Brunei Darussalam Journal of Health, 2016 6(2): 75-83

Investigation of toxic effect of moringa oleifera leaf-methanol extract on breast cancer cell
mcf-7

Nazia Hossain, Mohamed Elwathig Saeed Mirghani and Raha Raus

Nazia Hossain, B.Eng of Biochemical-Biotechnology Engineering, Faculty of Biochemical-Biotechnology

Engineering, International Islamic University Malaysia, Malaysia.

Associate Professor Dr. Mohamed Elwathig Saeed Mirghani, Department of Biotechnology Engineering, Kulliyyah
(Faculty) of Engineering, International Islamic University Malaysia, 53100, Jalan Gombak, Malaysia.
International Institute for Halal Research and Training (INHART), IIUM, Gombak, Malaysia

Assistant Professor Raha Bt. Raus, Department of Biotechnology Engineering, Kulliyyah (Faculty) of Engineering,
International Islamic University Malaysia, 53100, Jalan Gombak, Malaysia.

Abstract

Moringa oleifera (M. oleifera) leaf extracted with methanol was screened for anti-breast cancer activity.
Microtitrate tetrazolium (MTT) assay was used to determine the anti-cancer activity. MTT assay was evaluated on
two types of cell lines normal healthy cell-line VERO and breast cancer cell-line MCF-7. Both cell-lines were seeded
at 2 x 105 cells per milliliter (ml) in a 100μl of media (Dulbecco Modified Eagle Medium 90% and serum 10%) per
well in 96 well plates incorporation with 4μl/ml M. oleifera leaf extract at wavelength A570nm and 37 ͦ C in CO2
incubator (with 5% CO2 and 95% air) which is the physiological temperature. The highest cancer cell inhibition rate
was 88.39% (1.77 x 105 cells per ml). Moreover, M.oleifera leaf extraction conditions were optimized in terms of
temperature and incubation time by sonication method using Design of Experiment (DoE) software with response
surface methodology. The optimum condition for leaf extraction was measured to be temperature 50⁰C and
incubation time 45 min with medium frequency and it obtained 87.13% (1.74 x 10 5 cells per ml) cancer cell growth
inhibition and 13.92% (27.8 x 103 cells per ml) normal cell inhibition by this condition.

KEYWORDS: Moringa medicinal potential, Anti-Cancer Activity, Breast Cancer Inhibition, Optimization of Moringa
leaf extract, Anti-Breast Cancer Drug Discovery, Toxic Effect of Moringa leaf against MCF-7

Corresponding Author:
Nazia Hossain, B.Eng of Biochemical-Biotechnology Engineering
Faculty of Biochemical-Biotechnology Engineering, International Islamic
University Malaysia, Malaysia.
Email: [email protected] & [email protected]

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INTRODUCTION

Breast cancer is the most leading cause of women mortality minerals, β-carotene, benzyl isothiocyanate, niazimicin,
in whole world. 7.6 million people are dying worldwide by pterygospermin, benzyl isotiocyanate, benzyl
cancer every year where 1.38 million of breast cancer cases, glucosinolate. Besides nutritional benefits, M. oleifera is
the highest death rate cause. The World Health used for the treatment of rheumatism, ascites, infection,
Organization (WHO) of the United Nations (UN) projects hiccough influenza and internal abscess (Anwar, 2007).
that without immediate action, the global number of
deaths from cancer will increase by nearly 80% by 2030, Few researches were gone through different types of
with most occurring in low and middle income country. cancer cells incorporation with ethanol extraction of
(Abeloff, 2008) & (GLOBOCAN, 2014) Moringa leaves and they showed strong cytotoxicity against
cancer cells such as hepatocarcinoma and coloractal
DNA carries genes that are the blueprints for protein
adenocarcinoma. That’s why Moringa leaves extraction
produced in the body and proteins work to suppress the
was experimented to inhibit breast cancer cells (MCF-7) in
growth. But sometimes mutation occurs in some particular
this study (Hossain & et al., 2015) (Charoensin, 2014).
genes because of radiation, Ultra-violet radiation, smoking,
Compare to ethanol extraction, methanol extraction is
alcohol or other carcinogens what can cause the abnormal
easier, less time consuming. Methanol was normally
production of proteins and the combination of these
preferred in this study as the organic solvent since it is more
abnormal proteins create cancer as a result (Blask, 1988).
polar as compared to other types of alcohol such as
Cancer cells flock and build up tumors that may compress,
ethanol, acetone etc and in many cases methanol
invade and cause destruction of normal tissues. Sometimes,
extraction produced the best extraction results. Especially
cells break away from the tumor and flow through the
to extract leaves in different conditions by sonication (cell
bloodstream or the lymph system to others organs of body
disruption by ultra-sound), only methanol showed stable
parts and settle down somewhere and stay permanently to
extraction where ethanol and acetone caused cover-
build up ‘colony tumor’ and the cells start to grow over
bursting effect with high temperature (King, 1984).
there. This new site of tumor forming is known as
‘Metastasis’ (American Society of Clinical Oncology, 2009)
To mention, differents parts of M. oleifera have been
(Shishodia, 2004).
experimented to investigate its’ effect on various types of

