TNU Journal of Science and Technology
230(05): 190 - 196
http://jst.tnu.edu.vn 190 Email: jst@tnu.edu.vn
THE EXTRACT OF OROXYLUM INDICUM (L.) KURZ INHIBITS CELL
PROLIFERATION AND INDUCES APOPTOSIS IN HEPG2 LIVER CANCER
CELLS THROUGH THE MODULATION OF ROS PRODUCTION
Nguyen Phu Hung1, Le Thi Thanh Huong2, Hoang Van Hung3*, Can Dinh Quang Hung1,
Nguyen Hoang4, Do Nhu Tien3, Le Thi Ngoc Thuy1
1TNU - Center for Interdisciplinary Science and Education, 2TNU - University of Sciences,
3Thai Nguyen University, 4TNU - Lao Cai Campus
ARTICLE INFO
ABSTRACT
Received:
06/11/2024
Oroxylum indicum (L.) Kurz (O. indicum) is a medicinal plant used in
traditional medicine in various countries worldwide. This study aimes to
evaluate the anti-hepatocellular carcinoma effect of ethanol extracts from
the leaves, stem bark, and root bark of O. indicum. MTT assay was
performed to measure cell proliferation inhibition, DAPI staining to detect
apoptosis via nuclear morphology changes, and H2DCFDA fluorescence
staining to assess reactive oxygen species (ROS) production. The results
indicated that the extracts from the leaves, stem bark, and root bark of O.
indicum significantly reduced HepG2 cell proliferation and induced
apoptotic phenotypes in a dose-dependent manner. Among these, the leaf
extract exhibited a markedly stronger effect compared to the other two
extract samples. The leaf extract induced an excessive production of ROS
in the cells (47.2 ± 9.5% compared to 6.0 ± 2.2% in the control, p < 0.0001),
which was indicated as a mechanism of apoptosis induction and inhibition
of cell proliferation. Combined with previously published anticancer data,
this study further elucidates the potential of O. indicum against
hepatocellular carcinoma cells.
Revised:
21/01/2025
Published:
22/01/2025
KEYWORDS
Apoptosis
Oroxylum Indicum
Liver cancer
HepG2
ROS
DCH CHIẾT CỦA CÂY OROXYLUM INDICUM (L.) KURZ ỨC CHẾ
SỰ TĂNG SINH VÀ CẢM ỨNG APOPTOSIS ĐỐI VỚI TẾ BÀO UNG THƯ
GAN HEPG2 THÔNG QUA ĐIỀU CHỈNH SỰ SẢN SINH ROS
Nguyễn Phú Hùng1, Lê Thị Thanh Hương2, Hoàng Văn Hùng3*, Cấn Đình Quang Hưng2,
Nguyễn Hoàng4, Đỗ Như Tiến3, Lê Thị Ngọc Thuý1
1Trung tâm Khoa học và Giáo dục liên ngành – ĐH Thái Nguyên, 2Trường Đại học Khoa học - ĐH Thái Nguyên,
3Đại học Thái Nguyên, 4Phân hiệu Đại học Thái Nguyên tại Lào Cai
TÓM TẮT
Ngày nhận bài:
06/11/2024
O. indicum là một cây thuốc được sử dụng trong y học cổ truyền ở nhiều
nước khác nhau trên thế giới. Nghiên cứu này nhằm đnh gi hiu qu
chống lại tế bào ung thư gan HepG2 của dch chiết ethanol t l, v thân
và v r của cây O. indicum. Sàng lọc MTT được áp dụng để phân tích
tc động của dch chiết lên sự tăng sinh tế bào, apoptosis được phát hin
qua nhuộm nhân tế bào với DAPI và sự sn sinh ROS được đnh gi
thông qua nhuộm huỳnh quang với H2DCFDA. Kết qu cho thy, dch
chiết t l, v thân và v r của O. indicum đ làm gim đng kể sự tăng
sinh tế bào HepG2 và gây ra kiểu hình apoptosis tuỳ thuộc vào liều lượng.
