Reagents and chemicals
Analytical grade chemicals used: sodium carbonate, sodium nitrite, dosium dihydrogen, hydrogen peroxide, ferrous chloride, 2-deoxyribose, potassium ferricyanide, sulphuric acid were bought from Merck. 1,1-diphenyl-2-picryl-hydrazyl, potassium persulphate, 2-ethylbenzothiazoline sulfonic acid, rutin, nitro blue, Folin-Ciocalteu’s reagent, phenazine methosulphate, trichloroacetic-acid and tetrazolium were obtained from Sigma Chemicals Co., St. Louis USA. Oxidized glutathione (GSSG), (DTNB), glutathione (GSH),1,2-dithio-bis-nitroLbenzoic-acid (DTNB), glucose-6-phosphate, thiobarbituric acid (TBA), trichloroacetic acid (TCA), sodium tungstate, perchloric acid (PCA), 2,6-dichlorophenolindophenol, reduced glutathione (GSH), sodium hydroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH), sodium tungstate, glucose-6-phosphate, rutin, catechin, gallic acid, caffeic acid, apigenin, quercetin, myricetin, and kampferol were bought from Sigma Chemicals Co., USA were used.
Plant sampling
Alnus nitida stem bark was collected from Charbagh town of district Swat, Pakistan during March–April (2015.) The spot of collection was at 34.842727° north latitude and 72.431089° east longitude; at an elevation of 1000 m. Flora of Pakistan [23] was used for the identification of plant and further authenticated by Dr. Sumaira Sahreen, Associate Curator, Pakistan Museum of National History. The authenticated specimen (127963) was held at Pakistan Museum of National History.
Extract preparation
At room-temperature for 2 weeks the bark of A. nitida was air-dried under-shade-and with the help of Willy Mill granulated to 80-mesh size. Bark powder (5 kg) was drenched in-15 L of 95 %-methanol and reprises-the soaking thrice-and filtered the-extract-with WhatmanLNo.L1 filter paper. Rotary evaporator was utilized to dry the filtrate (ANM) under vacuum. Partial purification or separation was done by solvent-solvent extraction in escalating polarity. In the distilled water the extract was suspended and the solvents i.e., n-hexane (ANH), chloroform (ANC) and ethyl acetate (ANE) were used in augmentation order of polarization and as an aqueous extract residual (ANA) was used. Rotary evaporator was used for the evaporation of solvents of all the fractions, and then preserved at 4 °C.
Phytochemical analysis
Different qualitative tests were employed to identify the phytochemical classes present in the crude methanol extract and various fractions of the stem bark of A. nitida.
Assessment of phenols
For the presence of phenols previously reported methodology was followed [24]. Each sample (1 mg) was suspended in 2 ml of distilled water containing 10 % ferric chloride. The confirmation sign for the presence of phenol was the development of blue or green color.
Assessment of flavonoids
In order to investigate the presence of flavonoids in each sample Trease and Evans protocol [24] was employed. Briefly, 1 mg of every sample was allowed to react with 1 ml of 2 N sodium hydroxide. Appearance of yellow color was considered as the confirmation signal of flavonoid presence.
Assessment of coumarins
An amount of 1 mg of each sample was blended with 1 ml of 10 % sodium hydroxide. Appearance of yellow color in the test tube was the evidence of coumarins presence in the sample [24].
Assessment of saponins
Each sample (2 mg) was suspended in 2 ml of distilled water and vigorously mixed. The formation of a soapy layer of almost 1–2 cm was the indication of saponins presence [24].
Assessment of tannins
The confirmative signal of tannins presence was the development of dim blue or greenish dark shading on the mixing of 1 mg of every sample and 3 ml of 5 % ferric chloride [24].
Assessment of terpenoids
Each sample 0.5 mg was mixed with 3 ml of chloroform and 3 ml of concentrated-sulphuric acid. The appearance of red brown colored layer in the middle of two layers confirmed the existence of terpenoids [24].
Assessment of anthraquinones
Development of red color was considered as indication for the presence of anthraquinones after mixing of 1 mg of each sample with 2 ml of diluted 2 % hydrochloric acid [24].
