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1 UK Journal of Pharmaceutical and Biosciences Vol. 2(5), 27-33, 2014 UK Journal of Pharmaceutical and Biosciences Available at RESEARCH ARTICLE ISSN: Evaluation of Antidiabetic Activity of Isolated Compound from Ougeinia oojeinensis Bark Extract in Diabetic Rats Manohar Lal Samyal 1,, Anil Ahuja 1 Zabeer Ahmed 2*, 1 Department of Pharmacy, Sunrise University, Alwar , (Rajasthan), India 2 IIIM Jammu, Canal Road, Jammu , (J&K), India Article Information Received 5 Sept 2014 Received in revised form 25 Oct 2014 Accepted 1 Nov 2014 Keywords: Ougeinia oojeinensis Streptozotocin Total cholesterol Triglycerides Glibenclamide Isolated fractions * Corresponding Author: zabeerahmed@gmail.com Tel.: Abstract Ougeinia oojeinensis is used by tribal people for treatment of diabetes. The present aim of this study was to isolate different fractions from O. oojeinensis bark extracts, and evaluated the potency of antdiabetic activity of fractions. The nine different fractions were collected from ethanol extracts of O. oojeinensis bark. The fraction F4, F6, F7 and F8 were administered orally in Streptozotocin (STZ)-induced diabetic rats. After the administration of fractions, blood glucose levels were monitored at specific intervals and it was found that they were significant lowered. The effect of fractions on induced hyperlipidemia was analyzed where the fraction 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. Moreover, the fraction treated rats exhibited the significant rise in serum insulin level compared with streptozotocin- induced diabetic rats. The findings demonstrated that fraction isolated from ethanol extracts of O. oojeinensis bark has significant antidiabetic activity in streptozotocin-induced rats compared to standard drug.. 1 Introduction Diabetes mellitus is metabolic diseases and characterized by hyperglycemia that leads from absolute or relative deficiency of insulin secretion, impaired insulin action, or both. There are numerous factors that enhance the blood-glucose level in human some of them are consumption of carbohydrate-rich diets, upsurge in immobile life style and overweight. International Diabetes Federation documented that India has the highest number diabetic patient, and India is considered to be diabetes capital of the world. It is deliberated that the diabetes is the third most life-threatening disease whose death rate is just after cancer and cardiovascular disease. Now in prior basis, we applied effort to control the diabetes, because it is assuming that more than 400 million people of the world will be effected from diabetes by ,4. Today synthetic drug namely acarbose, miglitol and voglibiose, sulphonylureas, biguanides etc are used for the ailment of hyperglycemia. The synthetic drugs are associated with various side effects such as causing hypoglycemia at higher doses, dermatological reaction, liver problems, nausea and vomiting, generalized hypersensitivity reactions, lactic acidosis and diarrhea 5,6. The secondary complication of synthetic drugs leads to limitation in its use; and creating major medical issues in management of diabetes. Hence, health-care professionals are considering alternative medicines system, comprising many herbal medicines for the ailment of diabetes. Now the demand of herbal drugs has increased because of their effectiveness, less side effects and relatively low cost. Therefore more researches are required to develop new antihyperglycemic agents with high efficiency and low toxicity from traditional medicines. Ougeinia oojeinensis (Roxb.) Hochr belongs to family Fabaceae and in Hindi it is known as Tinsa. The traditional healers of Chhattisgarh state prescribed O. oojeinensis in the treatment of various diseases such as inflammation, jaundice, diabetes, skin diseases, leprosy etc 7,8. Apart from this many researchers worked on different parts of O. oojeinensis and reported their various pharmacological activities 9. The chemical components present in medicinal plant play chief role in imparting pharmacological activity. The efficacy of pharmacological activity of herbal drugs depends upon the quantity and nature of secondary metabolite available in plants. The

