HAEMATO-BIOCHEMICAL CHANGES IN SULPHADIMIDINE TOXICITY IN RATS

HAEMATO-BIOCHEMICAL CHANGES IN SULPHADIMIDINE TOXICITY IN RATS HEMANT KUMAR PANDEY, A.H. AHMAD 2, S.P. SINGH 3, K. KANNAN 4 Ph.D. Student, 2 Professor, 3 Prof. & Head, 4 SRF Department of Veterinary Pharmacology and Toxicology, C.V.A.Sc, Pantnagar-26345. Research Paper ABSTRACT Subacute toxicity of sulphadimidine, was investigated in rats following daily, i.p. @ 269, 34.5, 89.67 and 67.25 mg/ kg b.wt. to II, III, IV and V groups, and normal saline in control group I, respectively in rats. Rats did not exhibit any marked changes in their gross behavioural signs and symptoms during the period of study. Sulphadimidine caused significant decrease in all haematological parameters viz, haemoglobin, Packed cell volume, TEC & TLC. All the treated groups showed significant increase in total protein and decrease in albumin. The biochemical parameters such as Serum glucose, total cholesterol, urea, creatinine, calcium and enzymic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were increased at higher dose level in sulphadimidine treated groups in comparison to control group. Key words: Sulphadimidine, haemato-biochemical changes, subacute toxicity, rats INTRODUCTION Sulphadimidine is a sulphonamide with antimicrobial activity against Gram-positive cocci (hemolytic streptococci, pneumococci and some staphylococci), some Gram-negative bacteria (like Pasteurella spp., E. coli, Salmonella spp.) Rickettsiae and protozoa (Prescott and Baggot, 993). Sulphadimidine is rapidly absorbed from the site of application and slowly excreted by the organism, so in most animals a single daily dose is sufficient. In addition to adverse effects like crystalluria, hepatic necrosis and hypoprothrombinemia in dogs (Adams, 200) following administration of therapeutic dose, there is likelihood of toxic effects of sulphadimidine after repeated administration of therapeutic and high dose in animals. Further, there is no published report on hematological and biochemical alterations of sulphadimidine in rats. Keeping in view the clinical significance of sulphadimidine in animals, the present study was planned to evaluate the effect of sulphadimidine toxicity in rats. MATERIALS AND METHODS Experimental animals and drug Albino rats (Sprague Dawley) of 2 to 2.5 months of age, weighing between 50 to 200 gm, were procured from Laboratory Animal Resource Centre, IVRI, Izatnagar. The animals were kept in plastic cages and were acclimatized for two weeks in the experimental laboratory animal shed of the Department of Pharmacology, College of Veterinary and Animal Sciences, Pantnagar, under standard managemental conditions. Standard rat feed and water were provided ad libitum throughout the experimental period. The Institutional Animal Ethic Committee approved the experimental protocol. Sulphadimidine (Sulfadimidine- 333mg/ml solution) was procured from M/S Indian Immunologicals, Ltd. Hyderabad. Experimental design Rats were randomly divided into five groups (four test groups and one control group) with 8 rats in each group. Experiment was designed as per OECD guidelines. Repeated dose of sulphadimidine was obtained from acute toxicity study (LD 50 = 2690mg/kg).Group I was kept as control. Group II, III, IV and V received 269 mg/kg (LD 50/0 ), 34.5mg/kg (LD 50/20 ), 89.67mg/kg (LD 50/30 ) and 67.25 mg/ kg (LD 50/40 ) of sulphadimidine respectively. Rats were kept under observation for consecutive 28 days. On 29 th day, all the rats were sacrificed, blood samples was collected to evaluate the hematological and biochemical parameters. Blood samples collected were subjected for serum AST, ALT, ALP, Glucose, Urea, Creatinine, Cholesterol, Total proteins and Albumin estimation by using Diagnostic Kits procured from Span Diagnostics Ltd., Surat. Haematological parameters were estimated in blood as per the method given by Jain (986). Data obtained in the present study were statistically compared by analysis of variance (Snedecor and Cochran, 989). RESULTS AND DISCUSSION Table represents the data on haematobiochemical profile following subacute study sulphadimidine in rats. In the present investigation, the haematological parameters viz, Packed cell volume, hemoglobin, total erythrocyte count