Molecular Biology Reports

https://doi.org/10.1007/s11033-021-06466-y

ORIGINAL ARTICLE

Dichloromethane fraction of Moringa oleifera leaf methanolic

extract selectively inhibits breast cancer cells (MCF7) by induction
of apoptosis via upregulation of Bax, p53 and caspase 8 expressions
Umiey Fahietah Mohd Fisall1 · Noor Zafirah Ismail1 · Ismail Abiola Adebayo2 · Hasni Arsad1

Received: 2 January 2021 / Accepted: 1 June 2021

© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Moringa oleifera is a well-known medicinal plant which has anti-cancer and other biological activities. This research aims
to determine the cytotoxic and apoptotic effect of M. oleifera leave extract on the breast cancer (MCF7) cells. The extracts
were prepared using hexane, dichloromethane, chloroform and n-butanol by fractionating the crude 80% methanol extract
of the plant leaves. The cytotoxic effect of the extracts on MCF7 cells were determined using CellTiter 96® AQueous One
Solution Cell Proliferation (MTS) assay. The apoptosis study was conducted using Annexin V-FITC analysis and confirmed
by Western blotting using selected proteins, which are p53, Bax, cytochrome c and caspase 8. Our results showed that the
dichloromethane (DF-CME-MOL) extract was selectively cytotoxic to MCF7 cells (5 μg/mL) without significantly inhibiting
the non-cancerous breast (MCF 10A) cells. It had the highest selectivity index (SI) value of 9.5 among the tested extracts.
It also induced early apoptosis and increased the expressions of pro-apoptotic proteins Bax, caspase 8 and p53 in MCF7
cells. Gas chromatography-mass spectrometry analysis (GC-MS) analysis showed that the major compounds found in DF-
CME-MOL were benzeneacetonitrile, 4-hydroxy- and benzeneacetic acid, 4-hydroxy-, methyl ester among others that were
detected. Thus, DF-CME-MOL extract was found to inhibit the proliferation of MCF7 cells by apoptosis induction, which
is likely due to the activities of the detected phytochemical compounds of the extract.

Keywords MCF7 cells · MCF 10A · Moringa oleifera · Apoptosis · Cytotoxicity · p53 · Bax · Caspase 8

Introduction
Moringa is one of the genera found in Moringaceae family
and has been used traditionally to treat wounds and diseases
such as diabetes and colds. This genus is well-known as the
“horseradish” and “drumstick” family [1]. Among the 13
species, Moringa oleifera is one of the species that is widely
consumed particularly by Africans and Indians as folk rem-
edies. M. oleifera is known as “Munggai” or “Kelor” tree
in Malaysia. It is widely distributed throughout the world
specifically Africa, Latin America, Asia, Florida, the Pacific
* Hasni Arsad
hasniarsad@usm.my
1 manner.
Advanced Medical and Dental Institute, Universiti Sains
Malaysia, Bertam, 13200 Kepala Batas, Penang, Malaysia
2 et al. [4] that showed the solvent fractions with different
Department of Microbiology and Immunology, Faculty
of Biomedical Sciences, Kampala International University,
Western Campus, P. O. Box 71, Ishaka, Bushenyi, Uganda
Islands and the Caribbean Island [1, 2]. M. oleifera have
been reported to have significant antiproliferative effects on
several types of cancer cells such as breast, colon, alveolar
and pancreatic cells and induce apoptosis in human cervical
cancer (HeLa), hepatocellular carcinoma (HepG2), adeno-
carcinomic alveolar basal epithelial (A549), breast cancer
(MDA-MDB-231), colon carcinoma (HCT-8), malignant
melanoma (A375), metastatic melanoma (A2058) [3] and
pancreatic cancer (Panc-1) cells [4–8]. For example, Char-
oensin [9] carried out a two-stage of M. oleifera leaves
extraction process using dichloromethane and methanol
solvents. Both extracts were used to treat MCF7 cells, colo-
rectal cancer cell (Caco-2) and HepG2 to determine their
growth inhibition efficacy. The extracts significantly inhib-
ited the proliferation of the cancer cells in dose dependent
Besides that, there is also a study conducted by Adebayo
degree of polarities fractionated from ethanolic extract of
M. oleifera seeds exhibited profound inhibitory effects on

