Available online at www.pharmresfoundation.com ISSN: 2229-3787 ...

More documents

Recommendations

Info

Animals were randomized and divided into seven groups (1-7) of six mice in each group. Animals of Group-1 (control) received only vehicle (1 % Gum Acacia in distilled water). Animals of Group- 2 (Paracetamol-control) received acetaminophen 250 mg/kg only. Animals of Group-3 fed with acetaminophen 250 mg/kg and the standard drug Silymarin 50 mg/kg. Animals of Group-4 and 5 were fed with a single dose of acetaminophen 250 mg/kg and CIME of 200, 300 mg/kg. Similarly, animals of Group-6 and 7 were fed with a single dose of acetaminophen 250 mg/kg and CIPE of 200, 300 mg/kg. Plant extracts were administered three hours after the administration of acetaminophen. All the treatments were done by means of a gavage or gastric tube. The treatments were continued for seven consecutive days and on the eighth day of the experiment, animals were anaesthetized with ether and blood sample was collected by puncturing the retro orbital plexus and serum was separated by centrifugation at 1500 rpm for 10min. The serum was then estimated for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin level using assay kits according to the supplier (AGAPPE Diagnostics Pvt. Ltd., Maharashtra, India) specifications (Sabir and Rocha 2008). The estimation was done by using a photoanalyser instrument (FT-2; AMS); each determination was carried out with a suitable kit (AGAPPE Diagnostics Pvt. Ltd., Maharashtra, India) containing the necessary reagents at a predetermined wavelength for each parameter. Histopathological studies One animal belongs to the treated groups showing maximal activity as indicated by exhibit biochemical parameters from each test, positive Available online at www.pharmresfoundation.com ISSN: 2229-3787 control, hepatotoxin and control groups were selected for this purpose. The animals were sacrificed by ether anaesthesia and the abdomen was cut open to remove the liver, (5mm thick pieces) of the liver were treated in Bouin’s solution (mixture of 75 ml of saturated picric acid, 25 ml of 40% formaldehyde and 5ml of glacial acetic acid) for 12 hours, then embedded in paraffin wax and cut into 5 μm thick sections by using microtome and stained with haematoxylin-eosin dye and mounted in diphenyl xylene. Then the liver sections were observed under microscope for histopathological changes in liver architecture and photo microscopic were observed (100X) including necrosis, steatosis and fatty change of hepatic cells (Shai 2001; Saeed 2002). Statistical Analysis The results were recorded as Mean ± S.E.M. and statistical significance between treated groups with a control group was evaluated by One-way analysis of variance (ANOVA) followed by Dunnett’s t-test (Woodson, 1986). RESULTS Preliminary phytochemical analysis Results of different chemical tests on the CIME showed the presence of phytoconstituents, i.e., reducing sugars, flavonoids, phenolic compounds, alkaloids and gums; whereas CIPE showed a positive test for the presence of steroids and terpenoids. Effect of treatment with acetaminophen on serum ALT activities The estimation of all the biochemical parameters were carried out by using a photoanalyser (FT-2; AMS); serum ALT determination was done with a suitable kit (AGAPPE Diagnostics Reagent) containing the necessary reagents at a pre-determined 67
wavelength 340 nm where as the rate of oxidation of NADH was measured kinetically by monitoring the decrease in absorbance at 340 nm (Murray, 1994). Mice were treated with paracetamol in group 2 alone developed significant liver cell damage as was evident for a significant (P < 0.001) increase in serum alanine aminotransferase enzyme activities. The oral administrations of CIME and CIPE at the different dose levels of 200 and 300 mg/kg (groups 4 to 7), were shown the lower significantly (P < 0.01) activities of marker enzymes and comparable to the group 3 of standard drug silymarin (50 mg/kg; P < 0.01) as shown in Table 1. Effect of treatment with acetaminophen on the serum AST activities In the paracetamol treated group 2; there was a significant (P < 0.001) increase in aspartate aminotransferase activity compared to the normal group 1. AST activity of CIME and CIPE groups of two different doses 200 and 300 mg/kg (groups 4 to 7) significantly (P < 0.01) recovered the level of enzyme activities which is comparable with standard drug silymarin group-3 (P < 0.01) as shown in Table 1. Effect of treatment with acetaminophen on the levels of ALP activities The level of ALP activities were carried out using the kit AGAPPE Diagnostics Reagent analyzed in a photoanalyser (FT-2; AMS). By using a specific reagent at pH 10.3, alkaline phosphatase (ALP) catalyses the hydrolysis of colorless p-nitro phenyl phosphate (p-NPP) to yellow colored p-nitro phenol and phosphate. Change in absorbance due to yellow color formation is measured kinetically at 405 nm and is proportional to ALP activity in the sample (Moss and Henderson, 1994). Mice were treated with paracetamol (group 2) alone developed significant Available online at www.pharmresfoundation.com ISSN: 2229-3787 liver cell damage as was evident from a significant (P < 0.001) increase in serum enzyme levels of ALP compared to the normal group 1. CIME and CIPE groups of two different doses, 200 and 300 mg/kg significantly (P < 0.01 group 4, 5, 7; P < 0.05 group 6), recovered the ALP level as compared to the standard drug silymarin group 3 (50 mg/kg; P < 0.01) as shown in Table 1. Effect of treatment with acetaminophen on the serum bilirubin levels Serum bilirubin levels in paracetamol-induced liver damage were estimated using an analytical kit from AGAPPE Diagnostics Reagent. The assay principle is based on the fact that total bilirubin reacts in the presence of caffeine with diazotized sulfanilic acid to form azobilirubin. The determination of direct bilirubin at 546 nm is proportional to the concentration of bilirubin (Willard and Meites, 1982). A significant (P < 0.001) increase in serum bilirubin level was seen in paracetamol-treated animals in group 2 compared to that of the normal group 1. Bilirubin levels for CIME and CIPE groups of two different doses 200 and 300 mg/kg significantly (P < 0.01 group 4, 5, 7; P < 0.05 group 6) recovered against the paracetamol group 2 and both doses were comparable with the standard drug silymarin at 50 mg/kg (P < 0.01) as shown in Table 1. Histopathological studies Histopathological examination of liver sections of control group 1 and standard group 3 showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and central vein (Fig. 1 and 3). Disarrangement of normal hepatic cells with centrilobular necrosis, vacuolization of cytoplasm and fatty degeneration were observed in the paracetamol treated mice of group 2 in Fig. 2. 68
Page 1 and 2: Available online at www.pharmresfou
Page 3: under vacuum at the range of boilin
Page 7 and 8: where the plant extract showed a hi
Page 9 and 10: study are the consequence of parace
Page 11 and 12: modified prodrug of glutathione. J

Available online at www.pharmresfoundation.com ISSN: 2229-3787 ...

Create successful ePaper yourself

Delete template?

Save as template?