and total leukocyte count in the treated groups decreased significantly (p<0.05) as compared to control. The decrease in the Hb, PCV and TLC might be attributed to the fact that pooling of the circulating blood cells takes place in the spleen. All the treatment groups showed significantly (p<0.05) increase in values of total protein and decrease in values of albumin as compared to control. There exists significant changes in the level of globulin of groups I and II. An elevation in total serum proteins might be attributed to enhanced release of proteins from Journal of Veterinary Pharmacology and Toxicology/December 200/Vol.9/Issue -2/5-52 5

Pandey et al. Table : Effect on hemato-biochemical profile following daily i.p. administration of sulphadimidine for 28 days in rats. Parameters Treatment One way Group I Group II Group III Group IV Group V ANOVA Dose of sulphadimidine (mg/kg) --- 269 34.5 89.67 67.25 CD Value TEC(0 6 /µl) 7.82±0.6 5.02±0.09 a 6.02±0.09 a 6.85±0.4 a 7.03±0.3 a 0.36 TLC(0 3 /µl) 9.37±0.07 7.00±0.0 a 7.77±0.3 a 8.32±0.6 a 8.90±0. a 0.34 PCV(%) 55.38±0.32 47.3±0.52 a 50.25±0.45 a 52.38±0.50 a 54.00±0.33 a.24 Hb(g/dl) 3.38±0.8 9.05±0.2 a 9.34±0.25 a 0.78±0.4 a.34±0.5 a 0.55 Total protein (g/dl) 5.79±0.04 7.25±0.02 a 6.87±0.04 a 6.48±0.04 a 6.43±0.03 a 0. Albumin (g/dl) 4.59±0.03 3.53±0.02 a 3.5±0.03 a 3.76±0.07 a 4.20±0.02 a 0. Globulin (g/dl).20±0.06 3.72±0.03 a 3.36±0.05 a 2.72±0.07 2.23±0.04.52 A/G 3.87±0.20 0.94±0.0 a.05±0.02 a.39±0.06 a.89±0.04 2.03 Urea (mg/dl) 50.34±0.77 52.87±0.7 a 52.43±0.54 a 5.75±0.54 5.46±0.66.86 Creatinine (mg/dl).35±0.02.6±0.0 a.5±0.0 a.45±0.0 a.29±0.0 0.03 Glucose (mg/dl) 95.02±0.29 04.59±0.46 a 99.3±0.73 a 97.30±0.3 a 96.±0.4.35 Cholesterol (mg/dl) 35.67±0.20 38.30±0.5 a 37.76±0.35 a 37.6±0.20 a 36.33±0.24 0.66 Calcium (mg %) 9.05±0.7 6.00±0.4 a 6.43±0.5 a 6.65±0.05 a 7.94±0.27 a 0.49 AST (U/L) 46.3±0.72 52.25±.00 a 49.50±0.8 a 46.63±0.5 45.3±0.52 2. ALT (U/L) 64.5±.34 73.5±0.5 a 68.75±0.53 a 66.3±0.6 64.75±0.45 2.8 ALP (U/L) 54.95±0.7 45.75±.0 a 47.40±0.25 a 46.84±0.49 a 5.36±0.6 3.59 Values in table are Mean ± S.E. (n = 8), a= Significant (P<0.05) as compared to group I within same row liver into blood circulation under sulphadimidine induced stress in rats. All the treatment groups showed significant (p<0.05) increase in the level of creatinine. An increase in the serum creatinine level gave an indication of probable kidney damage (Cornelius, 989). The inhibitory effect on renal blood flow might be the major factor responsible for an increase in the serum creatinine level in subacute toxicity study of sulphadimidine in rats. In this investigation serum urea level was increased in sulphadimidine treated groups at higher dose level as compared to control group. The levels of glucose and cholesterol in the groups II, III and IV increased significantly (p<0.05) as compared to control. Increase in serum glucose level might be due to higher oxidative stress in the sulphadimidine treated groups as consequence to inhibition of insulin release from pancreatic cells in sulphadimidine treated rats. The serum calcium level in subacute study was found to be decreased significantly in all sulphadimidine treated groups as compared to control. Reduction in serum calcium level might have occurred due to both decreased absorption and metabolism or its mobilization from the calcified tissues in sulphadimidine treated rats. The enzymic activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was increased at higher dose level in sulphadimidine treated groups in comparison to control group. The level of alkaline phosphatase (ALP) was decreased in sulphadimidine treated groups. Increased activities of serum AST and ALT enzymes are considered as indicators of hepatic damage. The rise in activity of serum AST and ALT enzymes has been attributed to the damaged structural integrity of the liver. Being cytoplasmic in location, these enzymes are released into systemic blood circulation 52 Journal of Veterinary Pharmacology and Toxicology/December 200/Vol.9/Issue -2/5-52 after cellular damage of hepatocytes (Ahmed and Khater, 200). The results observed following intraperitoneal administration for 28 days indicate that the sulphadimidine at the dose rate of 67.25-269mg/kg produces