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Nature and Science 2010;8(3) Ahmed; Biochemical Studies on Nephroprotective
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41
Biochemical Studies on Nephroprotective Effect of Carob
(Ceratonia siliqua L.) Growing in Egypt
Mahgoub M. Ahmed
Molecular Drug Evaluation Dep., National Organization for Drug Control and Research (NODCAR). Egypt
dr_mahgoub1@yahoo.com
ABSTRACT: Reactive oxygen species and free radicals are involved in the nephrotoxicity induced by the
synthetic anticancer drug cisplatin. The nephroprotective effect of carob pods and leaves (100 and 200 mg/kg,
p.o.) was investigated using cisplatin (10 mg/kg body weight, i.p.) to induce oxidative renal damage in mice.
The results showed that cisplatin administration caused abnormal renal functions in all studied mice. Serum urea
and creatinine concentrations were significantly highered (P<0.5) in the cisplatin alone treated (control) group
compared to the normal group. The concentrations of serum creatinine and urea in the carob pods (200 mg/kg
body weight) treated group were reduced to 57.5% and 51.5%, respectively, with respect to the control group.
Also, cisplatin induced decline of renal antioxidant enzymes such as superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (GPx) activities, but the treatment of carob pods and leaves (100 and 200 mg/kg,
p.o.) significantly attenuated the cisplatin-induced nephrotoxicity. Both pods and leaves of carob at 100 and 200
mg/kg increased the concentration of reduced glutathione (GSH) and protected against the increase of cisplatin-
induced lipid peroxidation. In addition, treatment with cisplatin increased the activity of cathepsin D, RNase II,
DNase II and acid phosphatase. The treatment of carob pods and leaves (100 and 200 mg/kg, p.o.) improved the
activity of lysosomal enzymes nearly to the normal group. In conclusion, carob pods and leaves may be
effective to protect from oxidative renal damage and the leaves are the better nephroprotective agent than pods.
The protection may be mediated partially by preventing the decline of renal antioxidant statues.
[Nature and Science 2010;8(3):41-47]. (ISSN: 1545-0740).
Key words: nephrotoxicity; carob; cisplatin; antioxidant enzymes; lysosomal enzymes
INTRODUCTION
Cisplatin is a widely used antineoplastic
agent for the treatment of metastatic tumors of the
testis, metastatic ovarian tumors, lung cancer,
advanced bladder cancer and many other solid
tumors (Sweetman, 2002). The cytotoxic action of
the drug is often through its ability to bind DNA to
form cisplatin-DNA adducts (Goldstein and
Mayor, 1983). Although higher doses of cisplatin
are more efficacious for the suppression of cancer,
high dose therapy manifests irreversible renal
dysfunction and other toxicities yet (Simic and
Jovanovisc, 1986 and Halliwell and Cross, 1994).
Various data indicate that cisplatin induces
oxidative stress (Ajith et al., 2002), lipid
peroxidation (Bompart, 1989 and Matsushima et
al., 1998) and DNA damage (Liberthal et al.,
1996). Therefore administration of antioxidants has
been shown to ameliorate cisplatin-induced
nephrotoxicity in various species of animals
(Somani et al., 2000). The mechanism of
protective effects of antioxidants against cisplatin
nephrotoxicity is not fully known.
Ceratonia siliqua L., Fabacae (Carob) has
been widely cultivated in Mediterranean countries
for years. This tree was distributed by Arabs in the
Mediterranean area (Kumazawa et al., 2002). The
plant is grown locally in Egypt, and the pods are
used mainly for preparing a popular beverage.
Leaves and pods of carob exerted diverse
physiological function as antioxidant activity
(Kumazawa et al., 2002 and Rasheed, 2006).
Custódio et al. (2009) investidated the
Antioxidant activity and in vitro inhibition of tumor
cell growth of carob tree (Ceratonia siliqua). The
methanol extracts of the carob tree showed
significant radical scavenging activity and a
remarkable ability to inhibit tumor cell
proliferation. Carob pods and leaves extracts
contain antiproliferative agents that could be
practical importance in the development of
functional foods and/or chemopreventive drugs. In
addition leaves and pods of carob are rich in
polyphenols and flavonoids (Rasheed, 2006).
