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The present study was designed to investigate the effect of Oroxylum indicum …


Biology Articles » Ethnobiology » Hepatoprotective effect of root bark of Oroxylum indicum on carbon tetrachloride (CCl4) - induced hepatotoxicity in experimental » Materials and Methods

Materials and Methods
- Hepatoprotective effect of root bark of Oroxylum indicum on carbon tetrachloride (CCl4) - induced hepatotoxicity in experimental

Procurement of Plant material and extraction procedure

The fresh root bark of Syonakh was collected from Vanaushadhi Ektrikaran Udyan, Ahwa, Dang forest, Gujarat, India. The authentification of this plant was established by the taxonomist of Gujarat Ayurved University, Jamnagar, India. The voucher specimen (#404) was deposited in the Department of Pharmacognosy and Phytochemistry at L. M. College of Pharmacy, Ahmedabad. The root bark was sun dried and powdered to 60 mesh (approximately 250 µm diameter; by grinding with a porcelain pestle a shifting through appropriate mesh screens). The powder of root bark first defatted using petroleum ether (remaining post-extraction material constituted 0.32% [w/w] of the original solute [i.e., root bark powder] that underwent the extraction). The residual unextracted material was then air-dried; this defatted powder was then moistened with an ammonia (NH3) solution and then extracted with chloroform (post-extract remaining solute =0.78% [w/w] of pre-extracted material). After, drying the remaining unextracted material was then extracted with ethyl acetate (post-extract remaining solute =1.52% w/w). Finally, the thrice-extracted powder was then extracted with n-butanol (post-extract remaining solute=1.68% w/w). The solvent-specific eluents recovered in each extraction regimen were then air-dried and their corresponding powdered fractions stored in airtight container until usage.  

 

Phytochemical test

Phytochemical analysis of the extract was performed using standard methods. Specifically, the extract was analyzed for the presence of alkaloids (Sim, 1969), flavanoids (Geissman, 1955), saponins (Fischer, 1952), tannins (Robinson, 1964), anthraquinone, and carbohydrates (Freudenberg et al., 1962). Thin layer chromatography was employed to check for the presence of a baicalein. Quantification of the aglucone was performed using reverse phase HPLC (Khandhar et al., 2006, Zaveri et al., 2008) and extrapolation against a standard curve generated using purified baicalein.

 

Drugs and chemicals

All different organic solvents used for extraction were obtained from the S.D. Chemicals Private Limited (Mumbai, India), and were analytical grade. Fresh drug solutions were prepared in 1% carboxymethylcellulose (CMC) for oral administration. Hydrogen peroxide and ciocalteau phenol reagent were obtained from S.D. Fine Chemicals Limited (Mumbai). Trichloroacetic acid, thiobarbituric acid, phosphate buffer, Tris buffer, 5, 5'- dithibis-2-nitrobenzoic acid (DTNB), bovine serum albumin, epinephrine and silymarin were all obtained from Sigma-Aldrich (St Louis, MO). The kits for the estimation of Serum glutamic oxaloacetic transaminase (SGOT), Serum glutamic pyruvate transaminase (SGPT), alkaline phosphatase (ALP) and total bilirubin (TB) were purchased from Span Diagnostics Ltd.

 

Animals 

Wistar albino rats (Zydus Cadila Limited, Ahmedabad, India) of either sex weighing 170-225 g as well as male albino mice weighing 25-30 g were selected for the present study. Animals were provided a standard chow diet (certified Amrut brand rodent feed, Pranav Agro Industries, Pune, India) and filtered tap water was given ad libitum. The animalswere maintained under standard that was freely available under standard condition of a 12 h dark-light cycle, 60±10% humidity and a temperature of 21.5 ±10c. Coprophagy (and thus re-ingestion of any drug) was prevented by keeping the animals in cages with gratings on the floors. The distribution of animals in the groups, the sequence of trials and treatment allotted to each group was randomized. Freshly prepared solutions of drugs or chemicals were used throughout the study. After completion of the experiments, animals were sacrificed by over-anesthetization with ether. All experiments complied with University guidelines for animal experimentation. Throughout the entire study period, the rats were monitored for growth, health status, and food intake capacity to be certain that they were healthy.

 

Methodology 

The animals were randomly divided (to assure equal distribution of weights) into the following groups containing six animals each.

Group 1: (control) animals received only aqueous suspension of 1% CMC as vehicle   without any treatment once daily for seven days.

Group 2: (CCl4   treated) animals were intoxicated with CCl4 in arachis oil (1:1 v/v) (1 ml/kg of b.w., i.p., and twice a week, on 3rd and 6th day). The animals were sacrificed 24 h after the last CCl4   treatment.

Group 3: (Standard) CCl4 treated animals were received silymarin (25 mg/kg of b.w. suspended in 1% CMC solution, p.o.) once daily for seven days.

Group 4: (Drug treated) CCl4 treated animals were received 50% alcoholic extract and petroleum ether, chloroform, ethyl acetate, and n-butanol fractions (100 & 300 mg/kg of b.w., p.o.) once daily for seven days.  

 

Carbon tetrachloride (CCl4) - induced hepatotoxicity in experimental animals (Sarmistha et al., 1998)

Carbon tetrachloride (CCl4) (1 ml/kg body weight, twice a weak on 3rd and 6th day, i.p.) - induced acute hepatic necrosis in animals. Blood samples were collected separately under ether anesthesia through retro-orbital plexus 24 h after the last CCl4 injection. Blood was centrifuged at 2000 rpm for 30 min to separate the serum and analyzed for the assay of following marker enzymes and total protein. All the tests were carried out using serum diagnostic kits supplied by Span Diagnostic Ltd. Animals were sacrificed to collect the liver tissue for estimation of protein content, antioxidant enzyme activity, lipid peroxidation and for histopathological observations.