mild to moderate anemia and hepatotoxicity in rats. ACKNOWLEDGMENTS The authors are thankful to M/s Indian Immunologicals for providing drugs for this study. The authors gratefully acknowledge the technical assistance and facility provided by Director, experiment station, G.B. Pant University of Agriculture and Technology and Dean, College of Veterinary and Animal Sciences, Pantnagar. REFERENCES Adams. H.R. (200). Veterinary Pharmacology and Therapeutics 8 th Edn., Blackwell Publishing, Iowa, ISBN: 0-838-743-9.pp:796-806. Ahmed, M.B. and Khater, M.R. (200).The evaluation of the protective potential of Ambrosia maritima extract on acetaminophen-induced liver damage. J. Ethnopharmacol. 75: 69-74. Cornelius, C.E. (989). Liver function. In: Kaneko, J. J. ed. Clinical biochemistry of domestic animals. Academic press Sandiego, New York. pp. 386. Jain, N.C. (986). Schalm s Veterinary Haematology. 4 th Ed. Philadeiphia, Lea and Febringer. Prescott, J.F., and Baggot, J.D. (993). Sulfonamides, trimethoprim, ormetoprim and their combinations. In Antimicrobial Therapy in Veterinary Medicine, 2 nd ed., Ames: Iowa State Univ Press. pp.229-25. Snedecor. and Cochran (989). Statistical Methods. 8 th Edition. Iowa State University Press, Iowa.

ACUTE TOXICITY STUDY OF PANAX GINSENG ROOT IN RATS K. KANNAN, A.H. AHMAD 2, S.P. SINGH 3, H.K. PANDEY 4 SRF, Professor, 3 Prof. & Head, 4 Ph.D scholar. Department of Veterinary Pharmacology and Toxicology, C.V.A.Sc, Pantnagar-26345 Corresponding author: E-mail: dr.kannan09@gmail.com Research Paper ABSTRACT Acute toxicity study of ethanolic extract of Panax ginseng root was evaluated following its single oral dose @ g/kg, 2g/kg and 3g/kg. b.wt., for two weeks period in experimental albino rats. No apparent clinical manifestations and gross lesions in body organs of rats were recorded. A significant (P<0.05) decrease in Hb was observed in groups (ethanolic extract @ 2 and 3g/kg) as compared to control group. Serum glucose was significantly increased in groups (ethanolic extract @ 3g/kg). Groups III and IV (ethanolic extract @ 2 and 3g/kg) showed significant difference in serum creatinine, cholesterol, and ALT as compared to control group. Significant (P<0.05) increase was observed in the value of serum AST in all treatment groups. Rats of all groups showed no significant difference, but group IV (ethanolic extract @ 3g/kg) showed significant (P<0.05) decrease in level of RBC LPO in comparison to the untreated control. Key words: Panax ginseng root, acute toxicity, haematology, biochemical, rats. INTRODUCTION Ginseng is the root of the perennial herbs of Panax ginseng which contain a series of tetracyclic triterpenoid, saponins ginsenosides as active ingredients. It is considered a tonic or adaptogenic that enhances physical performance (including sexual), promotes vitality and increases resistance to stress and ageing. The adaptogenic properties of ginseng are believed to be due to its effects on hypothalamic-pituitary-adrenal axis, resulting in elevated plasma corticotropin and corticosteroids levels. When used appropriately, ginseng appears to be safe (Emilia et al., 2000). To the best of our knowledge, not much report is available with regards to the acute toxicity of ethanolic extract of Panax ginseng root in experimental animals. Therefore, the present study was conducted for the evaluation of toxicity of ethanolic extract of Panax ginseng root. MATERIALS AND METHODS Experimental animals Albino rats (Sprague Dawley) of 2 to 2.5 months of age, weighing between 80 to 20 gm, were procured from Laboratory Animal Resource Centre, IVRI, Izatnagar. The animals were kept in plastic cages and were acclimatized for two weeks in the experimental laboratory animal shed of the Department of Pharmacology, College of Veterinary and Animal Sciences, Pantnagar, under standard managemental conditions. Standard rat feed and water were provided ad libitum throughout the experimental period. The Institutional Animal Ethic Committee approved the experimental protocol. Plant material and preparation of extract The plant materials were procured from JKH Herbs and Spices, Navimumbai. The dried root of Panax ginseng was powdered. The powder was soaked in ethanol for 24 hours with continuous stirring