Plant-derived substances have recently achieved great diseases. Nevertheless, there is limited research for M.

interest owing to their versatile applications such as oleifera leaf extract on anti breast cancer cell and there is
Moringa oleifera of monogenic family, the Moringaceae. It no methanol leaf extraction on breast cancer cell has been

is a well famous plant cultivated in tropical or sub-tropical experimented. So, the goal of this research was to

areas such as Himalayan tracts of India, Pakistan, investigate anti-breast cancer activity of Moringa leaves

Bangladesh, Afganistan, Malaysia, Indonesia, Egypt, Sudan, with methanol extraction by sonication method and

Nigeria, Chad, Somalia, Tanzania and others. It is known as optimize it with different conditions run by Design of
‘drumstick tree’, ‘golden shower tree’, ‘horseradish tree’, Experiment (DoE) optimization software (Box, 2010)

‘ben oil tree’ and ‘benzoil tree’ (Musa, 2012). The leaves (Charoensin, 2014) .
contain nutrients especially essential amino acids, vitamins,

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MATERIALS AND METHODS RESULTS

Materials: Fresh Moringa oleifera leaves were collected Optimization of Process Conditions by Design of Expert
from a garden near to Terminal Putra LRT station, Gombak, (DOE)
Kuala Lumpur, Malaysia. Experimented samples both cell- To optimize the cancer cell inhibition it in terms of
lines breast cancer cell-line (MCF-7) & normal cell-line temperature and incubation time. 11 experiments were run
(VERO) and the chemicals accutase, phosphate buffer as designed using Design of Expert (DOE) software, version
solution (PBS) 10x solution, DMEM (Dulbecco Modified 6.0.8 to attain its optimal yield at 50⁰C, at an optimal
Eagle Medium) powder, 70% ethanol for sterilization, Fetal incubation time of 45 minute.
Bovine Serum (FBS), dimethyl sulphoxide (DMSO), NaOH
powder or pellet, HCl, Sodium carbonate powder, Anova Analysis
tryptophan blue dye & MTT assay were collected from IIUM
Response: Cell growth Inhibition
cell & tissue laboratory.
Analysis of Variance (ANOVA) for Response Surface
Methods: Fresh Moringa oleifera leaves were washed with Quadratic Model was used to determine whether the model
clean water, dried, ground and stored for experiment. The is significant or not. The coefficient p-value for linear,
Moringa leaf-methanol extraction process was optimized quadratic and interaction terms are shown in Table 1.
with 11 runs done by Design Expert software 6.0.8 (DOE)
The Model F-value of 22.79 implies the model is significant.
using response surface methodology. Extraction (11 runs)
There is only a 0.19% chance that a "Model F-Value" this
of the leaves was done by sonication method through an
large could occur due to noise. Values of "Prob > F" less than
ultrasonic bath at medium frequency at 40-60°C for 30-60
0.0500 indicate model terms are significant. In this case B,
minutes. 3g of each dried sample was dissolved in 30ml of
methanol. The crude extract was isolated through vacuum A2, B2 are significant model terms. Values greater than

filtration and rotary evaporation process. Then extract was 0.1000 indicate the model terms are not significant. The

transferred to a 15ml tube and 10ml DMSO was added to "Lack of Fit F-value" of 1.12 implies the Lack of Fit is not

preserve. Normal cell-line (VERO) and Breast cancer cell- significant relative to the pure error. There is a 50.42%

line (MCF-7) were cultured through stored frozen cells chance that a "Lack of Fit F-value" this large could occur due

thawing, media change, sub-culturing monolayer cells and to noise.

