum Dunn extract (PPDE) on mRNA expression of Bax, Bcl-2, caspase-3, caspase-8, caspase-9,
SOD1, and SOD2 in HepG2 human hepatoma cells (n�8).
a–d
Dierent lowercase letters indicate signicant dierences between the mean
values of each group (P<0.05).
Journal of Food Quality 7

were positively correlated with the concentration of PPDE,
while the expression levels of SOD1 and SOD2 genes were
negatively correlated with the concentration of PPDE.
4. Discussion
Excessive generation or accumulation of reactive oxygen
species (ROS) within cells can induce the peroxidation of
unsaturated fatty acids in cell membranes. is process leads
to an increased production of lipid peroxidation products,
such as malondialdehyde (MDA) and 4-hydroxynonenal,
which have toxic eects on cells [19, 20]. Hence, MDA is
commonly utilized as a marker to assess cell damage caused
by oxidative stress [21]. In this study, we observed that in-
cubation of HepG2 cells with PPDE resulted in higher levels
of total ROS and MDA in the hepatoma cells compared to
untreated Hep2 cells, suggesting that PPDE induced oxidative
stress in these cells through the generation of ROS. Conse-
quently, it was reasonable to predict that PPDE treatment
would promote the death or inhibition of the proliferation of
cancer cells, as a previous study demonstrated that P. praer-
uptorum Dunn had an inhibitory eect on the growth of
cervical cancer cells in vitro [2]. Similar results were obtained
in this study, in which PPDE was found to inhibit the pro-
liferation of cultured HepG2 hepatoma cells.
Under normal physiological conditions, cancer cells are
exposed to endogenous antioxidants such as enzymes (CAT,
SOD, and GSH-Px) and the small molecule glutathione
(GSH) that can induce oxidative stress, causing damage to
cancer cells, in the presence of the immune system. SOD is
an enzyme that converts O
2–
ions to H
2
O
2
, while CAT and
GSH-Px break down H
2
O
2
into water [22]. In addition,
GSH-Px utilizes GSH as a substrate to reduce H
2
O
2
,
alkylhydroperoxides, and organic hydroperoxides (ROOH)
into hydroxyl compounds (ROH) [23]. In this study, the
activities of the main antioxidant enzymes (CAT, SOD, and
GSH-Px), as well as the levels of c-GCS and GSH, in HepG2
cells all decreased after treatment with PPDE. Reducing CAT
and SOD activities would be expected to reduce the cells’
ability to protect themselves against oxidative stress and
increase lipid peroxidation, thereby promoting oxidative
stress-related damage to cancer cells [24, 25].
As a signicant nonenzymatic antioxidant in the body,
GSH is involved in reducing toxic lipid peroxides and H
2
O
2
directly by means of GSH-Px. Moreover, it also indirectly
inhibits free-radical chain reactions [26]. As the rate-limiting
enzyme in GSH biosynthesis, c-GCS plays a crucial role in
regulating the intracellular levels of GSH. In this study, PPDE
was found to decrease the levels of GSH and c-GCS in HepG2
cells, thereby preventing the cancer cells from regulating
intracellular oxidative stress. erefore, the reduced viability
and proliferation of the HepG2 cells after exposure to PPDE
might be a result of the eect of PPDE on reducing the
activities and levels of these antioxidant enzymes and small
molecules. In addition, PPDE downregulated the mRNA and
protein levels of SOD1 and SOD2 in HepG2 cells. By reducing
the expression of SOD1 and SOD2 at the transcriptional level
in cancer cells, PPDE decreased the overall SOD activity and
consequently promoted oxidative stress and damage [24, 27].