at 40ºC. The mixture was filtered through muslin cloth and whatmann filter paper no., the solvent was concentrated in a rotatory vacuum evaporator at 40-50ºC. The final extract was produced after drying the filtrate in incubator with fan (40ºC) and lyophilized. The percentage of yield of extract was calculated. Experimental design Rats of 2 to 2.5 months of age were randomly divided into four groups (three test groups and one control group) with 3 rats in each group. Experiment was designed as per OECD guidelines and /5 th dose (g.kg - b.w.) of oral LD 50 of Panax ginseng (>5 g.kg -, (Singh et al., 99) was administered in the first group as single oral dose in the form of gastric gavage using a plastic tube fitted to the tip of a plastic syringe. The dose was increased to 2 and 3g.kg - b.w. in the subsequent groups for study of acute toxicity. Rats were kept constantly observed for consecutive 4 days for appearance of specific clinical signs as well as mortality due to plant toxicity. On 5 th day, all the rats were sacrificed. The viscera and internal organs were examined for the gross lesions, samples of blood were collected to evaluate haematological, biochemical and antioxidative parameters. After completion of experiment, all rats were sacrificed and buried scientifically. Blood samples collected were subjected for serum AST, ALT, ALP, Glucose, Urea, Creatinine, Cholesterol, Total proteins and Albumin estimation by using Diagnostic Kits procured from Span Diagnostics Ltd., Surat. Haematological parameters were estimated in blood as per the method given by Jain (986). Comparisons Journal of Veterinary Pharmacology and Toxicology/December 200/Vol.9/Issue -2/53-55 53

Kannan et al. Table Acute toxicity study of ethanolic extract of Panax ginseng root in rats. No. Groups Treatment Parameters Control PRE 000 PRE 2000 PRE 3000 Clinical Observations No abnormalities No abnormalities Slight depression 2 a. Body weight.0 th day 83.33±.67 93.33±3.33 200.00±5.77 26.67±2.02 2. 7 th day 85.00±2.89 96.67±4.4 205.00±8.66 23.67±9.28 3. 4 th day 83.67±2.73 87.83±3.8 99.83±9.53 230.83±.08 b. Organ weight.relative liver weight 2.90±0.07 3.38±0.4 3.09±0.09 2.96 2.Relative kidney weight 0.82±0.0 0.65±0.03 0.75±0.04 0.62±0.09 a 3.Relative brain weight 0.73±0.09 0.80±0.0 0.75±0.04 0.59±0.0 3 Haematology.Haemoglobin (g/dl) 2.67±0.8 2.33±0.8.87±0.8 a.33±0.8 a 2.Packed Cell Volume (%) 45.67±.86 45.00±.73 46.67±2.33 42.67±.86 4 Serum Biochemical profile i.total Protein (g/dl) 7.32±0.07 7.38±0.6 7.48±0.03 7.62±0.20 ii. Albumin (g/dl) 4.6±0.05 4.2±0.09 4.25±0.04 4.2±0.04 iii. Globulin (g/dl) 3.6±0.04 3.7±0.24 3.23±0.07 3.4±0.9 iv. A: G ratio.32±0.02.35±0.4.32±0.04.24±0.07 v. Glucose (mg/dl) 95.44±.25 96.20±3.74 05.20±.8 5.28±2.09 a vi. Cholesterol (mg/dl) 49.93±0.9 53.27±3.53 57.60±2.40 a 58.63±0.68 a vii. Creatinine (mg/dl) 0.66±0.04 0.74±0.04 0.94±0.0 a 0.98±0.0 a 5 Serum Enzymic profile i.ast (IU/L) 48.28±0.66 63.0±0.5 a 63.3±0.54 a 65.35±0.65 a ii. ALT (IU/L) 32.38±.03 34.87±.66 36.3±0.47 a 39.66±0.89 a 6 Antioxidative parameters in RBC i.lpo (nm MDA/ml) 5.92±.06 3.9±0.33 4.53±0.76 2.85±.25 a ii.gsh (nm/ml) 0.85±0.09 0.8±0.09 0.9±0.03 0.8±0.0 Values in table are Mean ± S.E. (n = 3); a = Significant difference (P<0.05) as compared to control group among treated and untreated groups were made employing significant change was observed in protein profile of treated student t test. Statistically significant difference was rats in all the groups. A significant (P<0.05) increase in considered at 5 percent level. level of glucose was found to occur in rats of group IV. Acute hyperglycemia was reported to cause oxidative RESULTS AND DISCUSSION stress by various mechanisms in man (Marfella et al., Table depicts the data of clinical, 200) and rats (Kyselova et al., 2002). A significant haematobiochemical and oxidative parameters. Administration of ethanolic extracts (, 2 and 3g/kg b.wt) of Panax ginseng as a single dose to groups II, III and IV respectively, did not produce any apparent toxic signs in rats. Findings are in accordance with the findings of Hess et al. ( 982) where no toxic effect were recorded in beagle dogs fed with 0,.5, 5 or 5 mg ginseng extract per kg of body weight per day for 90 days. In the present study, significant