Food and Chemical Toxicology 49 (2011) 1270–1275

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Antiproliferation and induction of apoptosis by Moringa oleifera leaf extract

on human cancer cells
S. Sreelatha a,c,⇑, A. Jeyachitra b, P.R. Padma c
a
Faculty of Science, National University of Singapore, Singapore
b
Dept of Biochemistry, Madurai Kamaraj University, India
c
Dept of Biochemistry & Biotechnology, Avinashilingam University, Coimbatore, India

a r t i c l e i n f o a b s t r a c t

Article history: Medicinal plants provide an inexhaustible source of anticancer drugs in terms of both variety and mech-
Received 15 September 2010 anism of action. Induction of apoptosis is the key success of plant products as anticancer agents. The pres-
Accepted 1 March 2011 ent study was designed to determine the antiproliferative and apoptotic events of Moringa oleifera leaf
Available online 6 March 2011
extract (MLE) using human tumor (KB) cell line as a model system. KB cells were cultured in the presence
of leaf extracts at various concentrations for 48 h and the percentage of cell viability was evaluated by
Keywords: MTT assay. MLE showed a dose-dependent inhibition of cell proliferation of KB cells. The antiproliferative
Reactive oxygen species
effect of MLE was also associated with induction of apoptosis as well as morphological changes and DNA
Antiproliferation
KB tumor cells
fragmentation. The morphology of apoptotic nuclei was quantified using DAPI and propidium iodide
Apoptosis staining. The degree of DNA fragmentation was analyzed using agarose gel electrophoresis. In addition,
MLE at various concentrations was found to induce ROS production suggesting modulation of redox-
sensitive mechanism. Eventually, HPTLC analysis indicated the presence of phenolics such as quercetin
and kaempferol. Thus, these findings suggest that the leaf extracts from M. oleifera had strong antiprolif-
eration and potent induction of apoptosis. Thus, it indicates that M. oleifera leaf extracts has potential for
cancer chemoprevention and can be claimed as a therapeutic target for cancer.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction
Apoptosis, the process of programmed cell death, is now recog-
nized as a vital process in the regulation of tissue development and
homeostasis, which is highly conserved throughout evolution
(Ghobrial et al., 2005). Homeostasis between cell death and cell
proliferation is required to maintain normal state. Disruption of
this cellular balance or dysregulation of controlling mechanisms
can lead to human disease including cancer. Hence clinically many
diseases are the ultimate result of either deficient apoptosis or
excessive apoptosis (Thompson, 1995).Therefore apoptosis is nec-
essary for normal developmental processes, maintenance of
homeostasis, and elimination of damaged cells. During the past
two decades, the molecular mechanism of apoptosis has been
extensively studied and has gained recognition as an ideal way
to eliminate precancerous and cancer cells. Many studies have re-
ported associations between apoptosis and cancer, in as much as
the apoptosis-inducing agents potent in the treatment of various
cancer cells which are being appreciated as weapons for the man-
agement of cancer (Schmitt, 2003).
⇑ Corresponding author at: Faculty of Science, National University of Singapore,
Singapore. Tel.: +65 82834498.
E-mail address: sree_latha04@yahoo.com (S. Sreelatha).
Cancer is the largest single cause of death in both men and wo-
men. Recently, resistance to anticancer drugs has been observed.
Therefore, research and development of more effective and less
toxic drugs by the pharmaceutical industry has become necessary.
Chemical as well as biological agents that induce apoptosis have
been reported to be promising interventions in the management
of malignant cancer. Many substances derived from dietary or
medicinal plants are known to be effective and versatile chemo
preventive and antitumor agents in a number of experimental
models of carcinogenesis. There is an increasing evidence for an
association between a high consumption of fruits and vegetables
and reduced risk of cancer (La Vecchia et al., 1997; Morse et al.,
2000). Antiproliferative screening of models in vitro provide impor-
tant preliminary data to help select plant extracts with potential
antineoplastic properties for future study.
Moringa oleifera Lam. (Family: Moringaceae) is the most widely
cultivated species in tropics and subtropics of Asia and Africa. Also
known by the names as drumstick tree, horse radish tree and kelor
tree was utilized by the ancient Roman, Greeks and Egyptians. There
are many distinct species with distinct morphological types. OnlyM.
oleifera is cultivated widely in tropics. India has the prime position
in the cultivation and production of M. oleifera (Ramachandran
et al., 1980).Traditionally, almost all parts of this plant have been