2 phytoconstituents such as flavonoids, alkaloids, saponin and polyphenol compound are present extensively in different parts of this plant 10,11. The flavonoids impart chief role in regeneration of pancreatic β-cells, resulting in lowering the blood glucose level and lipid profile of diabetic patient 12,13. Singh et al. (2011) reported the antidiabetic potential of the methanol and aqueous extracts of leaves of O. oojeinensis 14. Samyal et al. (2014) documented significant antidiabetic and antihyperlipidemic activity of the ethanol extracts of bark and root of O. oojeinensis in streptozotocin-induced diabetic rats 15. In our earlier study we reported that ethanol extract of bark extract of O. oojeinensis exhibited maximum significant antidiabetic activity compared to root extract. The mechanisms of action of extracts for antidiabetic activity were not evaluated. In previous study, we demonstrated that various types of secondary metabolites were present in the extracts. The secondary metabolites which are responsible for antihypeglycemic activity were not investigated. Keeping the above information in view, we aimed to evaluate the antihyperglycemic activity of the different fraction of the ethanol extracts of bark of O. oojeinensis in streptozotocin-induced diabetic rats. 2 Material and Methods 2.1 Collection and identification of plant material The bark of O. oojeinensis were collected from the forest area of Purimandal (Jammu), India having altitude of 350 ft. The plant was authenticated by botanist Dr. Sumeet Gairola, Plant Systematic Section of 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 ). After authentication, plant material was dried at room temperature until it was free from the moisture. Finally barks were subjected to size reduction to get coarse powder. 2.2 Preparation of O. oojeinensis of ethanol extracts The powder of the bark of O. 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 was kept in air tight container for further study. 2.3 Qualitative chemical tests of plant extracts Qualitative chemical tests were performed to determine the presence of alkaloids, carbohydrates, glycosides, polyphenols, saponins, proteins and amino acids, fixed oils and fats, tannins and terpenoids 16. The O. oojeinensis extract was subjected to column chromatography using silica gel ( mesh size), and eluted with the following solvent ratios of Hexane: dichloromethane (DCM), 100:0, 80:20, 60:40, 40:60, 20:80, 0:100, then with 100:0, 90:10, 80:20, 70:30, 60:40, 50;50, 40:60, 30:70, 20:80, 10:90, 0:100, DCM: Methanol (Eth). The fractions (25 ml) were collected from the column. The elute collected were monitored by thin layer chromatography (eluent: DCM-MeOH, 9:1 and 3:2) for homogeneity and the similar fraction were pooled together. The Nine different fractions were collected and dried. The fraction F1, F2 and F3 were containing waxy material; the fractions F5 and F9 were powder but quantity was very little. The yield of fraction F4, F6, F7 and F8 were 130 mg, 320 mg, 260 mg and 380 mg respectively 17. The fractions were further analyzed for phytochemical screening and antihyperglycemic activity. 2.5 Qualitative chemical tests isolated compound Qualitative chemical tests were performed to determine the presence of alkaloids, carbohydrates, glycosides, polyphenols, saponins, proteins and amino acids, fixed oils and fats, tannins and terpenoids Oral glucose tolerance test (OGTT) of O. oojeinensis fractions The oral glucose tolerance test was performed in overnight fasted (18 hours) normal rats. The rats were divided into seven groups (n = 6). I served as normal control rats, administered drinking water daily; II had glucose control rats; III rats were administered standard drug Glibenclamide (0.5 mg/kg); IV rats were administered F4 (50 mg/kg); V rats were administered F6 (50 mg/kg); and VI rats were administered F7 (50 mg/kg); and VII rats were administered F8 (50 mg/kg). Glucose (2 g/kg) was fed to rats of II to VII, 30 minutes prior to the administration of the extracts and standard drug. Blood was withdrawn from the retro-orbital 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 12, Induction of non-insulin dependent diabetes mellitus (NIDDM) Non-insulin dependent diabetes mellitus was induced 12,14 in overnight fasted adult Wistar strain albino male rats weighing 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 > Isolation of compound from O. oojeinensis ethanol extracts UK J Pharm & Biosci, 2014: 2(5); 28

