EFFECT OF CITRUS LIMON JUICE AND TAMOXIFEN ON THE OXIDATIVE STRESS ACTIVITIES OF MCF-7 CELL INDUCED BREAST CANCER IN SPRAWGUE DAWLEY RATS.JOSEPH OYEPATA SIMEON

www.wjpps.com Vol 8, Issue 7, 2019. 76
Samson et al. World Journal of Pharmacy and Pharmaceutical Sciences
EFFECT OF CITRUS LIMON JUICE AND TAMOXIFEN ON THE
OXIDATIVE STRESS ACTIVITIES OF MCF-7 CELL INDUCED
BREAST CANCER IN SPRAWGUE DAWLEY RATS
Samson Adewale Oyebadejo*1
, Oyepata Simeon Joseph2
, Samson Oluwaseyi Adesite3
and
Emem Rapheal Abia4
1
Department of Biotechnology, Faculty of Biosciences, Shri Jagdish Prasad Jhabarmal
Tibrewala University, Rajasthan 333001.
2
Department of Pharmacology, Faculty of Pharmaceutical sciences, Bingham University,
Abuja, Nigeria.
3
Department of Histopathology, University of Uyo Teaching Hospital, Akwa Ibom, Nigeria.
4
Department of Physiology, Faculty of Basic Medical science, University of Uyo, Uyo,
Akwa Ibom, Nigeria.
ABSTRACT
In search of possible, safe and toxic anticancer drug for the treatment
of breast cancer, molecular pathological study of the activities of
Citrus limon juice on MCF-7 cell line induced breast cancer in Sprague
Dawley rats compared to Tamoxifen, Breast cancer reference drug was
carried out, over one hundred and twenty Sprague dawley rats of 40
days old, average body weight 180-220g were divided into ten (10)
containing of 12 animals per group, group 1 was control, fed only with
rat chow and water, group 2 was MCF-7 cell line induced rat alone
(BCIR only), group 3 Citrus limonj uice (CLJ) at 8.88%, Group 4
Citrus limon juice(CLJ) at 17.32%, group 5 Citrus limon juice(CLJ) at
25.98%, group 6 was given 0.2mg/kg of Tamoxifen alone, group 7
(BCIR+CLJ at 8.88%), group 8 (BCIR+CLJ at 17.32%), group 9
(BCIR+CLJ at 25.98%) and group (BCIR+ 0.2mg/kg of Tamoxifen).
Acute and sub – acute toxicity were carried out after the establishment of safety dose following
determination of LD50, at the end of the administration of the Citrus limon juice and Tamoxifen,
animal were sacrificed, fresh blood were taken into plain bottles without anticoagulants, serum was
collected and stored at 8o
c for Biochemical investigation of Oxidative stress activities; TAC, LDH, SOD
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 7.421
Volume 8, Issue 7, 76-92 Research Article ISSN 2278 – 4357
Article Received on
26 April 2019,
Revised on 16 May 2019,
Accepted on 06 June 2019
DOI: 10.20959/wjpps20197-14087
*Corresponding Author
Dr. Samson Adewale
Oyebadejo
Department of
Biotechnology, Faculty of
Biosciences, Shri Jagdish
Prasad Jhabarmal Tibrewala
University, Rajasthan
333001.