In the present study, the protective effects
of carob pods and leaves by two doses (100 and
200 mg/kg, p.o.) on cisplatin-induced renal damage
in mice were evaluated.
MATERIALS AND METHODS
Preparation of samples
Carob (C. Siliqua) pods and leaves
samples were obtained from Experimental Station
of Medicinal Plants, Giza. The pods and leaves
were grinded to fine powder before extraction.
Such powdered samples were kept in dark bottles.
Chemicals
Cisplatin (1mg/ml) Onco-Tain DBL was
from Mayne Pharma PLC, UK., Reduced
glutathione (GSH), 5,5-dithiobis (2-nitrobenzoic
acid) (DTNB), EDTA and thiobarbituric acid
(TBA) were from Sigma-Aldrich Co, St Louis,
Nature and Science 2010;8(3) Ahmed; Biochemical Studies on Nephroprotective
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42
USA. All other chemicals and reagents used were
of analytical grade.
Animals
Albino male mice (30±6 g) were used in
the present study. The mice were obtained from the
animal house of the National Organization for Drug
Control and Research (NODCAR), Egypt. The
animals were kept under standard laboratory
conditions of light/dark cycle (12/12h) and
temperature (25±2˚C). They were provided with a
nutritionally adequate standard laboratory diet.
Animals’ diet
The basal diet consists of casein 10%,
cotton seed oil 4%, salt mixture 4%, vitamin
mixture 1%, carbohydrates (sucrose, starch 1:1)
80.8% and choline choride 0.2% (American
Institue of Nutrition, 1980).
Plant extracts
One hundred gram of pods and leaves of
carob were separately extracted by percolation with
70% ethanol. The extracts were filltered,
concentrated under vacuum and freeze dried.
Experimental design
Animals were divided into 6 groups of 6
animals each.
Group I treated with vehicle (gum acacia, 1%) was
kept as normal.
Group II injected with a single dose of cisplatin
(CIS) (10 mg/kg b.wt., i.p.) was kept as control
Group III and IV were trearted with pods extract
(P.), 100 and 200 mg/kg b.wt.
Group V and VI were treated with leaves extract
(L.), 100 and 200 mg/kg b.wt.
The pods and leaves extracts were freshly prepared
as fine suspension in gum acacia and administered
by oral gavage one h before and 24 h and 48 h after
cisplatin injection. Seventy two hours after
cisplatin injection, animal were killed by cervical
decapitation. Blood was collected and the separated
serum was used for the estimation of creatinine
(Bartless et al., 1972) and urea (Patton and
Crouch, 1977). After decapitation, kidney was
rapidly removed and washed in cold isotonic saline.
The kidney was divided into two portions. The first
one was homogenized in 50 mM phosphate buffer
(pH 7) using an electronic homogenizer to prepare
10 % w/v homogenate. The homogenate was
centrifuged at 3000 rpm for 10 min. at 4˚C and the
supernatant was used for the estimation of total
protein (Lowry et al., 1951), lipid peroxidation
(TBARS) measured as malondialdehyde (MDA)
(Ohkawa et al., 1979), superoxide dismutase
(SOD) (Marklund and Marklund, 1974), catalase
(CAT) (Takahara et al., 1960), Glutathione
peroxidase ( GPx) (Rotruck et al., 1973), reduced
glutathione (GSH) (Ellman, 1959) and glutathione-
S-transferase (GST) (Habig et al., 1974). The
second portion was used for lysosomal isolation
according to Harikumar et al., (1989). The
activities of four lysosomal acid hydrolases were
measured. Cathepsin D, RNase II, DNase II and
acid phosphatase activities were determined
according to the method of Gianetto and de Duve
(1955) and Barett and Heath (1977).
Statistical analysis
The results are expressed as Mean±SEM.
The collected data were statistically analysed by
the least significant differences (LSD) at the level
5% of the probability procedure according to
Snedecor and Cochran (1980).
