Protective Effect of stem Bark Ethanol and Aqueous Extracts of ficus
Racemosa Against Cisplatin induced Nephrotoxicity in mice - by Shivalinge Gowda KP1 and Vrushabendra Swamy BM2*
a) Abstract b) Introduction
c)
Material and Methods
d)
Discussion
e)
Conclusion |
f)
References |
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Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is an antineoplastic drug used in the
treatment of many solid-organ cancers, including those of the head, neck, lung, testis, ovary,
and breast. While toxicities include ototoxicity, gastrotoxicity, myelosuppression, and
allergic reactions, the main dose-limiting side effect of cisplatin is nephrotoxicity. Reactive oxygen species and oxidative damage are the most important factors in cisplatin-induced acute renal failure. In the present study the protective effects of stem bark ethanol and aqueous extracts of Ficus racemosa in cisplatin induced mice were studied. The results of this study indicated that Ficus racemosa bark ethanol and aqueous extracts significantly protected
the cisplatin induced neprotoxicity in mice. The serum urea, creatinine, blood urea nitrogen
and lipid peroxidation levels in cisplatin alone treated groups were significantly elevated (P< 0.01) and catalase and renal glutathione (GSH) levels were declined when compared to control group. The serum urea, creatinine and blood urea nitrogen levels were reduced and, catalase and renal GSH levels elevated significantly (p<0.01) in the mice treated with stembark ethanol and aqueous extracts of Ficus racemosa (200 and 400 mg/kg, p.o). The results suggested that the stem bark extract of Ficus racemosa showed protective effect against cisplatin induced nephrotoxicity in mice, which may probably mediated by its antioxidant property.
Keywords: Ficus Racemosa, Cisplatin, Oxidative Damage, Lipid Peroxidation. |
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There are several nephrotoxic drugs in the
market for treating cancer and infectious
diseases caused by gram negative bacteria
namely cisplatin and gentamicin etc. Cisplatin
is a potent anticancer drug used in the
treatment various solid tumors. But the major
dose limiting side effect associated with
cisplatin is nephrotoxicity1. Cisplatin
(cis_diamminedichloroplatinum(II), CDDP) is
an antineoplastic drug used in the treatment of
many solid-organ cancers, including those of the head, neck, lung, testis, ovary, and breast.
While toxicities include ototoxicity,
gastrotoxicity, myelosuppression, and allergic
reactions, the main dose-limiting side effect of
cisplatin is nephrotoxicity. Cisplatin is cleared
by the kidney by both glomerular filtration
and tubular secretion. Cisplatin concentrations
within the kidney exceed those in blood
suggesting an active accumulation of drug by
renal parenchymal cells. Previous studies cells and isolated perfused proximal tubule
segments have provided evidence for
basolateral-to-apical transport of cisplatin2.
However there is lack of experimental to
justify its nephroprotective effect. Hence a
systematic study the neproprotective effect
against experimentally induced renal damage
has been carried out.The roots, bark-skin,
fruits, latex and leaves of Ficus racemosa have
great medicinal value.
The traditional uses of
Ficus racemosa are-wound healing, stomatitis,
sore throat, tooth ache, diarrhea, dysentery,
ulcerative colitis, anorexient, mouth wash,
haemoptysis3. The antidiabetic, hypolipidemic,
anticholinesterase, antibacterial, anthelmentic,
antioxidant, radioprotective, hepatoprotective,
and antidiuretic activities of Ficus racemosa
were reported. The stem bark of Ficus racemosa
contains tannin, wax, saponin gluanol acetate,
β-sitosterol, leucocyanidin- 3 – O – β – D -
glucopyrancoside, eucopelargonidin – 3 – O –
β – D - glucopyranoside, leucopelargonidin – 3
– O – α – L - rhamnopyranoside, lupeol, ceryl
behenate, lupeol acetate, α-amyrin acetate,
leucoanthocyanidin, and leucoanthocyanin
from trunk bark,lupeol, β-sitosterol and
stigmasterol4. |
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Collection and extraction
The stem barks of Ficus racemosa were
collected and identified and authenticated by a
qualified botanist. The shade dried and
powdered stem bark of Ficus racemosa (1 kg)
were extracted with 95% ethanol in a Soxhlet
apparatus (55°C; 25–30 cycles), followed by
water extraction on a hot water bath (70°C; 3–4
h). The ethanol and aqueous extracts were
concentrated to a small volume and then
evaporated to dryness. These extracts (EFR
and AFR) were kept in air tight containers.
Animals
Swiss albino mice (25-30g) were used for the
present study. Animals were housed in a
ventilated room under a 12/12 hr light/dark
cycle at 24±2 °C and had free access to water
and food. The animal care and experimental
protocols were made in accordance with
CPCSEA/ IAEC.
Method
The mice were selected and randomized into 6
groups, each group consisting of 6 mice.
Nephrotoxicity in mice was induced with the administration of cisplatin (12mg/kg). The
stem bark ethanol and aqueous extracts of
Ficus racemosa (EFR and AFR) were
administered (200, 400mg/kg) to mice orally 1
h before the administration of cisplatin and at
24h and 48h after cisplatin injection. The
parameters were studies 72 h after cisplatin
administration. Normal control group was not
administered with either extract or cisplatin.