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breast cancer cells. Adebayo and colleagues proved that the
fractionation of the crude extract concentrated active agents
in the plants by separating the unwanted and irrelevant com-
pounds and make the active fraction more effective antipro-
liferative agent. However, we are not aware of any reported
research work on the inhibitory effect of solvent fractions of
the crude M. oleifera leaf extract, even though, studies have
suggested that the crude extracts have significant inhibitory
effect against breast cancer [5]. Thus, this study aims to
investigate the cytotoxic effect of crude extract and fractions
of M. oleifera leaves on MCF7 cells, the apoptotic death
mechanism in MCF7 cells treated with the most effective
fraction and identify the compounds found in the selective
fraction using GC-MS analysis.
Methodology
Collection and identification of M. oleifera
M. oleifera leaves were purchased from HERBagus Trading,
Kepala Batas, Penang, Malaysia. The identity of the plant
was confirmed at School of Biological Sciences, Universiti
Sains Malaysia and it was deposited to the herbarium of the
school (Voucher No: 11729). The plant was also correctly
identified using DNA barcoding marker from the study by
Ismail et al. [10] as DNA barcoding markers give an extra
verification and confirmation about the plant obtained. The
DNA sequences were submitted to GenBank of the National
Center for Biotechnology Information (NCBI). The acces-
sion numbers (MK165484 and MK165485) were assigned
to the sequences by NCBI’s GenBank.
Plant extraction
M. oleifera leaves were weighed (500 g) and washed with
deionised water. The leaves were cut into small particles and
macerated. The macerated leaves were continuously mixed
with 80% methanol for 24 h with the aid of an orbital shaker
(Buch & Holm, Denmark) at 170 rpm. We used fresh leaves
as the raw material for crude extraction because we want to
avoid the leaves to degrade when exposed to high tempera-
tures. Tzanova et al. [11] stated that either fresh or dried
material can be used as a raw material for extraction. The
phytochemical content of the leaves was extracted according
to the method of Ismail et al. [12]. The crude extract was
filtered using Whatman filter paper No. 1 (Sigma-Aldrich,
United States). The leaves were soaked three times with the
same solvent and the extraction process was repeated each
time. Then, the solvent was completely evaporated using
rotary evaporator (Buchi, Unites States). The extract was
freeze-dried using freeze dryer (Martin Christ, Germany)
and stored at −20 °C for further used.
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Molecular Biology Reports
The crude extract was sequentially fractionated with the
following solvents; hexane, dichloromethane, chloroform
and n-butanol based on increasing polarity respectively.
The 30 g of crude extract was dissolved in 100 mL deion-
ised water and mixed with 300 mL hexane in the separating
funnel. The separating funnel was shaken vigorously. Then,
separating funnel was made to stand upright with the aid of a
retort stand for 1 h until the water (aqueous) and solvent lay-
ers were completely separated based on their densities. The
solvent together with its fraction was collected. The aqueous
layer was mixed with the next solvent and the fractionation
process was repeated. The process was repeated with the
remaining solvents. Then, the solvents were evaporated from
the fractions by evaporation using rotary evaporator (Buchi,
Unites States). All extracts were stored at −20 °C for further
used. Extraction yields were calculated using the following
formula: Extraction yield (%) = (Weight of the freeze-dried
extract/Weight of the original sample) × 100.
Cell culture
The epithelial breast cell lines MCF7 and MCF 10A were
purchased from American Type Culture Collection (ATCC),
United States. All supplemented media used to culture the
cells were purchased from Gibco, United States, The MCF7
and MCF 10A cells were cultured in supplemented Roswell
Park Memorial Institute 1640 (RPMI) and Dulbecco’s
modified eagle’s medium (DMEM) media, respectively, at
37 °C in 5% carbon dioxide ­(CO2) incubator (Nuaire, United
States). The MCF7 cells were cultured in the RPMI media
supplemented with 10% fetal bovine serum and 1% penicil-
lin/streptomycin (Penstrep). The MCF 10A were cultured
and maintained in DMEM media supplemented with 0.2%
epidermal growth factor (20 ng/mL), 5% (v/v) horse serum,
10 μg/mL insulin, 0.5 mg/mL hydrocortisone and 1% Pen-
strep. The viable cells that were proliferating exponentially
with over 80% viability were used in this study.
Cytotoxicity assay
Cytotoxicity assay was carried out using (MTS) Assay kit
(Promega, United States) according to the manufacturer’s
instruction. One hundred microlitres (100 µl) of medium
containing viable MCF7 and MCF 10A cells (5 × ­103 cells/
mL) was seeded into each well of 96 well-plates and incu-
bated in ­CO2 saturated incubator (Nuaire, United States)
for 24 h. After 24 h, triplicate treatments were applied for