RESULTS
Intravenous cisplatin administration
caused abnormal renal functions in all injected
mice. Serum urea and creatinine concentrations
were significantly increased (P<0.5) in the cisplatin
alone treated (control) group compared to the
normal group (Table 1). The concentrations of
serum creatinine and urea in the carob pods (200
mg/kg body weight) treated group were reduced to
57.5% and 51.5%, respectively, with respect to the
control group. Similarly, the concentrations of urea
and creatinine in the carob leave (200 mg/kg)
treated group were reduced to 62.8% and 65.2%,
respectively.
The activities of renal SOD, CAT and
GPx in the cisplatin plus carob pods or cisplatin
plus carob leaves administered group are given in
Table 2. Renal SOD activity was decreased
significantly (P<0.05) in the cisplatin alone treated
group compared to the normal group. The SOD
activity in the carob pods and leaves (200 mg/kg
body weight) administered groups were increased
significantly (P<0.05) when compared to that of
control group.
Nature and Science 2010;8(3) Ahmed; Biochemical Studies on Nephroprotective
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43
Table (1): Effect of carob pods and leaves on serum urea and creatinine in mice treated with cisplatin (CIS).
Values are Mean±SEM (n=6 animals). a p<0.05, (Student´s t test) significantly different from normal group. b p<0.05, significantly different
from control group. P, pods and L, leaves of carob.
Table (2): Effect of carob pods and leaves on renal SOD, CAT and GPx in mice treated with cisplatin (CIS).
Values are Mean±SEM (n=6 animals). a p<0.05, (Student´s t test) significantly different from normal group. b p<0.05, significantly
different from control group. ns, non significant different from control group. P, pods and L, leaves of carob.
The activity of CAT in the cisplatin alone
treated group was found to be decreased
significantly (P<0.05) when compared to the
normal group. Treatment of carob pods and leaves
effectivity prevented the cisplatin induced decline
of the CAT activity. Similarly, GPx activity was
decreased significantly in cisplatin treated group.
The enzyme activity was significantly increased
(P<0.05) except at low dose of carob pods that
could not prevent the decline of GPx activity.
The concentration of renal GSH was
significantly decreased (P<0.05) and that of
malondialdehyde was significantly increased
(Table 3) in cisplatin treated animals.
Administration of carob pods or leaves prior to
cisplatin injection increased GSH and decreased the
MDA concentrations.
Administration of cisplatin induced
significant decrese in renal GST activity in
comparison to normal value Table (3), whereas,
carob pods and leaves (200 mg/kg) significantly
ameliorated the effect of cisplatin compared to
cisplatin group.
The effects of cisplatin treatment on
lysosomal enzyme activities are presented in Table
4. Cisplatin treatment increased the activities of the
four lysosomal enzymes; cathepsin D, acid
phosphatase, DNase II and RNase II, significantly
(P<0.05) compared to normal group.
Administration of carob pods or leaves by two
doses prior to cisplatin injection significantly
(P<0.05) ameliorated the effect of cisplatin in all
enzyme activities, compared to control group.
Creatinine
(m
mol/L)
Urea
(m
mol/L)
Groups Mean±SE Mean±SE
28.1±4.2
260.4±50.2 a
152.6±13.6 b
110.7±27.9 b
108.2±10.9 b
90.5±12.2 b
6.8±1.1
23.1±2.1a
15.7±1.0b
11.2±1.2b
14.2±1.4b
8.6±1.0b
Normal
Control (CIS)
P100+ CIS
P200+ CIS
L100+ CIS
L200+ CIS
GPx
(nmol/min/mg protei )
CAT
(nmol/min/mg protein)
SOD
(nmol/min/mg protein)
Groups Mean±SE Mean±SE Mean±SE
253.1±6.1
130.7±3.5 a
157.2±3.0 ns
222.9±4.1 b
220.1±3.2 b
248.2±5.2 b
160.9±6.8
87.7±2.8 a
139.1±2.6 b
156.9±2.8 b
1452.8±3.3 b
158.1±5.2 b
22.2±2.6
10.6±1.9 a
15.2±2.8 ns
17.2±3.1 b
17.9±2.7 b
19.1±3.6 b
Normal
Control (CIS)
P100+ CIS
P200+ CIS
L100+ CIS
L200+ CIS
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Table (3): Effect of carob pods and leaves on renal GSH, TBARS and GST in mice treated with cisplatin (CIS).
