Estimation of Antihyperglycemic and Antihyperlipidemic Activity of Isolated Fractions from Ficus glomerata Bark Extract in Streptozotocin-Induced Diabetic Rats

Article Information Received 14 September 2014 Received in revised form 30 Oct 2014 Accepted 1 Nov 2014 Abstract Ficus glomerata is commonly used for the treatment of diabetes. We planned to isolate compound from the ethanol extract of F. glomerata bark, and also evaluate the antidiabetic activity of isolated fractions in Streptozotocin (STZ)-induced diabetic rats. Oral administration of ethanol extract of bark and root of F. glomerata at the doses of 200 and 400 mg/kg body weight was studied in STZ-induced diabetic rats. After administration of extract the fasting blood glucose levels were significantly decreased. The eight different fractions were collected from ethanol extract of F. glomerata bark and dried. The fraction F4, F5, F6, F7 and F9 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 streptozotocininduced diabetic rats. The findings demonstrated that fraction isolated from ethanol extracts of F. glomerata bark has significant antidiabetic activity in streptozotocin-induced rats compared to standard drug. These results indicated that F. glomerata possesses significant antidiabetic and antihyperlipidemic effect due to presence of these fractions.


Introduction
Every year the number of diabetic patients is growing alarmingly all over the World.Diabetes is a chronic disease characterized by derangement in carbohydrate, fat, protein metabolism.Most of the hypoglycemic agents used in allopathic 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 has 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 for using natural products with antidiabetic activity, on account of the side effects associated with the use of insulin and oral hypoglycemic agents.The available literature shows that there are more than 400 plant species showing hypoglycemic activity 2 .
Ficus glomerata belongs to the family Moraceae (Mulberry family), and is commonly known as Gular, Umar, Umber, and Udumbara.It is an herb found all parts of India.It is an evergreen tree; common throughout the State near villages and along streams and rivers; also planted along road-side.Phytochemical investigated on F. glomerata have reported the presence of cycloartenol, euphorbol, hexacosanate, triacetate, taraxerone, tetratriterpene, glauanolacetate, racemosic acid, glauanol, glucose, hentriacontane.

Collection and identification of plant material
The bark of Ficus glomerata was collected from the forest area of Raipur (Chhattisgarh).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.22231).After authentication, plant material was dried at room temperature until it was free from the moisture.Finally the barks were subjected to size reduction to get complete powder.

Preparation of F. glomerata ethanol extracts
The powder of the bark of F. glomerata, 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.

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 6 .

Isolation of compound from F. glomerata ethanol extracts
The F. glomerata extract was subjected to column chromatography using silica gel (60-120 mesh size), and eluted with the following solvent ratios of Hexane: dichloromethane (DCM), 100:0, 80:20, were containing waxy material; the fractions F3 and F8 were powder but quantity was very little.The yield of fraction F4, F5, F6, F7 and F9 were 120 mg, 360 mg, 180 mg, 230 mg and 465 mg.The fractions were further analyzed for phytochemical screening.The preliminary phytochemical screening of fractions was done 7 .The fractions were further analyzed for phytochemical screening and antihyperglycemic activity.

Qualitative chemical tests of 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 6 .

Oral glucose tolerance test (OGTT) of F. glomerata fractions
The oral glucose tolerance test 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 F4 (50 mg/kg); Group V rats were administered F6 (50 mg/kg); and Group VI rats were administered F7 (50 mg/kg); and Group VII rats were administered F8 (50 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 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 8,9 .

Induction of non-insulin dependent diabetes mellitus (NIDDM)
Non-insulin dependent diabetes mellitus was induced 8,9 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 2.8.1 Evaluation of antidiabetic activity of F. glomerata fractions
The animals were segregated into eight 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 F4 (50 mg/kg); Group V diabetic rats were administered F5 (50 mg/kg); and Group VI diabetic rats were administered F6 (50 mg/kg); and Group VII diabetic rats were administered F7 (50 mg/kg); and Group VIII diabetic rats were administered F9 (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 9 .

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 9 .

Effect of F. glomerata fractions on insulin level
The animals were segregated into eight 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 F4 (50 mg/kg); Group V diabetic rats were administered F5 (50 mg/kg); and Group VI diabetic rats were administered F6 (50 mg/kg); and Group VII diabetic rats were administered F7 (50 mg/kg); and Group VIII diabetic rats were administered F9 (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 10 .

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.

Phytochemical screening of ethanol extract of F. glomerata bark
The phytochemical investigation of ethanol extracts of Ficus glomerata bark revealed the presence of alkaloids, Saponins, glycosides, carbohydrates, tannins & phenolic compounds and flavonoids.

Preliminary phytochemical analysis of isolated fraction of ethanol extract of F. glomerata
The 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 (Group 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 F5 and F7 at dose of 50 mg/kg body weight significantly decreases the blood glucose level of diabetic rats on 7 th day.While the F6 and F9 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.The possible mechanism by which F. glomerata 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.From results it has been observed that the F5 showed maximum activity as compared to other fractions.

Anti-hyperlipidaemic activity of F. glomerata fractions
The outcomes of lipid profiles in control and experimental rats are exhibited in table 4. Comprehensively, it has been noticed that hyperlipidemia is a complexity associated with hyperglycemia.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 fraction F5, F6 and F7 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 and F9 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 F5 and F7 exhibited a potent blood glucose lowering properties in STZ diabetic rats.The potent antidiabetic effect of the fractions suggests the presence of potent antidiabetic active principles, which produced antihyperglycemic effect in diabetic rats 2,11 .

Effect on body weight by F. glomerata 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.

Effect on insulin by F. glomerata 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 (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.

of Pharmaceutical and Biosciences Available at www.ukjpb.com
5 Samyal et al.UK Journal ISSN: 2347-9442 Samyal et al.Estimation of Antihyperglycemic and Antihyperlipidemic Activity of Isolated Fractions UK J Pharm & Biosci, 2014: 2(5); 43(2014) reported significant antidiabetic activity of the ethanol extracts of bark and root of F. glomerata in streptozotocin-induced diabetic rats5.In our earlier study we reported that ethanol extract of bark extract of F. glomerata exhibited maximum significant antidiabetic activity compared to root extract.In the previous study we have not determined which type of secondary metabolite imparts antidiabetic activity in extracts.Hence in the present study we planned to investigate the antihyperglycemic and antihyperlipidemic activity of the various fractions of the ethanol extracts of F. glomerata bark in streptozotocin-induced diabetic rats.

Table 1 Preliminary phytochemical analysis of isolated fractions of ethanol extracts of F. glomerata
phytochemical investigation of F4 of F. glomerata bark revealed the presence of alkaloids, glycosides and carbohydrates.The F5 and F7 indicate the presence of tannins & phenolic compounds and flavonoids.The F6 indicate the presence of alkaloids, carbohydrates and tannins & phenolic compounds.The F9 exhibit the presence of alkaloids, glycosides and carbohydrates (Table1).

Table 2 Effect of isolated fractions of ethanol extracts of F. glomerata on oral glucose tolerance test Group Plasma glucose concentration (mg/dl)
Samyal et al.Estimation of Antihyperglycemic and Antihyperlipidemic Activity of Isolated Fractions UK J Pharm & Biosci, 2014: 2(5); 46antidiabetic effect in controlling the blood glucose level.The present experimental result indicated that F5 and F7 exhibited a potent blood glucose lowering properties in STZ diabetic rats.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.

Table 6 Effect of isolated fractions of extracts of F. glomerata in insulin level of STZ induced diabetes in rats Treatment Group Insulin Level (Mean ±SEM) In mg/dl
* 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