and LPD (MDA) were carried out, Result, Oxidative stress activities showed significant increase in
TAC, LDH, SOD and LPD (MDA) at P>0.001 in BCIR when compared to the BCIR+CLJ, BCIR+Tamoxifen
and non-BCIR groups. In conclusion, this study showed that Citrus limon juice posses strong
anitioxidative capacities with potential anticancer activities and does not pose pathological
conditions on the body during and after usage.
KEYWORDS: MCF-7 Cell lines, Breast cancer, Citrus limon Juice, Tamoxifen, Molecular
Pathology, Sprawgue Dawley Rats.
1.1 INTRODUCTION
It has been known that over 12.5% of death occur as a result of Breast cancer in Nigeria
given more death estimation than Human Immunodeficiency virus and Acquired Immuno
deficiency syndrome, Breast cancer in Nigeria are much younger than Caucasian females
(less than 35 years old), it is usually diagnosed at a more advanced stage due to the gender
factor and culture of silence secrecy. The Stigma - disclosure would jeopardize social
standing and marriage for the family members, the fear of mastectomy which leads in the
disfigurement, physical disability and the need to keep the body intact insufficient access to
quality health care providers such as physicians, screening scientists due to economical issues
and most of all, the lack of awareness on the importance of screening and usage of anticancer
drugs (American cancer research, 2007).
Breast cncer usually claim life of victim, one out of every ten will die of in live time, it has
been the common malignancy resulting into high mortality rate when compared to other form
of cancer affecting women. It is also found in men butnot verycommon.
Breast cancer are usually form from cancer cells or normal cells of the body that grow out of
control within the body as a result of direct or indirect insult on the cell, there cells may
eventually form tumor cells based on the lost of the normal function within the precursor
lining in the tissue surrounding cells normal this tumor cells found in the breast may travel to
other part of the body to become cancer cells taking their derivative from the tissues organ of
their new location. (Blackwell et al., 2000).
Breast cancer that spread from the lymph node may usually give rise to metastasis but not all
the metastasis are originated by the spread of cancer cells from the lymph nodes.

Modern anti-cancer therapies revealed positive response and survival, but the side or adverse
effects and high cost frequently leads to discontinuation. Therefore, there is an imperative
need of highly effective compounds with tolerable adverse effects that are affordable and at
the patient’s disposal (Decensi et al., 2005).
Oxidative stress and damages resulted from possible assault from the biological or chemical
or physical agent inflicting injuries on the cells or tissues or organs in the body, they are
characterized as the measure of free radical reduced in the body when these assault occiured
on the individual cells, however the disturbances created in cell normal redox states could
lead to toxic effect by producing peroxides and free radicals in the body capable of damaging
the cell componenents wich could comprise of lipid, protein, carbonhydrates and DNA as a
result of of oxidative stress that damages and break the DNA strand given rise to oxidative
stress (Sies, H, 2015).
Despite the usage of Tamoxifen for the treatment of Breast cancer, it has been implicated in
the many conditions that have practically made it not suitable as reliable theraupeutic form of
drugs (Bachelor et al., 2012).
MCF-7 is a form of cell lines isolated from the breast of a 69 years old Caucasian woman in
the year 1970, the woman was operated due to symptoms of cancer in the two breasts and she
was later treated with radiotherapy and hormonotheraphy. In the first year of subjection to
Breast surgery, it was diagnosed to be Benign tumor and after five years the second breast
undergo metastasis which resulted into adenocarcinoma where the pleural effusion sucked
from the site of metastasis yielded MCF-7 cell lines which was harvested and culture in a
suitable medium.
MCF-7 cell line make use of estrogen in the production of estradiol and to process through
estrogen receptors in the cell cytoplasm and estrogen receptor ER positive control cell lines,
it has been useful for in-vitro studies of breast cancer with many characteristics particular to
the mammary epithelium (www.altogen.com).
Citrus limon is very rich in chemicals such as tarpene (D-Limonene) with characteristic smell
and taste of limon; they are rich in citric acid with low pH characterized with sour taste, rich
in ascorbic acid which has made lemon to be consider as a good source of daily vitamin C
intake. It is believed and experimented that consumption of Citrus limon on daily basis has

