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SCIENTIFIC JOURNAL
OF MEDICAL RESEARCH
Scientific Journal of Medical Research
Vol. 1, Issue, 1, pp.6 - 12 , Winter, 2017
RESEARCH ARTICLE
STUDY OF USING KOJIC ACID AS ANTICANCER IN DIFFERENT
CONCENTRATION TO TREATMENT CANCERED RATS Rattus norvegicus BY
CYCLOPHOSPHAMIDE
Jasim A. Abdullah
Clinical Laboratories Department, Applied Medical Sciences College, Karbala University ,Karbala , Iraq.
ARTICLE INFO
Article History:
Received 20 August 2016
Received in revised form
17 September 2016
Accepted 24 September 2016
Published online 1 March 2017
Key words:
Kojic acid
Secodary Metbolites
Cyclophosphamide
Anticancer
ABSTRACT
Objective: This study was conduct to investigate effect of kojic acid to treatment ofcancered
rate.
Methods: Different concentration 0.5, 1, 2 mg/kg used as treatment through 30 days in some
histological and functional criteria in cancered rats Rattus norvegicus by cyclophosphamide
and the parameters that studied were WBC, PCV, HB , platelets , AST, ALT, and Bilirubin .
Results: The result showed there is significant difference in blood parameters (WBC ,
platelets) at P≤ 0.01 and there is no significant difference in (HB , PCV) when compared with
control group in addition T3, T4 , TSH levels showed no significant difference in all group of
rats that treated with kojic acid when compared with control group at P≥ 0.01 also showed
significant difference in ALT, AST, Bilirubin when compared with control group at P≤ 0.01 that
lead to increase of ALT, AST, Bilirubin related to increase with kojic acid and then we showed
significant change in the liver and kidney tissues in concentration 2 mg/kg of kojic acid when
compared with control group and finally.
Conclusion: We can consider the kojic acid is very good substance may be useful as antibiotic
or antioxidant in some researches because it is very safe on the body .
Corresponding author:
Jasim A. Abdullah.
Email: [email protected]
Applied Medical Sciences College,
Karbala University, Karbala, Iraq.
Copyright©2017, Jasim A. Abdullah. This is an open access article distributed under the Creative Commons Attribution License, which permits
unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.
Citation: Abdullah J.A. “Study of using kojic acid as anticancer in different concentration to treatment cancered rats Rattus norvegicus by
cyclophosphamide”, Scientific Journal of Medical Research. 2017 ; 1 (1) : 6 - 12.
INTRODUCTION
Kojic acid is an organic component and consider
secondary metabolite secreted by several microorganism
of aspergillus genus such as Aspergillus oryzae ,
Aspergillus tammorri , Aspergillus parasiticus , and
Aspergillus flavus1. The chemical structure was
determined as 5-hydroxy-2-hydroxymethy-4-pyrone2,
acute and sub chronic toxicity resulting from oral dose
has not been reported and chronic feeding studies in
mice and rats indicated the development of thyroid
follicular cell adenomas primarily3,4. Kojic acid
investigated the three endpoints of genotoxicity , gene
mutations ,structural chromosome alterations and
aneuploidy5, kojic acid it is not significant acute oral
toxicant in mice and rats with LD50 value greater than
6
.
1g/kg , at large doses lethargy, piloerection, ataxia,
depressed respiratory rate , loss of righting reflex,
increased salivation , body tremors were observed and
convulsions prior to death were reported 6 . Kojic acid it
is multifunctional and having weak acidic property and
the natural origin of kojic acid confirms it is nonhazardous bio-degradation7 , it is also used as chelating
agent and activator in insecticide production , Recently,
a new application of kojic acid is found in the cosmetic
industry8 , it is also used as skin whitening agent and
ultraviolet filter in skin care products widely available in
cosmetic market9, also used as food additive for
preventing enzymatic browning10,11 and kojic acid can
be used as antioxidant inon chelator for topical treatment
of wound healing12. Kojic acid loaded nanotechnology
Abdullah J.A., 2017
based drug delivery system can modulate drug
permeation through the skin and improve the drug
activity13, in our experiment we used a kojic acid as drug
or anticancer to treatment the cancered rats by
cyclophosphamide and explain the activity of kojic acid
in some histological and functional criteria .
