EFFECT OF EXACUM WIGHTIANUM ARN., AN ENDEMIC MEDICINAL PLANT ON THE ANTI DIABETIC ACTIVITY

The study revealed the antidiabetic activity of the ethanol extract of Exacum wightianum Arn. (Gentianaceae) in streptozotocin induced diabetic rats. The pilot studies were carried after oral administration at doses of 100, 200, 500 and 1000 mg/kg b.wt. in sub-acute study. In diabetic induced rats fed with the extract at 100 and 200 mg/kg body b.wt. The fasting plasma glucose levels were reduced to near normal body and liver weight were found to be increased. Whereas blood glucose, protein, albumin and creatinine levels were estimated after two weeks. The extract significantly inhibited the induction of albuminuria, proteinemia and uremia. The present study clearly indicated a significant antidiabetic activity with the ethanol extract of E. wightianum supports the traditional usage of the plant by Ayurvedic physicians for the control of diabetes. The extract could be useful in preventing the incidence of long term complications of diabetes mellitus.


INTRODUCTION
Diabetes, as one of the most common global diseases, affects approximately 200 million individuals worldwide and approximately 300 million people worldwide are at risk of diabetes (McCune and Johns, 2002). There are two types of diabetes: type 1 and type 2. In type 1 diabetes insulin deficiency originated from allergic reactions in genetically susceptible people that eventually destroy the pancreatic β-cells producing insulin. Type 1 diabetics are insulin dependent. Type 2 diabetes is the most common form of diabetes accounting for 90% of cases worldwide. Patients with type 2 diabetes are not dependent to use insulin. It is found that the main factor to improve vascular diseases seen in type 2 diabetics is a decreased plasma antioxidant level (Kelble, 2005). The approach to the management of type 2 diabetes is based on weight management and life-style modification. In modern medicine, no satisfactory effective therapy is still available to cure the diabetes mellitus. Now-a-days insulin therapy is encouraged for the management of diabetes mellitus, but there are several drawbacks like insulin resistance. The herb Exacum wightianum Arn. belongs to the family Gentianaceae. It has a long history of use in Ayurvedic medicine (the traditional medicine of India). It is extensively used as bitter tonic and febrifuge in the Ayurvedic system of medicine (Sharma, 1993). The extract has long been used in folk medicine for the treatment of hepatitis, cholecystitis, pneumonia, malaria, dysentery and spasm; whereas the recent investigations have shown that some xanthones possess a marked hypoglycemic activity when administered to rats. In the present investigation the effect of ethanolic extract of E. wightianum were studied on streptozotocin-induced diabetic male Wistar albino rats.

Collection of plant material
All aerial parts and roots of Exacum wightianum (Gentianaceae) was collected during blooming season August, 2010 from Naduvattam, Uthagamamdalam, the Nilgiri Hills, Western Ghats, Southern India, Tamil Nadu. The plant was identified and authenticated by a plant taxonomist, Mr. M. Murugesan, Scientist, SACON, Coimbatore.

Preparation of extract
500 g of shade dried and pulverized whole plant powder of E. wightianum was defatted with petroleum ether and the residue was then reextracted with ethanol by using soxhlet apparatus. This extract was stored at 4ºC and used for further studies.

Experimental Animal
Male Wistar Albino rats weighing 180-250 g were obtained from Agricultural University, Animal house lab, Trissur, Kerala. All the animals were maintained in polycarbonated cages in an animal room with 12 h light/12 h dark cycle at temperature of 22 ±2ºC and humidity of 45-60%. They were fed with commercial pelleted rats chow and free access water during the entire period of experiment. The experiments were performed according to ethical guidelines for the investigation of experimental pain in conscious animals (659/02/a/CPCSEA).

Induction of experimental diabetes
Diabetes was induced by administering intraperitonial injection of a freshly prepared solution of Streptozotocin monohydrate dissolved in 0.1 M cold citrate buffer (pH 4.5) to the overnight fasted rats (Sekar et al., 1990) and the STZ solution is made cold using cold citrate buffer immediately before administration. The drug induced hypoglycemia in all rats was controlled by treating 5% glucose solution. The blood glucose above 250 mg/dl on the third day after injection was considered as diabetic rats. At this time the treatment was started on the fifth day after injection and considered as first day of treatment.

Experimental design
The streptozotocin induced rats were divided into five groups each contains five numbers of rats. Group I served as normal rats without any treatment, Group II served as diabetic control rats. Group III diabetic rats given glibenclamide (600 μg/kg b.wt.).Group IV served as diabetic rats given ethanolic extract of E. wightianum (100 mg/kg b.wt.).Group V served as diabetic rats given ethanolic extract of E. wightianum (200 mg/kg b.wt.). At the end of the experiment all the animals were deprived of food overnight, anesthetized and sacrificed by cervical dislocation. Blood was collected in heparinised tubes and used for the further estimation of biochemical studies.
2.6. Toxicity study E. wightianum ethanolic extract was orally administrated at a concentration of 250,500,750 and 1000 mg/kg body weight/ day for a period of 14 days. The toxic effects were measured by body weight and morphological changes.

