Abstract
Context: Terminalia arjuna Roxb. (Combretaceae), commonly known as Arjuna, is a large tree grown throughout the Indian peninsula and used traditionally for several medicinal purposes.
Objective: To evaluate antihyperglycemic and antioxidant role of methanol extract of T. arjuna leaf (META) in Wistar rats.
Materials and methods: Hyperglycemia was induced in rats by single intraperitoneal injection of streptozotocin (STZ, 65 mg/kg body weight). Three days after STZ induction, the hyperglycemic rats were treated with META orally at the dose of 100 and 200 mg/kg body weight daily for 15 days. Glibenclamide (0.5 mg/kg, orally) was used as reference drug. The fasting blood glucose levels were measured on every fifth day during the 15-day treatment. Serum biochemical parameters such as serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), cholesterol, and total protein were estimated. Antioxidant properties were assessed by estimating hepatic lipid peroxidation, reduced glutathione (GSH), and catalase (CAT).
Results and discussion: META at the dose of 100 and 200 mg/kg orally significantly (P < 0.001) and dose-dependently reduced and normalized blood glucose levels as compared with that of STZ control group. Serum biochemical parameters were significantly (P < 0.001) restored toward normal levels in META-treated rats as compared with STZ control. META treatment also significantly (P < 0.001) decreased lipid peroxidation and recovered GSH level and CAT activity toward normal as compared with STZ control.
Conclusion: The present study infers that T. arjuna leaf demonstrated remarkable antihyperglycemic activity in STZ-induced diabetic rats. The potential antihyperglycemic action is plausibly due to its underlying antioxidant role.
Introduction
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, glycosuria, and negative nitrogen balance and it is mainly due to absolute deficiency or diminished effectiveness of insulin. It is the most prevalent disease in the world affecting 25% of the population and afflicts 150 million people and is predicted to rise to 300 million by 2025 (CitationVats et al., 2000). It causes numerous complications such as retinopathy, neuropathy, and peripheral vascular insufficiencies (CitationChehade & Mooradian, 2000).
Diabetes is still not completely curable by the present antidiabetic therapy. Insulin therapy is the only satisfactory approach in diabetic mellitus, even though it has several drawbacks like insulin resistance (CitationPiédrola et al., 2001), anorexia, brain atrophy, and fatty liver in chronic treatment (CitationWeidmann et al., 1993). There are several oral hypoglycemic agents used therapeutically but certain adverse effects and weak effectiveness of them have led to the search for more effective agents. Investigation in the plant kingdom culminated in the discovery of many natural antihyperglycemics (CitationPari & Maheswari, 1999).
Terminalia arjuna Roxb. (Combretaceae), commonly known as Arjuna, is a large tree grown on the banks of rivers, streams, and dry watercourses throughout the Indian peninsula. In India, the plant has been traditionally used for several medicinal purposes. The fruits of the plant are used as a tonic (CitationChopra et al., 1956). Externally, its leaf paste is used as a cover on sores and ulcers. The bark is antidysenteric, antipyretic, astringent, cardiotonic, lithotriptic, and tonic; a powder of the bark acts as diuretic in cirrhosis of liver and gives relief in symptomatic hypertension (CitationAnon., 1976; CitationChatterjee & Pakrashi, 1994). A decoction of bark made with milk is given every morning on an empty stomach, or its powder with milk, as a cardiotonic (CitationDastur, 1962). The powder of the bark is also given with honey in fractures and contusions with echymosis. Beside this, the extract of the bark as astringent is used for cleaning sores, ulcers, cancers, and so on (CitationDhiman, 2006). The extract of the bark is prescribed in scorpion stings and lowering blood glucose (CitationChopra et al., 1958). No pharmacological investigation is still reported on T. arjuna leaf. Present study was therefore aimed to investigate the antihyperglycemic effect of methanol extract of T. arjuna leaf (META) against streptozotocin (STZ)-induced diabetic Wistar rats.
Materials and methods
Plant material
The leaves of T. arjuna were collected during January 2008 from Nadia, West Bengal, India. The plant species was authenticated by Dr. Lakshmi Narashimhan, Scientist, Botanical Survey of India, Central National Herbarium, Howrah, West Bengal, India. The voucher specimen [CNH/I-I/(216)/2008/Tech.II/216] was maintained in our laboratory for future reference. The leaves were shade-dried with occasional shifting and then powdered with mechanical grinder passing through sieve no. 40, and stored in an air-tight container.