Apoptosis was another mechanism underlying the cy-
totoxic eects of PPDE on cancer cells that was explored in
this study. Apoptosis, or programmed cell death, is tightly
regulated by the Bcl-2 family of proteins [28]. Bcl-2 prevents
the release of cytochrome C from mitochondria and pro-
motes cell proliferation [29]. Conversely, Bax, a typical
apoptosis promoter, plays a crucial role in maintaining the
balance of Bcl-2 in the body and inuences the ecacy of
cancer treatments [30]. Bcl-2 is expressed in lower quantities
in early embryonic tissues, mature lymphocytes, epithelial
cells, and neurons compared to cancer cells, wherein its
expression is highly upregulated [31]. In contrast, Bax is
widely distributed in normal tissues and cells throughout the
human body, but it is expressed in very low quantities in
cancer tissues. Consequently, the levels of Bcl-2 and Bax
expression have been used as biomarkers for evaluating
cancer prognosis [20, 29]. In our study, we found that PPDE
signicantly decreased Bcl-2 expression and increased Bax
expression, potentially leading to the promotion of apoptosis
in these cells.
Members of the caspase family of proteases, such as
caspases 3, 8, and 9, play crucial roles in apoptosis as both
mediators and executors of programmed cell death [32].
Caspase-8 and caspase-9 are upstream caspases in the ap-
optosis pathway that can activate downstream caspase-3
[33]. Caspase-3, in turn, is responsible for cleaving sub-
strates that induce cell cycle arrest, inhibiting enzymes in-
volved in cancer cell repair and breaking down the cancer
cell system. In fact, most apoptosis processes, including
those involving mitochondria, endoplasmic reticulum, and
death receptors, require the active participation of caspase-3
[34]. Many of these processes are initiated and amplied by
caspase-9 [35]. Caspase-8 can activate Bax, leading to lysis of
the mitochondria and the release of Bcl-2, Bid, and cyto-
chrome c. ese released factors further activate caspase-9
and caspase-3 [36, 37]. Simultaneously, these factors in-
tegrate signals from both the death receptor and mito-
chondrial pathways, amplifying the apoptosis signal and
promoting apoptosis [38]. In our discovery, we observed
that PPDE increased the expression of caspase-3, -8, and -9
in HepG2 cells, highlighting an additional possible mech-
anism through which this extract exerted an anticancer eect
in vitro.
Following HPLC analysis of the composition of the
PPDE, caeic acid, isochlorogenic acid C, myricetin, bai-
calin, luteolin, and kaempferol were the six active substances
that comprised the extract. All of these compounds have
demonstrated antioxidant eects, and several studies have
shown that they inhibit the growth and viability of cancer
cells in vitro [39–44]. Considering these ndings, the po-
tential of these substances to inhibit liver cancer cells in vitro
was mentioned in the PPDE, presumably due to their
combined eects.
5. Conclusions
While PPDE inhibited the growth of HepG2 cells in vitro, it
had no obvious toxic eect on L02 normal hepatocytes.
Furthermore, PPDE promoted the apoptosis of HepG2 cells
8Journal of Food Quality

in a dose-dependent manner, with the highest concentration
(200 µg/mL) of PPDE demonstrating the highest rate of
apoptosis. Further experimental results conrmed that
PPDE reduced the viability and proliferation of HepG2 cells
by inhibiting the expression and reducing the activities of
antioxidant enzymes and small molecules that regulate in-
tracellular oxidative stress. Taken together, these results
demonstrated that PPDE is a biologically active plant extract
with potential anticancer properties, warranting further
scientic exploration into its clinical applications.
Data Availability
e datasets generated for this study are available upon
request to the corresponding author.
Conflicts of Interest
All authors declare that they have no conicts of interest.
Authors’ Contributions
Shiwen Hu and Pan Wang performed the majority of the
experiments and wrote the manuscript. Yongchun Chen,
Gang Kuang, Cun Wang, and Jing Luo contributed to the
data analysis. Shaocheng Chen designed and supervised the
study and checked the nal manuscript. e authors Shiwen
Hu and Pan Wang contributed equally to this work.
Acknowledgments
is research was funded by the Science and Technology
Project of Chongqing Education Commission (KJZD-
M201901601) and Chongqing University Innovation Re-
search Group Project (CXQTP20033), China.
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