MATERIALS AND METHODS
In our experiments used 12 rats and divided it into 1 rat
control positive and 2 rats control negative and the other
rats divided into three groups each group contain three
rats and then give the rats carcinogenic substance
cyclophosphamide 50µl/kg by oral administration 1ml
one time only .
Then treatment the rats that taking carcinogenic
substance by kojic acid every day for 30 days in
different concentration for each group .
After 30 days killing the rats by using chloroform and
draw the blood for measurements blood components by
using Ruby instrument .and measurementsT3,T4 and
TSH by ELISA instrument , but GPT ,GOT ,Bilirubin
by using strip method (manual).
After that anatomist all the rats and extracted organs
(liver, spleen, lung and kidney) for are preparation
histological section, cutting the organs into small pieces
and placed in solution 10% formalin for fixation,
washing the organ with tap water for 24 hour to remove
the formalin and then pass the organs in progressive
concentration of ethanol for 30 minute to remove water
from them (dehydration) and then liking samples
inXylene for 20 minutes to remove the alcohol from the
tissue (clearing ) and then placed the samples in the soft
wax and xylene for one hour and place the sample twice
in soft wax only for 1.5 – 2 hour in temperature 56-60cₒ
to remove the xylene from the sample (embedding ) .
buried sample in special molds container on the molten
paraffin wax and leave harden in the refrigerator to be
cut , then the sample cut in 5µm in thickness using
Microtome instrument and proven models on glass slide
, then put the slide at room temperature for the next day
to dry (sectioning) .
Staining section by hematoxeline and eosin stain as
passed the slide in xylene twice for 30 minutes each
time an then passed in concentration regressing of
ethanol for 3-5 minutes for each concentration , pass in
hematoxeline for 15-20 minutes , wash the sample by
distal water for 5 minutes , pass in eosin dye for 20- 30
second , and then passed up ward concentration of
ethanol for 3-5 minutes for each concentration and the
pass in xylene for 15 minutes (staining) in the end
placed cover slide using canda balsam (mounting) and
after being diagnosis histological section by Microscope.
Statistical Analysis
We used (SPSS) program in 18th version and enter the
data to get the statistical analysis process then find the
relationship between the coefficients dealing through
complete random testing and extracted LSD values and
compared with differences between medians and find
standard error values14.
RESULTS
Effect of kojic acid on T3,T4 and TSH
From the results of this study we showed there is no
significant differences of T3,T4 and TSH in all groups
of rats that treated with kojic acid for each concentration
when compared with control group and we show this
result in Table 1 So we showed no high different in the
concentration of T3,T4 and TSH that affected by kojic
acid and cyclophosphamide , also we showed there is
increase in the weight of thyroid gland of rats that will
be treated with kojic acid when compared with control
group.
Table 1. Effect of kojic acid on T3 ,T4 and TSH in rats.
Concen.
mg/Kg
T4
(ng/l)
TSH
(ϻu/ml)
Cont.+
(n=1)
0.0441±0.0013
A
0.0625±0.005
B
0.037±0.002
C
Cont.(n=2)
0.0442±0.0013
A
0.0655±0.005
B
0.039±0.002
C
0.5
(n=3)
0.0448±0.0014
A
0.0593±0.00219
B
0.037±0.0031
C
1
(n=3)
0.0466±0.0016
A
0.0583±0.0014
B
0.039±0.041
C
2
(n=3)
0.047±0.0032
A
0.0593±0.0018
B
0.038±0.0023
C
* The number refer to mean ± Stander error.
* Similar vertically capital letters refer to no significant difference
(P≥0.01) between hormones for each concentration.