The mice were selected and randomized into 6
groups, each group consisting of 6 mice. After
72h of last treatment the mice were
anaesthetized by chloroform and sacrificed.
Blood was then collected by cardiac puncture
and kidneys were dissected out immediately.
Post mitochondrial supernatant (PMS)
preparation
The kidneys were perfused immediately with
ice-cold normal saline and homogenized in
chilled potassium chloride (1.17%w/v) using a
homogenizer. The homogenate was
centrifuged at 800 g for 5 min at 4° C in a
refrigerated centrifuge to separate the nuclear
debris. The supernatant so obtained was
centrifuged at 10,500 g for 20 min at 4° C to get
the post mitochondrial supernatant (PMS)
which was used to assay LPO activity,
glutathione, superoxide dismutase(SOD) and
catalase7.
Estimation of serum urea
During the metabolism of protein in the body,
the liver creates ammonia, which is broken
down into a by-product called urea. Kidneys
filter excess urea into the urine and in sweat,
but some goes into the blood stream as serum
urea. Serum urea concentration is important to
determine if the kidneys are functioning
correctly. A high level of serum urea means
the kidneys are not filtering properly. Urea
reacts directly with diacetyl monoxime under
strongly acidic conditions to give a yellow
condensation product. This reaction is
intensified by the presence of ferric ions and
thiosemicarbazide and the resulting red
colored complex formed is a measure of the
urea concentration. The serum urea present in
the mice blood samples was estimated using
semi autoanalyzer.
Creatinine estimation
Creatinine reacts with picric acid in alkaline
medium to produce reddish yellow colored
compound. The alkaline medium is provided
by sodium hydroxide. In Jaffe’s reaction, thesodium salt of picric acid, sodium picrate is
formed in the first phase. The sodium picrate
reacts with creatinine to form raddish yellow
crystals of creatinine picrate and upon
acidification with HCl these crystals are
dissolved. The intensity of the color is directly
proportional to the amount of creatinine
present in the sample5. Creatinine present in
the mice serum was estimated using semi
automatic analyzer.
Lipidperoxidation (LPO)
Lipid peroxides are unstable and decompose
to form a complex series of compounds
including reactive carbonyl compounds.
Polyunsaturated fatty acid peroxides generate
malondialdehyde (MDA) and 4-
hydroxyalkenals (HAE) upon decomposition,
and the measurement of MDA and HAE has
been used as an indicator of lipid
peroxidation. The method in this assay is
designed to assay either MDA alone (in
hydrochloric acid) or MDA in combination
with HAE (in methane sulfonic acid.) The
assay is based on the reaction of a
chromogenic reagent, N-methyl-2-
phenylindole (R1), with MDA and 4-
hydroxyalkenals at 45°C. One molecule of
either MDA or 4-hydroxyalkenal reacts with 2
molecules of reagent R1 to yield a stable
chromophore with maximal absorbance at 586
nm. The MDA content was assayed by TBARS
method. The reaction mixture was prepared
by mixing 0.2ml of 8.1%w/v sodium lauryl
sulphate, 1.5 ml of 20% v/v acetic acid. The
pH was adjusted by adding NaOH, to this
1.5ml of 0.8% w/v aqueous solution of
thiobarbituric acid and 0.2ml of 10w/v of PMS
were added. The resultant was made up to
4ml with distilled water. The mixture was
heated at 95o C for 60min. It was cooled with
tap water and 1ml distilled water and 5ml
mixture of n-butanol and pyridine (15:1) were
added and centrifuged. The organic layer was
separated and its absorbance measured at
532nm. The content of LPO was reported as
nmole MDA per mg protein. Tissue protein
was estimated using the Biuret method of the
protein assay.
Glutathione estimation
Reduced glutathione was determined using
the modified method of Ellman (1951). An
aliquot of 1.0 ml of supernatant of liver
homogenate was treated with 0.5 ml of
Ellman’s reagent (19.8 mg of 5, 5’-dithiobisnitro benzoic acid (DTNB) in 100 ml
of 0.1 % sodium nitrate) and 3.0 ml of
phosphate buffer (0.2 M, pH 8.0). The
absorbance was measured at 412 nm. The
percentage inhibition of GSH was calculated
using the following equation:
% reduced glutathione inhibition = (Ao - A1) /Ao × 100
Where; Ao is the absorbance of the control and
A1 is the absorbance of the sample extract.
Catalase estimation
1ml H2O2 is added to 1.95ml of phosphate
buffer (0.05M, pH 7.0) and 0.05 ml of PMS
(10%). Changes in absorbance were measured
in spectrophotometer at 240nm. The catalase
activity was measured by using its extinction
coefficient. One unit of activity is equal to one
mM of H2O2 degraded/min and is expressed
as units/mg of protein6. |
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In conclusion, findings of the present study
show that Ficus racemosa is a protective agent
against gentamicin-induced nephrotoxicity in
rat. However, the exact protective
mechanism(s) of Ficus racemosa is unknown
and its mechanism is need to be investigated.
Table 1: Table Showing Serum Urea, Creatinine, LPO in Control, Cisplatin,
EFR and AFR Treated Mice
Table 2: Table showing reduced glutathione (GSH), catalase in control,
cisplatin, EFR and AFR treated mice.
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