cell counting respectively. The cells were seeded at 2 x 105

The "Pred R-Squared" of 0.7142 is not as close to the "Adj R-
cells per ml for breast cancer cell in a 100µl of media
Squared" of 0.9159 that was very much expected. A ratio
(DMEM 90% and serum 10%) per well in 96 well plates.
greater than 4 is desirable (Box, 2010). Here, the ratio of
Both cell-lines were incorporated with 4µl/ml Moringa
13.743 indicates an adequate signal for successful optimized
leaves-methanol extract in different plates and finally MTT
design. Mean is the average value of all outcomes and here
assay was added on the incorporation and the absorbance
mean cancer cell growth inhibition percentage by Moringa
at A570 nm was recorded immediately by an ELISA plate
oleifera leaves extract is 87.13 what is very positive with the
reader. Then the data was analyzed.
project.
𝑆𝑡𝑑. 𝐷𝑒𝑣
C. V. % = X 100%
𝑀𝑒𝑎𝑛

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Table 1: Sequential Model Sum of Squares

Source Sum of df Mean Square F value P value Prob > F

Squares

Model 163.58 5 32.72 22.79 0.0019 Significant

A-Temperature 001.68 1 01.68 01.17 0.3289

B-Incubation 088.26 1 88.26 61.49 0.0005 Significant

AB 003.70 1 03.70 02.58 0.1692

A2 019.11 1 19.11 13.31 0.0148 Significant

B2 064.59 1 19.11 45.00 0.0011 Significant

Residual 007.18 5 64.59

Lack of fit 004.50 3 01.50 01.12 0.5042 Not Significant

Pure Error 002.68 2 01.34

Core Total 170.76 10

Table 2: Model characteristics

Std. Dev. 1.20 R-Squared 0.9580

Mean 87.13 Adj R-Squared 0.9159

C.V.% 1.37 Pred R-Squared 0.7142

PRESS 48.81 Adeq Precision 13.743

Table 3: VERO cell-line growth Inhibition by different conditions

Temperature Incubation Time Cell Growth

(⁰C) (min) Inhibition (%)

50 45 19.40

50 30 6.08

40 60 12.17

50 45 13.92

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In this project, C.V. % was very low 1.37 what good sign for For normal cell-line (VERO), four experiments were done
result stability. The predicted residual sum of squares (PRESS) with different conditions based on DoE table to check M.
was 48.81 that was less than 50. oleifera leaves extract affect normal cells or not. These all

Diagonostic experiments showed normal cell inhibition by average cell

In the diagonostic, Figure 1 (predicted vs. actual response) inhibition 12.89% and in optimum extraction condition at

showed that the distribution of points are closed to linear temperature 50⁰C and incubation time 45 min with

line (R2 = 0.9580) and Figure 2 shows the 3 dimensional view medium frequency, cell growth inhibition is 13.92% (27.8 x

of the whole optimization process and optimized point as 103 cells per ml).

well.
Experiment Result There were 11 runs of experiments done on MCF-7 by

The results were taken using ELISA microplate reader at Moringa leaves extract with different conditions based on

570nm. In order to validate the result obtained from time and temperature in terms of DOE table and analyzed

triplicate for each run. Thus using the same concentration the results. It is shown in Table 4..

range, three 96 well plates were done for each run of each
cell-line. Average was taken to obtain the final value and cell Table 4 has shown high cancer cell growth inhibition by the

viability and cell growth inhibition percentage for both types extract. Average range of growth inhibition has been 80% -

of cells were calculated by the equations: 90% (mean value 87.13%) with various conditions and in
𝑂𝐷𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 optimum extraction condition at temperature 50⁰C and
Growth = X 100%
𝑂𝐷𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠 incubation time 45 min with medium frequency, cell growth
inhibition is 88.39% (1.77 x 105 cells per ml).
𝑂𝐷𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠
Growth inhibition = 100% − X 100%
𝑂𝐷𝑢𝑛𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑐𝑒𝑙𝑙𝑠

OD untreated cells = Media with cells without extract

OD treated cells = Media with cells with extract

Figure 1: Predicted vs. Actual values

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Figure 2: 3D view plot for desirability