Effect of Kojic Acid on Biochemical Criteria
This study will be indication there are significant
difference at P≤ 0.01 in the concentration of both GPT,
GOT enzymes and in the concentration of bilirubin in all
groups of rats that will be treated with kojic acid for
each concentration when compared with control groups
.we showed there are significant increase at P≤ 0.01 in
the level of GOT enzyme in all groups of rats will be
treated with kojic acid in concentration 0.5 , 1 , 2 mg/kg
that mean an increase in the concentration of GOT
related with increase in the concentration of kojic acid .
also the concentration of GPT will be affected with kojic
acid in different concentration 0.5 , 1 , 2 mg/kg observed
significant increase in each rats will be treated with kojic
acid but the highest value recorded at concentration
2mg/kg of kojic acid So GPT concentration will be
increase with increase in the concentration of kojic acid ,
Scientific Journal of Medical Research, Vol. 1, Issue, 1, pp.6 - 12 , Winter, 2017
.
Parameter
T3
(pg/m)
7
also the concentration of bilirubin in groups of rats that
treated with kojic acid will be increase in both
concentration of kojic acid 1, 2 except 0.5 mg/kg when
compare with control group that mean there are slightly
increase in the concentration of bilirubin in treated rats
and these result can be show in Table 2.
Table 2. Effect of kojic acid on some of biochemical criteria
in rats compare with control group.
Concen.
mg/Kg
Parameter
GOT
U/L
GPT
U/L
Bilirubin
mg/dl
Cont.+
(n=1)
7±2.3
D
8.5±4.17
D
21±4.66
B
Cont.(n=2)
10±2.3
D
14.75±14.17
D
32±4.66
B
0.5
(n=3)
23±1.5
B
25±0.34
B
18.3±3.33
B
1
(n=3)
28±4.88
A
19.6±2.185
C
24.3±2.8
B
2
(n=3)
22±0.5
C
42.6±12.19
A
36.3±0.8
A
LSD0.01
13.10
21.94
15.27
*The number refer to mean of enzyme concentration (U/l)±stander
error
*Various vertically capital letter refers to present significant difference
(P≤0.01) between enzymes for each concentration .
Effect of Kojic Acid on Blood Criteria
In this study we showed there are no significant effects
of kojic acid in concentration 0.5, 1, 2 mg/kg on Hb and
PCV in rats that will be treated with kojic acid at P≥
0.01 when compared with control group , but we showed
there are significant difference in both platelets and
WBC in rats treated with kojic acid when compared with
control group also WBC have slightly increase that
mean WBC will be increase when the concentration of
kojic acid will be increase , whereas platelets at control
group =710 but when treated with kojic acid about
0.5mg/kg =786 and in1mg/kg =855.6 and in 2mg/kg
=912.3 and that mean platelets will be increase when
increase of kojic acid at P≤ 0.01 , these result show in
Table 3.
observed some effects like glomerularnephritis and
occur alienation in lining tissue of renal tubules of
Table 3. Effect of kojic acid on blood criteria in rats
compare with control group.
Concen.
mg/Kg
Parameter
Cont.+
(n=1)
Platelet
(×109/l)
710±50.50
C
WBC
(×109/l)
7±0.51
C
Hb
(g/dl)
13±1.75
A
P.C.V
(g/dl)
36±1.15
B
Cont.(n=2)
786.5±50.50
C
5.6±0.51
C
9.33±1.75
A
33±1.15
B
0.5
(n=3)
786.6±52.67
C
7.36±0.178
11.51±0.248
A
37.3±0.88
B
1
(n=3)
855.6±34.66
B
8.066±0.61
B
11.27±0.69
A
37.3±1.185
B
2
(n=3)
912.3±33.34
A
11.7±0.70
A
11.95±0.80
A
39.9±2.9
B
LSD0.01
132.6
2.58
NS
NS
* The number refer to mean of blood components (g/dl) ± stander error
*Various vertically capital letter refers to present significant difference
(P≤0.01) between blood parameter for each concentration .