72 h at different concentration in 96 well-plates. Cells were
exposed to Tamoxifen (Nacalai tesque, Japan) (0 – 15 μM)
as positive control and 200 μL of crude and fraction extracts
(0 – 300 μg/mL). After 72 h of incubation, 20 μL MTS was
added into each well. The plates were incubated for 4 h
Molecular Biology Reports
at 37 °C. The absorbance was measured at 490 nm using
microplate reader (BMG Labtech, Germany).
The percentage of cell viability was calculated with the
following formula: Cell viability (%) = [(Absorbance of
treated cells – Absorbance of blank) ÷ (Absorbance of con-
trol cells – Absorbance of blank)] × 100. The half maximal
inhibitory concentration (­ IC50) values were obtained from
the dose response (variable slope) curve by line graph.
Selective index (SI) value was used to measure the cytotoxic
selectivity of the extracts tested. The SI value was deter-
mined using the following equation [12]: SI = ­IC50 (MCF
10A normal cells)/IC50 (MCF7 cancer cells).
Detection of apoptosis by flow cytometry
Following seeding of MCF7 cells (2.5 × ­104 cells/mL) for
24 h in six well plates, the cells were treated with selected
extract, negative control and positive control for 72 h. The
cells were stained with Annexin V-FITC and propidium
iodide (PI), using the Annexin V-FITC kit (Miltenyi Bio-
tec, Germany) according to the manufacturer’s instruction.
The apoptotic analysis was carried out using BD FACS-
Calibur flow cytometer (Becton Dickinson, United States).
The analysed cells were then distributed in to four quadrants
(Q1–Q4) of a graph plots to differentiate live and non-apop-
totic cells (lower-left quadrant), early apoptotic cells (lower-
right quadrant), late apoptotic cells (upper-right quadrant)
and necrotic cells (upper left quadrant) in the analysed cell
population.
Western blotting analysis
MCF7 cells (2.5 × ­104 cells/mL) in 10 mL were seeded in
75 ­cm2 flask and allowed to proliferate for 24 h in C
­ O2 satu-
rated incubator (Nuaire, United States) at 37 °C. Then, the
cells were treated with selected extract for 72 h. The cells
were harvested and rinsed with cold phosphate buffer saline
(Amresco, United States). The proteins were extracted using
radioimmunoprecipitation assay (RIPA) buffer (Cell Signal-
ing Technology, United States) supplemented with 1% pro-
tease inhibitor cocktail (Cell Signaling Technology, United
States). Protein quantification assay was performed using
Quick Start Bradford Protein Assay kit (Bio-Rad, United
States) according to the manufacturer’s instruction. The
proteins were electrophoresed in 12% SDS-PAGE gel. The
separated proteins in the gel were transferred to a polyvi-
nylidene fluoride (PVDF) western blotting membrane (Axon
Scientific, Malaysia). After blocking with 5% blocking
buffer (nonfat milk) (SunLac, Malaysia), the membrane was
blotted with primary antibodies purchased from Cell Signal-
ing Technology, United States, which are cytochrome c (cat.
no. ad 90529), p53 (cat. no. ab 1431), cleaved caspase 8
(cat. no. ab 25901), β-actin (cat. no. 4970) and Bax (cat. no.
ab7977) diluted in 0.1% Tris Buffered Saline with Tween-
20 (TBST) as recommended by the manufacturer overnight
at 4 °C. The membrane was washed thrice with 0.1% TBST
and blotted with peroxidase-conjugated goat anti-rabbit IgG
(secondary antibody) (cat. no. ab6721) for 1 h. For imaging,
the membrane was incubated with enhanced chemilumines-
cence (ECL) clarity reagent (Bio-Rad, United States) for
5 min with vigorous agitation. Bands of targeted proteins
were captured by Versa Doc Imaging System (Bio-rad,
United States) and the band intensities and fold changes
were obtained using Image Lab 5.2. 1 software (Bio-Rad,
United States). The calculations were made using the follow-
ing equations: Normalized band intensity = band intensity of
target protein ÷ band intensity of β-actin.
Statistical analysis
Numerical values are expressed as mean ± standard devia-
tion of three replicates of the experiment. The significant
levels of the results were determined by calculating p value
using pair t-test and One-Way ANOVA in IBM Statistical
Package for the Social Sciences (SPSS) Version 20 software.
GC‑MS analysis
GC-MS analysis of the selected extract was carried out in
a GC system (Agilent 6890 N series) equipped with a split/
splitless injector and auto-sampler attached to a polar HP-
5MS (5% phenyl polymethyl siloxane) capillary column
(Agilent 19091S-433; 30 m × 0.25 mm × 0.25 μm film thick-
ness). The flow rate of the carrier gas, helium (He) was set to
be at 1.0 ml.min−1 in splitless mode. The injector tempera-
ture was set at 250 °C. The oven temperature was initiated
at 70 °C for 2 min and increased to 200 °C (held for 5 min
at 8 °C ­min−1) and 200 °C to 250 °C (at 10 °C ­min−1 with
2 min hold time).
Results
Extraction yields of M. oleifera leave extract and its
fractions
The extraction yields of crude extracts of M. oleifera leaves
and its fractions were presented in Table 1. The crude extract
(CME-MOL) yielded was 6.84% (34.22 g) of the weight
of the fresh leaves (500 g). From 30 g of CME-MOL, the
