Effect of Garlic Oil on the Levels of Various Enzymes in the Serum and Tissue of Streptozotocin Diabetic Rats

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1 Bioscience Reports, Vol. 21, No. 1, 2001 Effect of Garlic Oil on the Levels of Various Enzymes in the Serum and Tissue of Streptozotocin Diabetic Rats O. C. Ohaeri 1 Receiûed April 17, 2000 Levels of red cell, serum acid, and alkaline phosphatases, serum amylase, alanine and aspartate transferase and bilirubin were examined in streptozotocin-induced diabetic rats treated with garlic oil and compared with the corresponding levels in diabetic control rats, normal rats and normal rats on garlic oil. Values of tissue amylase and total protein were also assessed from the pancreas, liver, and kidney. Treatment of diabetic rats with garlic oil significantly decreased the red cell phosphatase ( pf0.01), serum acid and alkaline phosphatase ( pf0.001) when compared to diabetic control rats. Serum alanine and asparate transferases were significantly ( pf0.001) decreased as well as serum amylase ( pf0.002) in garlic oil treated diabetic rats as compared with diabetic control rats. When treated with garlic oil, however, diabetic and normal rats showed significant increase ( pf0.05) in the amylase levels of the pancrease, liver, and kidney. KEY WORDS: Enzymes; garlic oil; streptozotocin diabetes. INTRODUCTION Garlic oil has been reported to alter phase I and phase II biotransformation enzymes in rats [1]. Adoga and Ibrahim [2] reported on the effects of garlic oil on some biochemical parameters in streptozotocin (STZ) diabetic rats. The protective effects of garlic extracts on liver microsomal membranes against lipid peroxidation is already known [3]. These studies therefore state that the use of garlic oil as treatment for diabetes mellitus could have important implications in situations where there seems to be cell impairment. What may not be obvious to the consumer of garlic oil is the possible toxic effects due to the long ingestion period required. Given in sufficient dosage streptozotocin causes a diabetes-like disease with severe β-cell necrosis [4]. The aims of the present work were therefore to look at the effect of ingesting garlic oil on the levels of carefully selected enzymes and nonenzymic markers of tissue damage such as serum amino transferases (AST and ALT), alkaline phosphatase (ALP) amylase, protein, and bilirubin. Department of Biochemistry, Faculty of Medical Sciences, University of Jos, P.M.B. 2084, Jos, Nigeria. 1 Present address: College of Biological and Physical Sciences, Federal University of Agriculture, P.M.B. 7267, Umudike, Umuahia, Abia State, Nigeria $ Plenum Publishing Corporation

2 20 Ohaeri MATERIALS AND METHODS White male albino rats (Wistar strain) weighing between g were purchased from the Animal House Unit, University of Jos. The rats were divided into four groups of eight and housed in metallic cages. They were fed ad-libitum on normal pellets (Pfizer, Kaduna, Nigeria) and water throughout the 28 days of study. Fresh garlic cloves (Allium satiûum) bought from Jos main market were used to prepare the garlic oil. The raw cloves of garlic were sliced, crushed, and soaked in diethyl ether for 48 hr. The supernatant was decanted. Extraction using diethyl ether was repeated three times. The oil was recovered from the combined extracts by distilling off the diethyl ether at 40 C. The oil was shaken with 20 volumes of redistilled petroleum ether (boiling point 40 C) and centrifuged at 1200 for 15 min. The clear supernatant was removed and the petroleum-soluble fraction of the oil was prepared by distilling off the solvent at 60 C. The yield was 500 mg kg 1 wet weight of garlic with a ph of 6.5 and F0.01% protein as contaminant. Diabetes was induced by injecting 60 mg kg 1 body weight of freshly prepared streptozotocin (a gift from UpJohn Company, Kalamazoo, MI) in 0.1 M citrate buffer ph 4.5 into test diabetic (group 1) and diabetic control (group 2) rats. The induction and confirmation of diabetes mellitus was as previously described [5]. Group 3 (solvent control) rats were injected with the same 0.1 M citrate buffer used to dissolve the streptozotocin while group 4 rats were retained as normal control. Test diabetic (group 1) and solvent control (group 3) animals were