NS refer to no significante.
cortex but in medulla occur interstitial bleeding and
expansion in loop of Henley . therefore , when treated
the rats by kojic in different concentration 0.5 , 1 ,
2mg/kg as treatment we showed no change in tissue of
liver and kidney that treated by kojic acid 0.5 , 1mg/k
except concentration 2mg/kg of kojic acid we showed
change of tissue in both liver and kidney when
compared with control group and we can show decrease
in effect of cyclophosphamide in this organs
respectively, these results can be show from Figure 110.
Histological examination of liver and kidney
We showed from this study the cyclophosphamide
possess high effects on liver and kidney in rats that
treated with cyclophosphamide in concentration 50µl
/kg we can observed vacuolization in cytoplasm of
hepatocyte and increase size of Kupffer cell because
enters much more amounts of water to cell that’s lead to
formation vacuoles in same cell, in addition occur
congesting in some liver veins . but in kidney we
8
.
Fig. 1 T.S in liver tissue appears normal structure that composed
of
H (Hepatocyte), K (Kupffer cells), S (Sinusoids),
HE(Hepatoplate) and G (Glycogen granules) positive control
group (E&H 40X).
Abdullah J.A., 2017
Fig. 2 T.S in kidney tissue appears normal structure that
composed of R(Renal corpuscle ), G(Glomerulus) ,D(Distal
convoluted tube) ,P(Proximal convoluted tube) and GA
(Guxtaglomerulappartus) positive control group (E&H 100X) .
Fig. 5 T.S in liver tissue treated with kojic acid 0.5 mg/kg appears
abnormal structure that composed of A (congesting in hepatic
vein) and B (expansion in sinusoid of liver) (E&H 40X).
Fig. 3 T.S in liver tissue treated with cyclophosphamide 50µl/kg
appears abnormal structure that composed of A (congesting in
hepatic vein) and B (expansion in sinusoid of liver) negative
control group (E&H 40X).
Fig.6 T.S in kidney tissue treated with kojic acid 0.5 mg/kg
appears abnormal structure that composed of A (shrinking of
glomerulus) , B (necrosis in proximal and distal renal tubules) and
C (Alienation of lining tissue in proximal and distal renal tubules )
(E&H 100X).
Fig. 4 T.S in kidney tissue treated with cyclophosphamide 50µl/kg
appears abnormal structure that composed of A (congesting in
branch of renal artery) , B (shrinking of glomerulus ) and C
(necrosis in proximal and distal renal tubules ) negative control
group (E&H 100X) .
Fig. 7 T.S in liver tissue treated with kojic acid 1mg/kg appears
abnormal structure that composed of A (congesting in hepatic
vein) and B (expansion in sinusoid of liver) C (enlargement of
hepatocyte) and D (necrosis in hepato plates) (E&H 40X).
Scientific Journal of Medical Research, Vol. 1, Issue, 1, pp.6 – 12, Winter, 2017
.
9
Fig. 8 T.S in kidney tissue treated with kojic acid 1 mg/kg appears
abnormal structure that composed of A (interstitial bleeding) , B
(expansion in loop of Henley ) and C (Alienation of lining tissue in
proximal and distal renal tubules) (E&H 40X).
Fig. 9 T.S in liver tissue treated with kojic acid 2 mg/kg appears
normal structure that composed of H (Hepatocyte) , K (Kupffer
cells) , S (Sinusoids) , HE(Hepatoplate) and C (Central vein) (E&H
40X).
Fig. 10 T.S in kidney tissue treated with kojic acid 2 mg/kg
appears normal structure that composed of R(Renal corpuscle ),
G (Glomerulus) , D(Distal convoluted tube) , P(Proximal
convoluted tube) and GA (Guxtaglomerulappartus) (E&H 100X).
