Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 4, 2011, pp. 455-459

Indian Journal of Pharmacology, Vol. 43, No. 4, July-August, 2011, pp. 455-459

Short Communication

Antidiabetic activity of ethanolic extract of tubers of Dioscorea alata in alloxan induced diabetic rats

V Maithili¹, SP Dhanabal¹, S Mahendran², R Vadivelan³

¹ Department of Phytopharmacy and Phytomedicine, JSS College of Pharmacy, Ooty, Tamil Nadu, India
² Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Ooty, Tamil Nadu, India
³ Department of Pharmacology, JSS College of Pharmacy, Ooty, Tamil Nadu, India

Correspondence Address: S P Dhanabal Department of Phytopharmacy and Phytomedicine, JSS College of Pharmacy, Ooty, Tamil Nadu India dhanabalkana@yahoo.com

Date of Submission: 12-Aug-2010
Date of Decision: 07-Sep-2010
Date of Acceptance: 26-Apr-2011

Code Number: ph11120

DOI: 10.4103/0253-7613.83121

Abstract

Keywords: Antihyperglycemic, alloxan, Dioscorea alata, diabetes mellitus, oral glucose tolerance test

Introduction

Diabetes mellitus (DM) is a chronic disease caused by inherited and/or acquired deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin produced. Such a deficiency results in increased concentrations of glucose in the blood, which in turn damage many of the body′s systems, in particular the blood vessels and nerves. ^[1] Medicinal plants continue to provide valuable therapeutic agents, in both modern medicine and in traditional system. ^[2] Discorea alata Linn. (family Dioscoreaceae), commonly known as ′yam′, is distributed mainly in the tropical and sub-tropical regions. In Indian traditional medicine, the tuber is used as a diuretic, aphrodisiac, anthelminitic and antidiabetic. ^[3] Thus, the present study was undertaken to evaluate the antidiabetic activity of ethanolic extract of Discorea alata (DA) tubers in alloxan induced diabetic rats.

Materials and Methods

Collection and Authentication of Plant Material

The tubers of the plant material were collected from Dharmapuri, Tamil Nadu and authenticated by Dr. P. Jayaraman, Botanist, Research Institute, Chennai, Tamil Nadu. A voucher specimen (JSSCPDP/2008/157) has been deposited at the Department of Phytopharmacy and Phytomedicine, JSS College of Pharmacy, Ooty.

Extraction and Preliminary Phytochemical Analysis

The tubers were dried under the shade and made to fine powder using a laboratory mill. The powdered plant material (1.8 kg) was extracted with ethanol (95%) using soxhlet apparatus for 24 hours. The solvent was then removed under reduced pressure and controlled temperature (40 - 50°C) in a rotavapor. Finally, a dark brown colored crude extract was obtained (yield: 7.66% w/w). The extract was subjected to qualitative chemical tests for the identification of various phytoconstituents. ^[4] For standardization, estimation of total phenol and total flavonol content was carried out by a standard procedure.

Analysis of Total Phenolic and Flavonol Content

The total phenolic content of plant extract was determined using Folin-Ciocalteu reagent. ^[5] Ethanol extract (100 μl) was mixed with 500 μl of the Folin-Ciocalteu reagent and 400 μl of (0.7 M) sodium carbonate. After shaking, it was kept for 2 hour reaction time and the absorbance at 765 nm was determined. The total phenolic content was expressed as mg of gallic acid equivalents (GAE) per gram of extract, using a standard curve of gallic acid. All measurements were carried out in three replicates.

Total flavonol content was determined by aluminium chloride method. ^[6] 0.5 ml of the extract was mixed with 1.5 ml methanol, 0.1 ml 10% AlCl _3, 0.1 ml of 1 M potassium acetate, and 2.5 ml of distilled water. After incubation at room temperature for 30 min, the absorbance of the reaction mixture was measured at 415 nm. All determinations were carried out in triplicates. The flavonol content was calculated using a standard curve obtained from various concentrations of rutin. The total flavonol content was expressed as mg of rutin equivalents (RE) per gram of extract, using a standard curve of rutin.

Animals

Healthy Wistar rats of either sex, weighing 180-220 g, were procured from the animal house, JSS College of Pharmacy, Ooty, India. The animal house was well ventilated and animals had 12 ± 1 hour day and night schedule. The animals were housed in large, spacious, hygienic cages during the course of the experimental period and the room temperature was maintained at 25 ± 1 °C. The animals were fed with standard rat feed and water ad libitum. The experiments were conducted as per the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Chennai, India (approval no. JSSCP/IAEC/PH.D/PHYTO/01/2008-09).

Preparation of Drug Solution

The ethanolic extract of DA and standard metformin were dissolved in 0.9% sodium chloride in water and administered orally to the animals with the help of an intragastric catheter.

