Advances in Cancer Biology - Metastasis 6 (2022) 100072

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Advances in Cancer Biology - Metastasis

journal homepage: www.journals.elsevier.com/advances-in-cancer-biology-metastasis

Anticancer effect of Moringa oleifera leaves extract against lung cancer cell
line via induction of apoptosis
Kinjal Bhadresha a, b, Vaidehi Thakore a, Jpan Brahmbhatt a, Vinal Upadhyay a, Nayan Jain a,
Rakesh Rawal a, *
a
Department of Life Sciences, School of Sciences, Gujarat University, Ahmedabad, 380009, India
b
Hematology/Oncology Division, School of Medicine, Indiana University, Indianapolis, IN, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Since ancient times, Moringa oleifera has been a common vegetable in many nations. It has a large number of
Moringa oleifera phenolic compounds with a diverse range of biological activity. It has anticancer properties that can be exploited
Anticancer drug to create novel medications for the treatment of various malignancies. The current study was conducted to
Apoptosis
evaluate the in vitro anticancer activities of M. oleifera leaves extracts. The M. oleifera leaves extracts significantly
Lung cancer
inhibited cell proliferation in the human cancer cell line A549 in a dose-dependent manner. Morphological
studies indicated that the extract of moringa leaves stimulated apoptosis as demonstrated by cell shrinkage,
blebbing, chromatin condensation, and nuclear fragmentation. Quantitative RT-PCR analyses of Bax and Bcl-2
showed abnormal expression profiles of these genes under various treatment conditions. This study demon­
strates that M. oleifera leaves may have the ability to suppress the growth of cancer cells while also enhancing
human health and developing new food ingredients. The phytochemicals from M. oleifera leaves can be employed
as the primary medications to cure cancer, according to in vitro studies.

1. Introduction
Lung cancer (LC) is one of the most significant and dangerous health
issues today, and it is also a major cause of death [1]. Small cell lung
cancer and non-small cell lung cancer (NSCLC) are two types of lung
cancer [2]. The most successful treatment for early-stage lung cancer is
surgery; however, patients with advanced lung cancer, for whom sur­
gery is no longer an option, frequently turn to radiotherapy, chemo­
therapy, and targeted therapy as their final options [2,3]. Ongoing drug
therapies have many side effects and other are being sought at present,
so this malignancy still remains incurable, requiring traditional medi­
cine as an alternate source to treat lung cancer and reduce the death rate
[4]; Gericke N et al., 2001. Therefore, it is urgent to find more efficient
potential antitumor drugs for lung cancer therapy.
Plants have been thought to be used as medicinal agent since ages
due to their lower toxicity and side effects than synthetic drugs; more­
over, complex cellular pathways are supported by phytochemical mol­
ecules [5,6]. Moringa oliefera belongs to the Moringaceae family. The
Moringa genus is fast growing and its 13 species have spread around the
world including Northeast Africa, Southwest Africa, Southwest Asia and
Madagascar [7,8]. Each part of the Moringa tree holds the nutritional
values as in various important phytochemical compounds. Prior studies
believed that moringa have 7 times more vitamin C than an orange, 9
times more protein than yoghurt, 10 times more vitamin A than carrot,
17 times more calcium than milk, 15 times more potassium than ba­
nanas, and 25 times more iron content than spinach [9]. Various works
on moringa discovered promising properties such as that it shows
anticancer activity, when increased reactive oxygen species that induce
p53, caspases and cleavage of PARP-1 resulted in apoptosis in cancer cell
lines [10]. Another study showed that its leaves extract induces
apoptosis in KB carcinoma cells [11]. In addition, Moringa Oliefera leaves
extract also has been shown to interrupt the proliferation of cancer cells
[12]. A recent study also suggested that moringa extract has significant
cytotoxic and cell vitality in PC3 cell line [13].
The aim of this study was to assess the potential anti-cancer effects of
Moringa oleifera leaves against lung cancer cells. The extracts of leaves
tested in our current study induced a significant level of apoptosis in
lung cancer cell line. In addition, a remarkable change in the normal
phenotypic properties of the cells was also observed. It was hypothesised
that M.oleifera leaf extracts induces cell death as a result of oxidative

* Corresponding author.
E-mail address: rakeshrawal@gujaratuniversity.ac.in (R. Rawal).