demonstrated to be more potent in efficacy than 500mg of vitamin C administered to the
body, thus made Citrus limon to posses great health benefit to body.
Citrus limon fruit as juice to posses antioxidant and anti-cancer properties, this may be due to
its fruit constituent such as essential oil or d-limonene, citric acid which could be nonamal,
decamal, dodecamal, yarcuyl, linanyl, citronelyl flavonoids, neohesperidine, rutin, erioatin
anthronil acid, limonins and methyl ester (Calomme et al., 1996).
Due to ongoing progress of the chemotherapeutic agents in cancer management, it still
remains to be 5 chemopreventive drugs for breast cancer such as Tamoxifen. Yet, there is
limited finding or no scientific backing on the curative effect. However, the use of the juice
has been attributed to posse’s giardiacidal activity, antioxidant activity, pediculicidal
activities, and antibacterial activities. Despite these activities there is limited or no scientific
based evidence on oxidative capacities and anticancer activaties of the juice in-vivo wise
using animal model and the need for toxicity study using animal induced model is also
encouraged in this study.
MATERIALS AND METHOD
Chemicals, Reagents and Equipments
All the chemicals, reagents, and equipments used in this study are of international standard
organization grade stardandardised by ISO and analytical grade without any form of
impurities.
Citrus Limon fruit Collection
The Citrus limon fruit samples of the same species and varieties were collected from a the
local farm in Uyo, Akwa Ibom, Nigeria within the month of October and December 2016 in
sterilized conditions from the same set of trees in sterilized polythene bags, stored at 4oC in a
refrigerator until use, they were authenticated by Botanist at the Agricultural Biotechnology
unit, Derindam Research Institute of Biotechnology.
Acute Toxicity Study of Citruslimon (L) juice
Acute toxicity study of Citrus limon (L) juice was carried out based on Lorke’s method
(Lorke, 1983), LD50 values of the Citrus Limon (L) at 10%, 20% and 30% were 8.88%,
17.32% and 25.98% and they were considered and used as Low, Middle and High dose
respectively.

Drugs
Tamoxifen citrate tablets (Cipla Ltd., Goa 403 722. India).
Animals and Management
Experimental rats used were approved by Animal Care and Use Committee (IAUC) of
Derindam Research Institute of Biotechnology, Nigeria based on the rules guiding the use of
laboratory.120 virgin-female Sprague-Dawley (SD) rats (40 days old) with weight of 180-
220g were obtained from the Animal house of the Institute, DRIB. The animals were divided
into Ten (10) groups of 12 rats per group. Animals are housed two rats per plastic cages and
allowed to acclimatize in standard conditions (under a 12 hours light and dark reaction, free
access to distilled water and commercialized food throughout the experiment.
Preparation Breast cancer (MCF-7) Cell lines
MCF-7 (Breast cancer) Cell lines was obtained from NCCS, Pune. Cells were cultured in
Duilecco’s modified Eagle’s medium, 10% Fetal Bovine Serum complete medium
supplemented with antibiotics, cells were maintained at 37°C in a 5% CO2 incubator and the
media were changed regularly throught the experiment. 90% of the cells wereconfluent, the
medium was removed and the cells washed with Phosphate buffer solution, dead cells were
removed by the addition of EDTA to detach the sticked cells. Cells obtained immediately
were centrifuged at 1000 rpm for 10 minutes at 4o C, the cells were washed twice with
Phosphate Buffer Solution and dispersed
Extractin of Citrus Limon (L) Juice
Fruits were washed with Distilled water, the juice were extracted manually, by cutting the
fruits in halves and carefully squeezing to extract the juice and sieved using cloth of muslin
of 4 fold into the beakers for the admistration during the exerimal procedures.
Breast Cancer / Mammary Tumor Induction
Experimental animals were anesthesized using 150mg/kg ketamine and 10mg/kg of xylazine
mixture by injection via intraperitoneal respectively. The injection site was properly cleaned
and sterilized with ethanol. The cell suspension, 600000 cells in 300µl PBS was drawn into
1c or 1ml TB syringes without needles to minimize damage, lysis and death to the cells. The
cell suspension was inoculated subcutaneously into the mammary fat pad (right flank) of the
Sprauge- Dawley (SD) rats using a TB syringe with #26-gauge needle, cell suspension of
300ul was injected by positioning the needle at 2mm posterioly and 2.5 mm laterally, inserted
through the skin and then lowered 5 mm into the mammary fat pad. The beds of rats were