given 50 mg kg 1 body weight daily of garlic oil supplement intragastically through stomach tubes. The animals were sacrificed on the 29th day and the blood was quickly but carefully put into sterile plastic tubes and allowed to clot at 37 C for 30 min, centrifuged at 3000g for 15 min and platelet-free unhemolyzed serum was collected for the tests. The kidney, pancreas, and liver were excised from the rats according to group and placed in labeled beakers. They were weighed and extracted in a 1:10 ratio using 0.25 M sucrose solution. Each homogenate was centrifuged at 5000 rpm for 10 min at 4 C and the supernatant was used for estimation of total protein and amylase of the pancreas, liver, and kidney. Serum aminotransferases were determined according to the methods of Reitman and Frankel [6]. Acid and alkaline phosphatase and amylase were determined according to the methods of Henry and Chiamori [7, 8]. Total protein estimation was based on the method of Reinhold [9]. Determination of serum bilirubin was based on the method of Stroev and Makarova [10]. When using Student s t-test, a value pf0.05 was considered statistically significant. RESULTS The induction and confirmation of diabetes mellitus using 24 hr fasting blood and urine glucose levels was as previously described [5]. Glucose was significantly ( pf0.001) higher in diabetic rats as compared with normal control rats (Table 1). Diabetic rats treated with garlic oil showed a significant reduction in red cell acid phosphatase ( pf0.001), serum acid and alkaline phosphatases ( pf0.001),

3 Garlic Oil and Serum Enzymes in Diabetic Rats 21 Table 1. Induction of Diabetes Mellitus as Shown by Serum and Urinary Glucose Levels in Wistar Strain Rats (MeanJSEM of Four Determinations) Serum glucose 24 hr Urine glucose 24 hr after administration after administration of streptozotocin of streptozotocin Groups (mmol L) (mmol L) Experimental group (ng10) 16.5J0.20 a 11.03J0.34 a Control (ng5) 5.40J J0.03 a As compared with control rats pf0.001 (Student s t-test). aminotransferases ( pf0.001), and amylase ( pf0.002) levels as compared to the diabetic control rats (Table 2). Concentrations of amylase in the pancreas, liver, and kidney of diabetic rats treated with garlic oil showed a significant increase ( pf0.05) as compared to diabetic control rats. In contrast there was no significant difference in the tissue protein levels of diabetic and normal rats with and without garlic oil treatment (Table 3). There was also no significant difference in the serum bilirubin levels of diabetic and normal rats with or without garlic oil (Table 4). DISCUSSION Hyperglycemia has been hypothesized to stimulate increases in pancreatic β- cell mass in animal models of diabetes [11]. Serum acid phosphatase is released in man mostly from the prostate although some is found in the lysosomes of other tissues and in erythrocytes [12]. The precise metabolic function of alkaline phosphatase is not well understood. It has been suggested to facilitate the transfer of metabolites such as glucose and fatty acids across the membranes and participates in the calcification process during bone formation [13]. Streptozotocin is known to have some toxic effects on the liver and kidney [14]. Several studies have reported that garlic oil possesses antimutagenic and anticarcinogenic properties [15]. It is possible therefore that the observed reduction in phosphatases by garlic oil is unconnected with the degree of control of hyperglycemia Table 2. Effects of Garlic Oil on Serum Enzyme Levels in Streptozotocin-Induced Diabetes Mellitus in Rats (MeanJSEM of Four Determinations: ng8) Alkaline Red cell acid Serum acid Alanine Aspartate Amylase phosphatase phosphatase phosphatase transferase transferase (somogyi Treatment (iu l) (iu l) (iu l) (iu l) (iu l) unit 100 ml) DiabeticCgarlic 134.5J1.20 c 103.5J1.70 a 15.6J0.31 a 47.9J3.08 a 24.3J0.63 a 138.5J1.00 b Diabetic 142.8J J J J J J0.76 NormalCgarlic 103.3J2.00 h 85.3J1.70 h 13.3J0.25 h 39.3J2.26 g 21.9J1.61 h 127.1J0.80 Normal 109.0J0.90 a,d 91.0J0.90 a,f 14.2J0.15 a,f 49.7J J J0.98 e As compared with diabetic control rats: a pf0.001, b pf0.002, c PF0.01. As compared with diabetic rats on garlic oil: d pf0.001, e pf0.002, f pf0.01. As compared with normal control rats: g pf0.05, h pf0.01 (Student s t-test).