https://doi.org/10.1016/j.adcanc.2022.100072
Received 23 July 2022; Received in revised form 15 October 2022; Accepted 18 October 2022
Available online 26 October 2022
2667-3940/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
K. Bhadresha et al.
stress in the cancerous cells.
2. Materials and methods
2.1. Collection of plant material
The leaves of M.oleifera were collected from the Botanical Garden,
Department of botany, Bioinformatics, and Climate Change Impact
Management, Ahmedabad, Gujarat, India. Geographically the latitude
and longitude of the city are 23◦ 2′ 1.9068′′ N and 72◦ 35′ 6.0792′′ E. The
leaves of M.oleifera were thoroughly washed, then chopped into small
pieces and spread onto tissue paper then aluminum trays. Further
samples were air-dried and then powdered and store for further analysis.
2.2. Preparation of the extract
Extraction was performed using the Soxhlet apparatus. 10g of dried
leaves were grinded separately into course powder and taken into a
round bottom flask in 100 ml of water; was continued for 18 h until it
dissolved into a solvent. The soluble extract was kept for evaporation in
the oven. The extract was collected and stored at 4 ◦ C. The stock solution
for the cell-based assays in culture was prepared by taking 1 mg in 1 ml.
2.3. Phytochemical screening
Prior qualitative phytochemical screening including; flavonoids, al­
kaloids, steroids, terpenoids, glycosides, saponins, catecholic tannins,
anthocyanin, carbohydrates and amino acids was traced in aqueous
extract of M. oleifera was done using standard procedures [14].
2.4. Fourier transform infrared spectroscopy (FTIR)
First, we performed FTIR for the spectral measurement of M. oleifera
powder samples. The FTIR spectra were recorded in an FTIR instrument
(Model/Maker: F25, Bruker) with PC-based software controlled instru­
ment operation and data processing. A small amount of powdered leaf
samples was taken and infrared transmittance was collected over a
number that ranged from 4000 cm − 1 to 500 cm− 1. The spectral data
were analyzed [15].
2.5. Cell proliferation assay (MTT assay)
The cytotoxicity study of M. Oliefera leaves extract was studied on
cultured cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo­
lium bromide (MTT) Assay. A549 Cells were seeded in 96 well plates in
DMEM supplemented with 10% FBS. The cells were grown in 96 well
plates for the establishment of monolayer till 70–80% confluences after
an incubation period for 24hrs in humidified 5% CO2 at 37 ◦ C. After
having the desired cell density, treatment was given with the different
dilution at 100 μg/ml, 200 μg/ml, 300 μg/ml, 400 μg/ml and 500 μg/ml
concentrations and was further incubated further for 24 h, then after
next day, 20 μl of MTT solution (5 mg/ml) was added to each well, plate
was kept for re-incubation for 3hrs. 100 μl of DMSO was added to
dissolve the formazon crystals. Later, the absorbance of the plate was
measured at 570 nm by using 96 well micro plate readers [16].
2.6. Morphological assessment of apoptotic cells by DAPI staining
4 0,6-diamidino-2-phenylindole (DAPI) staining was carried out ac­
cording to the method described by Papi et al. [17] with slight modifi­
cations. A549 cells were grown on sterile glass slides overnight and
treated for 24 h with Moringa extract (in serum-free medium) at a
concentration of 100 μg/ml and 400 μg/ml concentration. The cells
were incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 ◦ C.
At the end of the incubation, cells were fixed with 4% paraformaldehyde
and then permeabilized with Triton X-100 (0.1% in PBS). The cells were
2
Advances in Cancer Biology - Metastasis 6 (2022) 100072
finally stained using DAPI in PBS (2.5 μg/mL) and allowed to stand for
20 min under dark conditions. Finally, the morphological changes were
viewed using fluorescence microscopy (Zeiss, Oberkochen, Germany).
2.7. Morphological assessment of apoptotic cells by acridine orange (AO)
propidium iodide (PI) double staining
The AO/PI morphological assessment of apoptotic cells was carried
out according to the method of Arbab et al. [18] with minor modifica­
tions. This double staining method was performed on A549 cells treated
with Moringa extract (in serum-free medium) at 100 μg/ml and 400
μg/ml concentration for 24 h, and examined by fluorescence micro­
scopy. Treatment-free cells were grown as a negative control. Cells were
harvested with trypsin (Sigma-Aldrich, Germany), centrifuged at 300×g
for 10 min and kept cool on ice. Separately, 5 μl AO (10 mg/ml) was
mixed with 5 μl PI (10 mg/ml) making a double staining dye of AO/PI
and kept under an ice bath in dark conditions. An aliquot of the cell
suspensions (10 μl) was added to the dye mixture and was transferred (5
μl) onto a glass slide for fluorescence viewing. Slides were observed
under a UV-Fluorescence microscope within 30 min.
2.8. Real-time PCR (qRT- PCR)
Total RNA was extracted with Trizol (Life Technologies, USA) and
then cDNA was synthesized with 2 μg of total RNA using a High Capacity
cDNA Reverse Transcription Kit (Life Technologies, USA). Real-time
PCR was performed on the 20 ng of resulting cDNA using the Power
SYBR® Green Master Mix (Agilent Technology, USA), 10 mmol/L of
each primer (Table 1) in the QuantStudio® 5 Real-Time PCR system
(Applied Biosystems, USA). Amplification was carried out under the
following cycling conditions: 1 cycle of 5 min at 95 ◦ C for the initial
denaturation step and 40 cycles of 1 min at 95 ◦ C for the denaturation
step, 40 s at 60 ◦ C for the annealing and extension step. Melting curve
analysis was performed following the amplification to distinguish real
amplicon from nonspecific products or primer dimers. The expression of
fold change was evaluated using the ΔΔCT or 2− ΔΔCT method with
β-actin as endogenous control. All experiments were performed in trip­
licates independently and the average CT value was calculated for the
quantification fold change analysis.
2.9. Statistical analysis
Statistical evaluation was performed using T-test and ANOVA using
Graph Pad PRISM v8.7 software. P<0.05 was considered statistically
significant. All experiments were performed in triplicate.
3. Results
3.1. Phytochemical screening of aqueous extract of Moringa oleifera
leaves
The aqueous extract of suspected leaves revealed the presence of
various beneficial components which included; flavonoids, alkaloids,
steroids, terpenoids, glycosides, saponins, catecholic tannins, anthocy­
anin, carbohydrates and amino acids (Table 2). (In words to describe
Table 1
Primer list.
Gene Forward primer Reverse primer
name
BAX 5′ -GCCCTTTTGCTTCAGGGTTT-3′ 5′ - TCCAATGTCCAGCCTTTG-3′
BCL2 5′ -CGGAGGCTGGGATGCCTTTG- 5′ TTTGGGGCAGG CATGTTGAC-
3′ 3′
β-actin 5′ -GAGACCTTCAACACCCCAGCC- 5′ -AGACGCAGGATGGCATGGG-
3′ 3′
K. Bhadresha et al. Advances in Cancer Biology - Metastasis 6 (2022) 100072