supported with suitable heat lamp to avoid loss of body heat during the procedure. The
temperature, breathing and heart rated of animals were monitored closely. The rat were were
swang backward and over continouesly for 30 seconds to generate warnt as this facilitate
theier breathing rate and they became stabilized shortly after this procedure.
Experimental Design for In - vivo Anticancer Study
One hundred and Twenty Sprague –Dawley rats,40 days old, average body weight 180 -220g
were dividedinto ten (10) groups ( labeled as group 1-10) containing Ten (12) animals per
group. Cancer was induced using MCF-7 cell lines (Roghayehet al., 2010) in groups
2,7,8,9,10,11 and 12, after twenty one days of development of Breast cancer, animals were
treated with various concentrations of Citrus limon juice and Tamoxifen respectively for
Twelve (12) weeks as indicated below:
 Group 1: Control animal fed with feed and water only.
 Group 2: MCF-7 Cell line Induced Breast cancer rats only (BCIR)
 Group 3: Citrus limon Juice (CLJ), 8.88% (Low dose)
 Group 4: Citrus limon Juice (CLJ), 17.32% (Middle dose)
 Group 5: Citrus limon Juice (CLJ), 25.98% (High dose)
 Group 6: Tamoxifen 20mg/kg only
 Group 7: MCF-7 Cell line Induced Breast cancer rats + Citrus limonJuice, 8.88% (Low
dose)
 Group 8: MCF-7 Cell line Induced Breast cancer rats + Citrus limonJuice, 17.32%
(Middle dose)
 Group 9: MCF-7 Cell line Induced Breast cancer rats + Citrus limon Juice, 25.98%
(High dose)
 Group 10: MCF-7 Cell line Induced Breast cancer rats + Tamoxifen 0.2mg/kg
3.5.4 Preparation of Tamoxifen doses for Administration
Tamoxifen was prepared using the formulae stated below:
Rat weight (kg) X Dosage (mg/kg)
Tamoxifen Administered (ml) = --------------------------------------------------
Concentration of Tamoxifen (mg/ml)
3.5.5 Tumor Study

Following the Breast cancer induction, all the animals were monitored on daily baisis for any
form of tumour growth and development, areas found to be of abnormal groth in Tumour
mass were measured using the formulae of Carisson: V = (ab2
)/2 to calculate the following
following the measuremt of Length and Breast and witdth of the area of the tumour, where
‘a’ and ‘b’dennotes Length and Breath tumour distance covered or measured by use of the
caliper.
3.5.6 X-ray Imaging
Experimental animals were shved toward the area of mammallry pad and aneasthesised
following the induction of tumour,the of Xray machine a 44 kilvolts for 3 miiliseconds
made in Taiwan the advanced radiographs was used to observe the photographs to the
prensence tumour formed and the location of the Tumour as a confirmatory test the precence
of Mammary tumour induced.
Gross – Morphological Analysis
Feed and water consumption and body weight measurement
The amount of feed and water consumed was measure daily from the quality of feed and
water supplied, the amount remaining and individual animal body weight were recorded daily
in all the groups till the end of the experiment.
Organ weight
The various organs were excised from the animal and weighed. The weights of the organs
such as liver, kidney, lungs, uterus and ovaries were recorded and studied for any abnormal
gain or loss of weight. The weights of the organs expressed as relative weights or
organosomatic index are g/100g per body weight were calculated by following formula:
Absolute organs weight (g)
Relative organ weight = --------------------------------------------------------- x 100
Body weight of Animal on sacrificed day (g)
The initial and final weight of the animal and weight of organs in each group were taken
using theweighing balance. The values of all the morphometricdata were analyzed, computed
and compared statistically using Graphpad prism 6 version 2, all data were expressed as
Mean ± SEM. One way analysis of variance (ANOVA) was used to test for difference among

the groups and t-test was used to compare the significant differences between the means.
Values were considered significant at P<0.001.
Preparation of Cell lines
MCF-7 Breast cancer cell lines were cultured in DMEM 10% FBS complete medium. 10%
heated inactivated fetal bovine serum was added, antibiotics, and the cells were maintained
in 5% CO2 incubator at 37 degree celcius and the media were changed regularly through out
the experiment.
Sterility Test of Breast cancer (MCF-7) Cell lines
Sterility test was carried out from the onset to verify the Citrus limon juice capacity of
contamination free, 35 mm culture dish was plated with MCF-7 cell suspension in 2ml of
DMEM media, cells were allowed to adhere, the Citrus limon juice were added into the
culture dishes and incubated in 5% CO2 Incubator for 24 hours.
Biochemical Analysis of Oxidative Stress Activities
Blood samples were collected from the healthy and tumor-bearing rats by cardiac puncture
using #23 gauge needles before and after development of the tumor. After collection, the
samples were allowed to clot at room temperature and centrifuged at 300 g for 10min. Serum
were for the analysis stipulated below.
Determination of Total Antioxidant Activity
According to the method of WinnikS et al., 2011, solubilize Trolox standards were used to
prepare Trolox standard curve (4-20 nmol/well), samples were prepared in optimal dilution to
fit the standard curve readings. Cu2+
working solution was prepared by dilution of 1:50 in
Assay diluents provided dilute Microplate well were set in duplicate for standard at 100ul and
samples at 100ul, Cu2+
solutions were added to all the standard and samples with protein
mask, mixed, incubated at room temperature for 90 minutes and absorbance was read at
570nm using the formulae below:
Sample TAC = (Ts/Sv) D
Where Ts= TAC amount in the samplewell calculated from standard curve (nmol)
Sv = Sample volume added onto the wells (ul)
D= sample dilution factor

