ANTIDIABETIC ACTIVITY AND PHYTOCHEMICAL EVALUATION OF ARTEMISIA INDICA LINN

Abstract

Diabetes mellitus (DM) is a metabolic disorder of multiple etiologies characterized by chronic hyperglycemia due to impaired function/secretion of insulin or both thus resulting in disturbances of carbohydrate, fat and protein metabolism. There are two main types of DM, Type –I and Type-II DM. This classification is based on the etiology of the insulin secretion/function. Type I diabetes is responsible for 5-10% of all diabetic cases. It occurs as a result of autoimmune mediated selective destruction of > 90% of the insulin secreting beta cells of the pancreas and characterized by complete absence of insulin production, whereas Type-II DM is characterized clinically by hyperglycemia and insulin resistance. The treatment of the DM depends upon the type of the diabetes. Several oral hypoglycemic drugs (isolated either from plants sources or synthetic drugs) are used for the treatment of Type-II DM whereas for the treatment of type-I DM insulin preparations (from animal source) are available. Various oral hypoglycemic agents are available in the market for the treatment of DM. Even insulin cannot permanently restore normal pattern of glucose homeostasis and metformin approved for the treatment of non-insulin dependent diabetes mellitus (NIDDM) has been derived from plant Galega officinalis and was historically used in medieval Europe for the treatment of diabetes mellitus. Traditional plant based remedies are still the first choice in the developing countries for the management of Diabetes as these are cheap, cost effective, easily available and are associated with less or no adverse effects. Various Artemisia species have been extensively used for the treatment of Diabetes in folkloric medicine in various communities. Keeping in view the immense medicinal importance of the plants and folkloric use in developing countries, this study was planned to explore the antidiabetic activity of Artemisia indica (Asteraceae) on scientific ground. In this study the methanolic crude extracts, chloroform, ethyl acetate, n-butanol and n-hexane fractions of aerial parts of Artemisia indica were tested for their antidiabetic potential in Streptozotocin (STZ) (50 mg/kg. i.p) induced Diabetic Sprague Dawley rats. A daily oral dose of the methanolic crude extracts (200 and 400 mg/kg b.w) and chloroform fraction (200 mg/kg b.w) of Artemisia indica for 15 days showed a significant reduction in blood glucose level which was comparable to the standard antidiabetic drug, glibenclamide (500 μg/kg, p.o) (p˂0.01, n=8; one way ANOVA with Dunnett’s posthoc test). The crude methanolic extract (200 & 400 mg/kg) and chloroform fraction (200 mg/kg) also significantly (p˂0.001, n=8; one way ANOVA with Dunnett’s posthoc test) reversed STZ-induced loss in body weight. Furthermore, the crude methanolic extract (200 & 400 mg/kg) and chloroform fraction (200 mg/kg) also showed significant (p˂0.01, n=8; one way ANOVA with Dunnett’s posthoc test) reduction in total cholesterol, triglycerides and low density lipoproteins (LDL) and serum creatinine level. The ethyl acetate, n-butanol, and n-hexane fractions (200 mg/kg) also significantly (p˂0.05, n=8; one way ANOVA with Dunnett’s posthoc test) reduced total cholesterol and low density lipoproteins (LDL) and increased high density lipoproteins (HDL) in diabetic rats. Triglycerides level was significantly reduced (p˂0.05, n=8; one way ANOVA with Dunnett’s posthoc test) with n-butanol and n-hexane fractions (200 mg/kg) but not with ethyl acetate fraction (200 mg/kg). The crude methanolic extract (200 & 400 mg/kg) and chloroform fraction (200 mg/kg) also caused significant reduction (p˂0.001, n=8; one way ANOVA with Dunnett’s posthoc test) in serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in diabetic rats which suggests the possibilities, that the major function of the extracts are on the protective effects on the major tissues kidney, liver and pancreas, thereby reducing the incidence of diabetes in experimental animals. Preliminary phytochemical investigation of the crude methanolic extract and chloroform fraction indicated the presence of terpenoids, alkaloids, flavonoids, glycosides, saponins and tannins. Since the