increase (P<0.05) in relative weight of kidney was noticed in rats treated with Panax ginseng (3g/kg.b.wt). This increase in kidney weight may be attributed to proliferation of smooth endoplasmic reticulum. No significant change was observed in value of PCV as compared to control in groups II, III, IV, which received the single oral dose of Panax ginseng. But significant decrease in the value of hemoglobin was observed at higher dose levels. Haemolysis and shrinkage in RBC might be the reason for decline in haemoglobin in treated rats. No (P<0.05) increase in serum cholesterol level was recorded in groups III and IV. Liver is the major site of cholesterol, bile acid and phospholipids synthesis and metabolism. Hepatic cholesterol homeostasis is maintained by equilibrium between the activities of hydroxyl methyl glutaryl CoA (HMG-CoA) reductase and that of acyl CoA, cholesterol acyl transferase (Hochgraf et al., 2000). Thus, alteration in cholesterol level might have been due to effect on metabolism of cholesterol in this study. The serum concentrations of creatinine in groups III and IV were significantly higher (P<0.05) as compared to control group (Table ). Higher level of creatinine is suggestive of nephrotoxic effect (Cornelius, 989). A significant increase in the activity of serum AST in treated groups might be due to hepatic damage caused by Panax ginseng in this study. The rise in serum levels of ALT and AST activity has been attributed to the loss of structural integrity of hepatocytes as these enzymes are located in the 54 Journal of Veterinary Pharmacology and Toxicology/December 200/Vol.9/Issue -2/53-55

Therapy of endometritis in cows cytoplasm and are released into circulation after cellular damage (Ahmad and Khater, 200). There was significant reduction in lipid peroxidation (LPO) RBC status in group IV in comparison to control which suggested that Panax ginseng did not produce oxidative stress in erythrocytes of Panax ginseng treated rats. The reduction in LPO in RBCs in the present study evidenced that the extract administration at higher doses resulted in the decline in production of malondialdehyde (MDA). No significant changes were observed in GSH profile of RBC tested (Table ). It could be concluded from acute oral toxicity study of Panax ginseng in rats that it produces mild haemotoxic and moderate hepatotoxic effect at the dose level as high as 2g.kg - b.w. p.o. Panax ginseng did not elevate the oxidative stress in erythrocytes. ACKNOWLEDGMENT The authors are thankful to Life Science Research Board, New Delhi for providing financial support and technical assistance and other facility provided by Director, experiment station, G.B. Pant University of Agriculture and Tech and Dean, college of veterinary and animal sciences, Pantnagar, for this study. REFERENCES Ahmed, M.B. and Khater, M.R. (200).The evaluation of the protective potential of Ambrosia maritima extract on acetaminophen-induced liver damage. J. Ethnopharmacol. 75: 69-74. Cornelius, C.E. (989). Liver function. In: Kaneko, J. J. ed. Clinical biochemistry of domestic animals. Academic press Sandiego, New York. pp. 386. Emilia Nocerino, Marianna Amato, Angelo A. IzzoU. (2000). The aphrodisiac and adaptogenic properties of ginseng. Fitoterapia. 7:S-S5. Hess, F. G., Jr., Parent, R. A., Stevens, K. R., Cox, G.E. and Becci, P. J. (983). Effects of subchronic feeding of ginseng extract G5 in beagle dogs. Food Chem.Toxicol. 2: 95-97. Hochgraf, E., Cogan, U. and Shoshama, M. (2000). Dietary oxidized linoleic acid enhances liver cholesterol biosynthesis and secretion in rats. J. Nutr. Biochem. : 76 80. Jain, N.C. (986). Schalm s Veterinary Haematology. 4 th Ed. Philadeiphia, Lea and Febringer. Kyselova, P.; Zourek, M.; Rusavy, Z.; Trefil, L. and Racek, J. (2002). Hyperinsulinemia and oxidative stress. Physiol. Res. 5: 59-595. Marfella, R.; Quagliaro, L.; Nappo, F.; Ceriello, A. and Giugliano, D. (200). Acute hyperglycemia induces an oxidative stress in healthy subjects. J. Clin. Invest. 08: 635-636. Singh N., Verma P., Mishra N and Nath R. (99). A comparative evaluation of some anti-stress agents of plant origin. Indian J.f Pharmacol. 23: 99-03. Journal of Veterinary Pharmacology and Toxicology/December 200/Vol.9/Issue -2/53-55 55