Determination of Lactose dehydrogenase (LDH)
Based on Stentz Ret al., 2010, Enzyme linked Immuno-absorbent Assay method was used it
involeved the use of the microplate pre- coated with antibody specific to LDH, Standard
,samples were added to the appropriate microplate wells and combined with the specific
antibody, then Horadish-Peroxidase conjugated antibody specific to LDH was added to each
microplate well and incubated for 1 hour, free component were washed away, TMB substrate
solutions were added to each well in microplate, those well containing LDH and HRP
conjugated antibody appeared Blue in colour and later turned yellow after the addition of stop
solution, the absorbance were measured with microplate reader at wavelength of 450nm, the
absorbance measured was directly proportional to the concentration of LDH in the serum
samples and it was calculated by comparing the concentration of the LDH to the standard
curve obtained from the serial dilution of the standard concentration.
Determination of Superoxide dismutase (SOD)
Applying Stentz Ret al., 2010, Enzyme linked Immuno-absorbent Assay method which
utilized the microplate pre- coated with antibody specific to SOD, Standard ,samples were
added to the appropriate microplate wells and combined with the specific antibody, then
Horadish-Peroxidase conjugated antibody specific to SOD was added to each microplate well
and incubated for 1 hour, free component were washed away, TMB substrate solutions were
added to each well in microplate , those well containing SOD and HRP conjugated antibody
appeared Blue in colour and later turned yellow after the addition of stop solution, the
absorbance were measured with microplate reader at wavelength of 450nm, the absorbance
measured was directly proportional to the concentration of SOD in the serum samples and it
was calculated by comparing the concentration of the SOD to the standard curve obtained
from the serial dilution of the standard concentration.
Determination of Lipid Peroxidation
Buege and Aust, 1978 utilised Lipid peroxidation acted as indicator for tissue injury which
are induced by reactive oxygen species was measured as Thiobarbituric acid reactive
substances (TBARS), 0.5ml of samples were reacted with 2mls TBA reagents containing
0.375% of TBA and 15% of Trichloroacetic acid and 0.25NHCL,samples were boiled for 15
minutes ,cooled and centrifuged at 10000 revolution per miunute for 10 minutes, Absorbance
of the supernatants were spectrophotometrically measured at 532 nm wavelength, the TBARS