chloroform fraction of Artemisia indica retained the hypoglycemic activity of the crude methanolic extract, therefore the chloroform fraction was further subjected to bioassay guided isolation of phtytochemical constituents. This led to the isolation of eleven compounds from the chloroform fraction of Artemisia indica. These compounds included ursolic acid, carnosol, oleanolic acid, stigmasterol, methyl β-orsellinate, 2-hydroxybenzoic acid, gallic acid, 1,5-dihydroxy-3-methylanthraquinone, ethyl gallate, 4-hydroxybenzoic acid and benzoic acid. To the best of my knowledge this is the first study that shows the presence of these compounds in Artemisia indica. Carnosol is the main active constituent of Rosmarinus officinalis and Salvia pachyphylla and previously the hypoglycemic effects of aqueous extracts of these plants have been attributed to carnosol and carnosic acid content of these plants. However no study has been carried out to investigate the hypoglycemic activity of pure carnosol. In the present study carnosol was isolated from the chloroform fraction of Artemisia indica in sufficient quantity to be able to carry out in-vivo studies to determine its hypoglycemic potential in STZ-induced diabetic rats. The effects of carnosol on body weight and various biochemical parameters such as serum lipid profile and activities of liver enzymes were also determined. The results showed that oral administration of carnosol (1, 10, 30 and 100 mg/kg, b.w) caused a significant (p˂0.01, n=8; one way ANOVA with Dunnett’s posthoc test) reduction in blood glucose level compared to diabetic control. Carnosol 1, 10, 30 and 100 mg/kg decreased blood glucose level from 465.7±15, 446.7±53, 443±52 and from 437±30 to 264.3±15**, 260.6 ±16**, 230.8 ±13** and 220.3±12** mg/dl respectively. Furthermore carnosol (1, 10, 30 and 100 mg/kg) significantly (p˂0.01, n=8; one way ANOVA with Dunnett’s posthoc test) reversed STZ-induced loss in body weight (13.8%) in diabetic rats. Carnosol 1, 10, 30 and 100 mg/kg caused 9.9, 10.5, 11.2 and 12 % improvement in body weight. Carnosol (1, 10, 30 and 100 mg/kg, b.w) also caused a significant (p˂0.01, n=8; one way ANOVA with Dunnett’s posthoc test) reduction in total cholesterol, triglycerides, low density lipoproteins and serum creatinine level. Furthermore, carnosol (1, 10, 30 and 100 mg/kg) also significantly reduced (p˂0.001, n=8; one way ANOVA with Dunnett’s posthoc test) serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT) and alkaline phosphatase (ALP) in diabetic rats. The effects of carnosol (10 and 50 mg/kg, b.w.) on histopathological changes in the pancreas of STZ-induced diabetic rats were also investigated. STZ reduced dimensions of the islets of Langerhans, shrunken islets and significant reduction in the number of pancreatic beta (β) cells of islets in STZ-induced diabetic Sprague Dawley rats. Carnosol (10 and 50 mg/kg, b.w.) reversed these changes and caused partial restoration of normal cellular population, increased the size and caused hyperplasia of the islets of Langerhans. Furthermore the islets were less shrunken compared to the untreated diabetic group. These effects were similar to those observed with glibenclamide (500 μg/kg, b.w). In conclusion, the crude methanolic extract and chloroform fraction of Artemisia indica possess significant hypoglycemic activity and antidiabetic potential. These extracts also caused improvement in body weight and significantly reduced the lipid profile. Carnosol isolated from the chloroform fraction also exhibited antidiabetic activity. The crude methanolic extract, chloroform fraction and carnosol caused significant reduction in serum creatinine, serum SGPT, serum SGOT and ALP in STZ-induced diabetic rats and thus can have improvement in the condition of diabetes mellitus. Furthermore carnosol reversed STZ-induced changes in the pancreatic islets of Langerhans and caused regeneration and restored the integrity of pancreatic islets of Langerhans which may be responsible for its antihyperlgycemic effect. However further studies are required to investigate the hypoglycemic effects of other constituents of Artemisia indica and elucidate other possible molecular mechanism/s involved in the hypoglycemic effect of Artemisia indica and carnosol.