Evaluation of antidiabetic and antihyperlipidemic effects of Ougeinia oojeinensis in diabetic rats

Article Information Received 2 December 2013 Received in revised form 5 Feb 2014 Accepted 5 Feb 2014 Abstract Ougeinia oojeinensis is a medicinal plant, used by herbalists for treating various diseases, one of which is diabetes mellitus. The aim of this study was to investigate the antidiabetic activity of Ougeinia oojeinensis bark and root extracts. The different doses of ethanol extracts of bark and root were administered orally in Streptozotocin (STZ)-induced diabetic rats. After the administration of extracts, blood glucose levels were monitored at specific intervals and it was found that they were significant lowered. The effect of extracts on induced hyperlipidemia was analyzed where the extracts significantly lowered the elevated total cholesterol, triglycerides (TGL) and low density lipoprotein (LDL) level while increased the High density lipoprotein (HDL). Glibenclamide was used as a standard drug at a dose of 0.50 mg/kg body weight. The experimental data revealed that extract has significant antidiabetic activity in streptozotocininduced rats compared to standard drug.


Introduction
The scientific data revealed that every year the number of diabetic patients is growing alarmingly all over the World.It is induce by derangement in carbohydrate, fat, protein metabolism.Currently physician prescribed allopathic medicines for the treatment of diabetes, but these medicines are reported to have side effects in the long run.Therefore, there is a need to search for effective and safe drugs for diabetes 1 .The use of herbal medicines for the treatment of diabetes mellitus has gained importance throughout the world.The World Health Organization also recommended and encouraged this practice especially in countries where access to the conventional treatment of diabetes is not adequate.There is an increased demand to use natural products with antidiabetic activity due to the side effects associated with the use of insulin and oral hypoglycemic agents 2,3 .
Ougeinia oojeinensis (Roxb.)Hochr (Fabaceae) known in Hindi as Tinsa, is a deciduous trees, found in the outer Himalayas and sub-Himalayan tracts from Jammu to Bhutan up to an altitude of 1500m and extending through the whole of northern and central India into the greater part of Deccan peninsula 4 .The extract of the whole plant O. oojeinensis were scientifically evaluated for anti-inflammatory, hepatoprotective and analgesic activities in previous studies.
Phytochemical investigated on O. oojeinensis have reported the presence of lupeol, hydroxlupeol, betulin and isoflavanones such as dalbergioidin, homoferreirin and ougenin [5][6][7] .Yet there is no paucity of information regarding the antidiabetic activity of O. oojeinensis of bark.This study was undertaken to fill the lacuna in this regard.

Plant material
The bark and root of Ougeinia oojeinensis was collected from the forest area of Purimandal (Jammu)

Preparation of Ougeinia oojeinensis ethanol extracts
The powder of the bark and root of Ougeinia oojeinensis, was packed separately in the Soxhlet apparatus and extracted with ethanol, until the completion of the extraction.The extract was filtered while hot, and the resultant extract was distilled in vacuum under reduced pressure in order to remove the solvent completely, and later dried in a desiccator.After that ethanol extract of bark (EOOB) and root (EOOR) were kept in air tight container for further study.

Animals
Male Wistar albino rats having a weight of 170 -220 g were kept in quarantine for 10 days under standard husbandry conditions (27.3 o C, Relative humidity 65 ± 10%) for 12 hours in dark and light cycle, respectively, and were given standard food and water ad libitum.The study was permitted by the Institution Animal Ethical Committee.