Assessment of anthocyanins and betacyanins
Each sample (1 mg) was boiled for 10 min in 2 ml of 1 N sodium hydroxide. Formation of bluish green color was the sign of anthocyanin and yellow color formation of betacyanin presence [25].
Assessment of alkaloids
An amount of 2 mg of each sample was mixed with concentrated sulphuric acid. The reaction mixture was allowed to react with Mayer’s reagent. Appearance of green color or formation of white precipitates was the symbol of alkaloid presence [25].
Quantitative analysis
Total phenolic as well as flavonoid contents were quantified by the following narrated procedures.
Total phenolic contents (TPC)
Spectrophotometric analysis was performed for the analysis of total phenolic content [26]. Each sample (1 mg/ml) was briefly blended with 9 ml of distilled water and 2 ml of Folin Ciocalteu reagent. The acquired mixture was mixed vigorously for 10 min and 10 ml ofL 7 % Na2CO3 was further mixed in to the mixture. Mixture’s final volume was raised to 25 ml by adding distilled water and then placed into the incubator at room temperature for 60 min. Absorbance of the reaction mixture was measured at wavelength of 750 nm in triplicates for each sample. Gallic-acid was kept as standard, the estimation of TPC was done as mg of gallic acid equivalents (GAE) per gram of dryLextract/fraction.
Total flavonoid content (TFC)
For the TFC evaluation in the test samples, 0.3 ml of each sample was mixed with 0.25 M NaNO2 (0.25 ml) -followed by the addition of 0.1-ml of 0.3 MLAlCl3.6H2O, and 3.4 ml of-30 % methanol [27]. After 5 min interval 2 ml aliquot of 1 M NaOH was added to it. At the wavelength of 506 nm the absorbance of reaction amalgam was measured against the reagent blank. Content of total flavonoid as-mg rutinLequivalentsLper gram of dryLextract/fraction was assessed by employing the calibration-curve of rutin.
High performance liquid chromatography (HPLC-DAD) analysis
HPLC analysis of ANM and selected plant fractions (ANE and ANA) was performed using HPLC-DAD (Agilent 1200, Germany) equipped with Zorbex RXC8 (Agilent, USA) analytical column with 5 μm particle size and 25 ml capacity using previously reported method [28]. Each sample was diluted with HPLC grade methanol. Mobile phase was consisted of eluent A, (acetonitrile- methanol--water- acetic acid-/5: 10: 85: 1)-and eluent B (acetonitrile-methanol-acetic acid/40: 60:-1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 21–25 min (50 to 100 % B), 26–30 min (100 % B) and 31–40 (100 to 0 % B) at flow rate ofL1 ml/min. The standards and samples were prepared in HPLC grade methanol (1 mg/ml), filtered through 0.45 μm-membrane filter andL20 μl was injected for the analysis. Among the standards rutin was investigated at 257 nm, catechin and gallic acid at 279 nm, caffeic acid and apigenin at 325 nm while quercetin, myricetin and kampferol were analyzed atL368 nm [29]. The analysis was performed in triplicate and the column was reconditioned for 10 min after each run. Quantification was done by the integration of the peak by using the external standard method.
In vitro antioxidant assays
The in vitro antioxidant assays were carried out by preparing the plant samples (1 mg/ml) in 95 % methanol and then making its serial dilutions. The specific protocol was followed for finding specific scavenging activities of the plant samples.
