AMELIORATIVE EFFECT OF CURCUMIN ON AFLATOXIN - INDUCED TOXICITY IN SERUM OF MICE

Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 65 No. 3 pp. 339ñ343, 2008
ISSN 0001-6837
Polish Pharmaceutical Society
AMELIORATIVE EFFECT OF CURCUMIN ON AFLATOXIN-INDUCED
TOXICITY IN SERUM OF MICE
NEETA MATHURIA and RAMTEJ JAYRAM VERMA*
Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad ñ 380 009, INDIA
Abstract: The present investigation was an attempt to evaluate the ameliorative effect of curcumin on aflatox-
in-induced toxicity on serum and blood of mice. Aflatoxin was obtained by growing Aspergillus parasiticus in
SMKY liquid medium. Pure curcumin (97% purity) was purchased from Hi-Media Laboratories Pvt. Ltd.,
Mumbai, India. Young adult male albino mice were orally administered with low dose and high dose (750 and
1500 µg/kg body weight) with and without curcumin (2 mg/0.2 mL olive oil/animal/day) for 45 days. On 46th
day the animals were sacrificed by cervical dislocation. For serum parameters blood was collected in non-
EDTA containing vails from heart of the dissected mice. Serum parameters are creatinine, protein, AST and
ALT. The results revealed dose dependent increase in creatinine, AST and ALT and decrease in protein in
serum parameters of mice. Treatment with curcumin along with aflatoxin ameliorates aflatoxin-induced
changes in serum parameters.
Keywords: aflatoxin, curcumin, serum, creatinine, protein
Aflatoxins are a group of closely related sec-
Other pathological features in cattles are blood
ondary toxic fungal metabolites produced by
coagulation defects, which may involve impairment
Aspergillus flavus and A. parasiticus, of relatively
of prothrombin, factors VII and X and possibly fac-
low molecular weight organic compounds character-
tor IX. A single dose of aflatoxin causes an increase
ized by their diversity, their frequent specificity with
in plasma enzymes (aspartate aminotransferase, lac-
regard to the taxonomy of the producing organisms
tate dehydrogenase, glutamate dehydrogenase,
and their production during the stationary phase of
gamma-glutamyltransferase and alkaline phos-
the batch cultures. During early period of recogni-
phatase) and in bilirubin, probably reflecting liver
tion it was considered as a part of storage flora of
damage. Other abnormal clinical findings are pro-
inadequate post-harvest storage and poor storage
teinuria, ketonuria, glycosuria and hematuria (3).
conditions during distribution of commodities such
Similar changes in blood coagulation parameters
as maize, groundnuts, peanuts, barley etc. However,
have been reported in dogs (3).
it has now become increasingly recognized that the
Serum glutamate-pyruvate transaminase and
contamination of some commodities especially
glutamate oxaloacetate transaminase (mitochondri-
peanuts and maize with aflatoxin is a far more com-
al) activities were elevated. Hypoglycemia and low
plex phenomenon which may involve infection and
cerebrospinal fluid glucose were observed. The
aflatoxin production in the field. Although contami-
onset of the illness included coughing, rhinorrhea,
nation of crop in field is undoubtedly influenced by
sore throat, earache, slightly enlarged, firm yellow
insect damage, A. flavus is also known to infect
liver, and a pale, slightly widened renal cortex. A
intact kernels of maize by colonizing and growth
high rate of mortality (81% of the diagnosed cases)
down the external silk (1).
occurred. Since Reyeís Syndrome is characterized
Aflatoxins also decrease the production of vita-
by abnormal mitochondrial structure and function
min A in the liver. This has secondary effects such
(4), it is of interest to note that aflatoxin B causes
1
as decreased blood calcium levels, decreased bone
abnormal mitochondrial structure and function (5).
strength and a decreased tissue and serum toco-
Inhibition of protein synthesis caused by aflatoxins
pherol level. This decrease in tocopherol level can
alters serum protein composition, resulting in the
lead to vitamin A and E deficiencies (2)].
suppression of the production of non-specific
* Corresponding author: e-mail: ramtejverma2000@yahoo.com; mathuria_25@yahoo.co.in; zooldeptgu@satyam.net.in
339

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340
NEETA MATHURIA and RAMTEJ JAYRAM VERMA
humoral substances important to native defense (6).