following fractions were yielded; aqueous residue frac-
tion extract (AR-CME-MOL) (22.48 g), n-butanol fraction
extract (nBF-CME-MOL) (3.71 g), chloroform fraction
extract (CF-CME-MOL) (2.57 g), hexane fraction extract
(HF-CME-MOL) (0.71 g) and dichloromethane fraction
extract (DF-CME-MOL) (0.22 g). The highest fraction yield
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Table 1  Extraction yield of M. oleifera extracts
Crude extract Initial weight of sample (g)
CME-MOL 500
Fraction
Initial weight of CME-MOL for partition is 30 g
HF-CME-MOL
DF-CME-MOL
CF-CME-MOL
nBF-CME-MOL
AR-CME-MOL
CME-MOL crude methanolic extract of M. oleifera leaves, HF-CME-MOL hexane fraction of CME-MOL, DF-CME-MOL dichloromethane
fraction of CME-MOL, CF-CME-MOL chloroform fraction of CME-MOL, nBF-CME-MOL n-butanol fraction of CME-MOL, AR-CME-MOL
aqueous residue of CME-MOL
obtained was AR-CME-MOL while the lowest fraction yield
was DF-CME-MOL.
Cytotoxic effect of M. oleifera extracts and fractions
on MCF7 and MCF 10A cells
The cytotoxic effects of the crude extract of M. oleifera
leaves and its fractions against MCF7 and MCF 10A cells
were evaluated after 72 h treatment using MTS assay. Both
cells were also treated with tamoxifen as a standard drug.
Figure 1a and b showed the results of the cytotoxic effects of
the crude extract and its fractions on both cell lines (MCF7
and MCF 10A), in which each of the six concentrations of
the extracts was plotted against its respective cell viabil-
ity (%). CME-MOL, CF-CME-MOL and DF-CME-MOL
greatly inhibited the growth of MCF7 cells. Sharp and highly
significant decreases were observed from 100% viability to
18.6 – 10.7% viability after the cells were exposed to 9.4 µg/
ml DF-CME-MOL and CF-CME-MOL. As the concentra-
tion of the fractions were increased the viability of the cells
continued to decline. However, 9.4 µg/ml, 18.8 µg/ml and
37.5 µg/ml concentrations of CME-MOL slowly decreased
the viability of the cells. When the concentration of the frac-
tion was increased to 75 µg/ml, the viability of the cells
rapidly reduced to 4.2%. HF-CME-MOL and AR-CME-
MOL moderately inhibited the growth of the cancer cells in
a dose dependent manner. nBF-CME-MOL hardly inhibited
the cancer cells. The viability of the cells was at 75% after
the cells were exposed to 300 µg/ml of nBF-CME-MOL.
The viability of MCF 10A cells was less affected when
exposed to the extract and fractions as compared to their
effects on MCF7 cells. None of the extracts significantly
inhibited the viability of the cells when they were exposed
to 9.4 µg/ml and 18.8 µg/ml concentrations of the fractions
and extract. However, the viability of the cells was reduced
below 50% when exposed to 37.5 µg/ml of CF-CME-MOL,
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Molecular Biology Reports
Yields (g) Percentage yield (%)
34.22 6.84
Yield (g) Percentage
yield (%)
0.71 2.37
0.22 0.73
2.52 8.40
3.71 12.37
22.48 74.93
75 µg/ml of DF-CME-MOL and 75 µg/ml of CME-MOL.
The viability of the cells continued to decrease as the
concentrations of these extracts are increased. HF-CME-
MOL, AR-CME-MOL and nBF-CME-MOL did not pro-
foundly inhibit the viability of the cells even at 300 µg/ml
concentration.
The ­IC50 values reported in Table 2 were extrapolated
from the curves shown in Fig. 1a and b. To determine the
selective cytotoxicity of each extract towards MCF7 cells,
the measurement of SI value also was estimated by using the
formula earlier stated. The fraction that had the lowest ­IC50
against MCF7 cells and highest SI value was selected for
further studies. Among those extracts, DF-CME-MOL had
the lowest I­ C50 (5 µg/mL) against MCF7 cells and highest
SI value (9.5) (Table 2). The DF-CME-MOL was also used
to treat MCF7 cells for 24 h and 48 h to further confirm that
the ­IC50 of DF-CME-MOL at 72 h was the lowest (highest
cytotoxicity) than the other lower incubation periods and
to determine whether the extract cytotoxic effect is time
dependent. The results showed that DF-CME-MOL had
cytotoxic effect on MCF7 cells with the following I­ C50 val-
ues; ­IC50 = 23.75 μg/mL at 24 h and ­IC50 = 13.75 μg/mL at
48 h (Fig. 1c). Thus, the ­IC50 of DF-CME-MOL at 72 h was
the lowest (highest cytotoxicity) and DF-CME-MOL cyto-
toxicity on MCF7 cells was time dependent. The One-Way
ANOVA revealed that the percentages of the cell viability
treated at different incubation times (24, 48 and 72 h) using
the same concentration of DF-CME-MOL are significantly
different (p < 0.05).
DF‑CME‑MOL induced apoptosis in MCF7 cells
In this study, double-stain annexin V-FITC conjugate and pro-
pidium iodide dye was used to distinguish the viable, apoptotic
and/or necrosis cells. As presented in the results, we used an
increasing range of concentrations (2.5, 5 and 10 ug/ml) to
Molecular Biology Reports
Fig. 1  Cytotoxicity effect of crude and fraction extracts of M. oleif-
era leaves. a Percentage of cell viability MCF7 cells treated with M.
oleifera extracts at 72 h. b Percentage of cell viability of MCF 10A
cell treated with M. oleifera extracts at 72 h. c Time-dependent anti-
