Evaluation of Antihyperglycemic Activity of a Polyherbal Formulation in Alloxan induced Diabetic Rats

ABOUT AUTHORS:
S Hamza*, Dr D. K. Suresh, R. Hasan, Md S. Khalid, R. Mistry
Department of Pharmacology,
Luqman College of Pharmacy,
Gulbarga - 585 102,
Karnataka, India.

ABSTRACT:
The study was to assess the antidiabetic activity ofa polyherbal formulation in alloxan induced diabetic rats. The antidiabetic activity of a polyherbal formulation in alloxan induced diabetic rats was assessed using Alloxan β-cytotoxin induced chemical diabetes in a wide variety of wistar albinorats. The polyherbal formulation contains thefollowing plants leaves of Gymnema sylvestre, Ginkgo biloba and plant seeds of Mucuna prurience.The extracts were prepared by continues hot soxhlet extraction using petroleum ether and water. Three different formulations were prepared for the study. The study reveals that the polyherbal formulations of plants leaves of Gymnema sylvestre, Ginkgo biloba and plant seeds of Mucuna prurience have antidiabetic activity and theactionmay be due to the restoration or regeneration of β-cells of pancreas.

REFERENCE ID: PHARMATUTOR-ART-1245

INTRODUCTION:
Since time immemorial man’s quest for medicinal agents that alleviate his sufferings has remained unquenched. Herbal medicine as major remedy in traditional system of medicine has been used in medical practice since antiquity. It is a major component of all indigenous system of medicine and a common element in Ayurvedic, Homeopathic, and Naturopathic medicine. Since time immemorial man’s quest for medicinal agents that alleviate his sufferings has remained unquenched. The instinctive behavior of primitive man helped him to realize the beneficial action of plants in the treatment of various ailments. Indian material medica includes 2000 natural products of therapeutic importance of which 400 are of mineral and animal origin and rest are of vegetable origin. There are approximately 1250 Indian medicinal plants which are used in formulating therapeutic preparations. The plants or other natural products have potential efficacy on human health. The effect of extracts and isolates of herbs are tested on pharmacological models (Sati et al., 2011).
As per World Health Organization (WHO) 74% plant derived medicine, used in modern medicine is in a way that correlates directly to their traditional use. This practice continues today because of its biomedical benefits as well as place in cultural beliefs in many parts of world and has made a great contribution towards maintaining human health (Wanzala et al., 2005).
Even today Phytopharmaceuticals like morphine, digitalis, glycosides, vincristine and vinblastin are still used as drugs of choice by allopathic physicians. About two third of the drugs of the modern system of medicine have been developed from natural sources mainly from plants (Elhardallou et al., 2011).
Today we understood that plant represents an immense respiratory of biochemical including pharmaceuticals, flavors and novel bioactive substances. Medicinal plants play an important sale in the health care of developing countries, currently 70% of the world population use herbal medicines and world health promotes the use of herbal remedies as they are not only pate and easily available at low lost but are also time tested (Selvam et al., 2010). Since ancient times, plants have been an exemplary source of medicine. Ayurvedic and other Indian literature mention the use of plants in treatment of various human alignments. India has about 45,000 plant species and among them, several thousands have been claimed to possess medicinal properties.
Diabetes is a predominant public health concern as it causes substantial morbidity and mortality and long term complications with increasing risk of childhood and adult obesity, diabetes is likely to become even more prevalent over the coming decades. Researches conducted in last few decades on plants mentioned in ancient literature are used traditionally for diabetes have shown antidiabetic property. The Indian traditionally system of medicine and have shown experimental and clinical antidiabetic property (Joseph et al., 2011).
For the treatment of diabetes and their complication the commonly used Indian plants are: Allium cepa, Allium Sativum, Aloe vera, Cojanus cajon, Coccinia indica, Caesalpinia, bonducello, Ficus bengalenesis, Gymnncona sylvestve, Momordica chorantio, Ocimum Sanctum, Pteorocarpus marsupium, Swertia chirayita, Tinospora cordifolia, Tinospora foenum graceum. All plants have been shown varying degree of hypoglycemic and anti hyperglycemic activity (Selvam et al., 2010).