Estimation of Insulin, Blood glucose, Urea and Creatinine
The blood glucose level was estimated by the method of O-toludine by Sasaki et al. (1972), Insulin was estimated by radio immuno assay kit purchased from stat Diagnostics, Mumbai, India (Anderson et al., 1993). Urea level was assayed according to the method of Varley (2012) and Creatinine level was estimated by Owen et al., (1954).

Statistical analysis
All data were expressed as means ± S.E. Significant differences among the groups were determined by one-way analysis of variance using the DMRT statistical analysis program. Statistical significance was considered at p<0.05.

RESULTS
The ethanol extract of E. wightianum was administrated orally to rats at the doses of 100, 200, 500 and 1000 mg/kg b.wt. and the mean death rats were observed. The results showed that numbers of deaths of rats were observed at the different dose levels. There was no morphological change like respiratory distress, hair loss, restlessness, convulsions, laxative, coma, weight loss etc. There was no lethality or any toxic reactions found at any of the doses selected till the end of treatment period (Table 1). Table 2 shows the effect of E. wightianum extract on the body weight and organ weight in normal and streptozotocin induced diabetic rats. A significant weight loss was observed in the diabetic control group (Group II). The body weight and organ weight were increased in the E. wightianum extract treated groups IV and V at two concentrations 100 and 200 mg/kg b.wt. A significant improvement was observed in the group III treated with the standard drug, glibenclamide (Fig.1 a,b). The table 3 shows the effects of E. wightianum ethanol extract administered on streptozotocin induced diabetes for 14 days drug treatment. E. wightianum ethanol extract at the doses of 100 and 200 mg/kg b.wt. treatment significantly decreased the blood glucose level in streptozotocin induced diabetes rats (Group II). There was a significant elevation in blood glucose level with significant decrease in serum insulin levels in streptozotocin diabetic rats, compared with normal rats. Administration of E. wightianum extracts 100 and 200 mg/kg b.wt. and glibenclamide treated group III bring blood glucose and serum insulin towards normal levels. The effect of E. wightianum extracts 200 mg/kg b.wt. was significantly better than 100 mg/kg b.wt. The administration of E. wightianum extract and glibenclamide showed a significant effect in lowering blood glucose and increasing serum insulin. In the diabetic control group II, the levels of serum creatinine was found to be increased in comparison with control. Treatment with E. wightianum extract significantly prevented the streptozotocin induced creatinine level. The diabetic rats administrated with E. wightianum extract at 100 and 200 mg/Kg b.wt. and glibenclamide at the dose of 600μg/Kg b.wt., daily orally for 14 days consequently orally by 1GC altered the values of insulin, glucose and creatinine when compare to control. The levels of the urea in streptozotocin diabetic rats were significantly higher than the control. When these diabetic rats treated with two concentrations of extracts (100 and 200 mg/kg b.wt.) decreased the levels when compare to group II (Fig.2-5). Gallagher et al., (2003) studied the ability to inhibit glucose diffusion using same in vitro method. They reported that agrimony and avocado represented the most inhibitory effect on glucose diffusion [more than 60%] and mushrooms, coriander, eucalyptus, juniper, lucerne, mistletoe decreased significantly (ranged 6-48%) and elder, nettle extracts did not significantly decrease glucose diffusion. In the present study, the ethanol extract of E. wightianum effectively decreased the blood glucose in streptozotocin-induced diabetic rats, which is even better than glibenclamide. The results of the present study indicate that E. wightianum extract brought back the body weight, liver weight, glucose, insulin, protein and antioxidant. Aqueous extract of Punica granatum has brought decreased body weight of diabetic rats to normal (Khalil, 2004). The ability of this extract to prevent the body weight loss seems to be due to its anti-diabetic activity. Prakasam et al., (2003) have reported that a reduction in body weight was observed in STZ diabetic animals, but when animals were treated with Casearia esculenta root extracts, the decrease in the body weight was minimized to almost nil. A significant weight loss was observed in allaxon induced diabetic rats than normal rats, when treated with aqueous extract of Laportea ovalifolia and Tolbultamide in streptozotocin induced diabetic rats. The body weight was improved when compared with the untreated diabetic rats (Pari and Saravanan, 2004).  The administration of Glibenclamide also decreased the levels of urea and creatinine to some extent. Stabilization of serum creatinine and urea levels through administration of the extract E. wightianum is further a clear indication of the improvement of the functional status of the liver cells. Our results thus clearly demonstrated that the ethanolic extract of E. wightianum has potent antihyperglycemic in STZ induced diabetic rats. Further studies are warranted to isolate and characterize the bioactive antidiabetic principles from this plant, which can therefore be used as an alternative remedy for the treatment of diabetes mellitus and oxidative stress associated diabetic complications.