1Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 350974
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Department of Biology, College of
Science, University of Baghdad,
Baghdad-Iraq
2 H.E.J. Research Institute of Chemistry,
International Center for Chemical
and Biological Sciences, Karachi
University, Karachi, 74200, Pakistan
*Corresponding
Shaymaa Fadhel Abbas Albaayit,
shaymaa.Albaayit@sc.uobaghdad.
edu.iq
Received: 22 August 2022
Accepted: 03 March 2023
Published: 27 February 2024
Evaluation of Cytotoxic effect of Moringa
peregrina seeds on Oral Cancer, CAL 27,
Cell Line and Red Blood Cells Hemolysis
Evaluación del efecto citotóxico de las semillas de Moringa
peregrina en el cáncer oral, CAL 27, líneas celulares y
hemólisis de glóbulos rojos
Shaymaa Fadhel Abbas Albaayit1 , Rukesh Maharjan2
ABSTRACT
Background: Moringa peregrina Forssk is a well-known plant in ethnomedicine due to its
widespread uses in various diseases like cough, wound healing, rhinitis, fever, and detoxification.
The plant seeds contain compounds that are cytotoxic to many cancer cells. During the
therapeutic use of plants via the oral route, some compounds present in the plants may be
cytotoxic to normal cell lines and red blood cells. Objective: This study was the first report of
investigation of the cytotoxic profile on oral cancer, CAL 27, cell line, and hemolytic activities
on human erythrocytes of Moringa peregrina seeds ethanolic extract (MPSE). Methods:
MPSE was screened for its cytotoxic effect against oral cancer, CAL 27, cell line using 3-(4,
5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT) assay. The toxicity of MPSE
on human erythrocytes was determined by in vitro hemolytic assay. Results: MPSE showed
significant anti-proliferative activity against oral cancer, CAL 27 cell line at lower concentrations
with half maximal inhibitory concentration (IC 50 ) value of 21.03 μg/mL. At 1,000 μg/ml of MPSE,
the maximum hemolysis was found to be 14.3% which is within safer limit. Conclusions: This
study revealed a potential anti-oral cancer of MPSE and provided a baseline for its potential
use in oral cancer treatment with minimum hemolytic effect on human RBCs.
Key words: CAL 27 cell line, Moringa peregrina seed extract, Erythrocyte hemolysis, Toxicity,
Anticancer
ORIGINAL ARTICLE
Published 27 February 2024
Doi: https://doi.org/10.17533/udea.vitae.v31n1a350974
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
JOURNAL VITAE
School of Pharmaceutical and
Food Sciences
ISSN 0121-4004 | ISSNe 2145-2660
University of Antioquia
Medellin, Colombia
Filliations
1 Department of Biology, College of
Science, University of Baghdad,
Baghdad-Iraq
2 H.E.J. Research Institute of Chemistry,
International Center for Chemical
and Biological Sciences, Karachi
University, Karachi, 74200, Pakistan
*Corresponding
Shaymaa Fadhel Abbas Albaayit,
shaymaa.Albaayit@sc.uobaghdad.
edu.iq
Received: 22 August 2022
Accepted: 03 March 2023
Published: 27 February 2024
Evaluation of Cytotoxic effect of Moringa
peregrina seeds on Oral Cancer, CAL 27,
Cell Line and Red Blood Cells Hemolysis
Evaluación del efecto citotóxico de las semillas de Moringa
peregrina en el cáncer oral, CAL 27, líneas celulares y
hemólisis de glóbulos rojos
Shaymaa Fadhel Abbas Albaayit1 , Rukesh Maharjan2
ABSTRACT
Background: Moringa peregrina Forssk is a well-known plant in ethnomedicine due to its
widespread uses in various diseases like cough, wound healing, rhinitis, fever, and detoxification.
The plant seeds contain compounds that are cytotoxic to many cancer cells. During the
therapeutic use of plants via the oral route, some compounds present in the plants may be
cytotoxic to normal cell lines and red blood cells. Objective: This study was the first report of
investigation of the cytotoxic profile on oral cancer, CAL 27, cell line, and hemolytic activities
on human erythrocytes of Moringa peregrina seeds ethanolic extract (MPSE). Methods:
MPSE was screened for its cytotoxic effect against oral cancer, CAL 27, cell line using 3-(4,
5-dimethylthiazol-2-yl)-2, 5,-diphenyltetrazolium bromide (MTT) assay. The toxicity of MPSE
on human erythrocytes was determined by in vitro hemolytic assay. Results: MPSE showed
significant anti-proliferative activity against oral cancer, CAL 27 cell line at lower concentrations
with half maximal inhibitory concentration (IC 50 ) value of 21.03 μg/mL. At 1,000 μg/ml of MPSE,
the maximum hemolysis was found to be 14.3% which is within safer limit. Conclusions: This
study revealed a potential anti-oral cancer of MPSE and provided a baseline for its potential
use in oral cancer treatment with minimum hemolytic effect on human RBCs.
Key words: CAL 27 cell line, Moringa peregrina seed extract, Erythrocyte hemolysis, Toxicity,
Anticancer
ORIGINAL ARTICLE
Published 27 February 2024
Doi: https://doi.org/10.17533/udea.vitae.v31n1a350974
2Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 350974Shaymaa Fadhel Abbas Albaayit, Rukesh Maharjan
RESUMEN
Antecedentes: La Moringa peregrina Forssk es una planta muy conocida en etnomedicina debido a sus usos generalizados
en diversas enfermedades como la tos, la cicatrización de heridas, la rinitis, la fiebre y la desintoxicación. Las semillas de la
planta contienen compuestos citotóxicos para muchas células cancerosas. Durante el uso terapéutico de las plantas por vía
oral, algunos compuestos presentes en ellas pueden ser citotóxicos para las líneas celulares normales y los glóbulos rojos.
Objetivo: Este estudio fue el primer informe de investigación del perfil citotóxico sobre el cáncer oral, CAL 27, línea celular, y
las actividades hemolíticas en eritrocitos humanos del extracto etanólico de semillas de Moringa peregrina (MPSE). Métodos:
Se examinó el efecto citotóxico del MPSE contra la línea celular de cáncer oral CAL 27 mediante el ensayo con bromuro de 3-(4,
5-dimetiltiazol-2-il)-2, 5,-difeniltetrazolio (MTT). La toxicidad del MPSE sobre los eritrocitos humanos se determinó mediante
un ensayo hemolítico in vitro. Resultados: MPSE mostró una actividad antiproliferativa significativa contra el cáncer oral, línea
celular CAL 27 a concentraciones más bajas con un valor de concentración inhibitoria media máxima (IC50) de 21,03 μg/mL.