3 mg/dl. Only those rats that were found to have permanent NIDDM were used for the study. 2.8 Experimental design Evaluation of antidiabetic activity of O. oojeinensis fractions The animals were segregated into seven groups of six rats each. The extract was administered for 28 days. I served as normal control rats, administered drinking water daily for 28 days; II had diabetic control rats, administered drinking water daily for 28 days; III diabetic rats were administered standard drug Glibenclamide (0.5 mg/kg); IV diabetic rats were administered F4 (50 mg/kg); V diabetic rats were administered F6 (50 mg/kg); and VI diabetic rats were administered F7 (50 mg/kg); and VII diabetic rats were administered F8 (50 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 Effect of O. oojeinensis fractions on insulin level The animals were segregated into seven groups of six rats each. The extract was administered for 28 days. I served as normal control rats, administered drinking water daily for 28 days; II had diabetic control rats, administered drinking water daily for 28 days; III diabetic rats were administered standard drug Glibenclamide (0.5 mg/kg); IV diabetic rats were administered F4 (50 mg/kg); V diabetic rats were administered F6 (50 mg/kg); and VI diabetic rats were administered F7 (50 mg/kg); and VII diabetic rats were administered F8 (50 mg/kg) for 28 days. Afterwards, blood samples were withdrawn in order to examine the insulin levels. Serum insulin was measured using a GLAZYME INSULIN-EIA TEST 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. 3 Results 3.1 Phytochemical screening of ethanol extract of O. oojeinensis bark The phytochemical investigation of ethanol extracts of O. oojeinensis bark revealed the presence of alkaloids, Saponins, glycosides, carbohydrates, tannins & phenolic compounds and flavonoids. 3.2 Preliminary phytochemical analysis of isolated fraction of ethanol extract of O. oojeinensis The phytochemical investigation of F4 of O. oojeinensis bark revealed the presence of alkaloids, glycosides and carbohydrates. The F6 and F8 indicate the presence of tannins & phenolic compounds and flavonoids. However, the F7 exhibited the presence of alkaloids, saponins and carbohydrates (Table 1). The secondary metabolites play important role in reducing the blood sugar level. The fractions obtained from the ethanol extract of O. oojeinensis exhibited various types of secondary metabolites. Apart from this the F6 and F8 containing polyphenol compound and these organic substances impart chief role in antidiabetic activity. Hence this result supports us to evaluate the antidiabetic activity of the fractions obtained from ethanol extracts of O. oojeinensis. Table 1 Preliminary phytochemical analysis of isolated fractions of ethanol extracts of O. oojeinensis Phytoconstituents F4 F6 F7 F8 Alkaloids Saponins Glycosides Carbohydrates Tannins & Phenolic compounds Flavonoids Steroids Proteins and Amino acids Triterpenoids Fixed Oils and Fats (+) Present, (-) Absent 3.3 Antidiabetic activity Oral glucose tolerance effects of O. oojeinensis fractions The effects of fractions of F4, F6, F7 and F8 of O. oojeinensis on the plasma glucose level are shown in table 2. After administration of glucose in rats the rise in glucose level was observed in glucose UK J Pharm & Biosci, 2014: 2(5); 29

4 control, extract treated and standard group. In rats treated with fractions F6 and F8, 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. However, the fractions F4 and F7 fail to significant decrease in in plasma glucose level. Table 2 Effect of isolated fractions of O. oojeinensis on oral glucose tolerance test Plasma glucose concentration (mg/dl) 0 min 30 min 90 min Normal Control 79.5± ± ±1.89 Glucose control 77.4± ±4.05 a 156.9±2.18 a Glucose + Glibenclamide ( ± ±3.74* 79.5±4.17* mg/kg) F4 (50 mg/kg) 85.2± ± ±3.16 F6 (50 mg/kg) 84.7± ±1.25* 79.8±4.28* F7 (50 mg/kg) 81.6± ± ±1.85 F8 (50 mg/kg) 83.8± ± ±1.24* 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 Effect on non-insulin dependent diabetes mellitus of O. oojeinensis fraction Induction of diabetes in experimental rats was confirmed by the presence of a high fasting plasma glucose level. The effect of fraction of O. oojeinensis, on serum glucose levels of normal and Streptozotocin-induced rats are shown in table 3. The animals treated with streptozotocin namely group II, a significant increase in serum glucose level was observed on 0, 7 th, 14 th and 28 th day when compared with normal group rats ( I). 