MATERIALS AND METHODS:
Collection of plants: The different plants were purchased from different places and list of names of plants and their suppliers have been depicted in the Table no. 1

Preparation of sample: The dried coarser powder of leaves of Gymnema sylvestre, leaves of Ginkgo biloba and dried coarser powder of seeds of Mucuna prurience were subjected to continuous hot soxhlet extraction.

Extraction Process:
Petroleum ether and Water Extraction: The coarse powdered plant material was extracted with petroleum ether (60°- 80° C) and water in a soxhlet extraction apparatus. The extraction was carried out until the process was completed. Completion of the extraction was determined by the absence of color in the side arm of soxhlet apparatus and testing the siphoned solution. The extract was evaporated under reduced pressure until all the solvent have been removed to give an extract sample.

Preliminary Phytochemical Studies: Various extracts obtain from three plants were subjected to chemical tests as follow (Raju Ratan et al., 2008).

Test for alkaloids:
A quantity of 5 mg of the residue to each extract was taken separately in 5 ml of 1.5% v/v hydrochloric acid and filtered. These filtrates were then used for test of alkaloids.
1.   Meyer’s reagent shows Cream precipitate.
2.   Dragendorff’s reagent gives Orange brown precipitate.
3.   Hager’s reagent shows Yellow precipitate.
4.   Wagner’s reagent gives Reddish brown precipitate.

Test for sterols:
1.   Salkowaski test shows Development of red color.
2.   Liebermann’s test gives blue color.
3.   Liebermann-Burchard test shows a transient color development from red to blue and finally green was obtained.

Test for saponins:
Foam test gives formation of a stable, characteristic honeycomb like forth.

Test for sugar:
1. Molisch's test shows Presence of red brown ring at the junction of two.
2. Barfoed's test shows formation of a red precipitate of cuprous oxide within two minutes.

Test for tannins:
A quantity of 5 mg of test residue of each extract was taken separately in 5 ml water, warmed and filtered. Tests were carried out with the filtrate using following reagents.
1.   Ferric chloride test gives dark green or deep blue colour.
2.   Lead acetate test shows presence of precipitate.
3.   Potassium dichromate test Addition of solution of potassium dichromate in test filtrate led to the development of dark color.

Test for flavonoids:
Shinoda test shows development of pink, crimson or magenta colour within a minute or two indicated the presence of flavonoids.

Test for proteins:
1. Biuret test gives violet or pink colour.
2. Millon's test (mercury nitrate solution) shows presence of white precipitate, which slowly turns, to pink.

Test for Coumarins:
Placed a small amount of oil in a test tube and cover the test tube with dilute sodium hydroxide solution. The covered test tube was then placed in a boiling water bath for several minutes, the paper removed and exposed to ultraviolet light. Coumarins were found present since a yellowish green fluorescence appeared within few minutes.

Chart table for phytochemical screenings for the dried extracts are given in Table no. 02.

Toxicity Study:
The acute toxicity of the extract was evaluated in 56 normal albino mice. They were grouped into seven and each group contains eight mice (four male and four female). Each group was kept for fasting for 24hrs, after which they were treated once orally with one of the increasing doses of extract: 0, 5, 10, 15, 20, 25 or 30 g/Kg/b. w. The volume of each administrated dose did not exceed one ml. the mice were then observed for at least 48hrs and up to seven days, for death, lethargy, jerkiness, sensitiveness to noise and touch, stools quality and frequency.

Diabetic Screening Methods:
Preparation of Animals: Normal adult albino rats of either sex weighing 200-250 g collected from Central Animal House, Luqman College of Pharmacy, Gulbarga and housed under standard condition of temperature (22±5°C) and 55% humidity with a 12hr dark/light cycle. The animals were fed with standard pellet diet and water ad libitum.

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Induction of diabetes in experimental rats:
Diabetes was induced in above rats of either sex by a signal intraperitonially injection of alloxan monohydrate (120 mg/kg) in 0.2 ml Tween 80 (Tas et al., 2010). Alloxan, a β-cytotoxin, induced chemical diabetes in a wide variety of animal species by damaging the insulin secreting pancreatic cell (β-cell), results in a decrease in endogeneous insulin release which paves the ways for the decreased utilization of glucose by the tissues. After 12 days, animals with serum glucose level 150 mg/dl (diabetic) were selected for the study. The animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages.