4 22 Ohaeri Table 3. Effects of Garlic Oil on the Protein Levels and Amylase Contents of the Kidney, Pancreas, and Liver in Streptozotocin-Induced Diabetes Mellitus in Rats (MeanJSEM of Four Determinations: ng8) Protein mg wet wt. of tissue Amylase somogyi unit g wet wt. of tissue Treatment Pancreas Liver Kidney Pancreas Liver Kidney DiabeticCgarlic 231.0J J J J0.31 a 14.36J0.12 a 19.49J0.23 a Diabetic 229.7J J J J J J1.37 NormalCgarlic 230.0J J J J0.14 g 19.34J0.13 f 26.04J0.36 f Normal 227.7J J J J0.18 c,d 17.58J0.15 c,e 21.30J0.18 b,d As compared with diabetic control rats: a pf0.05, b pf0.02, c pf As compared with diabetic rats on garlic oil: d pf0.01, e pf As compared with normal control rats: f pf0.001, g pf0.002 (Student s t-test). but due rather to its antitumorogenic effect. Garlic oil may also be acting through an unknown mechanism. Aminotransferases are elevated in some diseases such as myocardial infaction, infectious hepatitis or other damage to either the heart or liver [16]. Assays of alanine and aspartate aminotransferase activities in blood serum can be used both in the diagnosis and in monitoring the progress of a patient during treatment [17]. The evidence which links hyperglycemia with cardiovascular disease is unclear. Recent studies suggest that hyperglycemia and poor glycemia control are associated with an increased risk for cardiovascular disease in type 2 diabetic patients [18]. In type 1 diabetes, the diabetes control and complications trial convincingly demonstrated that insulin treatment of patients reduced microvascular complications [19]. The present work, allied to the fact that garlic oil reduced the hypercoagulation effect of diabetes in our previous study [20] encourages the view that the oil reduced a number of risk factors of cardiovascular disease in experimental diabetic rats. Amylases are a group of hydrolases that split complex carbohydrates and are present in a number of organs and tissues [21]. The amylase content of the pancreas is a reflection of exocrine function. Elevated serum amylase activity is associated with pancreatitis, pancreatic duct obstruction, and intra-abdominal disease [12]. Decreased amylase levels of the pancrease, kidney, and liver are reflections of exocrine dysfunction or changes in the level of enzyme production in these organs rather than selective inhibition. Table 4. Effects of Garlic Oil on Serum Bilirubin in Streptozotocin-Induced Diabetes Mellitus in Rats (MeanJSEM of Four Determinations: ng8) (Student s t-test) Conjugated Unconjugated Total Ratio of bilirubin bilirubin bilirubin conjugated Treatment (µmol L) (µmol L) (µmol L) unconjugated DiabeticCgarlic 5.5J J J0.3 1:1 Diabetic 6.0J J J0.6 1:1 NormalCgarlic 5.5J J J0.5 1:1 Normal 5.4J J J0.3 1:1

5 Garlic Oil and Serum Enzymes in Diabetic Rats 23 The decrease in serum amylase of diabetic rats treated with garlic oil is suggestive of diminished pancreatitis. Taken together there is considerable evidence that the observed changes in serum and tissue enzymes were due to the direct toxic effects of streptozotocin. The effect of garlic oil therefore on the metabolic disposition of these enzymic and non-enzymic markers of tissue damage was accordingly beneficial. ACKNOWLEDGMENT I am grateful to Professor G. I. Adoga and Professor Z. S. C. Okoye for their