Drugs and chemicals
The chemicals were obtained from the following manufacturers: STZ, 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione (GSH), nitroblue tetrazolium (NBT; SISCO Research Lab., Mumbai, India); thiobarbituric acids, trichloroacetic acid (TCA; Merck, Mumbai, India); potassium dichromate, glacial acetic acid (Ranbaxy, Mumbai, India); and glibenclamide (Hoechst, Mumbai, India). All other reagents used were of analytical grade obtained commercially.
Preparation of extract
The powdered plant material (400 g) was macerated at room temperature (24–26°C) with methanol (850 mL) for 4 days with occasional shaking, followed by re-maceration with the same solvent for three more days. The macerates were combined, filtered, and distilled off in reduced pressure. The resulting concentrate was vacuum-dried at 40°C to yield the dry extract (META, yield 21.45% w/w). The dry extract was kept in a vacuum desiccator until use. Preliminary phytochemical studies of META (CitationHarborne, 1998) revealed the presence of alkaloids, triterpenoids, tannins, and flavonoids.
Animals
Adult male Wistar albino rats weighing 170–200 g were used for the present investigation. They were housed in a clean polypropylene cage and maintained under standard laboratory conditions (temperature 25°C ± 2°C with dark/light cycle 12/12 h). They were fed with standard pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The animals were acclimatized to laboratory conditions for 1 week prior to experiment. All procedures described were reviewed and approved by the University Animal Ethics Committee, Jadavpur University.
Acute toxicity
The acute oral toxicity of META in male Swiss albino mice was studied as per reported method (CitationLorke, 1983).
Induction of diabetes
Diabetes mellitus was induced in overnight fasted rats by a single intraperitoneal injection of STZ (65 mg/kg body weight) (CitationPellegrino et al., 1998). After 3 days, fasting blood glucose (FBG) levels were measured and the animals showing blood glucose level ≥225 mg/dL were used for the present investigation (CitationEwart et al., 1975).
Treatment schedule and estimation of FBG level
The rats were divided into five groups (n = 6). Except group I, which served as normal nondiabetic control, all other groups were comprised of diabetic rats. Group II served as diabetic (STZ) control. Groups III and IV received META (100 and 200 mg/kg b.w., p.o., respectively) and group V received reference drug glibenclamide (0.5 mg/ kg b.w., p.o.) daily for 15 days. FBG was measured on day 0, 5, 10, and 15 by using a one touch glucometer (Accu-check®). Twenty-four hours after the last dose, blood was collected from overnight fasted rats of each group by cardiac puncture for estimation of serum biochemical parameters, viz., serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), serum alkaline phosphatase (SALP), serum cholesterol, and total protein. Then the rats were sacrificed by cervical dislocation for the study of liver biochemical parameters like lipid peroxidation, GSH, and catalase (CAT).
Body weight, liver and kidney weights
The body weight of rats of each group were measured just before and 15 days after META treatment. Liver and kidney weights of all rats were measured after posttreatment sacrifice.
Estimation of serum biochemical parameters
Collected blood was used for the estimation of serum biochemical parameters, viz., SGOT, SGPT (CitationBergmeyer et al., 1978), SALP (CitationKing, 1965), serum cholesterol (CitationBergmeyer & Brent, 1974), and total protein (CitationLowry et al., 1951).
Estimation of liver biochemical parameters
Lipid peroxidation, that is, thiobarbituric acid reactive substances (TBARS) was estimated by the method of CitationFraga et al. (1988) and expressed as mM/100 g of liver tissue. GSH was determined by the method of CitationEllman (1959) and was expressed as mg/100 g of liver tissue. CAT activity was assayed according to the method described by CitationSinha (1972) and expressed as µmoles of H2O2 consumed/min/mg of liver tissue.
Statistical analysis
The experimental results were expressed as mean ± standard error of mean (SEM). Statistical significance was analyzed by one-way ANOVA followed by Dunnett’s post hoc test of significance. P-values of <0.001 were considered as statistically significant.