concentration was calculated using 1.3 tetra-ethoxypropane as standard and the result were
expressed as micromol/ml.
RESULT
Effect of Citrus limon juice and Tamoxifen on the oxidative stress activities of MCF-7
cell induced breast cancer in Sprawgue Dawley rats
Superoxide Dismutase (SOD) concentration was significantly increase at p<0.0001 in BCIR
group compared to all the groups, but was significaly reduced when compared to the
BCIR+CLJ at 8.88%, BCIR+CLJ at 17.32%, BCIR+CLJ at 25.98% and BCIR+0.2mg/kg of
Tamoxifen group. Lactase Dehydrogenase (LDH) level was not significant where CLJ and
Tamoxifen alone were compared with control but revealed high level of significant when
BCIR group alone was compared with control, CLJ and Tamoxifen alone and BCIR+CLJ and
BCIR+Tamoxifen treated group at p>005, Similarly when compared CLJ and Tamoxifen
alone groups toBCIR+CLJ and BCIR+Tamoxifen there was significant different at p>0.005.
Lipid peroxidation concentration (LPD) in BCIR group was increase when compared to
control group but not significant when compared CLJand Tamoxifen alone group meanwhile
it was shown further that the BCIR alone was different from BCIR+CLJ and
BCIR+Tamoxifen treated group at p>0.005 while CLJand Tamoxifen groups alone were
extremely significant from BCIR+CLJ and BCIR+Tamoxifen group. (Table 1.0) Total
antibody capacity (TAC) revealed that BCIR was extremely significantly reduced at p>0.005
when compared to control group and was very significant asCLJ at 8.88% was not significant
when compared to control group but very significant when compared to the rest of the group
at p>0.002. (Table 1.0) Meanwhile the BCIR+CL5 and BCIR+Tamoxifen were not very
significant when compared to BCIR only and non-significant when compared to other groups
(Table 2.0).
Table 1.0: Effect of Citrus limon (L) juice and Tamoxifen on the oxidative stress
activities of MCF-7 cell induced breast cancer in Sprawgue Dawley Rats.
Groups SOD (mg/dl) LDH (mg/dl) LPD (mg/dl) TAC (mg/dl)
Control (Distilled water) 1.3960.048 0.6100.018 0.3370.010 19.31.147
BCIR (MCF-7) only 7.3310.165a
1.1460.067 b
0.7420.013 a
0.49201.887b
Citrus limon (8.88%) 0.9050.186ns
0.5530.021 ns
0.3280.004 ns
49.984.728 a
Citrus limon (17.32%) 0.7730.045b
0.5330.029 n s
0.2880.015 ns
57.682.673 a
Citrus limon (25.9%) 0.6070.034a
0.5720.022 ns
0.2740.017 ns
65.282.086 a

0.20mg/kg Tamoxifen 0.9170.035c
0.5630.046 ns
0.2260.011 c
32.482.495 b
BCIR + C. limon (8.88%) 5.8240.184a
1.0040.036 b
0.6360.017 b
35.980.838 b
BCIR + C. limon (17.32%) 4.4920.484c
0.8370.032 c
0.5040.047 ns
37.360.607 b
BCIR + C. limon (25.98%) 3.3840.253c
0.7540.038 ns
0.4620.032 ns
49.761.613 a
BCIR + 0.20mg/kg Tamoxifen 3.0310.413ns
0.7820.024 ns
0.4640.057 ns
21.2260.797 c
Values are Mean ± SEM of 5 rats in a group.
a
BCIR Significantly different compared to control group (p<0.0001), ns
Significantly
different compared to BCIR + CLJ and
BCIR + Tamoxifen treated groups (p<0.05).
Superoxide dismutase (SOD), Lipd Peroxidation (LPD), Lactase Ddehydrogenase
(LDH) and Total Antioxidant Capacity (TAC).
T r e a t m e n t
SOD(mg/dl)
G 1 G 2 G 3 G 4 G 5 G 6 G 7 G 8 G 9 G 1 0
0
2
4
6
8
1 0
C o n tro l (D is tille d W a te r)
B C IR (M C F -7 ) o n ly
C itru s lim o n (8 .8 8 % )
C itru s lim o n (1 7 .3 2 % )
C itru s lim o n (2 5 .9 8 % )
0 .2 0 m g /k g T a m o x ife n
B C IR + C .lim o n (8 .8 8 % )
B C IR + C .lim o n (1 7 .3 2 % )
B C IR + C .lim o n (2 5 .9 8 % )
B C IR + 0 .2 0 m g /k g T a m o x ife n
Graph 20: Superoxide Dismutase.