proliferative effect of DF-CME-MOL on MCF7 cells. The analysis
revealed DF-CME-MOL inhibited the proliferation of MCF7 in a
time dependent manner (24, 48, and 72 h). Significance difference
analysis carried out using One-Way ANOVA revealed that different
percentages of the viability of the cells treated for different incuba-
ensure that the treatment of the cells for the apoptosis effect
of the fraction was dose dependent. In Fig. 2a and b, results
showed that the highest population was early apoptotic pop-
ulation and there was a continuous increase in the popula-
tion of early apoptotic cells after treatment with DF-CME-
MOLThe cells were stained with apoptotic dyes (Annexin V
and PI) after the treatment and the results showed that the
doses of DF-CME-MOL were directly proportional (2.5 µg/
mL; 26.57% < 5 µg/mL; 54.77% < 10 µg/mL; 87.01%). How-
ever, there were dose-dependent deductions on late apoptotic
population (2.5 µg/mL; 3.24% > 5 µg/mL; 2.52% > 10 µg/mL;
0.39%) and fewer necrotic signs were shown, which is less than
1% of population. Therefore, the results strongly suggest that
DF-CME-MOL induced apoptosis in MCF7 cells.
Effect of DF‑CME‑MOL on apoptotic death
mechanism pathway in MCF7 cells
We assessed the expression levels of selected apoptotic pro-
teins; which are p53, Bax, cytochrome c and caspase 8 in
tion times (24, 48 and 72 h) using the same concentration of DF-
CME-MOL are significantly different (p < 0.05). Bars with differ-
ent alphabetical superscripts are significantly different at p < 0.05).
d Percentage of cell viability of MCF7 cells treated with Tamoxifen
(positive control) for 72 h. Values were presented as mean ± SD of
three replicates. Crude methanol extract of M. oleifera, its hexane,
dichloromethane, chloroform, butanol, and aqueous residue fractions
are represented as CME-MOL, HF-CME-MOL, DF-CME-MOL, CF-
CME-MOL, BF-CME-MOL, and AR-CME-MOL respectively
untreated and DF-CME-MOL-treated MCF7 cells by west-
ern blotting analysis. As presented in Fig. 3a, DF-CME-MOL
increased the expression levels of the apoptotic protein mark-
ers in MCF7 cells. After normality, protein band intensities
of untreated cells (control) and treated cells showed signifi-
cant differences for p53, Bax and caspase 8, but no signifi-
cant difference was observed for cytochrome c in both cells
(Fig. 3b). DF-CME-MOL induced a 1.87-fold increase in p53
expression, a 1.47-fold increase in Bax expression, a 1.05-fold
increase in cytochrome c and a 2.21-fold increase in caspase
8. Based on the results obtained, caspase 8 had the highest
protein expression level in DF-CME-MOL-treated MCF7
cells. These results suggested that DF-CME-MOL induced
apoptosis in MCF7 cells through the selected apoptotic regu-
latory proteins.
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Table 2  The ­IC50 value of crude and fraction extracts of M. oleifera
leaves on MCF7 and MCF 10A cells at 72 h treatment period
Type of extract IC50 on MCF7 IC50 on MCF Selective
(µg/mL) 10A index
(µg/mL) (SI)
4-OH TAM 0.83 – –
CME-MOL 170 225 1.3x
HF-CME-MOL 198 NA – two solvent phases [20]. Many studies have proved that in
DF-CME-MOL 5 47.5 9.5x
CF-CME-MOL 6.25 32.5 5.2x
nBF-CME-MOL NA NA – for the current study as AR-CME-MOL (extract from polar
AR-CME-MOL NA NA – solvent) yield was higher than DF-CME-MOL (non-polar
The MCF7 and MCF 10A were exposed to the crude and fraction
extracts of M. oleifera leaves for 72 h. Tamoxifen was used as posi-
tive control. MTS assay was used to determine the extracts’ cytotoxic
and antiproliferative effects, and ­IC50 values were obtained
NA No activity at dose concentration-dependent test, 4-OH TAM
tamoxifen, CME-MOL crude methanolic extract of M. oleifera leaves,
HF-CME-MOL hexane fraction of CME-MOL, DF-CME-MOL
dichloromethane fraction of CME-MOL, CF-CME-MOL chloroform
fraction of CME-MOL, nBF-CME-MOL n-butanol fraction of CME-
MOL, AR-CME-MOL aqueous residue of CME-MOL
GC‑MS profiling of volatile compounds
in DF‑CME‑MOL
The phytochemical compounds of DF-CME-MOL were pro-
filed using GC-MS analysis. The phytochemical compounds
were identified by comparing the spectra of the unknown
extract’s phytochemicals with the spectra of known chemi-
cal compounds in the National Institute of Standards and
Technology (NIST) library. Based on Table 3, eighteen com-
pounds were identified in DF-CME-MOL.
Discussion
In this present study, the difference in solvents’ polarities
might influence the solubility of the phytochemical con-
stituents in the plant and its extraction yield [12, 13]. The
obtained highest yield of AR-CME-MOL may be due to
high content of impurities like sugars, organic acids, solu-
ble proteins and some polar compounds. Bandar et al. [14]
reported that the impurities could disturb the identification
and quantification of the compounds. Polarity of solvent
plays a fundamental role in increasing the solubility of phe-
nolic compounds as the less polar solutes dissolve in the
less polar solvent, while the more polar solutes dissolve in
the more polar solvent [15, 16]. Since the crude CME-MOL