Trong đ, dch chiết t l cho tc dụng mạnh hơn r rt so với hai loại
dch chiết t v thân và v r. Dch chiết t l đ cm ứng sự sn sinh quá
mức của ROS trong tế bào (47,2 ± 9,5% so với 6,0 ± 2,2% của đối chứng,
p < 0,0001), được chỉ ra như là cơ chế gây ra apoptosis và kìm hãm sự
tăng sinh của tế bào. Kết hợp với các dữ liu khng ung thư được công
bố trước đ, nghiên cứu này đ làm r thêm tiềm năng chống lại tế bào
ung thư gan của O. indicum.
Ngày hoàn thiện:
21/01/2025
Ngày đăng:
22/01/2025
DOI: https://doi.org/10.34238/tnu-jst.11490
* Corresponding author. Email: hoangvanhung@tnu.edu.vn
TNU Journal of Science and Technology
230(05): 190 - 196
http://jst.tnu.edu.vn 191 Email: jst@tnu.edu.vn
1. Introduction
Cancer is a complex group of diseases that arise when cells undergo abnormal changes in their
division cycle and disruption in the regulatory mechanisms of cell development. This leads to
uncontrolled proliferation and expansion of abnormal cells, resulting in tumor formation. Internal
factors such as immune system capabilities, genetic mutations, and mechanisms for repairing
genetic material, and external factors such as exposure to UV radiation, ionizing radiation, and
chemicals have been identified as contributing to the increasing incidence of cancer [1]. In 2024,
it is estimated that nearly 900,000 new cases of liver cancer will occur worldwide, making it the
sixth most common cancer type, with nearly 800,000 recorded deaths by the end of July 2024 [2].
Common cancer treatments include surgical tumor removal, chemotherapy, and ionizing radiation,
all of which contribute to improved survival rates. However, these methods also have adverse
effects. Consequently, current research is focused on exploring alternative treatments derived from
natural sources. Studies on herbal medicine in cancer treatment have shown positive results,
including the reduction of chemotherapy side effects and enhancement of the immune system to
combat cancer [3] - [5]. Among potential herbs, O. indicum, a widely available plant in tropical
countries, has shown promise. In traditional Vietnamese medicine, this plant is known for its anti-
inflammatory, detoxifying, antipyretic, and antiseptic properties [4]. Modern research has
identified and isolated various chemical compounds from O. indicum that exhibit the ability to
inhibit cancer cells, such as PC3 (prostate cancer), HepG2 (liver cancer), and MDA-MB-231
(breast cancer) [6] - [8]. Despite the identification and isolation of bioactive compounds, their
concentrations vary among different plant parts, leading to differences in their anticancer efficacy.
Therefore, this study aimes to evaluate the inhibitory effect on HepG2 cell proliferation and the
induction of apoptosis from extracts obtained from various parts of Oroxylum indicum.
2. Materials and methods
2.1. Materials and chemicals
The HepG2 cell line was obtained from the Inserm U1053 Laboratory, National Institute of
Health, France. The RPMI 1640 medium used for cell culture was provided by Thermo Fisher
Scientific. The fluorescent dye DAPI (4,6-diamidino-2-phenylindole) was supplied by Invitrogen,
and H2DCFDA (2',7'-dichlorofluorescin diacetate) was purchased from Sigma Aldrich.
2.2. Preparation of extracts from Oroxylum indicum parts
Fresh samples of leaves, stems, and roots from O. indicum were collected from the buffer zone
of Tam Dao National Park (latitude: 21º 39′ 28.58′′ N, longitude: 105º 31′ 4.22′′ E) as shown in
Figure 1. The samples were cleaned, dried at 45 ºC for 72 hours, and then ground into a fine powder.
Fifteen grams of each powder type were soaked in 45 mL of 90% ethanol and stirred at 200 rpm
for 48 hours. The extract residues were removed by filtration through Whatman paper (Merck,
Germany). The extracts were used for the analysis of activity on HepG2 cells.
Figure 1. The sample of O. indicum at the collection site
TNU Journal of Science and Technology
230(05): 190 - 196
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2.3. Cultivation and evaluation of Oroxylum indicum extracts on HepG2 cell morphology and
proliferation
HepG2 cancer cells were cultured in 96-well plates with RPMI-1640 medium containing 7%
fetal bovine serum and 1% penicillin/streptomycin (P/S), at a density of 10,000 cells per well. After
24 hours of culture, cells were treated with extracts from leaves, stems, or roots of O. indicum at
concentrations ranging from 10 to 200 µg/mL, with a control group receiving no extracts. After 48
hours of treatment, cell morphology was captured using a Ts2 NIKON inverted microscope
(Tokyo, Japan) and cell proliferation was analyzed using the MTT assay as described [9], [10].