Table 2
Phytochemical screening of leaves of Moringa oleifera.
Phytochemical component Qualitative analysis

Flavonoids +++
Alkaloids ++
Phenolics acid +
Steroids –
Terpenoids +
Glycosides ++
Saponins ++
Catecholic tannins ++
Anthocyanin +
Carbohydrates ++
Amino acids ++

low high content).

3.2. Spectral interpretation using FTIR

Functional group analysis was identified by FTIR spectroscopy on the

peak values in the IR. The native powder was subjected to FTIR analysis. Fig. 2. The effect of Moringa oleifera extracts on the proliferation of A549
cell lines. Inhibitory effect of Moringa oleifera on A549 cell lines respectively
The figure shows the broad band at 3254.97 cm -1 which is the
after 24 h. Data are represented as mean ± SEM of three independent
stretching vibration of a phenolic hydroxyl group (-OH) representing the
experiments.
hydrogen bond (Fig. 1). A vibration stretch at wavelength 2933.73 cm-1
was appeared to have an aromatic (C-H) group, and another band was
3.4. Morphological assessment of apoptotic cells by DAPI staining
observed at wavelengths of 1604.99 cm-1 and 1516.62 cm-1 stretching
vibration of the aromatic (C=C) group, at 1033.49 cm-1, and assigned to
In general, DAPI and AO/PI staining methods clearly exhibited
have C=O bond of ether, ester or phenol which can be considered as
apoptosis stimulation in A549 cells after treatment with M. Oleifera leaf
derivatives for flavonoids [19,20].
extract treatment. A549 cells treated with moringa extract showed clear
apoptotic characteristics that became more apparent as the concentra­
tion of treatment increased (Fig. 3A). In the DAPI staining images, there
3.3. Effect of M. Oleifera extract on A549 cells
was an increase in the number of cells with small, condensed nuclei after
24 h treated cells, indicating an increasing number of apoptotic cells
The cytotoxicity of M. Oleifera leaves aqueous extracts was tested
with increasing the concentration of extract. Apart from nuclear
over lung cancer A549 cell lines at different concentrations of 100 μg/
condensation, DAPI staining showed that all treated A549 cells appeared
ml, 200 μg/ml, 300 μg/ml, 400 μg/ml and 500 μg/ml using MTT assay.
to lose cell structure with increasing concentrations. The control cells,
Data illustrated in (Fig. 2) show the percentage of viabilities of A549
however, remained intact and evenly shaped. Treatment with higher
cells after 24 h incubation with treatments versus controls. The results
concentration for 24 h, all cells were completely ruptured. The DAPI
revealed dose dependent decreases (P < 0.05) in cell viabilities of sen­
staining noticeably showed apoptotic morphological changes in mor­
sitive A549 cells to 400 μg/ml and 500 μg/ml reaching 30% and 15%
inga leaf extract treated cells in terms of both nuclear condensation and
cell death compared to the control. Surprisingly, there was no significant
cell structure loss.
cytotoxic effect was observed in other concentrations.