A 1.000 μg/ml de MPSE, la hemólisis máxima fue del 14,3%, lo que está dentro del límite de seguridad. Conclusiones: Este
estudio reveló un potencial anticancerígeno oral de MPSE y proporcionó una base para su uso potencial en el tratamiento del
cáncer oral con un efecto hemolítico mínimo en los glóbulos rojos humanos.
INTRODUCTION
Cancer is one of the major diseases responsible
for high mortality worldwide. Oral squamous cell
carcinoma (OSCC) is the most common cancer in
the oral cavity and ranked 6th and 10 th commonest
in males and females worldwide respectively (1,
2). In Iraq, studies showed a rapid increase in the
incidence of OSCC (3). On clinical inspection by a
dentist, it is comparatively easy to identify a tumor
by visual examination or through some OSCC tumor
markers, but due to ignorance by the patients at
the early stage led to the progression of this tumor,
and subsequently, OSCC would be diagnosed
at the advanced stages which then lead to high
mortality (4). Surgery and chemotherapy fail to
cure or prevent the recurrence and metastasis of
tumors and are often associated with side effects;
thus, many researchers are focused on natural
products to discover many new drugs for oral cancer
treatment from some potential candidates (5, 6, 7).
Natural products are well flourished for therapeutic
actions preventing and treating different ailments
(8-10). Many new anticancer therapies from natural
products were reported in basics researches (11). In
general preventive practice, cytotoxic and hemolytic
properties of natural sources were screened to
predict the possible toxic effects on mammalian cells
before developing them into a therapeutic agent for
future work (12, 13).
Moringa peregrina Forssk is a well-known,
traditionally used plant grown in Africa and Asia (10).
M. peregrina seeds contain highly pharmacologically
active components such as oleic acid, linoleic
acid, isothiocyanate, tocopherols, flavonoids,
and phenolic compounds. These compounds are
reported to have different biological activities like
antidiabetic (14), anti-Herpes simplex virus (15), anti-
inflammatory (16), antibacterial (14), and anticancer
against various cell lines (CACO-2, MCF-7, HeLa,
HepG2, and L929) (11).
Even though several pharmacological studies on M.
peregrina seeds have been reported in the literature,
the anti-proliferation effect of M. peregrina seeds
against oral carcinoma cells and the evaluation of
the hemolytic capacity towards erythrocytes have
not been scientifically reported yet. Thus, this study
was conducted to test cytotoxic activities on the
CAL 27 cell line and the effect of MPSE on human
erythrocytes.
MATERIALS AND METHODS
Preparation of plant
In 2013, M. peregrina seeds were collected and
submitted at the Department of Botany, University
of Khartoum, where Dr. Maha Kordofani, a resident
botanist, authenticated these seeds. These M.
peregrina seeds were air-dried for 5 days at 25-30 o C,
powdered, and macerated in 1:5 dried plant weights
to ethanol volume ratio. The collected filtrate was
dried under reduced pressure at 46 to 51°C using
a rotary evaporator to obtain crude M. peregrina
seed extract (MPSE) (9).
Cytotoxicity Analysis Using 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) colorimetric assay
The MPSE was tested on the CAL 27 Cell lines to
check the cytotoxic activity of this extract. CAL 27
Cell line was grown in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 1% penicillin/
streptomycin, 10% FBS, and in a humidified 5% CO2
RESUMEN
Antecedentes: La Moringa peregrina Forssk es una planta muy conocida en etnomedicina debido a sus usos generalizados
en diversas enfermedades como la tos, la cicatrización de heridas, la rinitis, la fiebre y la desintoxicación. Las semillas de la
planta contienen compuestos citotóxicos para muchas células cancerosas. Durante el uso terapéutico de las plantas por vía
oral, algunos compuestos presentes en ellas pueden ser citotóxicos para las líneas celulares normales y los glóbulos rojos.
Objetivo: Este estudio fue el primer informe de investigación del perfil citotóxico sobre el cáncer oral, CAL 27, línea celular, y
las actividades hemolíticas en eritrocitos humanos del extracto etanólico de semillas de Moringa peregrina (MPSE). Métodos:
Se examinó el efecto citotóxico del MPSE contra la línea celular de cáncer oral CAL 27 mediante el ensayo con bromuro de 3-(4,
5-dimetiltiazol-2-il)-2, 5,-difeniltetrazolio (MTT). La toxicidad del MPSE sobre los eritrocitos humanos se determinó mediante
un ensayo hemolítico in vitro. Resultados: MPSE mostró una actividad antiproliferativa significativa contra el cáncer oral, línea
celular CAL 27 a concentraciones más bajas con un valor de concentración inhibitoria media máxima (IC50) de 21,03 μg/mL.
A 1.000 μg/ml de MPSE, la hemólisis máxima fue del 14,3%, lo que está dentro del límite de seguridad. Conclusiones: Este
estudio reveló un potencial anticancerígeno oral de MPSE y proporcionó una base para su uso potencial en el tratamiento del
cáncer oral con un efecto hemolítico mínimo en los glóbulos rojos humanos.
INTRODUCTION
Cancer is one of the major diseases responsible
for high mortality worldwide. Oral squamous cell
carcinoma (OSCC) is the most common cancer in
the oral cavity and ranked 6th and 10 th commonest
in males and females worldwide respectively (1,
2). In Iraq, studies showed a rapid increase in the
incidence of OSCC (3). On clinical inspection by a
dentist, it is comparatively easy to identify a tumor
by visual examination or through some OSCC tumor
markers, but due to ignorance by the patients at
the early stage led to the progression of this tumor,
and subsequently, OSCC would be diagnosed
at the advanced stages which then lead to high
mortality (4). Surgery and chemotherapy fail to
cure or prevent the recurrence and metastasis of
tumors and are often associated with side effects;
thus, many researchers are focused on natural
products to discover many new drugs for oral cancer
treatment from some potential candidates (5, 6, 7).
Natural products are well flourished for therapeutic
actions preventing and treating different ailments
(8-10). Many new anticancer therapies from natural
products were reported in basics researches (11). In
general preventive practice, cytotoxic and hemolytic
properties of natural sources were screened to
predict the possible toxic effects on mammalian cells
before developing them into a therapeutic agent for
future work (12, 13).
Moringa peregrina Forssk is a well-known,
traditionally used plant grown in Africa and Asia (10).