DPPHL (1, 1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
DPPH scavenging capacity of injurious impacts of free radicals was dictated by following the methodology reported previously [30]. A volume of 100 ml of methanol was used as solvent for 24 mg of DPPH and the stock was kept at 20 °C temperature for further utilization. The dilutions of pre-made DPPH stock solutions were prepared in methanol by optimizing absorbance of DPPH was at 0.908 (±0.02) at wavelength 517 nm. Different concentrations (25–250 μg/ml) of 100 ml of plant samples were mixed with dilution of 3 ml of DPPH. The tubes were thoroughly mixed and placed for 15 min in incubator at room temperature. Absorbance of the reaction mixture was measured at wavelength of 517 nm. Ascorbic acid was utilized as standard to compare the antioxidant activity. Potential as an antioxidant was determined by using Eq. 1:
$$ \mathrm{DPPH}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}-\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{sample}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}} \times 100 $$
(1)
Nitric oxide scavenging assay
Nitric oxide scavenging activity of ANM and its derived fractions was estimated by using Griess reagent as the main ingredient [31]. DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. For the development of Griess reagent, equimolar amount of 0.1 % napthylenediamine in distilled water and 1 % of sulphanilamide in 5 % phosphoric acid was added. An aliquot of 0.2 ml of sample was mixed with 0.2 ml of sodium-nitroprusside (10 mM) being formulated in saline phosphate buffer followed by addition of 2 ml aliquot of the Griess reagent to the reaction blend. For 3 h the reaction blend was incubated at room temperature and the absorbance was measured at the wavelength of 546 nm spectrophotometrically utilizing ascorbate as a positive control. For assessing the percentage inhibition of nitric oxide radical formation equation 1 was used.
Hydroxyl radical scavenging assay
For measuring the scavenging ability of methanol extract and its fractions of A.nitida, Halliwell et al. [32] methodology was followed. DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. As per the procedure, 500 μl of 2.8 mM) 2-deoxyribose was prepared in 50 mM phosphate buffer and pH was maintained at 7.4. The reaction cocktail was made by addition of 0.1 ml of 0.1 M EDTA, 0.2 ml of ferric chloride (100 mM) and 0.2 ml of 200 mM H2O2 and 0.1 ml of plant sample. To begin the reaction 0.1 ml of ascorbic acid (300 mM) was added and for 1 h placed in incubator at 37 °C. After that 2.9 %-trichloroacetic acid (2 ml) and 1 % w/v thiobarbituric acid (2 ml) prepared in 50 mM NaOH were further added to the reaction cocktail and for 15-min the whole mixture was heated in water bath. The absorbance was measured at wavelength of 532 nm once the mixture temperature falls to room temperature. For the analysis of hydroxyl radical scavenging activity Eq. 2 was applied:
$$ \mathrm{Superoxide}\ \mathrm{scavenging}\ \mathrm{activity}\ \left(\%\right)=\left(1-\frac{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{sample}}{\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}}\right) \times 100 $$
(2)
β-Carotene bleaching assay
The ability of A. nitida methanol extract and its fractions for β-carotene bleaching was determined by using the scheme of Dapkevicius et al. [33]. DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. In 10 ml of chloroform 2 mg of β-carotene was added and subsequently added 100 mg of Tween 80Land 20 mg of linoleic acid. Once the chloroform evaporated form the reaction blend, 100 ml of-distilled water was added, and vivaciously vortexed to achieve a uniform emulsion of β-carotene linoleate. In freshly prepared 250 μl of emulsion, 30 μl of plant sample was added and optical density was measured at wavelength of 470 nm at 0 h. Finally the absorbance was measured and recorded after keeping the reaction mixture at 40 °C for 1 h. Catechin served as standard in this assay and % inhibition of β-carotene was determined by Eq. 3:
$$ \mathrm{Percentage}\ \mathrm{inhibition}=\frac{\mathrm{Absorbance}\ \mathrm{after}\ 2\ \mathrm{h}}{\mathrm{Initial}\ \mathrm{absorbance}} \times 100 $$
(3)
Chelating power assay
The iron (II) binding capability at multiple sites confers the antioxidant potential of plant samples [34]. Methanol was used as a solvent for the preparation of plant sample and for serial dilutions. A volume of 200 μl of each dilution was mixed with 900 μl of methanol and 100 μl FeCl2.2H2O (2.5 mM) and incubated for 5 min. To trigger the reaction 800 μl of ferrozine (6.0 mM) was introduced and afterL15 min of incubation the optical density was recorded at 562 nm using EDTA as standard in comparison. For the evaluation of chelating power following Eq. 4 was employed:
$$ \mathrm{Chelating}\ \mathrm{effect}\ \%\kern0.5em =\kern0.75em \left[\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}\kern0.5em -\kern0.5em \mathrm{Absorbance}\ \mathrm{of}\ \mathrm{the}\ \mathrm{sample}/\mathrm{Absorbance}\ \mathrm{of}\ \mathrm{control}\right] \times 100 $$
(4)
Reducing power assay
DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. Briefly, 4 ml of plant extract was taken and mixed with 4 ml of 0.3 M phosphate buffer (pH 6.8) and 4 ml of potassium ferricyanide (10 mg/l) and the reaction cocktail was placed in the incubator for 20 min at 80 °C for 10 min. After addition of 4 ml of trichloroacetic acid (200 mg/l) in the reaction cocktail, 2 ml was of it was diluted with 4 ml of distilled water and 0.6 ml of FeCl3 (0.2 %). Optical density of the reaction mixture was taken at 700 nm after 10 min of incubation. Gallic acid was appropriated as a standard [35].