Fig. 1. Fourier-Transform Infrared (FT-IR) spectra of aqueous leaf extract of Moringa Oleifera.

3
K. Bhadresha et al. Advances in Cancer Biology - Metastasis 6 (2022) 100072

Fig. 3. Images of stained A549 cells treated with Moringa extract in a dose dependent manner. (A) DAPI fluorescence images of apoptotic A549 cells with
chromatin condensation in the cell nucleus; (B) Representative images showing live, early, late and necrotic cells of all cell lines. Live cells were stained by AO (green)
while PI used to stain the nucleus (red). Images were observed under a fluorescent microscope. (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)

3.5. Quantification of apoptosis by acridine orange and propidium iodide

double staining

The AO/PI staining images displayed apoptotic changes in the cells

treated with M. Oleifera leaf extract treated cells in terms of colour and
morphology. The apparent fluorescent colouration in the untreated cells
compared to the treated cells was the first noticeable change. The con­
trol cells showed a bright green colouration and an intact nuclear
structure, indicating healthy viable cells, while the treated cells
exhibited a bright orange stain, signifying the presence of apoptotic
cells. With increasing concentration, the relative number of green cells
gradually decreased, while that of orange cells increased, indicating a
shift from viable to apoptotic cells. Similar to the DAPI staining, AO/PI
staining also revealed a change in cell structure. After 24 h of incubation
with M. Oleifera leaves extract, there were clear indications of chromatin
condensation and blebbing of the A549 cell membrane. Upon increasing
the concentration, small apoptotic bodies and dead cells were identified. Fig. 4. The effect of moringa extract on mRNA expression of apoptosis
The morphological changes and the shift from green to orange fluores­ markers. mRNA expression of BCL-2 and BAX was analyzed by real time PCR.
cent cells that were observed from the AO/PI staining were sequential Moringa extract showed the potential for apoptosis process. Data were analyzed
for 24 h of treatment with moringa leaves extract, indicating gradual cell by comparative Cq method.
death by apoptosis with increasing concentration (Fig. 3B).
distinguish between tumor and normal cells. This frequently affects the
effectiveness of the therapy, making it impossible to heal cancer pa­
3.6. The effect of M.oleifera extracts on the expression of the Bcl-2 and
tients. Cancer cell elimination via cell cycle arrest and/or activation of
Bax genes
apoptosis with minimum side effects on normal cells is one of the re­
quirements for cancer therapy [21–23].
Bax and Bcl-2 mRNA levels were performed for defining the
A widespread vegetable plant in many Asian and South East Asian
apoptotic effects of M. Oleifera extract treatment (100 and 400 μg/mL)
nations, M. oleifera is home to a number of chemicals that have
on A549 cells after 24 h. As shown in Fig. 4, after treatments, increased
outstanding anti-oxidant and anti-cancer capabilities [24]. By disrupt­
Bax gene expression was observed and an important reduction was seen
ing the signal transduction cascade that encourages cancer cell growth
in the expression of Bcl-2 as compared with the untreated cells. Gene
and development, the plant demonstrates anti-cancer potential [25,26]
expressions were normalized to β-actin (reference gene). A noteworthy
The presence of eugenol, a phenolic natural chemical that targets
variation was observed between the gene expression of Bax and Bcl-2 at
E2F1/survivin in cancer cells [27], D-allose [28], isopropyl isothiocya­
various concentrations of extract, but a significant difference was
nate (Matsuda H et al., 2017), etc. Is primarily responsible for the pre­
observed between the gene expression of Bax and Bcl-2 at higher con­
vention of cancer cell proliferation. In light of M. oleifera anti-cancer
centrations (Fig. 4).
capabilities, we postulated that utilizing locally cultivated plants,
would be a successful treatment for lung cancer malignancies. In this
4. Discussion study, we evaluated the anti-cancer properties of M. oleifera leaves ex­
tracts against the lung cancer A549 cell line.
Chemotherapy’s fundamental drawback is that it cannot protect Our first and foremost aim was to obtain the details of the chemical
normal cells from damage because its blinded components are unable to