M. peregrina seeds contain highly pharmacologically
active components such as oleic acid, linoleic
acid, isothiocyanate, tocopherols, flavonoids,
and phenolic compounds. These compounds are
reported to have different biological activities like
antidiabetic (14), anti-Herpes simplex virus (15), anti-
inflammatory (16), antibacterial (14), and anticancer
against various cell lines (CACO-2, MCF-7, HeLa,
HepG2, and L929) (11).
Even though several pharmacological studies on M.
peregrina seeds have been reported in the literature,
the anti-proliferation effect of M. peregrina seeds
against oral carcinoma cells and the evaluation of
the hemolytic capacity towards erythrocytes have
not been scientifically reported yet. Thus, this study
was conducted to test cytotoxic activities on the
CAL 27 cell line and the effect of MPSE on human
erythrocytes.
MATERIALS AND METHODS
Preparation of plant
In 2013, M. peregrina seeds were collected and
submitted at the Department of Botany, University
of Khartoum, where Dr. Maha Kordofani, a resident
botanist, authenticated these seeds. These M.
peregrina seeds were air-dried for 5 days at 25-30 o C,
powdered, and macerated in 1:5 dried plant weights
to ethanol volume ratio. The collected filtrate was
dried under reduced pressure at 46 to 51°C using
a rotary evaporator to obtain crude M. peregrina
seed extract (MPSE) (9).
Cytotoxicity Analysis Using 3-(4,
5-dimethylthiazol-2-yl)-2, 5-diphenyl
tetrazolium bromide (MTT) colorimetric assay
The MPSE was tested on the CAL 27 Cell lines to
check the cytotoxic activity of this extract. CAL 27
Cell line was grown in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 1% penicillin/
streptomycin, 10% FBS, and in a humidified 5% CO2
3Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 350974
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
chamber at 37°C; the cells were seeded at 5 × 103 cells/
well into 96-well plates. The vehicle used to prepare
the initial stock of MPSE was 0.1% dimethylsulfoxide
(DMSO). After 24 h, these cells were treated with
MPSE at different concentrations (12.5-100 μg/mL)
and incubated for 48 h. After incubation, cell
viability was measured by adding 20 μL of MTT dye
(5 mg/mL) to all wells and further incubated for 4 h.
The percentage of cytotoxicity of the extract was
calculated by measuring the absorbance of each
well at a wavelength of 540 nm using an ELISA plate
reader (Tecan, California, USA) (17).
Hemolytic Activity
After getting approval from the Independent Ethics
Committee of ICCBS, University of Karachi, with
reference no ICCBS/IEC-047-HB-2019/Protocol/1.0,
the hemolytic assay was carried out.
The hemolytic effect of MPSE was determined
using the method described by Wiradharma et al.,
2011 (22). Human blood (3 mL) withdrawn from a
volunteer was collected in an EDTA-anticoagulated
venoject tube and centrifuged at 700 ×g, and the
packed erythrocytes were separated from the
plasma. The erythrocytes were washed thrice with
1X PBS and then diluted with PBS to obtain a 4%
erythrocyte suspension. 100 μL of erythrocyte
suspension were placed in triplicates in the wells of
the 96-well cell culture plates. The erythrocytes in
the respective wells were treated with 250, 500, and
1000 μg/mL of MPSE, and the plate was incubated at
37ºC for 1 h to allow for hemolysis. The plate was then
centrifuged at 800 ×g for 15 min. From each well,
100 μL of supernatants were transferred to a new
96-well plate, and the absorbance was determined in
a microplate reader (MultiSkanGo, Thermo Scientific)
at 576 nm. Erythrocytes treated with Triton X-100
(0.5%) and PBS served as the positive and negative
control, respectively. Erythrocytes in 2% DMSO were
the solvent control.
RESULTS
Effects of MPSE on CAL 27 Oral cancer cell line
MPSE inhibited in vitro proliferation of CAL 27 cell
lines. At 100 μg/mL, MPSE showed more than 80%
inhibition (Figure 1). IC 50 of MPSE was found to be
25 μg/mL, significantly higher than doxorubicin
IC 50 = 0.5 μg/mL (15). The extract was active below
100 μg/mL, demonstrating the presence of the
potential compounds in the extract and showing
anti-cancer activity.
0
50
100
12,5 25 50 100
% Cell proliferation inhibition
MPSE
μg/ml
Figure 1. Cell proliferation inhibition (%) of the oral cancer cell line CAL 27 after
treatment with M. peregrina seed extract (MPSE) for 48 h.
The hemolytic effec t of MPSE on human
erythrocytes
The hemolytic effect of MPSE augmented with
increasing treatment concentrations. MPSE caused
14.3 % hemolysis at 1,000 μg/mL. This hemolysis
was below 20%, demonstrating the non-hemolytic
property of this extract. The positive (Triton X-100
(0.5%) control showed full hemolysis (100%), while
the negative control (PBS-treated RBC cells) did not
observe hemolysis (0%) (Figure 2).
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
chamber at 37°C; the cells were seeded at 5 × 103 cells/
well into 96-well plates. The vehicle used to prepare
the initial stock of MPSE was 0.1% dimethylsulfoxide
(DMSO). After 24 h, these cells were treated with
MPSE at different concentrations (12.5-100 μg/mL)
and incubated for 48 h. After incubation, cell
viability was measured by adding 20 μL of MTT dye
(5 mg/mL) to all wells and further incubated for 4 h.
The percentage of cytotoxicity of the extract was
calculated by measuring the absorbance of each
well at a wavelength of 540 nm using an ELISA plate
reader (Tecan, California, USA) (17).
Hemolytic Activity
After getting approval from the Independent Ethics
Committee of ICCBS, University of Karachi, with
reference no ICCBS/IEC-047-HB-2019/Protocol/1.0,
the hemolytic assay was carried out.
The hemolytic effect of MPSE was determined
using the method described by Wiradharma et al.,
2011 (22). Human blood (3 mL) withdrawn from a
volunteer was collected in an EDTA-anticoagulated
venoject tube and centrifuged at 700 ×g, and the
packed erythrocytes were separated from the
plasma. The erythrocytes were washed thrice with
1X PBS and then diluted with PBS to obtain a 4%
erythrocyte suspension. 100 μL of erythrocyte
suspension were placed in triplicates in the wells of
the 96-well cell culture plates. The erythrocytes in
the respective wells were treated with 250, 500, and
1000 μg/mL of MPSE, and the plate was incubated at
37ºC for 1 h to allow for hemolysis. The plate was then
centrifuged at 800 ×g for 15 min. From each well,
100 μL of supernatants were transferred to a new
96-well plate, and the absorbance was determined in
a microplate reader (MultiSkanGo, Thermo Scientific)
at 576 nm. Erythrocytes treated with Triton X-100
(0.5%) and PBS served as the positive and negative
control, respectively. Erythrocytes in 2% DMSO were
the solvent control.