Phosphomolybedenum assay
The methodology of Prieto et al. [36] was used to assess the antioxidant capabilities of the plant samples. DMSO was used as a solvent for the preparation of plant sample and for serial dilutions. Accordingly, 0.2 ml of the plant sample was mixed with 2 ml of the reagent solution (prepared by adding 28 mM Na3PO4 and 0.6 M H2SO4 with that of 4 mM ammonium molybdate). After incubation at 90 °C in a water bath for 80 min the reaction mixture was cooled down at room temperature and optical density was recorded at 765 nm.
In vivo CCl4 induced hepatotoxicity in rats
Animals
Six weeks old (180–200 g) Sprague–Dawley-male rats were acquired from the Animal House situated at the Quaid-i-Azam University Islamabad, Pakistan. The animals were maintained at 24 ± 3 °C with a 12 h dark/light cycle at Primate Facility of the Quaid-i-Azam University Islamabad, Pakistan. The animals were bred with basal diet with water ad libitum and were sustained in standard laboratory conditions. The basal diet was composed of 20 % protein (casein), 10 % sucrose, 5 % corn oil, 2 % choline chloride, 1 % vitamin mixture, 3.5 % salt mixture and 5 % fibers (cellulose). The remainder was corn starch up to 100 %. Prior to the research propagation ethical approval (Bch#0275) was obtained from Ethics Committee Quaid-i-Azam University Islamabad. Experiments on animals were performed in accordance with the guidelines of the institute of animal ethical committee, NIH, Islamabad.
Acute toxicity test
Six week old Male Sprague Dawley rats were kept in fasting conditions for overnight with just water availability. Three animals were intra-gastrically administered with dose-of 50 mg/kg bw and were monitored for mortality rate for 2 weeks. DMSO was used as a solvent for the preparation of extract/fraction samples. No initial progression of toxicity was observed, but the methodology was subsequently followed with augmented amount of doses i.e., 100,-200, 400, 1000, 2000, 3000 and 4000 mg/kg bw of the maximum dose of the extract. Three animals were used for each treatment. Mortality was not noticed at the highest dose of 4000 mg/kg, thus 200 and 400 mg/kg bw doses were selected for the evaluation of hepato-protective propagation activities [37].
Experimental design
Eight groups (each having 7 rats) of Sprague–Dawley male-rats were made to contemplate the hepatoprotective effects of ANM. Group I was taken as a control and remained untreated. Olive oil and DMSO (1:1; v/v) at a dose of 1 ml/kg bw was administrated to the Group II orally. Group III was intraperitoneally administered with CCl4 (1 ml/kg bw; CCl4:Olive oil; 2:8 v/v). An amount of 50 mg/kg bw of silymarin (in DMSO; w/v) as a reference chemical was given to Group IV after 24 h of CCl4 treatment. Group V and VI were initially treated with CCl4 and then after 24 h, they were orally administered with 200 and 400Lmg/kg bw (in DMSO) of ANM. Animals of Groups VII and VIII received only ANM in DMSO at a dose-of-200 and 400 mg/kg bw, respectively. Once the experiment was accomplished, weight of the animals was recorded. Then the animals were euthanized after ether anesthesia. Blood was collected from heart (atrium), immediately removed the liver and transferred to the chilled saline solution and weighted. One liver portion was prepared for-histopathalogical studies, while the second portion was preserved in liquid nitrogen-and stowed at −80 °C for-added enzymatic and DNA damage investigation.