4
K. Bhadresha et al.
compounds present in M. oleifera leaves. The phytochemical screening
and FTIR analyses revealed numerous anti-cancer compounds present in
the extracts of leaves of M. oleifera. Furthermore, the FTIR analyses of
M. oleifera leaves extracts showed a number of phyto-constituents in the
spectra. Most of these constituents possess anticancer activity against
cancer cell lines and/or in vivo models. In summary, the FTIR analyses
demonstrate that the extracts of M. oleifera leaves have a number of
bioactive anti-cancer constituents which might be responsible for its
strong anti-cancer activity against the A549 cancer cell line.
Furthermore, when we tested these compounds against A549 cell
survival, a remarkable decrease in viable cells was detected in the MTT
assay. Within a small fluctuation in the number of viable cells, the effects
of the leaf extracts on cell survival were significantly effective. The
difference in the cellular absorption mechanism in this particular set of
experiments may be the cause of the variation in the number of viable
cells. What we discovered was that the presence of these extracts dras­
tically reduced cell survival when compared to the matching control.
Current research also indicated that the M. oleifera solvent fractions
possess in vitro anticancer activity against HeLa cancer cell line [29].
Furthermore, Al-Asmari AK et al. also proposed that the M. oleifera ex­
tracts act as an anti-cancer agent by decreasing cell motility and colony
formation in colorectal and breast cancer cell lines [30]. In addition, it
was previously established that the water-soluble fraction of M. oleifera
leaves induced apoptosis in lung cancer cells and is consequently a novel
type of potential anticancer candidate compound [31].
An essential feature of chemo-preventive agents is the ability to
selectively induce the cell death of cancer cells via apoptosis and not
through necrosis [32]. The nature of the apoptotic mechanism impor­
tant to cell death is dangerous in defining a favorable anticancer com­
pound. Identification of the physical self-destruction phases convoluted
in apoptosis is used to confirm potential anticancer drugs [33]. Consis­
tently, all morphological assessment assays in the current study revealed
clear apoptotic changes in A549 cells treated with M. oleifera extract,
including loss of cell integrity, condensation of cell nuclei, cell
shrinkage, membrane blebbing, and the formation of apoptotic bodies
[34]. also demonstrated that leaf extracts of M. oleifera induce the
apoptosis of HepG2 cells (Jung, I. L. et a, 2015).
The Bcl-2 family of genes consists of the pro-apoptotic and anti-
apoptotic members such as bax and bcl-2, respectively [35]. In this
study, an apparent increase in the expression of Bax and a related
decrease in Bcl-2 mRNA expression levels, as determined by quantitative
RT-PCR, were observed in the M. oleifera extract treated A549 cells. In
accordance with the quantitative RT-PCR analysis, an increase in Bax
protein expression was observed in A549 cells treated with M. oleifera
extract. More significantly, the expression levels of Bcl-2 protein were
simultaneously downregulated after M. oleifera extract treatment;
therefore the ratio of pro-apoptotic proteins to the anti-apoptotic pro­
teins was altered in favour of apoptosis. Thus, the results suggest that an
up-regulation of Bax and the corresponding down-regulation of Bcl-2
proteins observed in this study may be one of the critical mechanisms
through which M. oleifera extract induces apoptosis in A549 cells.
In summary, M. oleifera extract extracts act as anti-cancer agents by
decreasing cell proliferation and exhibiting apoptosis-mediated cell
death in A549 cancer cell line. Additionally, low cell survival and high
apoptosis were detected upon treatment with the extracts of M. oleifera
extract compared to control cells. Finally, our findings provide growing
evidence supporting the promising role of M. oleifera extract as anti-
cancer candidate and open a new vista for molecular analysis of their
actions on the predominant signaling mechanisms responsible for lung
cancer development.
Funding
The study was supported by the Indian Council of Medical Research
(Ortho/2018/NCD-I).
5
Advances in Cancer Biology - Metastasis 6 (2022) 100072
Declaration of competing interest
The authors declare no conflict of interest. The funding sponsors had
no role in the design of the study; in the collection, analyses, or inter­
pretation of data; in the writing of the manuscript, and in the decision to
publish the results.
Data availability
Data will be made available on request.
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