Animals of Group 4 and 5 were orally administered
Curcumin (Diferuloylmethane) was first isolated by
aflatoxins at a dose of 25 (low dose; LD) and 50
Vogel and Pelletier (7) and its chemical structure
(high dose; HD) µg/0.2 mL olive oil/animal/day
was determined by Roughley and Whiting (8). It has
(750 and 1500 µg/kg body weight), respectively, for
a melting point at 176-177OC, forms a reddish-
45 days. Group 6 and 7 animals were orally admin-
brown salt with alkali and is soluble in ethanol, alka-
istered low dose and high dose of aflatoxin along
li, acetone, acetic acid and chloroform. Curcumin is
with curcumin (2 mg/0.2 mL olive oil/animal/day),
found to be the biologically active compound in
respectively, for 45 days. All the treatments were
turmeric.
given orally using a feeding tube attached to a hypo-
Curcumin reduces carbon tetrachloride and D-
dermic syringe.
galactosamine-induced glutamate oxaloacetate
On completion of the treatment, the mice were
transaminase and glutamate pyruvate transaminase
weighed and were sacrificed by cervical dislocation.
levels (9). Curcumin has capacity of lowering cho-
The blood from heart was collected in non-EDTA
lesterol, fatty acids and triglycerides in alcohol-
added bulbs, allowed to clot and centrifuged at 1000
induced toxicity (10).
g for 10 min to collect non-hemolyzed serum. The
In vitro curcumin can significantly inhibit the
serum was diluted and used for various biochemical
generation of reactive oxygen species (ROS) like
analyses such as creatinine, estimated by method of
superoxide anions, H O and nitrite radical genera-
Varley (19), AST and ALT, by the method of
2
2
tion by activated macrophages, which play an
Reitman and Frankel (20) and protein by the method
important role in inflammation also. Curcumin low-
of Lowry et al. (21).
ers the production of ROS in vivo (11). Curcumin
For all the parameters a minimum of 10 repli-
reduces oxidized proteins in amyloid pathology in
cates were used and the data were statistically ana-
Alzheimer transgenic mice (12). It also decreases
lyzed using one way analysis of variance (ANOVA)
lipid peroxidation in rat liver microsomes, erythro-
followed by Tukey test. The levels of significance
cyte membranes and brain homogenates (13).
were accepted at p < 0.05. Comparisons of p-values
Curcumin shows anticoagulant activity by inhibiting
between different groups were performed.
collagen and adrenaline-induced platelet aggrega-
tion in vitro as well as in vivo in rat thoracic aorta
RESULTS AND DISCUSSION
(14). Both turmeric and curcumin decreases blood
sugar level in alloxan-induced diabetes in rat (15).
Table 1 shows the effect of aflatoxin as well as
Curcumin also decreases advanced glycation end
aflatoxin plus curcumin treatment on serum param-
products induced complications in diabetes mellitus
eters in mice. Also percent changes from vehicle
(16). In patients undergoing surgery, oral applica-
control (Group 2) are shown in Figures 1.1 ñ 1.4. No
tion of curcumin reduces post-operative inflamma-
significant change was observed between different
tion (17).
control groups (Groups 1 ñ 3).
Aflatoxin treatment for 45 days caused, as
EXPERIMENTAL
compared to vehicle control (Group 2), a dose-
dependent increase in creatinine (LD: 60.04%; HD:
Preparation of aflatoxin extract was the same
165.58%; Fig. 1.1) and activities of serum aspartate
as described in the preceding paper (18). Curcumin
aminotransferase (AST) (LD: 38.94%; HD: 62.01%;
was purchased from Hi-Media Laboratories Pvt.
Fig.1.2) and serum alanine aminotransferase (ALT)
Ltd., Mumbai, India.
(LD: 54.12%; HD: 126.30%; Fig. 1.3), however,
Young inbred, Swiss strain male albino mice
protein content was significantly decreased (LD:
(Mus musculus), weighing approximately 37-40 g,
48.83%; HD: 60.26%; Fig. 1.4) in serum of mice.
were obtained from Cadila Health Care,
Oral administration of curcumin along with
Ahmedabad, India. They were provided feed and
aflatoxin caused significant amelioration in aflatox-
water ad libitum and maintained under laboratory
in-induced effects in the serum parameters as com-
conditions. Seventy such animals were divided into
pared to the aflatoxin alone treated groups. This
seven groups and caged separately. Group 1 (con-
amelioration was almost complete in curcumin plus
trol) animals were maintained without any treat-
low dose aflatoxin-treated group (Group 6), while it
ment. Animals of Group 2 (vehicle control) received
was partial in curcumin plus high dose aflatoxin-
olive oil (0.2 mL/animal /day). Animals of Group 3
treated group (Group 7).
received curcumin (2 mg/0.2 mL olive oil/ani-
A significant decrease in serum protein was
mal/day) for 45 days and served as positive controls.
noted in aflatoxin-fed mice (Table 1). Almost all

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Ameliorative effect of curcumin on aflatoxin-induced toxicity in serum of mice
341
the serum proteins are invariably secreted by liver.