T r e a tm e n t
LDH(mg/dl)
G 1 G 2 G 3 G 4 G 5 G 6 G 7 G 8 G 9 G 1 0
0 .0
0 .5
1 .0
1 .5
C itru s lim o n (8 .8 8 % )
C itru s lim o n (1 7 .3 2 % )
C itru s lim o n (2 5 .9 8 % )
B C IR + C .lim o n (8 .8 8 % )
B C IR + C .lim o n (1 7 .3 2 % )
B C IR + C .lim o n (2 5 .9 8 % )
B C IR + C .lim o n (2 5 .9 8 % )+ 0 .2 8 m g /k g
T a m o n x ife n
Graph 21: Lactase Dehydrogenase.
T re a tm e n t
LPD(mg/dl)
G 1 G 2 G 3 G 4 G 5 G 6 G 7 G 8 G 9 G 10
0.0
0.2
0.4
0.6
0.8
1.0
C o n tro l (D istille d W a te r)
C itru s lim o n (8 .8 8 % )
C itru s lim o n (1 7 .3 2 % )
C itru s lim o n (2 5 .9 8 % )
0 .2 8 m g /kg T a m o xife n
B C IR + C .lim o n (8 .8 8 % )
B C IR + C .lim o n (1 7 .3 2 % )
B C IR + C .lim o n (2 5 .9 8 % )
B C IR + C .lim o n (2 5 .9 8 % )+ 0 .2 8 m g /k g
T am o nxife n
Graph 22: Lipid Peroxidation.

T re a tm e n t
TAC(mg/dl)
G 1 G 2 G 3 G 4 G 5 G 6 G 7 G 8 G 9 G 10
0
20
40
60
80
C itru s lim o n (8 .8 8 % )
C itru s lim o n (1 7 .3 2 % )
C itru s lim o n (2 5 .9 8 % )
B C IR + C .lim o n (8 .8 8 % )
B C IR + C .lim o n (1 7 .3 2 % )
B C IR + C .lim o n (2 5 .9 8 % )
B C IR + 0 .2 0 m g /k g T a m o xife n
Graph 23: Total Antioxidant Capacity.
DISCUSSION
According to Hsia and Liu, 2010, variety of conventional therapies for cancer based on
chemotherapy, radiotherapy and surgery are limited in efficacy. Most current cancer
chemotherapy regimens are normally associated with very high significant levels of toxicity
and drug resistance.
In developing countries like Nigeria, India and many African and Asian countries cancer
showed more occurrence than the infectious diseases such as HIV/AIDS, tuberculosis and
malaria deaths when combined together and by year 2020, more than 35% causes
(10.5million) of cancer will emerge according to WHO, 2007 since death rate is 16 out of
way 1000 death globally, finding possible cure has been an embattle problem over the years
which is the major headache for health care provider and the patients as well.
Manufacturing of drugs employed in the treatment of breast cancer has not been easy as it
involve huge capital intensive, man-power and exploration of ideas from scientist, medical
expert across the globe.
Finding easily available alternative for the treatment, cure, prevention and management will
surely pave ways for targeted different types of cancer and making the treatment easy thereby
reducing the mortality rate of cancer in our society. Major challenges to reduce breast cancer

burden is to develop highly effective alternative drugs specifically for breast cancer with
minimal side effects or no side effect on the patients.
Superoxide dismutase is a major oxidative enzyme that plays a vital role in the oxidative
stress encountered in breast cancer. Breast cancer or the presence of tumour impaired the
DNA, the enzymes that regulate DNA activities were inhited, catalysed the reductases
reaction by oxidixation, the reactive oxygen species are said to be responsible for the
detoxification, they are released via mitochondria when the cells or tissues falls assault of
damages from its surrounding.
According to Kumari et al., 2009, SOD is an effective method of denaturing oxidative stress.
SOD being a famous enzyme that catalyses the dismutation of reactive oxygen species by
reducing the O2 to H2O which is a less reactive oxygen species, SODs are produced by a by-
product of metabolism of oxygen and they are regulated and when not regulated, they can
damage the cells, they are very important part of antioxidant defence in living body, increase
in the activities of SODs is suggestive of complex oxidative stress with the system, breast
cancer induced rat group showed high level of SODs activities due to the deletion effect of
the MCF-7 cell lines induced into the animal to to present breast cancer, while in the control
group, Ciitrus limon alone and Tamoxifen, there was no or less activity of SOD, while the
BCIR+Citrus limonjuice at 8.88%, 17.32% and 25.98% the activites of the SODs became
reduced gradually as the concentration of CLJ increases and this is similar to the Tamoxifen
post-treated group as reference drugs. It is now discovered that the more the deleterious
activities occurs within the cells of the cells, there is need for more activities of the SODs to
catalyse the dismutation of the oxidative stress activity thereby metabolizing and breaking
down into O2, provide and other forms in which cells can use for the activities (Kumari et al.,
2006).
Lactase dehydrogenase has known to be an up regulated gene marker that is used to
determine the prognosis of breast cancer, it is an enzyme used for anaerobic glycolysis
containing isoenzymes that associate with breast cancer. Increased in the LDH is a suggestion
of increase in the enzymatic activities of factors predisposed to breast cancer. Breast cancer
induced rat groups has shown to have high concentration compared to the control and citrus
limon juice different concentration alone and Tamoxifen alone groups while in the BCIR
post-treated with citrus limon juice at 8.88%, 17.32% and 25.98% there were drop in the