Values are Mean±SEM (n=6 animals). a p<0.05, (Student´s t test) significantly different from normal group. b p<0.05, significantly different
from control group. ns, non significant different from control group. P, pods and L, leaves of carob.
DISSCUTION
Cisplatin has been shown to cause
nephrotoxicity in patients (De Conti et al., 1973
and Daugarrd et al., 1988) as well as in a variety
of animal species (Mckeage et al., 1993 and
Badary et al., 1997 a,b). A minimum dose of
cisplatin (5 mg/kg b.wt, i.p.) was sufficient to
induce nephrotoxicity in rats (Boogaard et al.,
1991 and Ravi et al., 1995). A higher dose of
cisplatin (10 mg/kg b.wt. i.p) corresponds to that
currently being used in clinical practice.
Administration of cisplatin exerts significant
increase in serum urea and creatinine
concentrations compared to normal group, which
clearly indicates the acute renal failure. The effect
of cisplatin was similar to those previously
described (Heidemann et al., 1989; Mckeage et
al., 1993 and Somani et al., 2000). Carob pods and
leaves ameliorated cisplatin-induced nephrotoxicity
as indicated by significant less increase in serum
urea and creatinine concentrations.
Table (4): Effect of carob pods and leaves on renal Cathepsin D, Acid Phospha-tase, DNase II and RNase II in
mice treated with cisplatin (CIS).
Values are Mean±SEM (n=6 animals). a p<0.05, (Student´s t test) significantly different from normal group. b
p<0.05, significantly different from control group. P, pods and L, leaves of carob.
The renal antioxidant status, such as SOD,
CAT, GPx activities and GSH concentration are
significantly decreased in the cisplatin alone treated
group of animals compared to normal group. The
decline of antioxidant status partially explains the
mechanism of nephrotoxicity induced by cisplatin.
The renal accumulation of platinum and covalent
binding of platinum to renal protein could, also,
play a role in the nephrotoxicity (Litterst and
Schweitzer, 1988). Cisplatin induced suppression
of renal antioxidant enzyme activities was also
supported by the published experimental results
(Ajith et al., 2002 and Cetin et al., 2006).
Carob pods and leaves (200 mg/kg b.wt.
i.p.) along with cisplatin could significantly
improve the depletion of the renal antioxidant
system.
GSH depletion increases the sensitivity of
organ to oxidative and chemical injury. Studies
with a number of models show that the metabolism
of xenobiotics often produced GSH depletion
(Mitchell et al., 1973, Jollow et al., 1974 and
Ahmed and Zaki, 2009). The depletion of GSH,
also, seems to be a prime factor that permits lipid
peroxidation in the cisplatin treated group.
Treatment of carob pods and leaves reduced the
depletion of GSH levels and provided protection to
the kidney. The protection of GSH is by forming
the substrate for GPx activity that can react directly
with various aldehydes produced from the
peroxidation of membrane lipid.
The intiation and propagation of lipid
peroxidation in the cisplatin treated group could be
caused by the decreased SOD activity. Such
GST
(nmol/min/ mg protei n)
TBARS
(n mol/mg protein)
GSH
(n mol/ mg protein)
Groups Mean±SE Mean±SE Mean±SE
20.1±3.9
11.9±1.2 a
15.1±4.0 ns
18.9±3.5 b
17.1±5.1 b
19.0±3.0 b
1.5±0.20
3.6±0.26 a
3.1±0.16 ns
1.8±0.19 b
1.9±0.15 b
1.6±0.21 b
5.0±0.6
2.4±0.5 a
3.8±1.0 ns
4.2±0.7 b
3.9±0.2 b
5.1±1.1 b
Normal
Control (CIS)
P100+ CIS
P200+ CIS
L100+ CIS
L200+ CIS
RNase II
(nmol/min/ mg protein)
DNase II
(nmol/min/ mg protein)
Acid phosphatase
(nmol/min/ mg protein)
Cathepsin D
(nmol/min/mg protein)
Groups
0.30±0.04
0.81±0.09 a
0.62±0.05 b
0.48±0.06 b
0.53±0.08 b
0.44±0.07 b
0.10±0.02
0.52±0.01 a
0.26±0.07 b
0.21±.05 b
0.20±0.08 b
0.18±0.06 b
45±0.02
97±0.09 a
52±0.09 b
48±0.07 b
51±0.09 b
48±0.06 b
30.0±9.5
68.2±10.1 a
35.2±5.9 b
34.1±6.8 b
31.2±9.7 b
29.2±8.9 b
Normal
Mean±SE
Control (CIS)
Mean±SE
P100+ CIS
Mean±SE
P200+ CIS
Mean±SE
L100+ CIS
Mean±SE
L200+ CIS
Mean±SE
Nature and Science 2010;8(3) Ahmed; Biochemical Studies on Nephroprotective
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45
decreased activity may be either due to loss of
copper and zine, which are essential for the activity
of enzyme or due to reactive oxygen species-
induced inactivation of the enzyme protein
(Sharma, 1985 and De Woskin and Riviere,
1992).