Oral glucose tolerance test (OGTT)
The oral glucose tolerance test 8 was performed in overnight fasted (18 hours) normal rats.The rats were divided into seven groups (n = 6).Group I served as normal control rats, administered drinking water daily; Group II had glucose control rats; Group III rats were administered standard drug Glibenclamide (0.5 mg/kg); Group IV rats were administered EOOB (200 mg/kg); Group V rats were administered EOOB (400 mg/kg); and Group VI rats were administered EOOR (200 mg/kg); and Group VII rats were administered EOOR (400 mg/kg).Glucose (2 g/kg) was fed to rats of Group II to Group VII, 30 minutes prior to the administration of the extracts and standard drug.Blood was withdrawn from the retroorbital sinus after 0, 30, and 90 minutes of extract and standard drug administration, and the plasma obtained after centrifugation at 3000 rpm was estimated for fasting plasma glucose levels using a glucose oxidase-peroxidase glucose estimation kit.

Induction of non-insulin dependent diabetes mellitus (NIDDM)
Non-insulin dependent diabetes mellitus was induced 9,10 in overnight fasted adult Wistar strain albino male rats weighing 170 -220 g by a single intraperitoneal injection of 60 mg/kg Streptozotocin, 15 minutes after i.p. administration of 120 mg/kg of nicotinamide.Streptozotocin (STZ) was dissolved in a citrate buffer (pH 4.5) and nicotinamide was dissolved in normal saline.Hyperglycemia was confirmed by the elevated glucose levels in plasma, determined at 72 hours and then on day 7, after injection.The threshold value of fasting plasma glucose to diagnose diabetes was taken as > 126 mg/dl.Only those rats that were found to have permanent NIDDM were used for the study.

Experimental design
The animals were segregated into five groups of six rats each.The extract was administered for 28 days.Group I served as normal control rats, administered drinking water daily for 28 days; Group II had diabetic control rats, administered drinking water daily for 28 days; Group III diabetic rats were administered standard drug Glibenclamide (0.5 mg/kg); Group IV diabetic rats were administered EOOB (200 mg/kg); Group V diabetic rats were administered EOOB (400 mg/kg); and Group VI diabetic rats were administered EOOR (200 mg/kg); and Group VII diabetic rats were administered EOOR (400 mg/kg) for 28 days.The fasting glucose levels were determined on days 0, 7 th , 14 th and 28 th of extract administration.During the experimental period, the rats were weighed daily and the mean change in body weight was calculated.

Estimation of biochemical parameters
The biochemical parameters were determined on day 12 after the animals were sacrificed by cervical dislocation.Total cholesterol, triglycerides (TGL), high-density lipoprotein (HDL) and low-density lipoprotein (LDL), were determined by the glucose oxidase method, using an auto-analyzer 11 .

Statistical analysis
The results are expressed as mean ± SEM of six independent experiments.Statistical significance between the groups was evaluated by one-way analysis of variance (ANOVA) followed by Dunet's test.A P < 0.05 value was considered as statistically significant.

Oral glucose tolerance effects
The effects of ethanol extracts of Ougeinia oojeinensis on the plasma glucose level are shown in Table 1.After administration of glucose in rats the rise in glucose level was observed in glucose control, extract treated and standard group.In rats treated with barks and root extracts of Ougeinia oojeinensis, there was a significant reduction in plasma glucose level, while in glucose control rats the plasma glucose level increased.Meanwhile same results were observed in glibenclamide treated group.

Effect on non-insulin dependent diabetes mellitus
Induction of diabetes in experimental rats was confirmed by the presence of a high fasting plasma glucose level.The effect of bark and root extracts of Ougeinia oojeinensis, on serum glucose levels of normal and Streptozotocin-induced rats are shown in Table 2.
The animals treated with streptozotocin namely Group II, a significant increase in serum glucose level was observed on 0, 7 th , The Group III received glibenclamide (0.5 mg/kg p.o.) showed significant decrease in serum glucose level when compared with diabetic control rats.After the oral administration of EOOB in diabetic control rats, a significant reduction in blood glucose level was observed when compared with diabetic control rats.Moreover the administration of EOOR in diabetic control rats, also significantly decreased the serum glucose level compared with diabetic control rats.The outcomes exhibited that EOOB and EOOR at dose of 400 mg/kg body weight significantly decreases the blood glucose level of diabetic rats on 7 th day.While the EOOB and EOOR at dose of 200 mg/kg body weight significantly decreases the blood glucose level of diabetic rats on 14 th day.From results it has been observed that the bark extracts showed maximum activity as compared to root extract.