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 fraction in diabetic control rats, a significant reduction in blood glucose level was observed when compared with diabetic control rats. Moreover the administration of fractions in diabetic control rats, also significantly decreased the serum glucose level compared with diabetic control rats. The outcomes exhibited that F6 and F8 at dose of 50 mg/kg body weight significantly decreases the blood glucose level of diabetic rats on 7 th day. While the F7 at dose of 50 mg/kg body weight significantly decreases the blood glucose level of diabetic rats on 14 th day. While, F4 does not significantly reduced blood glucose level of diabetic rats. From results it has been observed that the F6 showed maximum activity as compared to other fractions Anti-hyperlipidaemic activity of O. oojeinensis fractions The outcomes of lipid profiles in control and experimental rats are exhibited in table 4. 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. The fraction F6, F7 and F8 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 F6 exhibited maximum antihyperlipidaemic activity on compared with other fractions. The F4 revealed insignificant significant decrease in total cholesterol, LDL, Triglycerides and significant increase in HDL when compared with diabetic control group. The present experimental result indicated that F6 and F8 exhibited a potent blood glucose lowering properties in STZ diabetic rats Effect on body weight by O. oojeinensis fractions During the study, the body weights of rats before and after induction of diabetes, and after treatment were measured (Table 5). 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. 3.4 Effect on insulin by O. oojeinensis fractions The serum insulin level was decreased in diabetic rats with streptozotocin-induction as compared with the normal control group. There was also a significant difference of this parameter between the fraction treated groups and diabetic group, except F4 and F8 (Table 6). After 28 days of fraction supplementation to diabetic rats, there was a significant increase in the serum insulin level with respect to the diabetic group, except F4 and F8. 4 Discussions Streptozotocin, a monofunctional nitrosourea derivative, derives diabetogenic activity due to its ability to induce oxidative stress and damage in β-cells. Streptozotocin can selectively attack pancreatic β-cells by producing free radicals of oxygen, nitrogen monoxide, and reducing intracellular NAD and NADP, which are crucial for the electron delivery and energy metabolism in β-cells. The diabetes was induced on rats after administration of streptozotocin. The fractions of O. oojeinensis were screened for streptozotocin-induced antidiabetic activity. The F6 and F8 significantly reduced the blood glucose level in STZ-induced-diabetic rats as compared to the diabetic control group. Moreover the F6 and UK J Pharm & Biosci, 2014: 2(5); 30

5 F8 increased the body weight of diabetic rats. The possible plasma by increasing either the pancreatic secretion of insulin from mechanism by which O. oojeinensis brings about its hypoglycemic the existing beta cells or by its release from the bound form. action in diabetic rat may be by potentiating the insulin effect of Table 3 Effect of isolated fractions of O. oojeinensis on fasting plasma glucose level in rats Fasting plasma glucose concentration (mg/dl) Day 0 Day 7 th Day 14 th Day 28 th Normal Control 78.2± ± ± ±4.15 Diabetic control (Streptozotocin) Glibenclamide (0.50 mg/kg) 141.6±5.17 a 178.3±2.63 a 212.5±3.48 a 253.2±1.89 a 131.4± ±4.19* 81.8±3.24* 71.5±2.76* F4 (50 mg/kg) 135.7± ± ±2.81* 161.2±3.56* F6 (50 mg/kg) 140.2± ±5.23* 106.7±4.25* 86.2±2.89* F7 (50 mg/kg) 139.5± ± ±3.72* 185.4±6.12* F8 (50 mg/kg) 138.4± ±3.41* 116.8±4.36* 98.5±3.15* 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 Table 4 Determination of biochemical parameters after treatment with isolated fractions of O. oojeinensis Lipid Profile (mg/dl) Triglyceride Total Cholesterol HDL LDL Normal control 85.7± Diabetic control (Streptozotocin) 171.4±3.24 a a a a Glibenclamide (0.50 mg/kg) 81.3±4.28* * * * F4 (50 mg/kg) 142.3± * F6 (50 mg/kg) 81.4±2.54* * * * F7 (50 mg/kg) 146.8± * F8 (50 mg/kg) 89.4±3.18* * * * 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 Generally, it has been observed that hyperlipidemia is a complication associated with hyperglycemia. During study it was observed increase in total cholesterol, triglycerides, LDL, and decrease in HDL in streptozotocin induced diabetic rats as compared to normal animals. The F6 and F8 showed significant reduction in total cholesterol, LDL, Triglycerides and significant rise in HDL when. The potent antidiabetic effect of the plant extract suggests the presence of potent antidiabetic active principles, which produced antihyperglycemic effect in diabetic rats 15,19. The outcomes of lipid profile confirmed the potent antidiabetic activity of bark and root of O. oojeinensis. 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. UK J Pharm & Biosci, 2014: 2(5); 31

6 In the present study, F6 and F8 of O. 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. Insulin resistance is a condition where normal or elevated insulin level produces an attenuated biological response. In diabetes, insulin resistance is the central pathophysiological event. Table 5 Effect of isolated fractions of O. oojeinensis on changes in body weight in rats Change in Body weight (gm) Before Induction After Induction After Treatment Normal control ± ± ±5.32 Diabetic control (Streptozotocin) Glibenclamide (0.50 mg/kg) 179.8± ± ± ± ± ±4.83 F4 (50 mg/kg) 190.6± ± ±3.72 F6 (50 mg/kg) 182.4± ± ±4.29 F7 (50 mg/kg) 189.7± ± ±3.27 F8 (50 mg/kg) 181.3± ± ±2.71 Table 6 Effect of isolated fractions of O. oogenesis in insulin level of STZ induced diabetes in rats Treatment Insulin Level (Mean ±SEM) In mg/dl Initial Reading Final Reading Normal control 0.88± ±0.03 Diabetic control (Streptozotocin) Glibenclamide (0.50 mg/kg) 0.89± ±0.02 a 0.91± ±0.06 * F4 (50 mg/kg) 0.81± ±0.07 F6 (50 mg/kg) 0.84± ±0.04 * F7 (50 mg/kg) 0.76± ±0.05 F8 (50 mg/kg) 0.79± ±0.08 * 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 5 Conclusions From the present study, it could be concluded that oral administration of fractions of O. oojeinensis exhibited significant antidiabetic effect in controlling the blood glucose level. From results it has been observed that the F6 showed maximum activity as compared to other fractions. Additionally, the isolated fractions decreased total cholesterol, triglycerides and LDL with increase in HDL at the end of the treatment. This confirms the potent antihyperlipidemic effect of fraction obtained from ethanol extracts. It can thus be concluded that this plant fractions promises an effective breakthrough in its potential development as a powerful oral therapeutic agent for controlling and managing diabetes mellitus. 6 References 1. Ackermann AM, Gannon M. Molecular regulation of pancreatic β-cell mass development, maintenance, and expansion. Journal of Molecular Endocrinology. 2007; 38: American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care. 2009; 32(Suppl 1): S62 S Xiaoming Chen, Jing Jin, Jia Tang, Zhongfu Wang, Junjun Wang, Liqin Jin, Jianxin Lu. Extraction, purification, characterization and hypoglycemic activity of a polysaccharide isolated from the root of Ophiopogon japonicas. Carbohydrate Polymers. 2011; 83: Lin GP, Jiang T, Hu XB, Qiao, XH, Tuo QH. Effect of Siraitia grosvenorii polysaccharide on glucose and lipid of diabetic rabbits induced by feeding high fat/high sucrose chow. Experimental Diabetes Research. 2007; Mukherjee PK, Maiti K, Mukherjee K, Houghton PJ. Leads from Indian medicinal plants with hypoglycemic potentials. Journal of Ethnopharmacology.2006; 106: Sivagnanam I, Kalaivanan P, Rajamanickam M. Antidiabetic activity of oncocalyxone a isolated from Prenanthes sarmentosus. International Journal of Pharmacy and Pharmaceutical Sciences. 2013, 5(4); Sahu RK, Roy A. Hepatoprotective activity of ethanolic extract of bark of Ougeinia oojeinensis (Roxb.) Hochr in CCl 4 treated male rats. Pharmacologyonline. 2009: 2(May- August): Sahu RK, Sharma U, Roy A, Dewangan D, Namdeo KP. Antioxidant activity of ethanolic extract of bark of Ougeinia oojeinensis (Roxb.) Hochr on CCl 4 induced hepatotoxicity in rats. Bioscience, Biotechnology Research Asia. 2008: 5(2): Sahu RK, Kulshrestha V, Kothiya S, Yadav P, Roy A. Healing potential of gel containing extract of Ougeinia UK J Pharm & Biosci, 2014: 2(5); 32

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