Biochemical studies of serum
Different liver marker enzymes were used to perform the liver function tests such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) and bilirubin in serum was evaluated by standard procedure of AMP Diagnostic kits (Stattogger Strasse 31b 8045 Graz, Austria). Serum level of high density lipoproteins (HDL), low density lipoproteins (LDL), total cholesterol and triglycerides were estimated by following the procedures available on the kits.
Biochemical studies of liver
Small portion of liver was homogenized by the use of homogenizer. Volume of homogenate was noted and accordingly the 10× homogenate was prepared by mixing with EDTA (1 mM) and phosphate buffer (100 mM). The homogenate was placed for 20 min at 4 °C and then centrifuged at 12,000 × g in order to assemble the supernatant. The protein concentration in theLsupernatant was evaluated-according to the method of Lowry et al. [38] using BSA as standard.
Catalase assay (CAT)
On the basis of decomposition of hydrogen peroxide CAT actions were analyzed by following the method of Chance and Maehly [39]. In short, 2.5 ml of phosphate buffer (50 mM; pH 6.6) as a solvent for of the supernatant (0.1 ml) and 0.4 ml of H2O2 (5.9 mM; pH 6.0), change in absorbance was noted for one minute at wavelength of 240 nm in a-spectrophotometer.-A change of 0.01 in-absorbance for one-minute was taken as one unit of CAT activity.
Peroxidase assay-(POD)
Activities of POD were evaluated based on guaiacol peroxidation [39]. To find the POD activity of supernatant the reaction mixture was prepared by adding 0.3 ml of guaiacol (20 mM), 0.6 ml of H2O2l(40 mM), and 0.2 ml of the supernatant to 3.5 ml of phosphate buffer (50 mM, pH 5.6) serially. Change in absorbance of the reaction solution at 470 nm was observed for one minute. One unit of POD activity was defined as an absorbance-change-of 0.01 of the-solution-per minute.
Superoxide dismutase assay-(SOD)
For the assessment of hepatic SOD activity, to the reaction blend after the centrifugation (2500 × g for 20 min-followed by 10,000 × g for 20 min) 300 μl of supernatant along with 1.4 ml of sodium-pyrophosphate buffer (0.053 mM; pH 7.3), 0.2 ml of phenazine-methosulphateL(188 μM) were added to the reaction mixture. To initiate the reaction 0.4 ml of NADH (785 μM) was added and-then stopped after 3 min by adding 2 ml of glacial acetic acid. The color intensity was measured at wavelength of 560 nm [40].
Glutathione-S-transferase assay (GST)
The principle of this methodology is based on the interaction of GSH andL1-chloro-2,4-dinitrobenzeneL(CDNB)Land the subsequent conjugate made is measured with a spectrophotometer [41]. Shortly, 1.575 ml of phosphate buffer (0.2 M, pH 6.8), 0.3 ml of GSH (2LmM), 0.028 ml of 1-chloro-2,4-dinitrobenzeneL(CDNB; 1LmM) were blended followed by 1.2 ml of supernatant. GST activity was estimated by noting-the change in absorbance at wavelength of 340 nm with a molar extinction-coefficient of 8.6 × 103 M−1 cm−1.
Glutathione reductase assay (GSR)
GSR analysis was done by following the method of Carlberg and Mannervik [42]. Supernatant samples (0.2 ml) were amalgamated with 0.1 ml of EDTA (0.5 mM), 1.68 ml of phosphate buffer,-0.04 ml of oxidized glutathione (1 mM), and 0.1 ml of NADPH (0.1 mM). The OD was measured atL340 nm after blending. GST activity was estimated as nM NADPH oxidized/min/mg protein, using a molar extinction coefficient of 8.22 × 103 M−1 cm−1.
Glutathione peroxidase-assay (GSH-Px)
The activity of glutathione peroxidase was assayed as described earlier [43]. Hepatic-supernatant (1.2 ml) of every rat was mixed with 1.46 ml phosphate buffer (0.2 M; pH 7.9), 0.1 ml of EDTA (2 mM), 0.2 ml of sodium azide (2 mM), 0.06 ml of glutathione reductase (1 IU/ml), 0.08 ml of GSH (2LmM), 0.2 ml of NADPH (0.4 mM),L0.01 ml of H2O2 (0.25 mM). The absorbance was noted at 340 nm, and GSH-Px activity was assessed by using a molar extinction coefficient of 6.23 × 103 M−1 cm−1.