DISCUSSION
Effect of kojic acid on T3,T4 and TSH
This result will be agree with15 indicate after repeated
oral doses of kojic acid in rats the main target organs
affected are thyroid gland and pituitary gland as well as
10
.
liver , an increase in the weight of thyroid gland showed
at doses about 0.95 mg/kg of kojic acid in diet given to
male and female rats for 28 consecutive days and
observed there are significant difference at P≤ 0.01 in
the number of colloid in thyroid follicles and follicular
cell hypertrophy in thyroid gland that’s suffer from
enlargement , in addition showed there are decrease in
the concentration of TSH . Also16 observed the increase
in the weight of thyroid gland that caused by kojic acid
may be lead to inhibition of iodine up take to organs and
lead to decrease in the concentration of T3 and T4 and
increase in the concentration of TSH , also indicate the
kojic acid do not have any carcinogenic effect on thyroid
gland specifically and body generally . While17 showed
kojic acid leads to inhibition of iodine up take by thyroid
gland also inhibition of iodine in the organs at high
doses of kojic acid and this cause lead to decrease in the
concentration of T3,T4 and increase in the concentration
of TSH as well as increase in the weight of thyroid
gland. Whereas18,19 ensure that kojic acid when treated
to rats that it is not have any carcinogenic effect on
thyroid gland but lead to inhibition of iodine up take that
result in decrease in concentration of T4 and T3 and
increase in the concentration of TSH . But20 showed
significant decrease in the concentration of T3 and T4 to
the male rats when treated with cyclophosphamide , and
this decrease will result from precipitation of
cyclophosphamide in the tissue of thyroid gland and
cause damage to colloid in thyroid follicles and
follicular cell of thyroid gland.
Effect of Kojic Acid on Biochemical Criteria
21
Indicate there are increase in the level of both GOT
and GPT enzymes at dose 500-1000mg/kg of kojic acid
also level of ALP enzyme may be increase and also
there are increase in bilirubin, cholesterol and calcium
concentration . Also22,23 showed significant increase in
the concentration of both GOT and GPT enzyme in male
of rats treated with cyclophosphamide and these results
caused by inflammation and fibrosis in liver and24
indicate there are increase in the level of bilirubin and
total cholesterol in male of rats treated with
cyclophosphamide. Whereas25,26 mention that increase in
the level of bilirubin result from intoxicating and
chronic inflammation in liver when treated with
cyclophosphamide in male of rats. While27 showed that
female of rats treated with doxorubicin may cause
increase in the level of both GPT and GOT and when
infected with hepatitis or other inflammation that will be
affected on liver that cause increase in GPT more than
increase in GOT. But28 indicate that the level of GOT
may be increase when occur injury or infection to the
heart muscle while the level of GPT do not affect
because of GPT is more specific for liver ,therefore,
when occur damage to the heart muscle that cause
increase in the activity of GOT and increase in this
concentration in heart muscle.
Abdullah J.A., 2017
Effect of Kojic Acid on Blood Criteria
29
Showed the changes in the functioning criteria in rats
can be occur depending on the high concentration of
kojic acid and the period of dose –time ,therefore,
showed lymphocytes and WBC decrease in male and
female of rats treated with kojic acid at concentration
500-1000mg/kg through 4weeks. While30 showed in
dose 1000mg/kg of kojic acid that will be cause decrease
in RBC also decrease in PCV and concentration of HB
in rats treated with kojic acid. Also31 indicate there are
significant differences in blood criteria and showed
decrease in number of WBC in rats treated with
doxorubicin agent also increase in number of neutrophils
and decrease in number of lymphocytes when compare
with control group and the number of neutrophils will be
increase five time compare with control group while
lymphocyte will be decrease two time compare with
control group and do not show any difference in the
RBC and Hb concentration between the groups of rats
treated with doxorubicin agent and control group.
CONCLUSION
We can consider the kojic acid is very good substance
may be useful as antibiotic or antioxidant in some
researches because it is very safe on the body.
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Abdullah J.A., 2017