Acute Oral Toxicity Study

Acute oral toxicity test was carried out according to the OECD (Organization for Economic Co-operation and Development) guidelines No. 423. Wistar albino rats of either sex (180-220 g weight) were used.. Rats were kept for overnight fasting prior to drug administration. A total of three animals were used, which received a single oral dose (2000 mg/kg, body weight [b.w.]) of DA ethanol extract. After the administration of extract, food was withheld for further 3-4 hours. Animals were observed individually at least once during the first 30 min after dosing, periodically during the first 24 hours (with special attention during the first 4 hours), and daily thereafter for a period of 14 days. Once daily, cage side observations included changes in skin and fur, eyes and mucous membrane (nasal) and also respiratory rate, circulatory (heart rate and blood pressure), autonomic (salivation, lacrimation, perspiration, piloerection, urinary incontinence, and defecation) and central nervous system (drowsiness, gait, tremors, and convulsion) changes. Mortality, if any, was determined over a period of 2 weeks. ^[7]

Oral Glucose Tolerance Test

The oral glucose tolerance test (OGTT) ^[8] was performed in overnight fasted (18 hours) normal rats. Rats were divided into four groups, each consisting of six rats were administered 0.9% (w/v) saline, metformin 250 mg/kg, and DA ethanol extract (100 and 200 mg/kg), respectively. Glucose (3 g/kg) was fed 30 min after the administration of extract. Blood was withdrawn from the retro orbital sinus under ether inhalation at 0, 30, 60, and 120 min of glucose administration and blood glucose level was estimated by glucose oxidase-peroxidase method.

Hypoglycemic Activity

Fasted rats were divided into four groups of six animals each. Group I animals received only vehicle (0.9% w/v saline) orally in a volume of 0.5 ml/kg and served as a control. Group II received metformin (250 mg/kg, p.o) as a reference drug suspended in vehicle. The DA extract, suspended in vehicle, was administered at doses of 100 and 200 mg/kg, p.o. to the animals of groups III and IV, respectively. Blood samples were collected from the retro orbital sinus under ether inhalation at 0, 30, 60, and 120 min for glucose estimation after dosing. ^[9]

Induction of Experimental Diabetes

Diabetes was induced in Wistar rats by single intraperitoneal injection of alloxan monohydrate in sterile normal saline to overnight fasted animals at a dose of 120 mg/kg b.w. The fasting blood glucose level was determined after 72 hours of alloxan injection. The rats having blood glucose levels above 200 mg/dl were used for the study. The diabetic animals were allowed free access to tap water, pellet diet, and were maintained at room temperature in plastic cages. ^[10]

Experimental Design

Overnight fasted diabetic rats were divided into four groups of six animals in each group. Group I animals served as normal control, which received 0.9% sodium chloride and group II served as diabetic control. Group III and IV, diabetic rats were treated with ethanolic extract of DA (100 and 200 mg/kg b.w., p.o.). Group V, which served as positive control, was treated with metformin (250 mg/kg b.w., p.o.). All the treatments were carried out for a period of 21 days. Body weight of the animals was recorded every week. The fasting blood samples were collected on days 1, 7, 14, and 21 to determine the glucose level by glucose oxidase method.

At the end of the treatments, the blood samples were collected by retro orbital puncture and serum was separated by centrifugation at 4000 rpm for 15 min. The separated serum was used for biochemical estimation. Triglycerides, total cholesterol, total protein, albumin, creatinine, and HDL- cholesterol were analyzed from the serum by auto analyzer using Ecoline kits (Merck, Mumbai, India). VLDL ^[11] (very low density lipoprotein)-cholesterol was calculated as: triglycerides/5; LDL ^[11] (low density lipoprotein)-cholesterol was calculated by the equation:

LDL-cholesterol = total cholesterol-(HDL+VLDL)

Statistical Analysis

All values are expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by Tukey′s multiple comparison tests. The results were considered statistically significant if P < 0.05.

Results

Preliminary Phytochemical Analysis

The percentage yield of ethanol extract was found to be 7.66% w/w. The qualitative phytochemical analysis of the ethanolic extract showed the presence of phytosterols, triterpenoids, proteins, glycosides, saponins, flavonoids, fats and oils, tannins, and phenolic compounds.

Analysis of Total Phenolic and Flavonol Content

The total phenolic and flavonol content of the ethanol extract of DA was found to be 100.70 ± 1.11 mg GAE/g and 33.07 ± 0.68 mg RE/g, respectively.

Effect of DA Ethanol Extract on Acute Toxicity Study

Acute toxicity study revealed the non-toxic nature of the extract. There was no mortality or any toxic reactions found at the maximum tested dose level of 2000 mg/kg.

Effect of DA Ethanol Extract on Oral Glucose Tolerance Test

The serum glucose levels of normal rats reached a peak at 60 min after the oral administration of glucose (3 g/kg) and gradually decreased to 127.30 mg/dl in 2 hours. The pretreatment with DA ethanol extract (100 and 200 mg/kg) and metformin (250 mg/kg) elicited decreased serum glucose level significantly (P < 0.001) as compared to the control group [Figure - 1].