 

Assessment of liver function:

Effect of drug administration on assay of serum enzymes 

Measurement of Serum glutamic oxaloacetic transaminase (SGOT), (Reitman and Frankels methods, 1957), Serum glutamic pyruvate transaminase (SGPT), Reitman and Frankels methods, 1957), alkaline phosphatase (ALP)(Kind and Kings Method, 1954) and total bilirubin (TB) levels (Malloy and Evelyn Method, 1937) was carried out. Protein content (PR) was estimated in liver homogenate by the method of Lowry et al., (1951). 

 

Effect of drug administration on liver antioxidant enzymes and on lipid peroxide levels

Liver in each case was dissected out quickly, blotted off blood, washed with ice-cold saline and a 10% homogenate was prepared in phosphate buffer (PH 7). The homogenate was centrifuged at 3000 rpm for 15 min at 4 0C and the supernatant was used for the estimation of the following parameters. The total protein concentration in each sample was determined (Lowry et. al., 1952). The effects of theroot bark extract on the activity of the antioxidant enzymes superoxide dismutase (SOD; in terms of mU/mg protein) (Misra and Frodvich, 1973), catalase (CAT; as U/min/mg protein) (Aebi, 1974) and on the levels of reduced glutathione (GSH; as µmole/mg protein) (Beutler et. al., 1963) in the homogenate were assayed. The levels of malondialdehyde (MDA) in each sample were estimated (expressed as µmole thiobarbituric acid reactive substances [TBARS]/mg protein) at 535 nm in a Shimandzu UV Spectrophotometer (Shimadzu, Japan) (Kiso et. al., 1984).

SOD activity in the samples was determined by mixing 0.1 ml of sample with 0.1 ml of EDTA (1 x 10-4 M), 0.5 ml of carbonate buffer (pH 9.7), and 1 ml of epinephrine (3 x 103 M) (Sigma). The optical density of the adrenochrome was assessed at 480 nm at 30 sec intervals for a total of 3 min. SOD activity was expressed as mU/mg of protein. One unit of activity was defined as the enzyme concentration required to inhibit the chromogen produced, by 50%, in one minute under the defined assay condition.

Catalase activity in each sample was measured by assessing the decomposition of hydrogen peroxide (H2O2) at 240 nm. In a cuvette, 50µl sample was mixed with 2.95 ml of reaction buffer (0.05 M phosphate buffer [pH 7.0] containing 30 mM H2O2) and the absorbance was measured at 15 sec intervals for 3 min. As the optical density measured reflects the peroxide concentration in the cuvette, the activity of catalase in the 3 min period was deduced and expressed as mM H2O2 consumed/mg tissue/min.

Reduced glutathione (GSH) content in each sample was measured after initial precipitation of proteins with 10% chilled trichloroacetic acid. After 30 min incubation, the samples were then centrifuged at 1000 g for 10 min at 40C. The GSH levels in the supernatant were then determined by mixing 0.5 ml of the material with 2.0 ml 0.3 M phosphate buffer (pH 7.0) and 0.25 ml DTNB reagent (40 mg/l00 ml in 1% sodium citrate buffer), and then measuring the absorbance at 412 nm. Standard solutions containing different concentrations of GSH were prepared in parallel to generate a standard curve. Results are expressed as µmoles of GSH/mg of protein.

The levels of malondialdehyde (MDA, representative of peroxidative damage to cell membranes) were measured by mixing 2 ml of 5% suspension of recovered, samples (in 0.1 M phosphate-buffered saline [pH 7.4]) with 2 ml of a 28% trichloroacetic acid solution. After thorough mixing, the mixture was then centrifuged at 10,000g at 4°C for 5 minutes and the supernatant was separated for estimation of MDA. For this, 4 ml supernatant was mixed with 1 ml of 1% thiobarbituric acid solution (TBA), and heated at 100°C for 60 min. The mixture was then cooled to room temperature and the absorbance was measured spectrophotometrically at 532 nm. After accounting for background absorbance using buffer blanks, the total TBARS (TBA-reactive substrate) concentration in each sample was derived from the TBA extinction coefficient Є = 1.56 x 105 M-1 cm-1. The level of MDA in each sample was calculated and data was expressed in terms of nmoles of MDA/mg of protein in each sample.

 

Histopathological studies

On day 7, the final day of the treatments, all the animals were sacrificed by overdose of ether anesthesia and several tissues recovered during necropsy for use in histopathological analyses. The liver tissues from both control and treated (standard and fractions) were preserved in 10 % formalin solution. Thereafter, after embedding in paraffin, 6 µm thick sections were cut and then stained with haematoxylin to permit histological examination. All tissues were then assessed for any morphological changes like necrosis, ballooning degeneration, fatty changes or inflammation of lymphocytes under a photomicroscope. Photomicrographs of representative sections were taken at 10X magnification using a Trinocular Research Zeisss Microscope (Gottingen, Germany) for histopathological observation.    

 

Statistical analysis  

All the results were expressed as Mean ± SEM. The significance of difference between mean values for the various treatments was tested using one way analysis of variance (ANOVA) followed by Tukey's multiple range test (Bolton, 1997). Differences between treatment groups were considered as statistically significant at p < 0.05. 


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