AMELIORATIVE ACTIVITY OF ASCORBIC ACID (AA) ON PARAQUAT (PQ) INDUCED SERUM BIOCHEMICAL ALTERATIONS IN EXPERIMENTAL RATS

1. Post-graduate, Department of Veterinary Pathology, College of Veterinary Science, PVNRTVU, Rajendranagar, Hyderabad-500030, India. 2. Assistant Professor and Head, Department of Veterinary Pathology, College of Veterinary Science, PVNRTVU, Mamnoor, Warangal-506166, India. 3. Professor and Head, Department of Veterinary Pathology, College of Veterinary Science, PVNRTVU, Rajendranagar, Hyderabad-500030, India. 4. Professor, Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, PVNRTVU, Rajendranagar, Hyderabad-500030, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 05 February 2020 Final Accepted: 07 March 2020 Published: April 2020

The present study aimed to investigate the protective effect of ascorbic acid on paraquat induced serum biochemical alterations in experimental rats. Forty-eight (48) male albino Wistarrats were randomized into four (4) groups consisting of twelve (12) in each. Group 1-Control. Group 2 -Paraquat at the rate of 40 mg/kg b.wt. Group 3 -Ascorbic acid at the rate of 250 mg/kg b.wt. Group 4 -Paraquat at the rate of 40 mg/kg b.wt + Ascorbic acid at the rate of 250 mg/kg b.wt. The treatment regimens were administered by oral gavage once daily for twenty-one (21) days. Significantly (P<0.05) increased activity of aspartate transaminase, alanine transaminase, alkaline phosphatase, blood urea nitrogen and serum creatinine and significantly (P<0.05) decreased activity of total protein was observed in group 2 on 7 th and 21 st day of experiment. Cotreatment with ascorbic acid showed a remarkable protection against the parameters investigated possibly via antioxidant defence mechanism.
Ascorbic acid (AA) being an antioxidant, is effective in scavenging oxygen free radicals (Okolonkwo et al., 2014). In the present study, protective effect of AA was evaluated against PQ induced serum biochemical alterations.

Materials and Methods:-Experimental animals:
Forty-eight (48) male albino Wistar rats weighing 200-250 grams were procured from Sanzyme Laboratories Ltd., Hyderabad. The rats were housed in solid bottom polypropylene cages at Ruska Labs, Hyderabad and were maintained in controlled environment (20-22 0 C) throughout the course of experiment. Sterile husk was used as standard bedding material. All the rats were provided with standard pellet diet procured from Vyas Labs, Uppal, Hyderabad and deionized water at ad libitum throughout the experimental period. The experiment was carried out according to the guidelines and prior approval of Institutional Animals Ethics Committee (IAEC-No.02-2019).

Drugs and chemicals:
Paraquat (GRAMOXONE ® -24% w/v solution) was procured from Seed Research and Technology Center, Professor Jayashankar Telangana State Agriculture University, Rajendranagar, Hyderabad which was manufactured by Syngenta India Ltd., Delhi. Ascorbic acid (Vitamin C) as L-ASCORBIC ACID was obtained from S.D. Fine-Chem Ltd., Mumbai, India. Aspen biochemical kits were purchased from Rapid Diagnostics Pvt. Ltd., Delhi to evaluate serum biochemical parameters.

Experimental design:
A total of 48 male albino Wistar rats were randomly divided into four (4) groups consisting of twelve (12) animals in each. Group 1 -Control Group 2 -PQ (@ 40 mg/kg b.wt) Group 3 -AA (@250 mg/kg b.wt) Group 4 -PQ (@40 mg/kg b.wt) + AA (@ 250 mg/kg b.wt) The dose regimens were administered per os (p.o.) once daily for a period of three (3) weeks. The rats were monitored for clinical signs and death.

Serum biochemistry:
Approximately, 2mL of blood was collected from each rat (retro-orbital plexus) through capillary tube into clot promoting vacutainers and allowed to clot for 3-4 hours, later centrifuged at 20,000 rpm for 10 minutes; serum was separated into Eppendorf tubes and stored at 20 0 C. The stored serum samples were used to estimate serum biochemical parameters by using semi-automatic biochemical analyser (star 21plus-Aspen Diagnostics Pvt. Ltd., Delhi). Serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), blood urea nitrogen (BUN) and creatinine were measured spectrophotometrically according to the protocol given by the commercial kits.

Statistical analysis:
Data obtained (serum biochemistry) was subjected to statistical analysis by applying one-way ANOVA and using statistical package for social sciences (SPSS) version 25.0. Differences between the means were tested by using Duncan's multiple comparison tests and significance level was set at P<0.05 (Snedecor and Cochran, 1994).  Table 1.

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The mean values of ALT in different groups (1, 2, 3 and 4) were ranged from 35.84±0.68 to 51.37±4.14 on day 7 th and 37.43±0.52 to 62.46±4.81 on day 21 st of experiment. A significant (P<0.05) elevation in the mean values of ALT were recorded in group 2 when compared with group 1, group 3 and group 4 rat serum samples on 7 th and 21 st day of experiment. The results were presented in Table 2.

Kidney function assays:
In the present study, the mean values of different serum biochemical parameters viz., AST, ALT, ALP, BUN and serum creatinine were significantly (P<0.05) increased whereas TP was significantly (P<0.05) decreased among group 2 rats when compared with group 1 rats on 7 th and 21 st day of experiment which is an indicative of hepatic and renal damage.  Vandenberghe (1995) who had explained the ALP inhibition due to oxidative damage to lipid membranes in the canalicular zone which interferes with the bile flow. According to Sastry et al. (1982) and Das and Mukherjee (2000), the depleted TP values could be due to impaired protein synthesis by liver or increased protein loss via kidney excretion.
According to Evers et al. (1983), an increased mean values of BUN and serum creatinine might be due to AKI because the highest concentration of PQ was observed in kidneys after lungs post intoxication. The PQ induced nephrotoxicity associated with direct tubular toxicity, inflammation, oxidative stress and apoptosis might be the cause for elevated BUN and serum creatinine levels (Kimbrough, 1974 andVale et al., 1987).
Significantly (P<0.05) reduced mean values of AST, ALT, BUN and serum creatinine, numerically reduced mean values of ALP and significantly (P<0.05) increased mean values of TP were observed in group 4 when compared to group 2 rats on 7 th and 21 st day of experiment which could be due to ameliorative effect of AA by protecting critical macromolecules from oxidative damage.

Conclusion:-
In conclusion, the present study revealed that the AA (250 mg/kg) supplementation can effectively ameliorate PQ (40 mg/kg) induced alterations of serum biochemical parameters possibly via antioxidant defence mechanism.
110 Acknowledgement:- The authors are thankful to P V Narsimha Rao Telangana Veterinary University for providing support and necessary facilities to carry out the research work.