Reduced glutathione assay (GSH)
The concentration of reduced glutathione was assessed by spectrophotometric technique [44]. The basis of this method-is based on the breakdown-of 1,2-dithio-bis nitro-benzoic acid (DTNB) by sulfosalicylic acid, as a result yellow-color is produced. The yellow color produced was read immediately at 412 nm on a-spectrophotometer and was expressed as μM GSH/g tissue.
Estimation of lipid-peroxidation assay (TBARS)
The analysis of lipid peroxides as thiobarbituric acid reactive substances (TBARS) was done by following protocol of Iqbal and Wright [45]. Shortly after the ascorbic acid and ferric chloride were mixed with the supernatant it was put in a shaking water bath at 37 °C for 1 h, after that trichloroacetic acid was added. 2.0 ml 0.69 % thiobarbituric acid was added and all the tubes were placed in a water bath to boil for 10 min, and then transferred to crushed ice bath before centrifuging at 2500 × g for 15 min. The quantity of TBARS manufactured in each of the samples was evaluated by measuring the OD of the supernatant atI535 nm against a reagent blank. The outcomes were expressed as nM TBARS/min/mg tissue-at 37 °C using a molar extinction coefficient of 2.58 × M−1 cm−1.
Hydrogen peroxide assay (H2O2)
Oxidation of phenol red in the presence of H2O2 mediated horseradish peroxidase was done to assay hydrogen peroxide (H2O2) [46]. Horse radish peroxidase (8.5 units), phenol red (0.29 nM), dextrose (6.6 nM) and phosphate buffer (0.04 M; pH 8.0), comprising solution was used for the suspension of homogenate sample (3.0 ml) and incubation was done at 37 °C for 60 min. A volume of 0.01 ml of NaOH (1 N) was mixed following centrifugation at 800 × g for 5 min. Supernatant was checked for absorbance at 615 nm against a reagent blank. H2O2/min/mg tissue-based concentration was shown on the basis of H2O2 oxidized phenol red of standard curve.
Nitrite assay
Equal quantity i.e. 100 μl each of both 5 % ZnSO4 and 0.3 M NaOH was used to remove the proteins of tissue samples (100 mg), which were then centrifuged at 6400 g for 15–20 min. The supernatant was collected by centrifugation at 6400 × g for 20 min. To the cuvette Griess reagent (1.0 ml) was added, and the spectrophotometer was blanked at wavelength of 540 nm, then the supernatant was added to the cuvette having Griess reagent. Nitrite concentration was computed using a standard curve for sodium nitrite [47].
Tissue protein estimation
The method of Lowry et al. [38] was used to determine the total amount of soluble protein in tissue homogenate. To the tissue homogenate 200 μl of 1.1 M potassium phosphate buffer-(pH 8.0) was added to dilute the tissue sample. A volume of 1 ml of alkaline copper-solution was added to-this blend, and placed at room temperature. After incubation for 20 min, 200 μl of Folin-Ciocalteu phenol reagent was added. Reaction tubes containing the test mixtures were then vortexed and incubated again atL37°C for 20 min. At 650 nm optical density was measured spectrophotometrically. Total soluble proteins of the tissue samples-were then detected using standard curve of bovine serum-albumin.
Histopathological studies
Histopathological examination was performed for the hepatic injuries. Samples were paraffin embedded, after fixation in fixative solution consisting of: formaldehyde 20 %, absolute alcohol 70 %, glacial acetic acidL10 %. Sections were made at 4–5 μm, stained with hematoxylin/eosin and were examined under light-microscope-(DIALUX 20 EB).
Statistical analysis
The parametric data were expressed as the mean- ± SD for the 07 rats in each group. Statistix 8.1 software was used for statistical analysis. After estimating the normality test of all the groups for various parameters the data was analyzed for one way analysis of variance to compute the differences between groups. Post hoc testing was carried out for inter-group comparisons using the least significant difference (LSD) test at P > 0.01.