was obtained from polar extraction solvent, the extraction
yield of DF-CME-MOL showed the least amount of yield
because the solvent is moderately polar. Possibly, the low
yield could be due to the lower polarity of dichloromethane.
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Molecular Biology Reports
Lin and Giusti [17] reported that extraction efficiency of dif-
ferent solvents may be influenced by their polarity. Nonethe-
less, the extraction yields also depend on the solubility of
solvent toward the amount of solute available in the crude
sample [18]. The percentage yields of M. oleifera extracts
were influenced by the interaction forces between solvent
and solute molecules [19] and hydrophobicity between the
different plant species, polar solvents produce a higher yield
of extract than non-polar solvent [14, 21]. This is also true
solvent) yield.
According to Chan et al. [22], they reported that the frac-
tion of plant extracts using various solvents may have a sig-
nificant influence on biological activity (like anticancer and
antioxidant) and gave varying results from a single plant
species. From the results obtained, the cytotoxicity of each
of the extracts was determined using the ­IC50 value, the ­IC50
was extrapolated from the cell viability exponential curve.
Generally, the lower I­ C50 value indicated the higher cyto-
toxic activity. SI value was used to evaluate the selectivity
of extract toxicity towards cancer cells compared to normal
cells. The SI value greater than six (6) indicates high selec-
tivity [23], hence DF-CME-MOL had fulfilled those criteria
with a highest potency (9.5). The guidelines from American
National Cancer Institute have set the criterion for effective
cytotoxic crude extracts that their I­ C50 must be less than
30 µg/mL after incubation time of 72 h [24–26]. According
to the United States National Cancer Institute plant screen-
ing program, a plant extract is generally considered to have
active cytotoxic effect if the ­IC50 value is less than 20 µg/
mL after 48 to 72 h treatment period [27]. Interestingly, DF-
CME-MOL showed very potent cytotoxic effect on MCF7
cells and it had fulfilled the criteria above as its ­IC50 value
is below 20 µg/mL after 48 h treatment.
M. oleifera extracts have been reported to have effec-
tive inhibitory activity against the growth of different type
of cancer cell lines such as A549 cells (human alveolar
epithelial cells) [28], HepG2 (liver hepatocellular carci-
noma) [9], Caco-2 (human Caucasian colon adenocarci-
noma) [9], cervical cancer (HeLa) cells, HCT116 cells
(human colorectal carcinoma) [8] and MDA-MB-231 cells
(breast cancer cell) [5]. However, different types of solvent
were used to extract the cytotoxic extracts in these stud-
ies. This is because extracts obtained by different type of
solvents might inhibit cancer cell proliferation differently
on the different cells. Therefore, this study had proved
that different solvent fractions from the same plant species
(M. oleifera) had varying cytotoxic effects on MCF7 cell
proliferation. Additionally, among all the tested extracts,
DF-CME-MOL with low polarity had the strongest potent
cytotoxic activity in MCF7 cells with I­ C50 value of 5 μg/
Molecular Biology Reports
Fig. 2  Flow cytometry analysis of MCF7 cells treated with 2.5, 5 and
10 μg/mL DF-CME-MOL at 72 h. a Representative figures showing
population of viable; lower left (Annexin V− PI−), early apoptotic;
lower right (Annexin V + PI−), late apoptotic; upper right (Annexin
V + PI +) and necrotic; upper left (Annexin V− PI +) cells. b Bar
mL. According to Moo-Puc et al. [29] and Guedes et al.
[30], plant extract with a low polarity could penetrates
cell membranes more easily than more polar extract and
it might be an evidence for the observed potent cytotoxic
effect of DF-CME-MOL on MCF7 cell in this study.
The previous study by Charoensin [9] also reported that
dichloromethane of M. oleifera extract had higher cyto-
toxic effect on Caco-2, HepG2 and MCF7 cancer cell lines
when compared to methanol extract. In addition, the find-
ings were similar to a study by Ismail et al. [12] and Haron
et al. [31], who reported that the dichloromethane fraction
chart showing increased proportion of early apoptotic cells after
exposure of DF-CME-MOL. The results of three independent experi-
ments are presented as a mean ± standard deviation. *Statistically sig-
nificant (P < 0.05); independent t-test
of Clinacanthus nutans extract was selectively cytotoxic
to MCF7 cells and HeLa cells.
Furthermore, Tiloke et al. [7] reported that aqueous
extract of M. oleifera had antiproliferative effect on A549
lung cell with I­ C50 166.7 μg/mL, which was in line with
the studies by Jung [28]. However, their findings are not in
agreement with the finding of this study as aqueous extract
(AR-CME-MOL) was not cytotoxic to MCF7 cells. The rea-
son for the different outcomes could be that the phytochemi-
cal compounds in AR-CME-MOL don’t have antiprolifera-
tive effect on MCF7 cells. Additionally, the different active
13
 Molecular Biology Reports