Results
Acute toxicity
The oral LD50 value of the META in mice was 900 mg/kg body weight.
FBG level
The FBG levels of normal, diabetic, and treated rats are summarized in . STZ at the dose of 65 mg/kg produced marked hyperglycemia as evident from significant (P < 0.001) elevation in FBG level in STZ control group as compared with normal control group. Administration of META in STZ-induced diabetic rats at the doses of 100 and 200 mg/kg b.w. produced significant (P < 0.001) and dose-dependent fall in blood glucose levels when compared with the STZ control group. The FBG-reducing effect by META at the dose of 200 mg/kg b.w. was found to be more potent than that of the reference drug glibenclamide (0.5 mg/kg b.w.).
Table 1. Influence of methanol extract of T. arjuna leaf (META) on fasting blood glucose level in hyperglycemic and non-hyperglycemic rats.
Body weight, liver and kidney weights
The body weight, liver and kidney weights of rats from STZ control group (after 15 days) were significantly (P < 0.001) decreased when compared with normal control group. META at 100 and 200 mg/kg b.w. significantly (P < 0.001) maintained the body weight, liver and kidney weights toward normal in a dose-related manner as compared with STZ control ().
Table 2. Influence of methanol extract of T. arjuna leaf (META) on body weight and weight of kidney and liver in hyperglycemic and non-hyperglycemic rats.
Serum biochemical parameters
Biochemical parameters like SGOT, SGPT, SALP, and serum cholesterol in the STZ control group were significantly (P < 0.001) elevated as compared with the normal control group. Treatment with META at the dose of 100 and 200 mg/kg b.w. significantly (P < 0.001) brought the SGOT, SGPT, SALP, and serum cholesterol levels toward the normal values in a dose-dependent manner. The total protein was found to be significantly decreased in the STZ control group as compared with the normal control group (P < 0.001). Administration of META in diabetic animals significantly (P < 0.001) increased the total protein content as compared with the STZ control group ().
Table 3. Influence of methanol extract of T. arjuna leaf (META) on serum biochemical parameters in hyperglycemic and non-hyperglycemic rats.
Liver biochemical parameters
The levels of TBARS were significantly (P < 0.001) increased in STZ control animals as compared with normal control group. Treatment with META at 100 and 200 mg/kg b.w. significantly (P < 0.001) reduced the TBARS levels when compared with STZ control animals in dose-related manner. The level of GSH was significantly (P < 0.001) depleted in STZ control group as compared with normal control group. GSH level was found to be significantly and dose-dependently (P < 0.001) elevated toward normal level on administration of META as compared with STZ control group. There was significant (P < 0.001) reduction in CAT activity in STZ control group compared with normal group. Administration of META significantly (P < 0.001) recovered CAT activity toward normal when compared with STZ control animals ().
Table 4. Influence of methanol extract of T. arjuna leaf (META) on liver biochemical parameters in hyperglycemic and non-hyperglycemic rats.
Discussion
The present work was aimed to study the antihyperglycemic activity of META in STZ-induced diabetic rats. The results of this study revealed that META at the doses of 100 and 200 mg/kg b.w. significantly normalized the elevated blood glucose level and restored serum and liver biochemical parameters toward normal values.
STZ is an antibiotic obtained from Streptomyces achromogenes. STZ possess diabetogenic properties mediated by pancreatic β-cell destruction; hence, this compound has been widely used to induce diabetes mellitus in experimental animals (CitationJunod et al., 1969). Once STZ enters into cells, it undergoes spontaneous decomposition to form an isocyanate and a methyldiazohydroxide compound. Isocyanate and methyldiazohydroxide compound cause intramolecular carboxylation and alkylation of cellular components, respectively (CitationVarva et al., 1960). The DNA damage of β-cells of pancreas is mainly by alkylation with carbonium ion produced by methyldiazohydroxide (CitationWright et al., 1999). STZ causes damage not only to the pancreatic β-cells but also to hepatocytes, nephrons, and cardiomyocytes.