Anti-hyperlipidaemic activity
The outcomes of lipid profiles in control and experimental rats are exhibited in table 3. The rats of diabetic control showed significant increase in serum TGL, total cholesterol and LDL while increase in HDL when compared with normal.The rat treated with glibenclamide also reduced TGL, total cholesterol, LDL, and increased HDL when compared with diabetic control group.The EOOB and EOOR showed significant decrease in total cholesterol, LDL, Triglycerides and significant increase in HDL when compared with diabetic control group.All these effects were observed on day 28 th .From result of lipid profile it has been observed that the barks extract exhibited maximum antihyperlipidaemic activity on compared with roots extract.The present experimental result indicated that ethanol extracts of barks and roots exhibited a potent blood glucose lowering properties in STZ diabetic rats.
During the study, the body weights of rats before and after induction of diabetes, and after treatment were measured (Table 4).The results exhibited that decreased in body weight of rats after induction of diabetes, and increased in body weight of rats after treatment with extracts.

Discussions
In our study, we selected Ougeinia oojeinensis to check its antidiabetic activity owing to its traditional uses.The diabetes was induced on rats after administration of streptozotocin.The EOOB and EOOR were screened for streptozotocin-induced antidiabetic activity.The EOOB and EOOR significantly reduced the blood glucose level in STZ-induced-diabetic rats as compared to the diabetic control group.Moreover the EOOB and EOOR increased the body weight of diabetic rats.The possible mechanism by which Ougeinia oojeinensis brings about its hypoglycemic action in diabetic rat may be by potentiating the insulin effect of plasma by increasing either the pancreatic secretion of insulin from the existing beta cells or by its release from the bound form.In recent years, considerable interest has been directed towards the investigation of plasma lipids and lipoproteins pattern in diabetes mellitus due to the fact that abnormal lipid level leads to the development of coronary artery disease in diabetic patients 8 .
In the present study, ethanol extract of bark and root of Ougeinia oojeinensis had significantly decreased total cholesterol, triglycerides and LDL with increase in HDL which is having a protective function for the heart compared with diabetic control group.
having altitude of 350 ft.The plant was authenticated by botanist Dr. Sumeet Gairola plant systematic section, plant biotechnology division, I.I.I.M, Jammu, India and a voucher specimen was deposited in the Herbarium of Department of Botany, IIIM Jammu (Acc.no.22271).After authentification, plant material was dried at room temperature until it was free from the UK Journal of Pharmaceutical and Biosciences Available at www.ukjpb.comSamyal et al.Antidiabetic Activity UK J Pharm & Biosci, 2014: 2(1); 8 moisture.Finally barks and roots were subjected to size reduction to get coarse powder.

Table 1 : Effect of ethanol extracts of Ougeinia oojeinensis on oral glucose tolerance test Group Plasma glucose concentration (mg/dl)
Ethanol extract of Ougeinia oogenesis of bark (EOOB), Ethanol extract of Ougeinia oogenesis of root (EOOR), Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at a P<0.05 when compared with normal control group, *P<0.05 when compared with diabetic control group

Table 3 : Determination of biochemical parameters after treatment with ethanol extracts of Ougeinia oojeinensis
Ethanol extract of Ougeinia oogenesis of bark (EOOB), Ethanol extract of Ougeinia oogenesis of root (EOOR), Values are expressed as mean ± SEM (Number of animals, n=6); significantly different at a P<0.05 when compared with normal control group, *P<0.05 when compared with diabetic control group

Table 4 : Effect of ethanol extracts of Ougeinia oojeinensis on changes in body weight in rats 5 Conclusions
This research was carried out in the Diabetes Pharmacology Division of Indian Institute of Integrative Medicine (CSIR), Canal Road,