The activity of CAT and GPx, also,
decreased in the cisplatin treated group, which in
turn increased the hydrogen peroxide concentration
and enhanced the lipid peroxidation. Hence the
concentration of MDA, as a result of lipid
peroxidation, increased in the cisplatin treated
group. Treatment with leaves and pods of carob
prevented the lipid peroxidation by enhancing the
renal SOD, CAT and GPx activities. It is well
known that many phenolic compounds, which are
found in carob, exert powerful antioxidant effects.
They, also, inhibit lipid peroxidation by scavenging
reactive oxygen species (ROS), such as OH0
(Shahidi and Wanasundara, 1992).
From the data presented (Table 4), it is
clear that cisplatin treatment in general resulted in
increase in the activity of all lysosomal enzymes
under study. In the carob pods and leaves treated
groups this effect was improved nearly to the
normal group.
There is a correlation between lipid
peroxidation and the release of lysosomal enzymes
from lysosomes. Hence the process of lipid
peroxidation activates phospholipases and removes
the peroxidized lipid from the membrane (Kappus,
1985). The oxidation of unsaturated fatty acids in
biological membranes by free radical leads to a
decrease in membrane fluidity and disruption of
membrane structure and function (Haragushi et al.,
1997).
The increase in activities of RNase II and
DNase II is a mater of concern, since this can lead
to indiscriminate degradation of RNA and DNA
ultimately resulting in necrosis of the cells in the
tissues, i. e. kidney and liver (Patel et al., 2005).
Also, Gianetto and de Duve (1955) reported that
the cathepsin D activity increased substantially in
experimental thyrotoxicosis. It was found that the
acid phosphatase activity increased after cisplatin
treatment (Table 4). Panya et al. (2003) showed
that lysosomal acid phosphatase preferentially acts
on nucleotides and that AMP is the preferred
substrate. The concerned action of activated
nucleases and acid phosphatase would lead not only
to the breakdown of nucleic acids but also to the
further dephosphorylation of mononucleotides,
thereby leading to the acceleration of the process of
cell degeneration.
Ethanolic extract of carob leaves
possessed strong radical scavenging activity in vitro
as measured by DPPH assay. Furthermore, the in
vivo studies confirmed the antioxidant efficacy of
this extract as well as its hepatoprotective activity
(Rasheed, 2006). Polyphenols in carob pods have
antioxidant activity (Kumazawa et al., 2002). In
addition the crude polyphenol extracts of carob
pods showed strong antioxidant activity (Harbore,
and Baxter, 1999). The protective effect of carob
pods and leaves, in the present study, against
cisplatin-induced nephrotoxicity is in harmony and
supports the previous reports indicating the
antioxidant and cytoprotective potential of carob
pods and leaves.
In conclusion, carob pods and leaves
improve the nephrotoxicity of cisplatin in mice.
The nephroprotective effects of carob pods and
leaves may be partially mediated by preventing the
cisplatin-induced decline of renal antioxidant status
and lysosomal membrane damage.
Correspondence to:
Mahgoub M. Ahmed
Molecular Drug Evaluation Dep., National
Organization for Drug Control and Research
(NODCAR), Giza, Egypt
6 Abou Hazem St., Pyramids Ave., Giza, Egypt
Telephone: 002-02-35850005/002-055-2774020,
Cellular phone: 002-012-2586031
Email: dr_mahgoub1@yahoo.com
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