Effect of DA Ethanol Extract in Fasted Normal Rats

Based on the anti hyperglycemic activity in OGTT, the ethanol extract was subjected to hypoglycemic activity at two dose levels (100 and 200 mg/kg) as shown in [Table - 1]. The ethanol extract of DA did not shown any hypoglycemic activity.

Effect of DA Ethanol Extract on Alloxan Induced Rats

On repeated oral administration of the vehicle, DA extract or metformin, for 21 days, a sustained and significant (P < 0.001) decrease in the serum blood glucose of the diabetic rats was observed at a dose of 100 (47.48% fall) and 200 mg/kg (52.09% fall), in a dose dependent manner as compared to the vehicle treated group. Metformin also showed a significant (P < 0.001) decrease in serum glucose (53.04% fall) at a dose of 250 mg/kg, p.o. as compared with the vehicle treated group [Figure - 2].

Normal control animals had an increase in their body weight, but diabetic rats showed significant reduction in body weight during the 21 days treatment. Alloxan caused body weight reduction, which was reversed significantly (P < 0.001) by ethanol extract of DA after 21 days of treatment [Figure - 3].

Serum triglycerides, creatinine, total cholesterol, LDL, and VLDL cholesterol levels were elevated significantly (P < 0.001) in alloxan induced diabetic rats as compared to normal rats. In alloxan induced diabetic rats, when treated with the DA extract/metformin, there was a significant (P < 0.001) reduction in the elevated levels of serum triglycerides, creatinine, total cholesterol, LDL, and VLDL cholesterol. Similarly, decreased HDL, albumin, and total protein in serum during alloxan induced diabetes were found to be significantly (P < 0.001) increased by DA extract and metformin treatment [Table - 2].

Discussion

The fundamental mechanism underlying hyperglycemia involves over-production (excessive hepatic glycogenolysis and gluconeogenesis) and decreased utilization of glucose by the tissues. ^[12] The results of the present study indicate that DA extract (100 and 200 mg/kg b.w.) was found to reduce the glucose level in glucose loaded animals and in alloxan induced diabetic animals. Alloxan has been shown to induce free radical production and cause tissue injury. ^[13] The pancreas is especially susceptible to the action of alloxan-induced free-radical damage. Dioscorin, a purified storage protein of yam, was previously reported to possess scavenging properties against free radicals. ^[14] Induction of diabetes with alloxan is associated with a characteristic loss of body weight, which is due to increased muscle wasting and due to loss of tissue proteins. ^[15] The differences in the body weights observed during the period of treatment of the rats treated with DA extract/metformin were increased as compared to the diabetic control, which may be due to its protective effect in controlling muscle wasting, i.e., reversal of gluconeogenesis and may also be due to proper glycemic control. In alloxanized animals, there was an increase in the value of total cholesterol (TCH), triglycerides (TG), LDL, VLDL, except HDL and protein values (albumin and total protein), while the extract treated group showed an increased value of HDL, protein values, and reduced VLDL, TC, and TG in a significant manner. This reduced the VLDL, TC, and TG; it may be presumed that the extract is responsible for the enhancement of the transcription of lipoprotein lipase similar to that of insulin, since in the untreated or under treated diabetes animals, the level of triglycerides and cholesterol increases due to increased production of VLDL and unavailability of protein lipase which hydrolyses the triglycerides to VLDL because of insulin deficiency. Diabetic nephropathy in uncontrolled diabetes is a serious micro-vascular complication leading to glycosylation of renal basement membranes and result in increased creatinine concentrations. ^[16] While the diabetic controls had significantly higher amounts of creatinine in the serum, the DA extract administered diabetic rats exhibited significantly lowered creatinine level. This reduction could be a result of improved renal function due to a reduced blood glucose concentration and subsequent glycosylation of renal basement membranes. Phytochemical studies of ethanolic extract revealed the presence of phenolic and flavonoid compounds. Hydro-Q chromene, gamma-tocopherol-9, alpha-tocopherol, coenzyme Q, 1-feruloylglycerol, dioscorin, cyanidine-3-glucoside, (+)-catechin, procyanidine, cyanidine, peonidin3-gentiobioside, alatanins A, B and C has been reported from the tubers of the plant. ^[17],[18] Any of these above flavonoid compounds could have induced the observed effects. However, it is reported that flavonoids constitute active biological principles of most medicinal plants with hypoglycemic and antidiabetic properties. ^[19] Thus, this active principle may be responsible for the observed antidiabetic effect of the DA extract.

In conclusion, the ethanolic extract of DA tubers was found to exhibit an antidiabetic activity in alloxan-diabetic rats. Further pharmacological investigations are needed to elucidate the mechanism of the observed antihyperglycemic effect.

Acknowledgements

The authors sincerely thank Dr. K.Elango, Principal, and JSS College of Pharmacy (A constituent college of JSS University, Mysore), Ooty for providing the necessary facilities and AICTE New Delhi for the financial assistance under QIP provided for this work.

References

Copyright 2011 - Indian Journal of Pharmacology

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