Decreased biosynthesis and secretion of protein
abcdef
might be due to formation of aflatoxin adducts
abcdef
abcef
abcef
with DNA, RNA and protein. Extensive hepato-
cellular necrosis, fatty infiltration and bile duct
High dose
proliferation have also been observed in aflatoxin-
5.302 ± 0.038
50.67 ± 0.14
58.21 ± 0.38
23.41 ± 0.22
fed mice. Aflatoxins have previously been shown
aflatoxin + curcumin
as compared to group 5, p <
to lower the total protein concentration in serum of
e
rabbits (22) and broilers (23). Biosynthesis and
abcdeg
abcdeg
abcdeg
abcdeg
secretion of serum protein becomes normalized as
7
no necrotic changes were observed in histopatho-
6
logical study of liver of aflatoxin plus curcumin-
Low dose
treated mice.
3.925 ± 0.012
58.06 ± 0.18
43.51 ± 0.22
17.51 ± 0.31
aflatoxin + curcumin
Oral administration of aflatoxin for 45 days
caused, as compared to the controls, significantly
higher level of creatinine in the serum of mice
abcdfg
abcdfg
abcdfg
abcdfg
as compared to group 4, p < 0.05;
(Table 1). Creatine is synthesized in the liver, pass-
d
es into circulation and is taken up almost entirely by
5
skeletal muscle for conversion to creatine phos-
High dose
aflatoxin
phate, which acts as an energy reservoir. Creatine
9.213 ± 0.002
25.38 ± 0.04
65.31 ± 0.73
32.61 ± 0.71
and its phosphate are converted spontaneously into
creatinine (24). The two substances are handled dif-
ferently by the kidney. Both are filtered at glomeru-
abcefg
abcefg
abcef
abcef
lus. Although there may be some additional secre-
tion of creatinine by renal tubules, creatine is reab-
4
as compared to group 3, p < 0.05
sorbed by the tubules at low plasma concentration.
Low dose
aflatoxin
c
This ensures that there is little, or no creatine in
5.552 ± 0.056
32.68 ± 0.01
56.01 ± 0.47
22.21 ± 0.48
Experimental Groups
urine (24). The heightened appearance (p < 0.05) of
creatinine in the serum of aflatoxin-fed mice indi-
cates the increased transformation of phosphocrea-
3
tine to creatinine in muscle which might be due to
control
lesser utilization of phosphocreatine in muscular
Curcumin
3.392 ± 0.001
63.46 ± 0.28
40.01 ± 1.89
13.21 ± 0.51
contraction. The kidney rapidly excretes creatinine.
Histopathological studies revealed glomerular dam-
as compared to group 2, p < 0.05;
age and tubular degeneration in the kidney of afla-
b
toxin-fed mice. Thus significant increase in creati-
nine concentration in serum could be due to
increased release from muscles and/or decrease
Vehicle
control
excretion from the kidney. Verma and Raval (25)
3.469 ± 0.006
63.86 ± 0.01
40.31 ± 0.29
14.41 ± 0.42
reported the occurrence of nephrotoxicity and the
elevation of creatinine in serum and urine of rabbits
receiving aflatoxin-contaminated feed (15 mg/kg)
12
for 60 days. Verma and Kolhe (26) showed time-
as compared to group 7, p < 0.05.
control
g
dependent rises in creatine and creatinine concentra-
Untreated
as compared to group 1, p < 0.05; a
3.427 ± 0.006
63.51 ± 0.09
39.61 ± 0.95
13.81 ± 0.51
tions in the serum and urine of aflatoxin-fed rabbits.
This suggests that aflatoxin causes adverse changes
in skeletal muscle and kidney at a very early stage.
They also suggested the occurrence of cumulative
toxicity during aflatoxicosis.
Curcumin alone treatment did not have any
effect on serum creatinine levels. However, curcum-
in when given along with aflatoxin, ameliorates
as compared to group 6, p < 0.05; f
aflatoxin-induced effects in the serum parameters as
Parameters
Creatinine (mg/100 mL)
Protein (mg/100 mL)
Serum aspartate
aminotransferase
activity (mU/mL)
Serum alanine
aminotransferase activity
(mU/mL)
compared to the aflatoxin alone treated mice. These
Table 1. Effect of curcumin on aflatoxin-induced changes in the serum parameters of mice.
Values are the mean ± S.E.M.; n = 10.
0.05;

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342
NEETA MATHURIA and RAMTEJ JAYRAM VERMA
Figure 1.1. Percent change in creatinine content (from vehicle con-
Figure 1.3. Percent change in aspartate aminotransferase activity
trol) in serum.
(from vehicle control) in serum.
Figure 1.2. Percent change in protein content (from vehicle con-
Figure 1.4. Percent change in alanine aminotransferase activity
trol) in serum.
(from vehicle control) in serum.
changes could be due to amelioration in aflatoxin-
ameliorating the toxicity induced by aflatoxin in
induced histopathological changes in kidney.
serum of mice.
The enzymes: serum aspartate aminotrans-
ferase (AST) and serum alanine aminotransferase
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