Anti Diabetic and Anti Dyslipidemia Activities of Cleome gynandra in Alloxan Induced Diabetic Rats

MATERIALS AND METHODS

Collection of plant materials: Fresh leaves of Cleome gynandra were collected and botanically identified. The leaves were washed with distilled water, shade dried, powdered, and stored in an air tight container until future use. Preparation of ethanolic extract- Preparation of plant extract was done The collected fresh leaves were thoroughly cleaned with distilled water, dried well and powdered. It was soaked in absolute ethanol in cold (72 h). After three days, the extract was filtered, and then it was evaporated at 400C in cylindrical water bath for the elimination of solvent. A semisolid extract (40 g) was obtained after complete elimination of alcohol under reduced pressure. It was stored in refrigerator until used.

Experimental animals: White male Wister rats weighing about 150-180 g were used. They were purchased from the Mahaveer enterprises Hyderabad. They were kept under observation for about 15 days before the onset of the experiment to exclude any intercurrent infection. The chosen animals were housed in plastic well aerated cages at normal atmospheric temperature (25±5°C) and normal 12 h light/dark cycle. Moreover, they had free access to water and were supplied daily with standard diet of known composition ad libitum. All animal procedures were in accordance with the recommendations of the ethical committee guidelines for Care and Use of Animals.

Chemical agents: Alloxan monohydrate (A) purchased from Sigma Chemical Company (St. Louis, MO USA). Cleome gynandra plant collected from chittur district of A.P, India. Metformine gift sample from Natco. Pharma. Hyderabad, India.

Induction and treatment of diabetes: Diabetes was induced by a single injection of alloxan (120 mg kg^-1 i.p ) after fasting for at least 16 h, in freshly prepared 1% sodium carboxy methyl cellulose, blood glucose levels were measured after 48 h alloxan administration, development of diabetes mellitus was proven by sustained hyperglycemia (diabetic rats had glycaemia>200 mg dL^-1). The diabetes developed rats were selected for the study and treated with the Ethanolic extract of CG 200 mg kg^-1 per oral (p.o)., Ethanolic extract of CG 400 mg kg^-1 (p.o.) in test groups and standard group treated with Metformine 25 mg kg^-1 (p.o) for 8 consecutive days (after alloxan administration).

Experimental design: The rats were randomly divided into 5 groups (n = 6) as follows:

Biochemical evaluation: Blood samples were collected from the retro orbital puncture of rat’s on 0 and, 8th days of control, diabetic and Ethanolic extract of CG (200 and 400 mg kg^-1) treated diabetic rats, centrifuged at 1000 rpm for 15 min. and determined the blood glucose levels (Trinder, 1969), Lipid profiles (Bucolo and David, 1973) and SGOP and SGPT (Reitman and Frankel, 1957) after 8 days treatment.

Statistical analysis: The data are presented as Mean±S.D Statistical comparisons were made by one-way analysis of variance (ANOVA) and followed by Student-Neuman-Keuls test. Data were considered significant when p values were lower than 0.05.

RESULTS

The effects of Ethanolic extract of CG 200 and 400 mg kg^-1 on blood glucose levels (0 and 8 days) of control, diabetic and Ethanolic extract of CG, treated diabetic rats were summarized in Table 1, respectively. Lipid profiles were represented in Table 2. The treatment with Ethanolic extract of CG 200 and 400 mg kg^-1 were significantly reduced the blood glucose concentration in diabetics groups (p<0.001).

Table 1:	Blood glucose, SGOT and SGPT levels were estimated in normal, diabetic control and treatment groups
Data was Mean±SD, **p<0.01, ***p<0.001 vs. diabetic and control groups

Table 2:	Lipid profile levels in normal, diabetic control and treatment groups
Data was Mean±SD, **p<0.01, ***p<0.001 vs. diabetic and treatment groups

The Ethanolic extract of CG was significantly reduced the triglycerides, LDL-Cholesterol, SGOT, SGPT and total cholesterol but increased HDL-cholesterol levels after treatment.

DISCUSSION

Plants may act on blood glucose through different mechanisms (Chakravarthy et al., 1980). Anti diabetic herbs stimulates beta cell in the pancreas and also regenerate pancreatic beta cells (Bopanna et al., 1997; Chorvathova et al., 1993). Fiber of plants also interferes with carbohydrate absorption, affecting blood glucose level.

Alloxan induced diabetic rats exhibited loss of body weight which is one of the threats associated with DM. Treatment with Cleome gynandra extract showed signs of recovery as comparable with the standard drug Metformin. Treatment with Cleome gynandra extract arrested elevation of glucose and lipid profiles. Administration of alloxan significantly increased the level of glucose when compared to control rats which might account for the cytotoxic effect on beta cells. Alloxan is relatively toxic to insulin producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter. This cytotoxic action is mediated by ROS source of generation of free radical is dialuric acid, a reduction product of alloxan. The free radicals undergo dismutation to H₂O₂. The action of ROS increase in cytosolic calcium concentration causes rapid destruction of beta cells (Szkudelski, 2001) and decreasing the secretion of insulin which in turn increase the blood glucose level.

Treatment with Cleome gynandra leaf extract produced significant improvement in the levels of ALT and AST are the specific markers to assess hepatocellular damage leading to liver cell necrosis (Amacher, 1998). In present study ALT and AST activities were assessed as it is the more specific index of liver cell damage in humans (Clark et al., 1973) and in experimental animals (Mitchell et al., 1974). Thus lowering of these enzymes content in serum is a definite indication of hepatoprotective action of a drug. High level of AST indicates hepato cellular damage. Activity of AST in serum was increased in alloxan intoxication Cleome gynandra extract afforded a significant protection against alloxan induced increase in the serum enzyme level. Ethanolic extract of Cleome gynandra extract may induce accelerated regeneration of liver cells by reducing the leakage of AST in to blood there by lowering its value to normal levels. ALT is more specific to the liver and a better parameter for detecting liver damage (Warnholtz et al., 2001). In the present study alloxan induced ALT level was brought back to normal by the administration of Cleome gynandra extract. In the present study indicates that diabetic animals had prior high blood glucose level. After administration of Cleome gynandra extract reduced the LDL-cholesterol, total cholesterol, triglyceride levels were observed in alloxan induced rats and plant containing flavonoids and other constituents were inhibited the dyslipidemea in our study is support of further findings of Jung et al. (2006) reported that can inhibit lipogenesis and lower plasmatic triglycerides levels by enhancing LDL receptors expression and increasing fat bile rejection from the results of clinical studies (Knekt et al., 2002). Insulin resistance was compensated by the enhanced insulin secretion, whereas persistently elevated FFAs may contribute to progressive β-cell failure (β-cell lipotoxicity) in individuals genetically predisposed to type 2 diabetes mellitus (Gu et al., 1992).

Cleome gynandra occurs throughout the tropic and subtropic regions. It contains chemical constituents such as triterpenes, tannins, anthroquinones, flavonoids, saponins, steroids, resins, lectins, glycosides, sugars, phenolic compounds and alkaloids (Narendhirakannan et al., 2005) and these are more beneficial in diabetes and its associated complications, holding hope of the new generation antihyperglycemic drug.

CONCLUSION

Our results shown that oral administration of cleome gynandra extract has a beneficial effect on the alloxan-induces diabetes rats by reducing hyperglycaemia, dyslipidamia and improving the HDL status. This study suggests that the induction of diabetes mellitus by alloxan in rats may be prevented by flavonoids of plant constituent’s administration. We hypothetized that this effect may be result of antiradical/chelatory properties of flavonoids. There is a need to continue to explore the mechanisms for anti diabetes.