The cell proliferation rate was calculated using the following formula:
%Cell proliferation = (OD value of AGE treated well) / (OD value of control well) × 100%.
IC50 values were determined using GraphPad Prism 9.5 (San Diego, California, USA).
2.4. Evaluation of apoptotic nucleus morphology using DAPI staining
To detect changes in nuclear morphology of HepG2 cells, cells were seeded in 24-well plates
at a density of 3×10^4 cells per well. After 24 hours, cells were treated with extracts from leaves,
stems, or roots of O. indicum at concentrations from 10 to 200 µg/mL. After 48 hours of treatment,
cell nuclei were stained with 5 µg/mL DAPI solution for 10 minutes. Nuclear images were captured
using a NIKON T2U fluorescence microscope and analyzed using ImageJ software.
2.5. Detection of ROS expression in cells using H2DCFDA staining
ROS expression was analyzed as previously described [10]. After treatment with the extracts,
the cells were washed twice with PBS. H2DCFDA solution (1 µg/mL) was used to stain the cells
for 10 minutes. Subsequently, nuclei were stained with Hoechst solution (5 µg/mL) for 10 minutes.
Cells were washed twice with 1X PBS before being observed and analyzed under a fluorescence
microscope. Data were processed using ImageJ and GraphPad Prism 9.5.
3. Results and Discussion
3.1. Effect of O. indicum extracts on cell proliferation
The impact of O. indicum extracts on cell proliferation was assessed using the MTT assay.
Figure 2 indicates that O. indicum extracts affected the proliferation levels of HepG2 cells. Cell
proliferation significantly decreased with increasing extract concentrations. Significant differences
in proliferation compared to the control were observed at 20 µg/mL for the leaf extract (p < 0.0001),
whereas significant differences were noted at 100 µg/mL for the stem (p < 0.01) and root (p <
0.001) extracts. The inhibition percentage calculated (Table 2) showed that the leaf extract
effectively inhibited cell proliferation at 20 µg/mL, with an inhibition rate of 33.15% ± 11.83%,
increasing to 76.9 ± 3.6% at 200 µg/mL. No significant differences in inhibition percentages
compared to the control were observed at concentrations below 100 µg/mL for the stem and root
extracts. Inhibition of cell proliferation was noted starting at 100 µg/mL, with rates of 29.2 ± 5.4%
for the stem extract and 44.8% ± 7.5% for the root extract (Table 1). The IC50 value for leaf extract
was 27.2 µg/mL, while stem and root extracts had IC50 values greater than 100 µg/mL.
Chiraatthakit and colleagues reported that ethanol extracts of O. indicum inhibited the
proliferation of MDA-MB-231 breast cancer cells at 600 µg/mL. Ethanol extracts from leaves and
fruits of O. indicum also demonstrated inhibitory effects on MCF-7 cell viability at concentrations
of 57.02 ± 2.85 μg/mL (leaf extract) and 131.3 ± 19.2 μg/mL (fruit extract) [11]. Buranrat and
Boontha observed that ethanol extracts from O. indicum seeds inhibited HeLa (cervical cancer) cell
proliferation with an IC50 of 50 µg/mL [12]. Based on our results and published studies, leaf
extracts of O. indicum demonstrated stronger antitumor activity than stem and root extracts.
TNU Journal of Science and Technology
230(05): 190 - 196
http://jst.tnu.edu.vn 193 Email: jst@tnu.edu.vn
Figure 2. Effect of O. indicum extracts on HepG2 cell proliferation. Viability rates of HepG2 cells at
various concentrations of ethanol extracts from leaves (a), stem bark (b), and root bark (c) of O. indicum.