RESULTS
Effects of MPSE on CAL 27 Oral cancer cell line
MPSE inhibited in vitro proliferation of CAL 27 cell
lines. At 100 μg/mL, MPSE showed more than 80%
inhibition (Figure 1). IC 50 of MPSE was found to be
25 μg/mL, significantly higher than doxorubicin
IC 50 = 0.5 μg/mL (15). The extract was active below
100 μg/mL, demonstrating the presence of the
potential compounds in the extract and showing
anti-cancer activity.
0
50
100
12,5 25 50 100
% Cell proliferation inhibition
MPSE
μg/ml
Figure 1. Cell proliferation inhibition (%) of the oral cancer cell line CAL 27 after
treatment with M. peregrina seed extract (MPSE) for 48 h.
The hemolytic effec t of MPSE on human
erythrocytes
The hemolytic effect of MPSE augmented with
increasing treatment concentrations. MPSE caused
14.3 % hemolysis at 1,000 μg/mL. This hemolysis
was below 20%, demonstrating the non-hemolytic
property of this extract. The positive (Triton X-100
(0.5%) control showed full hemolysis (100%), while
the negative control (PBS-treated RBC cells) did not
observe hemolysis (0%) (Figure 2).
4Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 350974Shaymaa Fadhel Abbas Albaayit, Rukesh Maharjan
DISCUSSION
Since ancient times, people from distinct civilizations
have used different plants from their surroundings
to treat various disorders. Recently, the emergence
of multidrug-resistant diseases, such as MDR
pathogens, cancer, diabetes, and other inflammatory
diseases, has motivated researchers to explore
different natural sources, such as animals and
plants, to find novel compounds to treat various
conditions. To date, many antibacterial, anticancer
compounds isolated from natural sources have been
extensively used due to their effective therapeutic
use (17-22). Recently, M. peregrina seed extracts
have gained popularity due to their antioxidant (11),
anti-inflammatory (13), antibacterial (14), anticancer
(15), antispasmodic (14) activities.
M. peregrina seed have been reported for its
cytotoxic effect on CACO-2, HeLa, AU565, MCF-7,
L929, HepG2, PC-3 cell lines (15, 19). Abou-Hashem
et al. 2019 (19) reported the chloroform fraction
of the ethanolic extract of M. peregrina seed
extract as the most active antitumor fraction. HPLC
analysis of this chloroform fraction reported many
polyphenols like vanillin, syringic acid, naringenin,
ferulic acid, quercetin, coumaric acid, daidzein, and
cinnamic acid. Syringic acid is reported to have an
antimitogenic effect on human colorectal cancer
cells (20). Coumaric acid (23) and vanillin (24) also
inhibit colon cancer cells by inhibiting the cell cycle
and inducing apoptosis. Naringenin and ferulic acid
inhibit gastric cancer cells and osteosarcoma cells
through apoptosis by downregulating the protein
kinase (AKT) pathway (25, 26). Daidzein induced
apoptosis and cell cycle arrest in human ovarian
cancer cells (27). Cinnamic acid inhibits human
melanoma cells through apoptosis by disrupting
the cytoskeleton (28). This fraction also contains
flavonoids like quercetin which interacts with DNA
and activates the mitochondrial pathway of apoptosis
in leukemia cancer cells (29). All these phenolic and
flavonoid compounds were reported to have an
anticancer effect on different cancer cell lines;
therefore, these compounds may act synergistically
on different targets of the complex cellular pathway
and induce cytotoxicity toward cancer cells. These
compounds induce apoptosis by activating various
apoptotic pathways like upregulating caspase3
with the release of cytochrome c, downregulating
anti-apoptotic genes (Bcl-2, Bcl-xL), cell cycle arrest,
premature aging, activating mitochondrial apoptosis
pathway, by enhancing the immune system to
destroy cancer cells, reducing proliferation,
angiogenesis, differentiation and metastasis of
cancers (30-32).
Although many anticancer compounds had potent
anticancer activity against various cancer cell lines,
they failed to advance in clinical trials because
they were cytotoxic to normal host cells and blood
cells. Some were highly hemolytic, limiting them
to topical use. Therefore, it is necessary to verify in
vitro the hemolytic effect of M. peregrina on human
erythrocytes since most anticancer agents damage
blood cells, causing anemia and myelosuppression
(33). Any compounds, formulations, or extracts with
less than 10% hemolysis are non-hemolytic, and those
with more than 25% are at risk of hemolysis (34).
0
20
40
60
80
100
PBS 250 500 1000 Triton X-100
% Hemolysis
Concentrations μg/ml
Figure 2. Hemolysis of human erythrocytes induced by M. peregrina seed
extract (MPSE) after 1 h incubation.
DISCUSSION
Since ancient times, people from distinct civilizations
have used different plants from their surroundings
to treat various disorders. Recently, the emergence
of multidrug-resistant diseases, such as MDR
pathogens, cancer, diabetes, and other inflammatory
diseases, has motivated researchers to explore
different natural sources, such as animals and
plants, to find novel compounds to treat various
conditions. To date, many antibacterial, anticancer
compounds isolated from natural sources have been
extensively used due to their effective therapeutic
use (17-22). Recently, M. peregrina seed extracts
have gained popularity due to their antioxidant (11),
anti-inflammatory (13), antibacterial (14), anticancer
(15), antispasmodic (14) activities.