useful comments and advice. REFERENCES 1. Dalvi, R. R. (1992) Alterations in hepatic phase I and phase II biotransformation enzymes by garlic oil in rats. Toxicol. Lett. 60: Adoga, G. I. and Ibrahim, M. B. (1990) Effects of garlic oil on some biochemical parameters in streptozotocin induced diabetic rats. Med. Sci. Res. 18: Horie, T. et al. (1989) Protection of liver microsomal membranes from lipid peroxidation by garlic extract. Planta Medica 55: Okamoto, H. (1990) The molecular basis of experimental diabetes. In: Molecular Biology of the Islets of Langerhans (H. Okamoto, ed.), Cambridge University Press, pp Ohaeri, O. C. (1999) Renal function and calcium metabolism in streptozotocin diabetic rats treated with garlic oil. Med. Sci. Res. 27: Reitman, S. and Frankel, S. (1957) Colorimetric method for the determination of serum transaminase. Amer. J. Clin. Pathol. 28: Henry, R. J. and Chiamori, N. (1959) Determination of acid and alkaline phosphatase. Clin. Chem. 5: Henry, R. J. and Chiamori, N. (1970) Saccharogenic method for amylase determination. In: Fundamentals of Clinical Chemistry (N. W. Tietz, ed.), W. B. Saunders Co., Philadelphia, pp Reinhold, J. G. (1962) Total protein, albumin and globulin. In: Standard Methods of Clinical Chemistry (D. Seligson, ed.), Academy Incorporated, New York, p Stroev, E. A. and Makarova, V. G. (1988) Determination of bilirubin and its fractions in blood serum. In: Laboratory Manual in Biochemistry (E. A. Stroev and V. G. Makarova, eds.), Mir Publishers, Moscow, pp Screenan, S., Pick, A. J., Levisetti, M., Baldwin, A. C., Pugh, W., and Polonsky, K. S. (1999) Increased β-cell proliferation and reduced mass before diabetes onset in the nonobese diabetic mouse. Diabetes 48: Eastham, R. D. (1982) In: Biochemical Values in Clinical Medicine (R. D. Eastham, ed.), John Wright and Son, Bristol, pp , Peach, H., Compston, J. E., Verdi, S., and Horton, W. L. (1982) Values of plasma, calcium, phosphorus and alkaline phosphatase measurements in the diagnosis of oeseomalacia. J. Clin. Pathol. 35: Sakamoto, T., Zhu, X. G., and Thompson, J. C. (1983) Hepatic inactivation of CCK-8 and CCK- 33 in dogs. Gastroenterol. 84: Unnikrishnan, M. C. and Kuttan, R. (1990) Tumour reducing and anticarcinogenic activity of selected spices. Cancer Lett. 51: Murray, R. K., Granner, D. K., Mayes, P. A., and Rodwell, V. W. (1993) Catabolism of proteins and of amino acid nitrogen. In: Harper s Biochemistry, 23rd edn. A Lange Medical Book. Prentice- Hall International Limited, London, pp Matthew, C. K. and Van Holde, K. E. (1993) Metabolism of nitrogenous compounds: Principles of biosynthesis, utilization, turnover, and excretion. In: Biochemistry, The Benjamin-Cummings Publishing Co. Inc., California, p. 682.

6 24 Ohaeri 18. Laakso, M. (1999) Hyperglycemia and cardiovascular disease in type 2 diabetes. Diabetes 48: The Diabetes Control and Complications Trail Research Group (1993) The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. New England J. Med. 329: Adoga, G. I. and Ohaeri, O. C. (1991) Effects of garlic oil on prothrombin, thrombin in and partial thromboplastin times in streptozotocin induced diabetic rats. Med. Sci. Res. 19: Bazin, R. and Lavin, M. (1979) Diet composition and insulin effect on amylase to lipase ratio in pancreas of diabetic rats. Digestion 19: