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Indian Journal of Pharmacology, Vol. 41, No. 1, January-February, 2009, pp. 19-22 Research Article Protective effect of Cassia glauca Linn. on the serum glucose and hepatic enzymes level in streptozotocin induced NIDDM in rats Farswan Mamta, Mazumder PapiyaMitra, Percha V Department of Pharmaceutical Sciences, SBS (PG) Institute of Biomedical Sciences, Balawala, Dehradun Date of Submission: 20-Jul-2007 Code Number: ph09005 Abstract Objective: The objective of the present study was to investigate the hypoglycemic and hepatoprotective effect of Cassia glauca leaf extracts on normal and non insulin dependent diabetes mellitus (NIDDM) in rats. The study was further carried out to investigate the effect of different fractions of the active extract of Cassia glauca, on normal and NIDDM rats, and the effect of active fraction on the blood glucose and hepatic enzymes level.Methods: Diabetes was induced by streptozotocin (STZ) at a dose of 90mg/kg, i.p. in neonates. Different extracts of cassia glauca (100mg/kg, p.o.) were administered to the diabetic rats. Acetone extract was found to lower the serum glucose level significantly in diabetic rats. Further, the acetone extract was subjected to column chromatography and four fractions were obtained on the basis of TLC. All the four fractions (100mg/kg, p.o.) were administered to the diabetic rats. Fraction 1 (F1) caused the maximum reduction in the blood glucose level. The results of the test were compared with the standard antidiabetic drug glibenclamide (5mg/kg, p.o.). Results: Fraction 1 of acetone extract caused a significant reduction in the levels of hepatic enzyme Aspartate transaminase (AST), alanine transaminase (ALT), creatine kinase (CK), and lactate dehydrogenase (LDH) in STZ-induced diabetic rats. Conclusion: Improvement in the blood sugar level and normalization of liver functions by Cassia glauca indicates that the plant has hepatoprotective potential, along with antidiabetic activity, and it provides a scientific rationale for the use of Cassia glauca as an antidiabetic agent. Keywords: β-sitosterol, blood glucose, Cassia glauca, hepatoprotective Introduction There is still an unmet need for medicinal plants and phytopharmaceuticals with scientifically proven antidiabetic activity. Diabetes mellitus is characterized by a progressive decline in insulin action (insulin resistance, followed by the inability of β cells to compensate for insulin resistance. [1] The b cells normally compensate insulin resistance by secreting more amount of insulin to maintain the glucose homeostasis. In non insulin dependent diabetes mellitus (NIDDM) this β cell function gets impaired leading to deterioration in glucose homeostasis and subsequent development of impaired glucose tolerance and frank diabetes. [2],[3] Hyperglycemia in the diabetics is associated with alteration of glucose and lipid metabolism and modification in liver enzymes level [4] . Liver is an important insulin dependent tissue which plays a pivotal role in glucose and lipid metabolism and is severely affected during diabetes. [5] In diabetes the levels of hepatic enzymes increases. The level of ALT increases due to hepatocellular damage and is always accompanied by an increase in AST activity. Moreover the ALT and AST activity has been used as an indicator of liver function. [6] The prevalence of diabetes is approximately 3% of the world population and is about 5% in the United States. [7] Approximately 10% of patients have type 1 diabetes mellitus (DM) and the remainder have type 2 Diabetes Mellitus. Insulin and oral hypoglycemics are the most widely used drugs for diabetes but they also have various side effects like hypoglycemia, weight gain (sulfonylurea), lactic acidosis with biguinides and all of these drugs may cause liver and renal damage. [8] From the beginning of last century, evidence of lipid lowering properties of medicinal plants has accumulated. [9] In recent years many traditionally used medicinal plants have been tested for their antidiabetic potential in experimental animals. [10] Ethnobotanical information indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes. [11] Cassia glauca (Family - Caesalpiniaceae) is about 10 ft high, evergreen shrub. Aerial parts of the plant are used as central nervous system depressant and as a diuretic. [12] Seeds of the plant are known to possess purgative and antimalarial properties. The leaves of the plant are pounded in sugar and milk and used to cure blennorrhagia. Bark and leaves are used in diabetes and gonorrhea in the folk lore medicine. This plant is also a good pollution tolerant and reduces chemical pollutants from the atmosphere. [13] Phytochemical investigation of the plant showed the presence of γ- sitosteroline, fatty acids, anthraquinones, tannins and alkaloids. The leaves contain β -sitosterol and lupeol . Different species of Cassia like Cassia alata , [14] Cassia auriculata [15] have also been studied well for their antidiabetic potential. Therefore the objectives of the present study were to evaluate the hypoglycaemic and hepatoprotective activity of active fraction of Cassia glauca leaf extracts on normal and NIDDM rats. The results obtained with Cassia glauca were compared with glibenclamide, a commonly used hypoglycaemic agent. Materials and Methods Cassia glauca leaves were collected from the forest of Dehradun. The plant was identified, authenticated and the voucher specimen (A-30) has been kept at the herbarium of Sardar Bhagwan Singh (PG) Institute of Biomedical Sciences Dehradun. Streptozotocin was purchased from Calbiochem, Germany. Glibenclamide was obtained as gift samples from Ranbaxy Research Laboratories. Analytical grade chemicals like various organic solvents (petroleum ether, chloroform, acetone, and methanol) from E. Merck India Ltd and Ranbaxy were used for the extraction and phytochemical study of the constituents. Preparation of Plant extracts Phytochemical study Fractionation of the acetone extract of Cassia glauca leaf Determination of blood glucose and lipid profile in diabetic rats Acute toxicity studies Induction of Diabetes Treatment protocol Group I: Normal animals received tween 80 in a dose of 1% suspension in distilled water. Group II: Diabetic animals received tween 80 in a dose of 1% suspension in distilled water. Group III: Diabetic animals received glibenclamide in a dose of 5 mg/kg, p.o. Group IV: Diabetic animals received Petroleum ether extract in a dose of 100 mg/kg, p.o. Group V: Diabetic animals received chloroform extract, in a dose of 100 mg/kg, p.o Group VI: Diabetic animals received acetone extract, in a dose of 100 mg/kg, p.o Group VII: Diabetic animals received methanol extract. in a dose of 100 mg/kg, p.o. At the end of experimental period the animals were fasted overnight and blood was taken from the retro orbital plexus under mild ether anesthesia, serum was separated out and blood sugar level was evaluated by the method of glucose oxidase- peroxides method using Span diagnostic kits. Preliminary pharmacological screening of the fractions and effect of active fraction on hepatic enzymes level in diabetic rats Group I: Normal animals received tween 80 in a dose of 1% suspension in distilled water. At the end of experimental period animal were fasted overnight and blood was taken from the retro orbital plexus under mild ether anesthesia, it was centrifuged and serum was separated out. Effect of each fraction on FBS level was evaluated. Fraction 1 caused maximum reduction in FBS in diabetic rats. Serum from the group receiving fraction 1 was taken to study the effect of fraction 1 on the lipid profile. AST, ALT, CK and LDH were determined by UV kinetic method 22 using span diagnostic kits. Results of the test were compared with that of the standard antidiabetic drug glibenclamide. Statistical analysis Results Phytochemical study Acute toxicity studies Effect of Cassia glauca leaf extracts and Fractions of active extract on fasting blood sugar and hepatic enzymes level in diabetic rats. [Table - 1] illustrates the effect of different extracts of Cassia glauca on serum glucose level in the diabetic rats. Results showed that all the extracts caused reduction in blood glucose level but maximum reduction was found in acetone extract. Acetone extract showed 52% reduction as compared to glibenclamide (67%) reduction in fasting blood sugar. Acetone extract of the leaf caused significant (p< 0.01) decrease in the fasting blood glucose. [Table - 2] illustrates the effect of different fractions of acetone extract of Cassia glauca on serum glucose level in the diabetic rats. Results showed that all the fractions of acetone extract of the leaf caused significant decrease in the fasting blood glucose. However F1 exhibited maximum reduction in fasting blood sugar (59%) as compared to glibenclamide which caused 67% reduction in FBS. [Table - 3] illustrates the effect of F1 and glibenclamide on AST, ALT, Creatine kinase and lactate dehydrogenase level. Fraction 1 of the acetone extract caused significant (p< 0.01) improvement in AST, ALT, CK and LDH levels in the diabetic rats after treatment. Discussion In the recent years various plant extracts have been claimed to be useful for the treatment of diabetes. [23] Administration of STZ caused rapid destruction of b cells which lead to a reduction of insulin release which slowly progress to impaired glucose stimulated insulin release and insulin resistance which are the marked feature of type II diabetes. The hypoglycemic effect of plant extracts is generally dependent upon the degree of b cell destruction. In diabetes, the increase in blood glucose level is usually accompanied by an increase in the activity of transaminases. More over the ALT, AST and LDH activities has been used as an indicator of liver function. Acetone extract caused significant reduction in the serum blood glucose level as compared to glibenclamide. Acetone extract was subjected to column chromatography in order to get fractions containing maximum hypoglycemic activity. Fraction 1 caused reduction in blood sugar which is more than the acetone extract. Fraction 1 also caused 31% reduction in the level of AST and 32% reduction in the level of ALT where as glibenclamide caused 23% reduction in the level of CK and 20% reduction in the level of LDH. The improvements in the levels of liver enzymes in diabetic animals could be beneficial in preventing diabetic complications, as well as improving lipid and protein metabolism in diabetic liver. The reversal of AST and ALT activity in Cassia glauca treated diabetic rats towards near normalcy is evidence of the prevention of cellular and tissue damage under diabetic conditions. Phytochemical investigation of Cassia glauca leaf showed the presence of g-sitosteroline, fatty acids, anthraquinones, tannin and alkaloids. On the chemical basis it may be concluded that b - sitosterol and tannins in the leaf are responsible for the antidiabetic activity of Cassia glauca as it is known that tannins and sterols containing drugs possess antidiabetic activity. [24] The present investigation revealed that acetone extract and fraction 1 of the acetone extract of Cassia glauca leaf has shown significant antidiabetic activity. Improvement in the liver enzymes level by Cassia glauca indiates that the plant has hepatoprotective potential along with antidiabetic activity. The active constituent in the fraction responsible for antidiabetic and hepatoprotective activity is not known at present and work is on process to isolate it. Acknowledgement The authors are immensely thankful to management, SBS (PG) Institute of Biomedical Sciences and Research, Balawala Dehradun (U.K.) for providing the requisite facilities and financial support.References
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