Collection of blood and determination of serum glucose:
Blood sample from rats were collected by tail vein technique Serum glucose level were determined by GOD-POD method (Sheth et al., 2011) by GOD-POD Kit obtained from Manish Chemicals, Gulbarga. Different Antidiabetic formulations have been figured out in Table no. 03

Experimental design: In the experiment, a total of 30 rats (24 diabetic surviving rats, 6 normal rats) were used. The rats were divided into five groups containing 6 rats in each group.

To check out the exact antihyperglycemic activity of formulations, rats were divided into following groups. The group 1 had normal rats (without diabetes). The diabetic rats were fasted overnight and divided into five groups (n=6)
Group I: Vehicle control without diabetes (1% Tween 80 of about 2ml)
Group II: Diabetic (Negative) control (Alloxan 100mg/kg. b. w. suspended in Tween 80)
Group III: Treated with polyherbal formulation 1 (300mg/kg. b.w. p.o.) + Alloxan 120mg/kg, b.w., suspended in Tween 80
Group IV: With formulation 2 (400mg/kg. b.w. p.o.) + Alloxan 120mg/kg, b.w., suspended in Tween 80
Group V: With formulation 3 (450mg/kg. b.w. p.o.) + Alloxan 120mg/kg, b.w., suspended in Tween 80
Group VI: Positive control (Alloxan 100mg/kg. b.w. suspended in Tween 80 + Glibenclamide 10mg/kg b. w.).

In Pharmacological evaluation, acute study involved withdrawal of blood at 0,1,3,5, and 7 hrs after administration of vehicle, glibenclamide, extract and formulation. The blood obtained from rats by retro orbital method was centrifuged and serum obtained to determine the glucose level was estimated.

Statistical Analysis: The data were expressed as mean±SEM obtained from the number of experiments (n) one way ANOVA followed by Dunnett’s post test was performed using graph pad software. Differences between groups were considered.

RESULTS:
Preliminary Phytochemical Analysis: These drugs show the presence of alkaloids, carbohydrates and glycosides, fixed oils and fats, tannins and phenolic compounds and flavonoids. The polyherbal formulation contains the plants leaves of Gymnema sylvestre, Ginkgo biloba and plant seeds of Mucuna prurience. The extracts were prepared by continuous hot soxhlet extraction using petroleum ether and water.
Effect of different formulations on serum glucose level (mg/dl) in Alloxan induced Diabetic Rats is given in Table no. 04
The results were compared with reference standard Glibenclamide. The single dose of polyherbal formulation 3 (450mg/kg. b.w.) has more significantly reduced the blood glucose level up to 93.431+1.51% as compared to 113.28+0.91% of polyherbal formulation 1(150mg/kg b.w.), when compared with the standard Glibenclamide. From the above result, our study indicates that the polyherbal formulation have antidiabetic activity. The number of functionally intact β- cells in the islet of Langerhans is of much importance for the development course of diabetic. The renewal for β-cells in diabetics has been studied in several animal models. It was also suggested that regenerations of β-cells have proven destruction by alloxan may be the primary cause of the recovery of alloxan injected rats from the effects of the drug (Sentman et al., 1999). From the above suggestion it can be concluded that the antidiabetic activity of the polyherbal formulation may be due to restoration of β-cells of the pancreas and this effect may be due to the presence of tannin (Laxmi et al., 2010), flavonoids, alkaloids are may be due to the synergistic effect of the above phytoconstituents in the polyherbal formulation.

DISCUSSION:
The primary objective of our work was to investigate the antidiabetic potential of the polyherbal formulation which is a mixture of Ethanolic extract of plants leaves of Gymnema sylvestre, Ginkgo biloba and plant seeds of Mucuna prurience. 450mg/kg dose of polyherbal formulation have provided significant restoration of β-cells of the rat within few hours. The therapeutic efficacy of the drug was determined by toxicity study and the effective dose was obtained. From the above results, our study reveals that the polyherbal formulations have antidiabetic activity and the action may be due to the restoration or regeneration of β-cells of pancreas. Further investigation is necessary to standardize the formulation and to find the active constituents present in the formulation responsible for the antidiabetic activity.

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