0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2011.03.006
 S. Sreelatha et al. / Food and Chemical Toxicology 49 (2011) 1270–1275
used to treat many diseases such as abdominal tumors, hysteria,
scurvy, paralytic attacks, helmintic bladder, prostate troubles, sores
and skin infections. The leaves are highly nutritious, which contain
more vitamin A than carrots, more calcium than milk, more iron
than spinach, more vitamin C than oranges and more potassium
than bananas and more protein than milk and eggs. M. oleifera is rich
in various phytochemicals like carotenoids, vitamins, minerals,
aminoacids, sterols, glycosides, alkaloids, flavonoids, moringine,
moringinine, phytoestrogens caffeoylquinic acids and phenolics in
flowers, leaves, roots, fruits and seeds (Guevara et al., 1999; Fuglie,
1999; Anwar et al., 2007). In recent decades M. oleifera leaves, flow-
ers, gums, roots and seeds are widely used for many diseases includ-
ing inflammation, cardiovascular and liver diseases, immune
boosting agent, regulate blood sugar and cholesterol (Limaye
et al., 1995; Faizi et al., 1994; Rao and Misra, 1998).
Taking into account the use in folk medicine of M. oleifera and
its widespread claims of the medical effectiveness and the lack of
experimental studies on its apoptotic activity, the present investi-
gation was undertaken to evaluate the antiproliferation and apop-
tosis of the aqueous extract of M. oleifera on KB human tumor cell
lines.
2. Materials and methods
2.1. Plant material and preparation of extracts
M. oleifera leaves were collected from the Horticulture Research Institute,
Periyakulam, Tamil Nadu Agricultural University, India, and a voucher specimen
was deposited in Botanical Survey of India, Coimbatore, India. (BSI/SC/5/25/05-06/
Tech-908). The air dried leaves of M. oleifera were made into a coarse powder. Five
hundred grams of dried powders of leaves were stirred with seven parts of distilled
water at 80 °C for 2 h separately and cooled down to room temperature. Each super-
natant was recovered after filtration through folded paper using a suction apparatus.
The respective solvent was completely removed by rotary vacuum evaporator. The
extract was freeze dried and stored in a vacuum desiccator for further use. The yield
of the extract was 32% with reference to the dry starting material. The concentration
of aqueous M. oleifera leaf extract was determined based on its maximum protection
against proliferation activity.
2.2. Cell line and culture medium
KB tumor cell line was obtained from the National Centre for Cell Sciences
(NCCS), Pune, India. The cells were maintained in DMEM supplemented with 10%
fetal bovine serum (FBS) and antibiotics (100 unit/mL of penicillin G and 100 lg/
mL of streptomycin sulfate) and cultured at 37 °C in 5% CO2. Confluent grown cells
were harvested by trypsinization (0.25%), collected into DMEM containing 10% FBS
and held on ice till the assay. Before the assay, the cells were spun down at
2000 rpm for 5 min at 4 °C in a microfuge and resuspended in Hank’s Balanced Salt
Solution (HBSS).
2.3. Determination of quercetin and kaempferol
As the leaves when subjected to phytochemical screening revealed the presence
of various phytoconstituents the leaf extracts were then examined for the specific
phenolic composition by the method of HPTLC (Nikolova et al., 2004). Quercetin,
kaempferol and M. oleifera leaf extracts were applied to 10  10 cm aluminium-
packed HPTLC plates coated with 0.2 mm layers of silica gel G60 F254 plates (Merck).
The mobile phase was toluene–dioxane–acetic acid 95:25:4. Compounds were visu-
alized by spraying with Naturstoff A reagent. The amounts of the compounds in the
samples were calculated by comparing the densitogram peak areas from the sam-
ples with those of the standards on the same plate.
2.4. Cell proliferation assay
Briefly, KB cells (10,000 cells/well) were cultured in collagen I (50 lg/mL) coated
96-well culture plates, in a total volume of 200 lL DMEM-F12 supplemented with
1% FBS. KB cells were once thought to be derived from an oral cancer but in fact they
were derived from glandular cancer of cervix (Masters, 2002).Cells were treated
with 0.2% DMSO (vehicle control) and leaf extracts (0–200 lg/ml) and Cisplatin
(0–10 lg/ml) for 48 h under the same conditions. Both treatment and control groups
were performed in 6–8 replicate wells. The relative number of viable cells was then
determined at 48 h after incubation, by adding 1 mg/mL of 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and incubating the cell further for
4 h. The formazan crystals formed were then solubilized with acid isopropanol for
1271
1 h. The absorption value of the solution at 595 nm directly represents relative cell
numbers. The cell decrease percent relative to the control group was then deter-
mined (Igarashi and Miyazawa, 2001). The percentage of cell viability was calculated
according to the following equation.
Absorbance of treated cells