M. peregrina seed have been reported for its
cytotoxic effect on CACO-2, HeLa, AU565, MCF-7,
L929, HepG2, PC-3 cell lines (15, 19). Abou-Hashem
et al. 2019 (19) reported the chloroform fraction
of the ethanolic extract of M. peregrina seed
extract as the most active antitumor fraction. HPLC
analysis of this chloroform fraction reported many
polyphenols like vanillin, syringic acid, naringenin,
ferulic acid, quercetin, coumaric acid, daidzein, and
cinnamic acid. Syringic acid is reported to have an
antimitogenic effect on human colorectal cancer
cells (20). Coumaric acid (23) and vanillin (24) also
inhibit colon cancer cells by inhibiting the cell cycle
and inducing apoptosis. Naringenin and ferulic acid
inhibit gastric cancer cells and osteosarcoma cells
through apoptosis by downregulating the protein
kinase (AKT) pathway (25, 26). Daidzein induced
apoptosis and cell cycle arrest in human ovarian
cancer cells (27). Cinnamic acid inhibits human
melanoma cells through apoptosis by disrupting
the cytoskeleton (28). This fraction also contains
flavonoids like quercetin which interacts with DNA
and activates the mitochondrial pathway of apoptosis
in leukemia cancer cells (29). All these phenolic and
flavonoid compounds were reported to have an
anticancer effect on different cancer cell lines;
therefore, these compounds may act synergistically
on different targets of the complex cellular pathway
and induce cytotoxicity toward cancer cells. These
compounds induce apoptosis by activating various
apoptotic pathways like upregulating caspase3
with the release of cytochrome c, downregulating
anti-apoptotic genes (Bcl-2, Bcl-xL), cell cycle arrest,
premature aging, activating mitochondrial apoptosis
pathway, by enhancing the immune system to
destroy cancer cells, reducing proliferation,
angiogenesis, differentiation and metastasis of
cancers (30-32).
Although many anticancer compounds had potent
anticancer activity against various cancer cell lines,
they failed to advance in clinical trials because
they were cytotoxic to normal host cells and blood
cells. Some were highly hemolytic, limiting them
to topical use. Therefore, it is necessary to verify in
vitro the hemolytic effect of M. peregrina on human
erythrocytes since most anticancer agents damage
blood cells, causing anemia and myelosuppression
(33). Any compounds, formulations, or extracts with
less than 10% hemolysis are non-hemolytic, and those
with more than 25% are at risk of hemolysis (34).
0
20
40
60
80
100
PBS 250 500 1000 Triton X-100
% Hemolysis
Concentrations μg/ml
Figure 2. Hemolysis of human erythrocytes induced by M. peregrina seed
extract (MPSE) after 1 h incubation.
5Journal Vitae | https://revistas.udea.edu.co/index.php/vitaeVolume 31 | Number 01 | Article 350974
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
Therefore, for the suitability of any compounds,
formulation, or extracts to treat diseases, hemolysis
must be less than 25%. In the present study, MPSE
showed a maximum of 14.3% hemolysis when treated
at 1,000 μg/mL, which is below 25%. Thus, this extract
showed non-hemolytic activity and can be used for
oral consumption for treating various diseases.
CONCLUSION
MPSE is toxic to oral cancer, CAL 27 cells, with a
low hemolytic property; thus, this seed possesses
hemocompatibility capacity to be used as a
therapeutic agent.
ACKNOWLEDGMENTS
The Corresponding author highly appreciate and
thankful to Prof. Dr. M. Iqbal Choudhary (Director,
ICCBS) for providing NAM-ICCBS fellowship
in ICCBS, University of Karachi, Pakistan. Author also
thankful to Rukesh Maharjan (ICCBS) for his help
REFERENCES
1. Abid AM, Merza MS. Immunohistochemical expression of Cyclin
D1 and NF-KB p65 in oral lichen planus and oral squamous
cell carcinoma (Comparative study). J Baghdad Coll Dent.
2014;26:80-7. https://jbcd.uobaghdad.edu.iq/index.php/jbcd/
article/view/301.
2. Tsai LL, Yu CC, Chang YC, Yu CH, Chou MY. Markedly increased
Oct4 and Nanog expression correlates with cisplatin resistance
in oral squamous cell carcinoma. J. Oral Pathol Med. 201;40(8):
621-628. DOI: https://doi.org/10.1111/j.1600-0714.2011.01015.x
3. Fuoad SAA, Mohammad DN, Hamied MAS, Garib BT. Oro-facial
malignancy in north of Iraq: a retrospective study of biopsied
cases. BMC Oral Health. 2021;21(1):1-10. DOI: https://doi.
org/10.1186/s12903-021-01521-3.
4. Usman S, Jamal A, Teh MT, Waseem A. Major Molecular Signaling
Pathways in Oral Cancer Associated with Therapeutic Resistance.
Front Oral Health. 2021;1. DOI: https://doi.org/10.3389/
froh.2020.603160.
5. Ali M, Abbas A, Drea A. Green synthesis of nano binary oxide
SiO2/V2O5 NPs integrated ointment cream application on wound
dressings and skin cancer cells. Baghdad Sci J. 2022;20(3):0734.
DOI: https://doi.org/10.21123/bsj.2022.7318.
6. Nazhvani AD, Sarafraz N, Askari F, Heidari F, Razmkhah M.. Anti-
cancer effects of traditional medicinal herbs on oral squamous
cell carcinoma. Asian Pac J Cancer Prev. 2020;21(2):479. DOI:
https://doi.org/10.31557/APJCP.2020.21.2.479.
7. Cardona-Mendoza A, Olivares-Niño G, Díaz-Báez D, Lafaurie
GI, Perdomo SJ. Chemopreventive and Anti-tumor Potential of
Natural Products in Oral Cancer. Nutr Cancer. 2022;74(3):779-795.
DOI: https://doi.org/10.1080/01635581.2021.1931698.
8. Al-Bahrani RM, Radif HM, Albaayit SFA. Evaluation of potent
silver nanoparticles production from Agaricus bisporus against
Helicobacter pylori. Pak J Agric Sci. 2020;57(4):1197-1201. DOI:
https://doi.org/10.21162/PAKJAS/20.9893.
9. Albaayit SFA, Rasedee A, Abdullah N. Zerumbone-loaded
nanostructured lipid carrier gel facilitates wound healing in
rats. Rev. bras. farmacogn. 2020;30(2):272-278. DOI: https://doi.
org/10.1007/s43450-020-00023-7.
10. Albaayit SFA, Rasedee A, Abdullah N, Abba Y. Methanolic
extract of Clausena excavata promotes wound healing via
antiinflammatory and anti-apoptotic activities. Asian PacJ Trop
Biomed. 2020;10(5):232-238. DOI: https://doi.org/10.4103/2221-
1691.281467.
11. Albaayit SFA. In vitro evaluation of anticancer activity of Moringa
peregrina seeds on breast cancer cells. Eurasia J Math Sci Technol
Educ. 2020;11:163-166. http://www.epstem.net/en/download/
article-file/1439710.
12. Albaayit SFA, Maharjan R, Khan M. Evaluation of hemolysis
activity of Zerumbone on RBCs and brine shrimp toxicity.
Baghdad Sci J. 2021;18(1):65-69. DOI: https://doi.org/10.21123/
bsj.2021.18.1.0065.
13. Albaayit SFA, Ozaslan M. Cytotoxic and urease inhibition
potential of Moringa peregrina seed ethanolic extract. Int
J Pharm. 2019; 15(1): 151-155. DOI: https://doi.org/10.3923/
ijp.2019.151.155.
14. Abd Rani NZ, Husain K, Kumolosasi E. Moringa genus: a review of
phytochemistry and pharmacology. Front Pharmacol. 2018;9:108.
DOI: https://doi.org/10.3389/fphar.2018.00108.
15. Soltan MM, Zaki AK. Antiviral screening of forty-two Egyptian
medicinal plants. J ethnopharmacol. 2009;126(1):102-107. DOI:
https://doi.org/10.1016/j.jep.2009.08.001.
16. Albaayit SFA, Al-Khafaji ASK, Alnaimy HS. In vitro macrophage
nitric oxide and interleukin-1 beta suppression by Moringa
peregrina seed. Turk J Pharm Sci. 2019; 16(3): 362-365. DOI:
https://doi.org/10.4274/tjps.galenos.2018.52244.
17. Albaayit SFA, Khan M, Abdullah R. Zerumbone induces growth
inhibition of Burkitt’s lymphoma cell line via apoptosis. Nat
Volatiles Essent Oils. 2021;8(3):56-63. https://doi.org/ 10.37929/
nveo.927770.
18. Albaayit SFA, Maharjan R. Immunomodulation of Zerumbone
via Decreasing the Production of Reactive Oxygen Species from
Immune Cells. Pak J Biol Sci. 2018;21(9):475-479. DOI: https://doi.
org/10.3923/pjbs.2018.475.479.
19. Albaayit SFA. Enzyme inhibitory properties of zerumbone. Pak
J Agri Sci. 2021;58(3):1207-1209. DOI: https://doi.org/10.21162/
PAKJAS/21.9759.
20. A lb aayit SFA . Evaluatio n of anti - met hicillin resis t ant
Staphylococcus aureus property of Clausena excavata leaves
by using atomic force microscopy and flowcytometry techniques.
Pak J Agri Sci. 2021;58(1):315-320. DOI: https://doi.org/10.3389/
froh.2020.603160.
21. Albaayit SFA, Maharjan R, Abdullah R, Noor MHM. Anti-
Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer
Studies on Clausena excavataBurum. f. (Rutaceae) Leaves. BioMed
Res Int. 2021. DOI: https://doi.org/10.1155/2021/3123476.
22. Albaayit SFA, Mohammed MK. Antiangiogenic activity and ROS-
mediated lung cancer cell line injury of zerumbone. J Fac Pharm
Ankara. 2023;47(3). DOI: https://doi.org/10.33483/jfpau.1112778.
23. Wiradharma N, Khoe U, Hauser CA, Seow SV, Zhang S, Yang YY.
Synthetic cationic amphiphilic α-helical peptides as antimicrobial
agents. Biomaterials. 2011;32(8):2204-2212. DOI: https://doi.
org/10.1016/j.biomaterials.2010.11.054.
24. Fang L, Gao L, Xie L, Xiao G. Eukaryotic translation initiation factor
5A-2 involves in doxorubicin-induced epithelial-mesenchymal
transition in oral squamous cell carcinoma cells. J Cancer.
2018;9(19):3479-3488. DOI: https://doi.org/10.7150/jca.26136.
Evaluation of Cytotoxic effect of Moringa peregrina seeds on Oral Cancer, CAL 27, Cell Line and Red Blood Cells Hemolysis
Therefore, for the suitability of any compounds,
formulation, or extracts to treat diseases, hemolysis
must be less than 25%. In the present study, MPSE
showed a maximum of 14.3% hemolysis when treated
at 1,000 μg/mL, which is below 25%. Thus, this extract
showed non-hemolytic activity and can be used for
oral consumption for treating various diseases.
CONCLUSION
MPSE is toxic to oral cancer, CAL 27 cells, with a
low hemolytic property; thus, this seed possesses
hemocompatibility capacity to be used as a
therapeutic agent.
ACKNOWLEDGMENTS
The Corresponding author highly appreciate and
thankful to Prof. Dr. M. Iqbal Choudhary (Director,
ICCBS) for providing NAM-ICCBS fellowship
in ICCBS, University of Karachi, Pakistan. Author also
thankful to Rukesh Maharjan (ICCBS) for his help
REFERENCES
1. Abid AM, Merza MS. Immunohistochemical expression of Cyclin
D1 and NF-KB p65 in oral lichen planus and oral squamous
cell carcinoma (Comparative study). J Baghdad Coll Dent.
2014;26:80-7. https://jbcd.uobaghdad.edu.iq/index.php/jbcd/
article/view/301.
2. Tsai LL, Yu CC, Chang YC, Yu CH, Chou MY. Markedly increased
Oct4 and Nanog expression correlates with cisplatin resistance
in oral squamous cell carcinoma. J. Oral Pathol Med. 201;40(8):
621-628. DOI: https://doi.org/10.1111/j.1600-0714.2011.01015.x
3. Fuoad SAA, Mohammad DN, Hamied MAS, Garib BT. Oro-facial
malignancy in north of Iraq: a retrospective study of biopsied
cases. BMC Oral Health. 2021;21(1):1-10. DOI: https://doi.
org/10.1186/s12903-021-01521-3.
4. Usman S, Jamal A, Teh MT, Waseem A. Major Molecular Signaling
Pathways in Oral Cancer Associated with Therapeutic Resistance.
Front Oral Health. 2021;1. DOI: https://doi.org/10.3389/
froh.2020.603160.
5. Ali M, Abbas A, Drea A. Green synthesis of nano binary oxide
SiO2/V2O5 NPs integrated ointment cream application on wound
dressings and skin cancer cells. Baghdad Sci J. 2022;20(3):0734.
DOI: https://doi.org/10.21123/bsj.2022.7318.
6. Nazhvani AD, Sarafraz N, Askari F, Heidari F, Razmkhah M.. Anti-
cancer effects of traditional medicinal herbs on oral squamous
cell carcinoma. Asian Pac J Cancer Prev. 2020;21(2):479. DOI:
https://doi.org/10.31557/APJCP.2020.21.2.479.
7. Cardona-Mendoza A, Olivares-Niño G, Díaz-Báez D, Lafaurie
GI, Perdomo SJ. Chemopreventive and Anti-tumor Potential of
Natural Products in Oral Cancer. Nutr Cancer. 2022;74(3):779-795.
DOI: https://doi.org/10.1080/01635581.2021.1931698.
8. Al-Bahrani RM, Radif HM, Albaayit SFA. Evaluation of potent
silver nanoparticles production from Agaricus bisporus against
Helicobacter pylori. Pak J Agric Sci. 2020;57(4):1197-1201. DOI:
https://doi.org/10.21162/PAKJAS/20.9893.
9. Albaayit SFA, Rasedee A, Abdullah N. Zerumbone-loaded
nanostructured lipid carrier gel facilitates wound healing in
rats. Rev. bras. farmacogn. 2020;30(2):272-278. DOI: https://doi.
org/10.1007/s43450-020-00023-7.
10. Albaayit SFA, Rasedee A, Abdullah N, Abba Y. Methanolic
extract of Clausena excavata promotes wound healing via
antiinflammatory and anti-apoptotic activities. Asian PacJ Trop
Biomed. 2020;10(5):232-238. DOI: https://doi.org/10.4103/2221-
1691.281467.
11. Albaayit SFA. In vitro evaluation of anticancer activity of Moringa
peregrina seeds on breast cancer cells. Eurasia J Math Sci Technol
Educ. 2020;11:163-166. http://www.epstem.net/en/download/
article-file/1439710.
12. Albaayit SFA, Maharjan R, Khan M. Evaluation of hemolysis
activity of Zerumbone on RBCs and brine shrimp toxicity.
Baghdad Sci J. 2021;18(1):65-69. DOI: https://doi.org/10.21123/
bsj.2021.18.1.0065.
13. Albaayit SFA, Ozaslan M. Cytotoxic and urease inhibition
potential of Moringa peregrina seed ethanolic extract. Int
J Pharm. 2019; 15(1): 151-155. DOI: https://doi.org/10.3923/
ijp.2019.151.155.
14. Abd Rani NZ, Husain K, Kumolosasi E. Moringa genus: a review of
phytochemistry and pharmacology. Front Pharmacol. 2018;9:108.
DOI: https://doi.org/10.3389/fphar.2018.00108.
15. Soltan MM, Zaki AK. Antiviral screening of forty-two Egyptian
medicinal plants. J ethnopharmacol. 2009;126(1):102-107. DOI:
https://doi.org/10.1016/j.jep.2009.08.001.
16. Albaayit SFA, Al-Khafaji ASK, Alnaimy HS. In vitro macrophage
nitric oxide and interleukin-1 beta suppression by Moringa
peregrina seed. Turk J Pharm Sci. 2019; 16(3): 362-365. DOI:
https://doi.org/10.4274/tjps.galenos.2018.52244.
17. Albaayit SFA, Khan M, Abdullah R. Zerumbone induces growth
inhibition of Burkitt’s lymphoma cell line via apoptosis. Nat
Volatiles Essent Oils. 2021;8(3):56-63. https://doi.org/ 10.37929/
nveo.927770.
18. Albaayit SFA, Maharjan R. Immunomodulation of Zerumbone
via Decreasing the Production of Reactive Oxygen Species from
Immune Cells. Pak J Biol Sci. 2018;21(9):475-479. DOI: https://doi.
org/10.3923/pjbs.2018.475.479.
19. Albaayit SFA. Enzyme inhibitory properties of zerumbone. Pak
J Agri Sci. 2021;58(3):1207-1209. DOI: https://doi.org/10.21162/
PAKJAS/21.9759.
20. A lb aayit SFA . Evaluatio n of anti - met hicillin resis t ant
Staphylococcus aureus property of Clausena excavata leaves
by using atomic force microscopy and flowcytometry techniques.
Pak J Agri Sci. 2021;58(1):315-320. DOI: https://doi.org/10.3389/
froh.2020.603160.
21. Albaayit SFA, Maharjan R, Abdullah R, Noor MHM. Anti-
Enterococcus Faecalis, Cytotoxicity, Phytotoxicity, and Anticancer
Studies on Clausena excavataBurum. f. (Rutaceae) Leaves. BioMed
Res Int. 2021. DOI: https://doi.org/10.1155/2021/3123476.
22. Albaayit SFA, Mohammed MK. Antiangiogenic activity and ROS-
mediated lung cancer cell line injury of zerumbone. J Fac Pharm
Ankara. 2023;47(3). DOI: https://doi.org/10.33483/jfpau.1112778.
23. Wiradharma N, Khoe U, Hauser CA, Seow SV, Zhang S, Yang YY.
Synthetic cationic amphiphilic α-helical peptides as antimicrobial
agents. Biomaterials. 2011;32(8):2204-2212. DOI: https://doi.
org/10.1016/j.biomaterials.2010.11.054.
24. Fang L, Gao L, Xie L, Xiao G. Eukaryotic translation initiation factor
5A-2 involves in doxorubicin-induced epithelial-mesenchymal
transition in oral squamous cell carcinoma cells. J Cancer.
2018;9(19):3479-3488. DOI: https://doi.org/10.7150/jca.26136.
6Journal Vitae | https://revistas.udea.edu.co/index.php/vitae Volume 31 | Number 01 | Article 350974Shaymaa Fadhel Abbas Albaayit, Rukesh Maharjan
25. Sadraei H, Asghari G, Farahnaki F. Assessment of hydroalcoholic
extract of seeds and leaves of Moringa peregrina on ileum spasm.
Res Pharm Sci. 2015;10(3):252-258. https://www.ncbi.nlm.nih.gov/
pmc/articles/PMC4621632/pdf/RPS-10-252.pdf
26. Abou-Hashem MM, Abo-Elmatty DM, Mesbah NM, Abd EL-
Mawgoud AM. Induction of sub-G0 arrest and apoptosis by
seed extract of Moringa peregrina (Forssk.) Fiori in cervical and
prostate cancer cell lines. J Integr Med. 2019;17(6):410-422. DOI:
https://doi.org/10.1016/j.joim.2019.09.004.
27. Abaza MS, Al-Attiyah RA, Bhardwaj R, Abbadi G, Koyippally
M, Afzal M. Syringic acid from Tamarix aucheriana possesses
antimitogenic and chemo-sensitizing activities in human
colorectal cancer cells. Pharm. Biol. 2013;51(9):1110-1124. DOI:
https://doi.org/10.3109/13880209.2013.781194.
28. Jaganathan SK, Supriyanto E, Mandal M. Events associated with
apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells.
World J Gastroenterol.2013;19(43):7726-7734. DOI: https://doi.
org/103748/wjg.v19.i43.7726.
29. Maiyoa F, Moodley R, Singh M. Phytochemistry, cytotoxicity and
apoptosis studies of β-sitosterol-3-oglucoside and β-amyrin from
Prunus africana. African Journal of Traditional. BMC Complement
Altern Med. 2016;13(4):105-112. DOI: https://doi.org/10.21010/
ajtcam.v13i4.15.
30. Bao L, Liu F, Guo HB, Li Y, Tan BB, Zhang WX, Peng YH. Naringenin
inhibits proliferation, migration, and invasion as well as induces
apoptosis of gastric cancer SGC7901 cell line by downregulation
of AKT pathway. Tumour Biol. 2016;37:11365-11374. DOI: https://
doi.org/10.1007/s13277-016-5013-2.
31. Albaayit SFA, Khan M, Abdullah R, Noor MHM. Ethyl acetate
extract of Clausena excavata induces growth inhibition of
non-small-lung cancer, NCI-H460, cell line via apoptosis. J
Appl Biomed. 2021;19(1):40-47. DOI: https://doi.org/10.32725/
jab.2021.007
32. Wang T, Gong X, Jiang R, Li H, Du W, Kuang G. Ferulic acid inhibits
proliferation and promotes apoptosis via blockage of PI3K/Akt
pathway in osteosarcoma cell. Am J Transl Res.2016;8(2):968-
980. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846940.
33. Hua F, Li CH, Chen XG, Liu XP. Daidzein exerts anticancer activity
towards SKOV3 human ovarian cancer cells by inducing apoptosis
and cell cycle arrest, and inhibiting the Raf/MEK/ERK cascade. Int
J mol med. 2018;41(6): 3485-3492. DOI: https://doi.org/10.3892/
ijmm.2018.3531.
34. Niero EL, Machado-Santelli GM. Cinnamic acid induces apoptotic
cell death and cytoskeleton disruption in human melanoma
cells. J Exp Clin. Cancer Res. 2013;32(1):1-14. DOI: https://doi.
org/10.1186/1756-9966-32-31.
35. Hashemzaei M, Delarami Far A, Yari A, Heravi RE, Tabrizian K,
Taghdisi SM, Sadegh SE, Tsarouhas K, Kouretas D, Tzanakakis
G, Nikitovic D. Anticancer and apoptosis-inducing effects of
quercetin in vitro and in vivo. Oncol Repo. 2017;38(2): 819-828.
DOI: https://doi.org/10.3892/or.2017.5766.
25. Sadraei H, Asghari G, Farahnaki F. Assessment of hydroalcoholic
extract of seeds and leaves of Moringa peregrina on ileum spasm.
Res Pharm Sci. 2015;10(3):252-258. https://www.ncbi.nlm.nih.gov/
pmc/articles/PMC4621632/pdf/RPS-10-252.pdf
26. Abou-Hashem MM, Abo-Elmatty DM, Mesbah NM, Abd EL-
Mawgoud AM. Induction of sub-G0 arrest and apoptosis by
seed extract of Moringa peregrina (Forssk.) Fiori in cervical and
prostate cancer cell lines. J Integr Med. 2019;17(6):410-422. DOI:
https://doi.org/10.1016/j.joim.2019.09.004.
27. Abaza MS, Al-Attiyah RA, Bhardwaj R, Abbadi G, Koyippally
M, Afzal M. Syringic acid from Tamarix aucheriana possesses
antimitogenic and chemo-sensitizing activities in human
colorectal cancer cells. Pharm. Biol. 2013;51(9):1110-1124. DOI:
https://doi.org/10.3109/13880209.2013.781194.
28. Jaganathan SK, Supriyanto E, Mandal M. Events associated with
apoptotic effect of p-Coumaric acid in HCT-15 colon cancer cells.
World J Gastroenterol.2013;19(43):7726-7734. DOI: https://doi.
org/103748/wjg.v19.i43.7726.
29. Maiyoa F, Moodley R, Singh M. Phytochemistry, cytotoxicity and
apoptosis studies of β-sitosterol-3-oglucoside and β-amyrin from
Prunus africana. African Journal of Traditional. BMC Complement
Altern Med. 2016;13(4):105-112. DOI: https://doi.org/10.21010/
ajtcam.v13i4.15.
30. Bao L, Liu F, Guo HB, Li Y, Tan BB, Zhang WX, Peng YH. Naringenin
inhibits proliferation, migration, and invasion as well as induces
apoptosis of gastric cancer SGC7901 cell line by downregulation
of AKT pathway. Tumour Biol. 2016;37:11365-11374. DOI: https://
doi.org/10.1007/s13277-016-5013-2.
31. Albaayit SFA, Khan M, Abdullah R, Noor MHM. Ethyl acetate
extract of Clausena excavata induces growth inhibition of
non-small-lung cancer, NCI-H460, cell line via apoptosis. J
Appl Biomed. 2021;19(1):40-47. DOI: https://doi.org/10.32725/
jab.2021.007
32. Wang T, Gong X, Jiang R, Li H, Du W, Kuang G. Ferulic acid inhibits
proliferation and promotes apoptosis via blockage of PI3K/Akt
pathway in osteosarcoma cell. Am J Transl Res.2016;8(2):968-
980. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846940.
33. Hua F, Li CH, Chen XG, Liu XP. Daidzein exerts anticancer activity
towards SKOV3 human ovarian cancer cells by inducing apoptosis
and cell cycle arrest, and inhibiting the Raf/MEK/ERK cascade. Int
J mol med. 2018;41(6): 3485-3492. DOI: https://doi.org/10.3892/
ijmm.2018.3531.
34. Niero EL, Machado-Santelli GM. Cinnamic acid induces apoptotic
cell death and cytoskeleton disruption in human melanoma
cells. J Exp Clin. Cancer Res. 2013;32(1):1-14. DOI: https://doi.
org/10.1186/1756-9966-32-31.
35. Hashemzaei M, Delarami Far A, Yari A, Heravi RE, Tabrizian K,
Taghdisi SM, Sadegh SE, Tsarouhas K, Kouretas D, Tzanakakis
G, Nikitovic D. Anticancer and apoptosis-inducing effects of
quercetin in vitro and in vivo. Oncol Repo. 2017;38(2): 819-828.
DOI: https://doi.org/10.3892/or.2017.5766.