Fig. 3  Effect of DF-CME-MOL

on apoptotic death mechanism
pathway in MCF7 cells. a Dif-
ferential expression of p53, Bax,
Cytochrome C and Caspase 8 in
MCF7 cells after treatment with
DF-CME-MOL. b Expression
levels of p53, Bax, Cytochrome
C and Caspase 8 in control
and treated of MCF7 cells.
The results of three independ-
ent experiments are presented
as a mean ± standard devia-
tion. *Statistically significant
(p < 0.05)

Table 3  List of phytochemical RT (min) Peak area (%) Compound name

constituents in DF-CME-MOL
by GC-MS 10.73 1.96 Phenol, 4-(methoxymethyl)-
11.01 1.01 2-Methoxy-4-vinylphenol
11.67 4.65 2H-Pyran-3,4,5-triol, tetrahydro-2-methoxy-6-methyl-
11.80 1.37 Benzaldehyde, 4-hydroxy-
12.52 5.17 Phenol, 2-propyl
13.22 0.73 Phenol, 2-methoxy-4-(1-propenyl)-, (Z)-
13.66 20.20 Benzeneacetonitrile, 4-hydroxy-
13.82 28.85 Benzeneacetic acid, 4-hydroxy-, methyl ester
14.37 0.39 Benzoic acid, 4-ethoxy-, ethyl ester
15.22 0.27 Megastigmatrienone
17.24 7.50 Benzaldehyde, 3,4-dimethoxy-
17.41 2.98 4-((1E)-3-Hydroxy-1-propenyl)-2-methoxyphenol
20.01 0.26 Hexadecanoic acid, methyl ester
20.93 4.19 Phenol, 2-ethyl-
21.53 0.35 Phenol, 2-[(4-hydroxyphenyl)methyl]-
22.17 12.47 1,4-Benzenediamine
23.01 2.04 Phenol, 4,4‟-methylenebis-
29.95 5.59 Benzeneethanol, 4-hydroxy-