The% of cell viability ¼  100
Absorbance of control cells
2.5. Observation of morphological changes of cells
Cells (2  105 cells/well) were incubated for 48 h in the absence or presence of
M. oleifera leaf extracts in 24-well plates. After incubation, the medium was re-
moved and cells in wells were washed once with HBSS. They were then observed
by phase contrast inverted microscope (Zeiss, Germany) at 400 magnification
(Chih et al., 2001).
2.6. Propidium iodide (PI) staining
PI can stain the nuclear changes of living and apoptotic cells. Briefly, cells
(2  105 cells/well) were incubated for 48 h with M. oleifera leaf extracts (200 lg/
ml) in 24-well plates. Cisplatin (10 lg/ml) was used as positive control. After
incubation, cells were permeabilized with a mixture of acetone: methanol (1:1)
at 20 °C for 10 min after treating with extract. Cells were washed with HBSS, then,
200 ll of 5 lg/ml PI was added into each well and incubated at 37 °C for 30 min in
the dark. Cells were detected by green filter of fluorescence inverted microscope
(Zeiss, Germany) at 400 magnification (Sarker et al., 2000).
2.7. Determination of apoptotic index
DAPI (40 -6-diamidino-2-phenylindole) forms fluorescent complexes with natu-
ral double stranded DNA and it is a useful tool in cellular studies. The apoptotic in-
dex was analyzed by DAPI staining as described by Rashmi et al. (2003). Cells
(3  106 cells) were exposed to the extract (200 lg/ml) for 48 h and were gently
scraped and harvested by centrifugation. The cells were then fixed with 3% para
formaldehyde and permeabilized with 0.2% Triton X -100 and incubated with DAPI.
The apoptotic index was scored by counting the cells with condensed chromatin
and fragmented under fluorescent microscope (Nikon, Japan).
2.8. Detection of DNA fragmentation
DNA fragmentation was analyzed by agarose gel electrophoresis as described by
Yang et al. (2000) with slight modifications. Cells (3  106 cells) were exposed to
the extract for 48 h and were gently scraped and harvested by centrifugation. The
cell pellets were incubated for 60 min at 50 °C in 100 ll lysis buffer (100 mM
Tris–HCl pH 8, 100 mM NaCl and 10 mM EDTA). Proteinase K (10 ll) was added
and further incubated for 30 min at 50 °C. RNase (3 ll) was then added and incu-
bated for 2 h at 50 °C. The DNA was extracted with Phenol–chloroform–isoamyl
alcohol, subjected to 2.0% of agarose gel electrophoresis, stained with ethidium bro-
mide and visualized under UV light transilluminator.
2.9. Reactive oxygen species assay
Intracellular reactive oxygen species (ROS) production was measured in both M.
oleifera extract-treated and control cells using DCFH-DA (Chang et al., 2001). Briefly,
2  105 cells/well were exposed to leaf extract and incubated. After incubation, cells
were detached with trypsin–EDTA and washed once with PBS. Treated and control
cells were resuspended in 0.5 ml PBS containing 10 M DCFH-DA at 37 °C for 30 min
and then incubated with 4 mM H2O2 (as inducer for ROS production) at 37 °C for
30 min. ROS productions of cells were evaluated by luminescence spectrophotom-
eter (Perkin–Elmer, MA).
2.10. Statistical analysis
All the parameters studied were subjected to statistical treatment using Sigma
Stat statistical package (Version 3.1). The data were expressed as mean ± SD (n = 6)
where ‘n’ represents the no of samples. One-way ANOVA, followed by post hoc anal-
ysis using Fischer’s LSD was adopted to all the parameters under study to test the
level of statistical significance. The difference was considered significant if p < 0.05.
3. Results and discussion
Apoptosis is a major form of cell death that plays an important
role in many diverse processes ranging from development to stress
responses (Levine et al., 2001).The inactivation of apoptosis is
central to many cancers. There fore induction of apoptosis seems
to be an effective strategy against tumor progression (Brown and
1272 S. Sreelatha et al. / Food and Chemical Toxicology 49 (2011) 1270–1275
Attardi, 2005). Screening of plants and plant derived products as
potential inducers of apoptosis have become the major strategy
in anticancer drug research. The present study was focused on
the ability of the M. oleifera leaf extract in influencing the process
of apoptosis in KB cells and its anti proliferative activity.
3.1. Antiproliferative activity
MTT dye is used to determine the cell viability in assays of cell
proliferation and cytotoxicity (shoemaker et al., 2004). Many her-
bals and phytochemicals have been reported for their cytoprotec-
tive effect using the MTT assay (Horokova et al., 2003). In the
present study, the effect of M. oleifera leaf extracts on the extent
of survival of KB tumor cells was followed using the MTT reduction
assay. MLE significantly inhibited the proliferation of KB cells in a
dose dependent manner (Fig. 1a and 1b). The results summarized
showed that the extracts used at non toxic concentration in normal
cells exhibited significant inhibitory effect on the proliferation of
KB cells greater than 85%. Similar result was observed when cis-
platin was used as a positive control. Morphological changes could
be seen after 48 h treatment with MLE and cisplatin characterized
by round cells and fewer adherent cells when compared with con-
trol group. Wang and Jiao (2001) has reported that the efficiency of
compounds to induce apoptosis in cancer cells indicates anticancer
activity. The results presented showed a decrease in the percentage
of cell viability and the leaf extract effectively inhibited the cell
proliferation. Therefore In light of all the above reports, it is con-
ceivable that M. oleifera leaves may possess antiproliferative
properties.
3.2. Morphological changes in KB cell line
Membrane blebbing is considered to be one of the key morpho-
logical alterations of apoptotic cells (Machuy et al., 2004). Several
studies have characterized membrane blebbing as a feature to
quantitate apoptotic death of cells. The Morphological changes in
the KB cells observed by phase contrast microscope revealed
changes in the refractive index of cell, followed by a series of
changes like cytoplasmic membrane shrinkage, loss of contact with
neighboring cells, membrane blebbing and apoptotic body as pre-
sented in Fig 2a and b. Marked morphological changes could be
seen after 48h treatment with both MLE and cisplatin character-
ized by rounding of cells and some sensitive cells detached from
the surface. Fischer et al. (2003) have reported that the key mor-
phological alterations during apoptosis including chromatin con-
densation, nuclear remodeling and membrane blebbing are
120
100
% Cell viability
80
*
60
* tracts demonstrated apoptosis in KB cells as evident by their in-
40
20
0 DAPI staining (Seo et al., 2001). This report is also in absolute
0 25µg/ml 50µg/ml 100µg/ml 150µg/ml
MLE
Fig 1a. Antiproliferative effects of MLE to KB cells. Viable cell number was
measured with MTT assay. Data were expressed as mean ± SD of 3 replicates.
(⁄p < 0.001 vs control cells).
120
100
%Cell viabilty
80
60
*
*
40
*
20
0
0 2µg/ml 4µg/ml 6µg/ml 8µg/ml 10µg/ml
Cisplatin
Fig 1b. Antiproliferative effects of Cisplatin to KB cells. Viable cell number was
measured with MTT assay. Data were expressed as mean ± SD of 3 replicates.
(⁄p < 0.001 vs control cells.).
determined by interplay of caspase substrate cleavage. In tune
with these studies, the results obtained in our investigation con-
firm that KB cells when treated with MLE showed apoptotic body
formation as a morphological mark of apoptosis.
3.3. Propidium Iodide staining
Cells undergoing apoptosis become increasingly permeable to
propidium iodide (PI) which is too large a molecule to enter live
and active cells. PI is membrane impermeant that is commonly
used for identifying cells. Therefore, PI staining is taken as an index
of the extent of apoptosis in the cells (England et al., 2004). KB cells
treated with leaf extracts was highly permeable to PI, indicating the
induction of apoptosis as presented in Fig 2c and d .Morphological
analysis of KB cells with PI staining displayed nuclear shrinking,
DNA condensation and fragmentation suggesting the induction of
apoptosis by the leaf extracts. These results indicate that M. oleifera
leaves can cause apoptosis of KB tumor cells. Plant extracts and
plant derived products have been reported to induce the process
of apoptosis in several cells, which is indicative of their pharmaco-
logical efficiency (Thatte et al., 2000). This gains importance of the
MLE extracts as potentially useful drug implicating cell death by
apoptosis.
3.4. DAPI staining
The apoptotic cell death is one of the mechanisms by which cell
growth is suppressed. Both DAPI and PI staining are fluorescent nu-
clear dye that binds strongly to DNA. DAPI staining has been used
to characterize the effect of plant extract induced apoptosis in can-
cer cells. DAPI is a fluorescent stain that is used to highlight the nu-
clear changes during apoptosis and also to assess the percentage of
apoptotic cells with condensed and fragmented chromatin (Choi
et al., 1999). The extent of nuclear changes during apoptosis, as
measured by DAPI staining in the control and treated groups are
presented in Fig 2e and f. As deducible from the results, the leaf ex-
*
creased permeability to DAPI with the presence of nuclear
apoptotic bodies and chromatin condensation. Induction of apop-
tosis by an ethyl acetate extract of the stem bark of Cudrania tric-
uspidata in HL-60 human leukemia cells was demonstrated by
200µg/ml
agreement with our observations, where in DAPI staining and
morphological changes correlated to indicate cellular apoptosis.
The results showed good correlation with cell morphology study
and propidium iodide staining which confirms the efficiency of
M. oleifera leaf extracts in inducing apoptosis.
 S. Sreelatha et al. / Food and Chemical Toxicology 49 (2011) 1270–1275 1273

(a) Control (DMSO) (b) MLE treated (membrane blebbing)

(c) Control (PI staining) (d) MLE treated (apoptosis)

(e) Control (DAPI staining) (f) MLE treated (Chromatin condensation

& nuclear fragmentation).
Fig 2. Morphological features of nuclei of KB cells treated with DMSO and MLE extracts for 48 h. Cells were observed by phase contrast microscope. PI staining and DAPI
staining showing Membrane blebbing, apoptosis, chromatin condensation & nuclear fragmentation.

3.5. DNA fragmentation in KB cells Marker Control Cisplatin 200µg/ml 150µg/ml 100µg/ml

Apoptosis in several cells is clearly characterized by the system-

atic cleavage of DNA to provide nucleosomal fragments of 200 bp
(or its multiples) forming a typical ladder on agarose gel (Bortner
et al., 1995). In order to gain more insight into cell death pathways,
DNA fragmentation of KB cells was detected on agarose gel electro-
phoresis. There was a significant DNA fragmentation or laddering
pattern in the leaf extract treated group (Fig 3) than the control
group which confirms the induction of apoptosis in KB cells. Previ-
ous scientific studies have reported that DNA fragmentation is a
late event in apoptosis and large fragments of 50–300 Kb are ob-
served in early stages and only later they get further fragmented
to 200 bp fragments or its multiples (Schliephacke et al.,
2004).The obtained synergistic results of MLE extracts showed a
strong characteristic feature of apoptosis.

Fig 3. Effect of MLE and Cisplatin on DNA fragmentation of KB cells detected by

3.6. ROS production of KB cancer cell line agarose gel electrophoresis.

Intracellular ROS was measured in terms of fluorescence by

DCFH-DA. KB cells incubated with the leaf extract showed a
dose-dependent increase in the intracellular ROS production when
compared to the control (Fig 4). At higher concentration the trea-
ted cells showed a remarkable increase of ROS level which presum-
ably revealed that most cells underwent induction of early
apoptosis caused by oxidative stress. Many of the agents that
induce apoptosis are oxidants or stimulators of cellular oxidative
metabolism, while many inhibitors of apoptosis show antioxidant
activity (Buttke and Sandstrom, 1994). Mitochondria are a source
of ROS during apoptosis and reduced mitochondria membrane
potential leads to increased generation of ROS and apoptosis
(Zamzami et al., 1995). ROS have been implicated as a second
1274
400
350
% of relative amount of ROS
300
250
200
150
100
50
0
Control 25µg/ml 50µg/ml 100µg/ml 150µg/ml
Fig 4. Effect of MLE on ROS production of KB cells. The values are mean ± SD (n = 3).
(⁄p < 0.001 vs control cells).
messenger in multiple signaling pathways (Apel and Hirt, 2004)
and can also play an important role in apoptosis by regulating
the activity of certain enzymes involved in the cell death pathway
(Garcia-Ruiz et al., 1997; Kruman et al., 1997). Growth inhibition
and ROS generation by M. oleifera leaf extracts in KB cells indicate
that ROS production probably causes apoptotic cell-death via the
mitochondrial pathway.
3.7. Phytochemical analysis
Phytochemical analysis was performed in order to identify the
chemical nature of active principles possibly rendering antioxidant
protection. Qualitative analysis of the extracts revealed the pres-
ence of phenolics, flavonoids and trace amounts of alkaloids. On
estimating the levels of polyphenolic compounds in each fraction
using standard quercetin the aqueous fraction was found to have
the higher polyphenolic content. Therefore this extract was used
for the study throughout. The leaf extract examined for phenolic
contents revealed the presence of quercetin and kaempferol. The
presence of quercetin and kaempferol in the aqueous extract of
M. oleifera leaves was verified as reported by Singh et al. (2009),
and the amount of quercetin and kaempferol observed in the leaf
extract was found to be 795 and 216 lg/g, respectively.
Natural products, including plants, provide rich resources for
anticancer drug discovery (Schwartzman et al., 2002). The effect
evidenced in KB cells may, in part, be correlated with the modula-
tion of redox-sensitive mechanisms that can be involved in cancer
cell proliferation. On the other hand, it has been reported that phe-
nolic phytochemicals can, paradoxically, induce the formation of
ROS to achieve an intolerable level of high oxidative stress only
in some cancer cells (Loo, 2003). Quercetin and Gallic acid each
generated H2O2 in time and concentration dependent manners
when added to cell culture media (Long et al., 2000). In our previ-
ous study, a number of phytochemicals, including phenolics and
flavonoids were quantified from the leaf extracts of M. oleifera (Sre-
elatha and Padma, 2009). These results imply that phenolics and
flavonoids present in the fractions of M. oleifera extracts, may act
as potential chemo preventive agents with respect to inhibition
of the proliferation of human carcinoma cancer through the induc-
tion of apoptosis in vitro. Quercetin and kaempferol that were pres-
ent in appreciable quantities in M. oleifera might be responsible for
their antiproliferation by induction of apoptotic cell death and
intracellular ROS production significantly.
In conclusion, M. oleifera exhibits an antiproliferative effect by
inducing loss of cell viability, morphology change, internucleoso-
mal DNA fragmentation and ROS generation in KB cells. Our data
confirm the potential of M. oleifera as an agent of chemotherapeutic
and cytostatic activity in human carcinoma cancer. These data sug-
gest that the leaf extracts may possess anticancer properties and,
therefore, may be potentially valuable for application in drug
products.
S. Sreelatha et al. / Food and Chemical Toxicology 49 (2011) 1270–1275
Conflict of Interest
*
*
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are grateful to Dr.Anandha Kumar, Alagappa Uni-
versity, India for providing research facilities and expert guidance
for this work.
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