LDH concentration indicating responsiveness of the BCIR group to applied treatment as a
result of regulation function of the LDH.
Lipid peroxidation is known to be breaking down of lipids as a result of loss of electrons
stimulated by force reduced, which leads to the damage of cell membrane, in breast cancer
studies the use of lipid peroxidation helps in determining the amount and rate of damage
occurring within the normal cells as a result of assault occurring in them by carcerogenic
agents, breast cancer induced rat group showed high level of cell membrane damages leading
to the lost of the cellular components of the normal cells while in other groups administered
only with Citrus limon juice concentration and Tamoxifen groups alone did not showed any
cell membrane damages or lost of cytoplasmic consist but in the group post-treated with
Citrus limon juice at various concentration after the breast cancer induction rats, showed
gradual and responsive recovery effect of the lipid peroxidation in concentration which was
reversed within normal range and was similar to the 0.2mg/kg Tamoxifen post-treated after
breast cancer induced group alone were post-treated (Buege and Aust, 1978).
Total antibody capacity is the analytic mechanism that has been designed or used to
determine or evaluate antioxidant response of a sample towards frees-radical produced in
disease conditions, any biological sample oxidative strength to get rid of free radical in a
disease condition is the total antioxidant capacity the sample exhibits, using Citrus limon and
Tamoxifen respectively at different condition revealed strong total antioxidant capacities
compared to the breast cancer induced group, though the antioxidant capacities is dependent
of the concentration aid the post-treated groups of Citrus limon juice and have high TAC than
the BCIR post-treated group of Tamoxifen while that of BCIR group only shown very little
or none antioxidant capacity, the Citrus limon juice at different concentration of TAC when
compared to the BCIR+ Citrus limon juice post-treated group, this showed that in disease, in
the breast cancer induced in the rats, Citrus limon was able to produce antioxidant response
against the free radical installed by the MCF-7 breast cancer cell line induced while the
0.2mg/kg, Tamoxifen group produce very less or minimal antioxidant capacity when
compared to both the Citrus limon juice alone at different concentration or control group but
higher than that of breast cancer induced group alone.
CONCLUSION
From this study, it was discovered that in search for safe and effective alternative measures
for the treatment of breast cancer, the use of plant such as Citrus limon juice obtained from

the fruit of the Citrus limon revealed strong antioxidant capacities and good inhibitory effect
on free radicals thereby reversing the possible oxidative damages and oxidatives stressed
imposed on the cells thereby increasing the protective mechanism of the cells administered
with the juice. As a result of this finding, it is revealed and concluded that high potency and
strong inhibition of Citrus limon juice enhanced and ameliorates oxidative stress effect
induced on the animals by MCF- cell lines paving ways for iproved cellular mechanism that
suppress the free radical activities which affect the cells of the animals.
CONFLICT INTEREST
The authors declared that they have no competing interests.
AUTHOR’S CONTRIBUTIONS
All the Authors contributed equally.
ACKNOWLEDGEMENTS
This research was not supported by any organization or funding body, however the authors
contributed collectively to carry out the experiment.
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EFFECT OF CITRUS LIMON JUICE AND TAMOXIFEN ON THE OXIDATIVE STRESS ACTIVITIES OF MCF-7 CELL INDUCED BREAST CANCER IN SPRAWGUE DAWLEY RATS.JOSEPH OYEPATA SIMEON

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EFFECT OF CITRUS LIMON JUICE AND TAMOXIFEN ON THE OXIDATIVE STRESS ACTIVITIES OF MCF-7 CELL INDUCED BREAST CANCER IN SPRAWGUE DAWLEY RATS.JOSEPH OYEPATA SIMEON