Hyperglycemia was observed after 3 days of STZ induction. Treatment with META in STZ-induced diabetic rats dose-dependently started reducing FBG levels after 5 days and made them normoglycemic after 15 days. The antihyperglycemic effect of META at 200 mg/ kg b.w. dose was found to be more effective than the effect exerted by the reference drug, glibenclamide at the dose of 0.5 mg/kg b.w. META also showed marked effect in controlling the loss of body weight, liver and kidney weights of diabetic rats.
Elevation of serum biomarker enzymes such as SGOT, SGPT, and ALP was observed in diabetic rats indicating impaired liver functions that may be due to hepatic damage. The decreased total protein content in STZ-induced animals also substantiated the hepatic damage by STZ. The diabetic complications such as increased gluconeogenesis and ketogenesis may be due to elevated transaminase activities (CitationGhosh & Suryawanshi, 2001). Fifteen days of treatment with META restored all the above-mentioned parameters toward the normal levels in a dose-dependent manner. It is well known that in uncontrolled diabetes mellitus, there is an increase in total cholesterol in blood, which may contribute to coronary artery diseases (CitationArvind et al., 2002). In the present study, the elevated serum cholesterol level in diabetic rats was normalized after treatment with META. This suggests that the extract may inhibit the pathway of cholesterol synthesis in diabetic rats.
Oxidative stress in diabetes mellitus has been shown to co-exist with a reduction in the endogenous antioxidant status (CitationBaynes, 1991). Several evidences suggest that STZ induces oxidative stress (CitationWright et al., 1999). Oxidative stress is caused by a relative overproduction of reactive oxygen species (ROS). ROS results in lipid peroxidation and subsequently increased in TBARS levels leading to degradation of cellular macromolecules. A marked increase in the concentration of TBARS in STZ-induced diabetic rats indicates enhanced lipid peroxidation leading to tissue injury and failure of the antioxidant defense mechanisms to prevent overproduction of ROS. Lipid peroxidation is usually measured in terms of TBARS as a biomarker of oxidative stress (CitationJanero, 1990). Treatment with META inhibited hepatic lipid peroxidation in diabetic rats as revealed by reduction TBARS levels toward normal levels. This indicated the inhibition in free radicals (ROS) generation in STZ-induced diabetic rats.
Glutathione plays an important role in the endogenous nonenzymatic antioxidant system. Primarily, it acts as reducing agent and detoxifies hydrogen peroxide in presence of an enzyme, glutathione peroxidase (CitationArias & Jakoby, 1976). The depleted GSH may be due to reduction in GSH synthesis or degradation of GSH by oxidative stress in STZ-induced hyperglycemic animals (CitationLoven et al., 1986). META treatment significantly elevated the reduced hepatic glutathione levels toward normal in diabetic rats. The results showed that the antihyperglycemic activity of META was accompanied with the enhancement in nonenzymatic antioxidant protection. These findings suggest that the META may exert its antihyperglycemic effect through the enhancement of cellular antioxidant system.
Enzymatic antioxidant mechanisms play an important role in the elimination of free radicals (ROS). CAT is a heme-containing enzyme catalyzing the detoxification of H2O2 to water and oxygen (CitationVenukumar & Latha, 2002). The inhibition of CAT activity as a result of STZ-induced hyperglycemia was reported earlier (CitationSabu & Kuttan, 2004), and the similar findings were observed in our present study. META treatment significantly recovered the CAT activity toward normal in a dose-dependent manner.
Preliminary phytochemical studies showed the presence of alkaloids, flavonoids, tannins, and triterpenoids in META. Flavonoids and tannins are well-known polyphenolic natural antioxidants, which may be responsible for the antioxidant role of META.
In the present study, administration of META to STZ-induced hyperglycemic rats demonstrated prominent reduction in blood sugar level, normalization of serum biochemical profiles comparing with STZ control rats. Also, META treatment resulted in significant modulation of lipid peroxidation, endogenous nonenzymatic (GSH), and enzymatic (CAT) antioxidant and detoxification systems. Therefore, it can be concluded that the META is remarkably effective against STZ-induced diabetes in Wistar rats plausibly by virtue of its augmenting endogenous antioxidant mechanisms.
Acknowledgement
The authors are thankful to the authority of Jadavpur University, Kolkata 700032, India for providing necessary facilities.
Declaration of interest
The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
Related Research Data
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