Table 4: Result Analysis by DOE

Std Run Factor 1 Factor 2 Response 1

A : Temperature (°C) B : Incubation Time Cell Growth Inhibition
(min) (%)

3 1 50 60 78.4659
4 2 60 60 80.8468
2 3 60 30 89.7957
1 4 40 30 89.7979
6 5 60 45 91.0828
9 6 50 45 87.3738
5 7 40 45 90.4039
10 8 50 45 89.6298
7 9 50 30 87.4286
3 10 40 60 84.6971
11 11 50 45 88.9549

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DISCUSSION methanol causes toxicity that may cause metabolic acidosis,

Optimization is a process to find an alternative with the most neurologic squealed and even death if ingested (Mukherjee,
cost-effective or highest achievable performance under the 2001). To avoid this impurity, DMSO is recommended to use
given constraints by maximizing desired factors and as solvent to re-dissolve the extract instead of methanol
minimizing undesired ones (Hossain & et al., 2015) (Porter & though using DMSO is expensive. For further research, dose
et al., 1997). To optimize this extraction process, DoE responsive experiment should be recommended.
statistical analyses was used in this study where average PRESS
was 48.81. Average PRESS value is the determination factor The MTT assay used to evaluate the cytotoxicity of
whether the model is over-fitted or under-fitted and PRESS methanolic leaf extract of Moringa oleifera on MCF-7 and
value less than 50 is desired for optimized design (Box, 2010). VERO cell lines showed that the extract is significantly
So, in this study optimized design was good overall. Besides, cytotoxic to MCF-7 cell line and considerably less so towards
predicted vs. actual response showed that the distribution of normal cell-line VERO. These findings highlights the
2
points are closed to linear line (R = 0.9580). As difference potentials of Moringa oleifera leaf extract in the treatment of
between the actual and predicted value is small, the result is breast cancer. Its therapeutic potential is huge and can be
counted better (Box, 2010). In this design, difference for cell used as alternative to or supplementation for the various
growth inhibition is very small in all runs that indicated the therapies. However there is a need to identify the actual
model is very good. These results proved that Response components responsible for this cytotoxicity and to isolate
Surface Methodology statistical analysis fitted this experiment them and to study their effect in vivo to ascertain their
for optimizing the extraction condition. efficacies and or any side effects. The concerns of side effects
with pharmacological agents, and growing interest in plant
Moringa oleifera extract work on different cancer cells in other bio-resources for treatment of cancers mean this extract
research (Charoensin, 2014). In this research, breast cancer could have important role in future studies on the
5
cell (MCF-7) growth inhibition was 87.13% (1.74 x 10 cells per management of breast cancer.
ml) in average that proved Moringa leaves extract was able to
inhibit breast cancer cell with very high percentage in all kinds CONCLUSION
of condition parameters. When mixture was diluted with
The Moringa leaf-methanol extract showed extremely high
media the growth inhibition was also reduced, which
anti-breast cancer activity that indicates the medicinal value
evidenced that Moringa leaves extract was very strong against
of M. oleifera in terms of cancer preventive drugs and
MCF-7 cell growth.
chemotherapy. The Moringa leaves extraction optimum
condition was found to be temperature 50⁰C and incubation
On the other hand, in this research, experiments on normal
time 45 min with medium frequency at sonication. It is
cell-lines (VERO) showed that Moringa extract inhibited VERO
strongly suggested that it could potentially be an ideal
cell-line with small percentage too. But based on previous
anticancer therapeutic candidate specific to cancer cells.
researches, it was proven that Moringa oleifera is good for
These results also suggested that extraction strategy adopted
health nutrition and its leaves extract usually does not kill
in this work could lead to several industrial applications and
normal cell-line (Anwar, 2007). In this case, it was suspected
the potential therapeutic implications of the soluble extract
that the presence of methanol with leaves extract might affect
from M. oleifera leaves extract in the treatment of various
this result by inhibiting normal cell growth. Because methanol
types of cancers.
is volatile alcohol similar with drinking alcohol and sometimes

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ACKNOWLEDGEMENT

This work supported by the Cell and Tissue Laboratory at Biochemical-Biotechnology Engineering Department
(BTE), Faculty of Engineering, International Islamic University Malaysia (IIUM

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Supplementary Data

Figure 1: Contour view plot for desirability

Figure 2: 3D view plot for desirability

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