Data measured by the MTT assay expressed as Mean ± SD. **P < 0.01, ****P < 0.0001, ANOVA multiple
comparison test
Table 1. Inhibition percentage of O. indicum on HepG2 cells
Concentration
10
20
50
100
200
IC50 (µg/mL)
%
inhibition
(Mean ±
SD)
Leaf
0.0
p > 0.05
33.1 ± 11.8
p < 0.001
52.6 ± 3.4
p < 0.0001
61.8 ± 3.1
p < 0.0001
76.9 ± 3.6
p < 0.0001
27.2
Stem
bark
0
p > 0.05
0
p > 0.05
18.7 ±8.6
p > 0.05
29.2 ± 5.4
p < 0.01
45.0 ± 2.8
p< 0.0001
> 100
Root
bark
0
P > 0.05
15.9 ± 7.2
P > 0.05
15.11 ± 1.4
p > 0.05
44.8 ± 7.5
p < 0.0001
54.4 ± 7.7
P < 0.0001
> 100
3.2. Impact of O. indicum extracts on cell nuclear morphology
To evaluate the effect of the three extract components on nuclear morphology, a concentration
of 100 µg/mL was utilized for each extract, as this concentration demonstrated effective inhibition
of HepG2 cells. Extracts from different parts of O. indicum induced apoptosis with varying degrees
of nuclear morphology (Figure 3A). In the control cells, with no O. indicum extract added, the
apoptosis nuclear morphology was minimal at 0.8 ± 0.3%. In contrast, the nuclear morphology
indicative of apoptosis was 27.3 ± 6.5% for leaf extract, and 14.3 ± 5.0% and 11.6 ± 3.2% for stem
bark and root bark extracts, respectively (Figure 2B).
TNU Journal of Science and Technology
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Figure 3. Effect of O. indicum extracts on apoptotic morphology of cells. (a) HepG2 cell nuclear
morphology after 48 hours of treatment with extracts (100 µg/mL) from various parts of O. indicum. (b)
Percentage of cells with apoptotic nuclear morphology for extracts from different parts of O. indicum
Previous studies have indicated that O. indicum extracts affect both intrinsic and extrinsic
apoptosis mechanisms in cancer cells, related to p53 and Vascular endothelial growth factor
(VEGF) signaling pathways, leading to changes in nuclear morphology [8]. Other studies have
provided evidence that O. indicum activates the caspase 3/7 pathway, resulting in DNA
fragmentation and apoptosis in HSC-3 cell lines [13]. Kumar and colleagues also demonstrated
that O. indicum extracts activate apoptosis through death receptor activation, leading to caspase-8
activation in breast cancer (MCF7, MDA-MB-231) and liver cancer (WRL-68) cell lines [14].
Additionally, evidence suggests that O. indicum activates caspase-8 through the binding of death
receptors with TNF-α, CD95L, and TRAIL ligands [15]. Thus, O. indicum may contain compounds
that induce strong apoptosis in various cancer cells, including liver cancer cells. Further research
is needed to fully elucidate its mechanisms and explore clinical applications.
3.3. Leaf extract of Oroxylum indicum induces overproduction of ROS in liver cancer cells
A critical mechanism of apoptosis is the overproduction of ROS in cells. Therefore, the effect
of O. indicum extracts on ROS production in liver cancer cells was assessed using H2DCFDA
fluorescence staining. The leaf extract (100 µg/mL) was selected for ROS expression analysis
because of its stronger cell inhibitory activity compared to stem and root extracts. The results
(Figure 4) indicate that the leaf extract from O. indicum significantly increased the presence of
green-fluorescent cells, which show high activity of reactive oxygen species including superoxide
anions (O2• −), hydroxyl radicals (HO•), nitric oxide (•NO), and lipid radicals. The percentage of
ROS-positive cells treated with leaf extract was 47.2 ± 9.5%, compared to 6.0 ± 2.2% in the control.
This demonstrates that the leaf extract from O. indicum induces excessive ROS expression. A
previous study on MCF7 breast cancer cell lines demonstrated that O. indicum leaf and fruit
extracts induce ROS formation, leading to enhanced apoptosis [11]. Additionally, in epithelial bile
duct cancer research, O. indicum leaf extract was shown to inhibit the cell cycle at the G2/M phase
and suppress epidermal growth factor receptor (EGFR) expression, with ROS production
enhancement being a key mechanism for cell cycle arrest and proliferation inhibition [15].
Moreover, increased ROS production associated with O. indicum has also been noted in oral cancer
[13]. The overexpression of ROS leads to genomic instability and promotes apoptosis, which is
considered crucial for the development of current anticancer drugs. Some drugs have been
developed with the characteristic of inducing high ROS production within cells, leading to nuclear
DNA breakage and macrophage induction [16]. Additionally, radiotherapy has also been developed