RT retention time

13
Molecular Biology Reports
compounds in the extracts and alteration in phenotype and
genotype of cancerous cells might be part of the reasons for
the observed varying results [9].
Kabeer et al. [32] reported that apoptosis is a crucial event
as a popular target of therapeutic strategy, which is known as
a hallmark for cell inhibition to occur. Early apoptosis trig-
gers cell death caused by microphages, resulting in the pro-
cess of phagocytosis [33]. The early apoptosis was strongly
induced by DF-CME-MOL after 72 h treatment. This result
is different from the results of previous studies, wherein it
was found that M. oleifera extracts strongly induced late
apoptosis in MDA-MB-231 [5] and HepG2 cells [34]. This
difference revealed that MCF7 cells are more sensitive to
induction of early apoptosis by DF-CME-MOL.
It has been shown from the present study, that the death
mechanism of MCF7 cells when treated with DF-CME-
MOL was through apoptosis by increasing the expression
of pro-apoptotic proteins. In related studies, there are several
research works that had proven the ability of M. oleifera leaf
to induce apoptosis in many cancer cells. Aqueous M. oleif-
era extract had proved its antiproliferative effect on A549
and oesophageal (SNO) cancer cells by inducing intrinsic
apoptotic pathway [7, 35]. In addition, a study by Yurina
et al. [36] concluded that M. oleifera leaf extract decreased
Hsp27 expression and increased the expression of caspase
9 in MCF7 cells, thus triggered the induction of apoptosis
in the cancer cells. In the present study, activation of p53
and Bax in treated MCF7 cells by DF-CME-MOL strongly
suggests that the mitochondrial signal pathways (intrinsic
pathway) was activated.
Caspase 8 was significantly upregulated by DF-CME-
MOL in MCF7 cells (2.21-fold). The activation of Caspase
8 is triggered by the binding of ligands to its specific death
receptor on the surface of cell [32, 37]. Activation of cas-
pase 8 (initiator caspase) initiated apoptosis signal by direct
cleavage of downstream effector caspases (caspase 3, cas-
pase 6 and caspase 7) [38]. Ju et al. [39] revealed that M.
oleifera extract activated caspase 8 and caspase 3 in pros-
tate cancer (PC-3) cells and upregulated their expressions.
Therefore, the present study also suggests that caspase 8 was
activated and its expression was upregulated by DF-CME-
MOL in MCF7 cells. This results to induction of apoptosis
in the cancer cells through the extrinsic pathway. This study
proposes DF-CME-MOL could be promising source of anti-
cancer agent especially in treating breast cancer. The above
results strongly demonstrated that DF-CME-MOL success-
fully inhibited the proliferation of MCF7 cells by apoptosis
induction via activated caspase 8.
Based on the research work reported by Mukunzi et al.
[40], difference in chemical constituents of M. oleifera is
possibly associated with the different environmental [41,
42] and climatic conditions of the geographical location (or
country) the plant is grown [43, 44]. For instance, a sample
of M. oleifera grown in China (CN) contained 61 volatile
compounds while another sample of the same plant grown
in Rwanda (RW) contained 59 volatile compounds. In the
present research 18 volatile compounds were found in DF-
CME-MOL extract. However, only two compounds namely,
hexadecanoic acid and benzaldehyde found in M. oleifera
extract by Mukunzi et al. [40] were similarly detected in
DF-CME-MOL. In another study by Al-Asmari et al. [5],
the ethanol extracts of the leaves and seeds of M. oleifera
had twelve and seventeen volatile compounds respectively.
Isopropyl isothiocyanate, D-allose and cetene were exclu-
sively detected in the leaves extract while eugenol, dibu-
tyl phthalate, 2-chloropropionic acid and 5-eicosene, were
exclusively detected in the bark extract. These compounds
have been found to have anticancer activities. However, none
of the compounds reported by Al-Asmari et al. [5] was found
in this present study. This might be due to the different sol-
vents used for extraction and the polarity of the solvents. The
GCMS analysis revealed that DF-CME-MOL has several
biologically active phytochemical substances, which may
exhibit great antioxidant and anticancer activities. GC-MS
analysis has revealed the existence of many bioactive phy-
tochemicals in Moringa sp. plant extracts such as alkaloid,
alcohols, aldehyde, aromatic compounds, fatty acid methyl
esters, terpenoids, flavanoid, phenolics and steroids that can
be postulated for antioxidant and anticancer activity [45].
Previous studies have found that M. oleifera leaves are
potential antioxidant and anticancer agents [7, 46]. Based
on GC-MS analysis compared to NIST library, the following
compounds identified in DF-CME-MOL have antioxidant
activity: Phenol, 4-(methoxymethyl)-, 2-Methoxy-4-vi-
nylphenol, Phenol, 2-propyl, Phenol, 2-methoxy-4-(1-pro-
penyl)-, (Z)-, 4-((1E)-3-Hydroxy-1-propenyl)-2-meth-
oxyphenol, Hexadecanoic acid, methyl ester, Phenol,
2-[(4-hydroxyphenyl)methyl]-, Phenol, 4,4‟-methylenebis-
and Benzeneethanol, 4-hydroxy- [5, 47]. Other identified
compounds, which are Benzaldehyde, 4-hydroxy-, Benze-
neacetonitrile, 4-hydroxy-, Benzaldehyde, 3,4-dimethoxy-,
Hexadecanoic acid, methyl ester, Phenol, 2-ethyl- and Phe-
nol, 4,4‟-methylenebis- have been reported to have anti-
cancer properties [5, 48]. Thus, the results suggest that the
presence of these constituents in DF-CME-MOL could be
responsible for its antioxidant and anticancer activities. Sup-
portively, DF-CME-MOL also induced apoptosis as revealed
by the flow cytometry and western blotting analysis, which
further buttresses the fact that DF-CME-MOL could be con-
sidered a potential source that can be exploited for antican-
cer drug development [49].
The findings of this study showed that DF-CME-MOL
extracts exerts the most cytotoxic effect with lowest ­IC50
value (5 μg/mL) on MCF7 cells among all the tested extracts
of M. oleifera. The cytotoxic effect is responsible for its
antiproliferative activity. The DF-CME-MOL extract also
13

inhibits the growth of MCF7 cells via induction of early
apoptosis, which can be clearly observed after 72 h treat-
ment period. In addition, the DF-CME-MOL extract induces
apoptosis by upregulating pro-apoptotic proteins p53, Bax
and caspase 8. These three proteins are key to regulating the
apoptotic signalling pathway. Also, GC-MS analysis reveals
the presence of potential phytochemical components in DF-
CME-MOL that might have contributed to its strong anti-
proliferative and antioxidant properties. For future studies,
we would like to give additional information and interesting
facts on other proteins such as Bcl-2, caspase 3 and cas-
pase 9. Moreover, it would be worthwhile if we identify and
isolate the active ingredient in DF-CME-MOL for future
studies.
Acknowledgements The second author gratefully acknowledges Uni-
versiti Sains Malaysia for awarding her fellowship under the USM Fel-
lowship Scheme.
Author contributions UFMF and HA designed the experiment.
UFMF performed the experiments. UFMF, NZI and IAA analysed the
data. NZI and IAA wrote the manuscript. The authors reviewed and
approved this manuscript.
Funding This research was funded by Universiti Sains Malaysia Bridg-
ing Grant, Grant Number 304/CIPPT/6316239.
Declarations
Conflict of interest The authors have declared that there is no conflict
of interest.
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