Antihyperglycemic activity of ethanolic extract of leaves of Dioscorea japonica in STZ-induced diabetic rats

 

Budhwani Ashok K. ¹*, Shrivastava B.², Singhai A.K.³, Gupta Prashant¹

¹Daksh Institute of Pharmaceutical Science, Chhatarpur (M.P.)

²Jaipur National University, Jaipur (Raj)

³Laxmi Narayan College of Pharmacy, Bhopal (M.P.)

*Corresponding Author E-mail: ashokbudhwani@yahoo.com

 

 

ABSTRACT:

To evaluate the antihyperglycemic activity of ethanolic extract of leaves of Dioscorea japonica in STZ induced diabetic rats. STZ-induced (60 mg/kg, i.p.) diabetic mice given ethanolic leaf extract (200, 300, 350 and 400 mg/kg, p.o.,n=6) of D. japonica and in vehicle (distilled water, 10 ml/kg.,p.o.) and in standard drug metformin (500 mg/kg.,p.o.) respectively for 14 days. The dose of 350 mg/kg body weight (bw) reduced the fasting blood glucose (FBG) level by 6.0% within 1 hr, whereas, the peak blood glucose at 1 hr during glucose tolerance test (GTT) was reduced by 17.1% in normal rats. The same dose of ethanolic extract reduced FBG by 13.0% and improved glucose tolerance by 37.2 and 60.6% at 1and2 h, respectively, during GTT in STZ-diabetic rats. The dose of 350 mg/kg body weight of ethanolic extract in 14-day treatment of a group of STZ-diabetic rats produced 73.3% fall in FBG level and no sugar was observed in fasting urine. It brought about fall in the level of total cholesterol (TC) by 49.3% with increase of 30.3% in high-density lipoprotein (HDL) and decrease of 71.9 and 28.7% in low-density lipoprotein (LDL) and triglycerides (TG) levels, respectively. The ethanolic leaf extract of D. japonica (200, 300, 350 and 400 mg/kg, p.o) showed significant (P<0.01) reduction of serum glucose level in STZ-induced diabetic mice at 14^th day. It is concluded that D. japonica has significant antihyperglycemic activity as it lowers serum level of glucose in diabetic mice and significantly increase glucose tolerance

.

 

1. INTRODUCTION

Utilization of natural products in medicine is an interesting aspect and has been very popular in primary health care for the treatment of several diseases ^[1,2]. Numerous Ayurvedic herbal drugs have been indicated for their effectiveness in treatment of diabetes and related disorders. Natural products possess numerous advantages as drugs with the main being their low cost and ease of availability in the local     market^[3-6].  Alarming increase in the number of diabetic patients around the world has shifted the research focus to find herbal alternatives that are safe as well as effective. Uncontrolled hyperglycemia can lead to progressive secondary complication in diabetes such as nephropathy, neuropathy, retinopathy and cardiovascular diseases^[7].  Furthermore, after the recommendation made by WHO on diabetes mellitus, investigations on hypoglycemic agents from medicinal plants have become more important^[8].

 

Dioscorea japonica (family: Dioscoreaceae), is available throughout the tropical and warm temperate regions of the india, mainly for its edible fruit. This plant is reputed to possess several medicinal properties ^[9]. Various phytochemical and biological activity studies have been carried out with the plant ^[10,11]. From the leaves of Dioscorea japonica, several flavonoids^[12]  and a tetrahydroisoquinoline alkaloid with cardiotonic activity^[13] have been isolated. Many workers have reported its use as an insecticidal agent^[14], antimicrobial and insecticidal activity^[15], mosquitocidal effect against Culex quinquefasciatus^[16], anti-cancerous activity^[17]. Ayurvedic practitioners use stem and leaves extract as an indigenous uterotonic drug^[18]. The aqueous extract of Dioscorea japonica seeds do not interfere with the reproductive performance of pregnant rats^[19]. Post-coital anti-fertility activity^[20] is reported in the seed extract of Dioscorea japonica, while the aerial parts are inactive. The objective of this investigation of antidiabetic potential of Dioscorea japonica, we have investigated effect of extracts its leave on fasting blood sugar levels and urine biochemical analysis in STZ-induced diabetic rats.

 

2. MATERIAL AND METHODS

2.1. Chemicals

Streptozotocin (STZ) was purchased from Sigma-Aldrich Co., USA. Glucose, total cholesterol (TC), high-density lipoprotein (HDL) and triglyceride (TG) were assayed using standard kits from Ranbaxy Diagnostics, New Delhi, India. One-touch gluco-meter (Accu-check sensor) of Roche Diagnostics, Germany, and Uristix of Bayer Diagnostics India Ltd. were used in the experiment.

 

2.2. Animals

All the animal experimentation were carried out after prior permission from the institutional ethical committee of Daksh Institute of Pharmaceutical Science, Chhatarpur (M.P.), India. The protocol approval reference number is DIPS/IAEC/11/PN-05 The guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India were adhered to during the whole experimentation.

 

Adult Wistar strain of albino rats aged 3-4 months, weighing about 150-200 g were obtained from the Institutional animal house and used in the experiments. Animals were kept at room temperature of 25–30◦C and at 45–55% relative humidity for 12 h, each of dark and light cycle. Animals were fed with Standard diet purchased from Gold Pellet, India Ltd.  Animals were provided with food and water ad libitum and maintained at 25-28°C.

 

2.3. Preparation of the plant extract

Leaves of Dioscorea japonica were collected in the month of April and May from the gardens of Indian Agriculture Research Institute, Bhopal, India and were identified by taxonomist, Prof. Zia-ul-hassan, Department of Botany, Safia college of science, Bhopal, India. A voucher specimen has been kept at the herbarium of the college. The voucher specimen no. 298/bot/safia/11.  The leaves were washed with water and shade-dried. About 500 g of crushed leaves were extracted twice with 5 L of boiling ethanol for 6 h. The resulting extract was cooled and filtered. The filtrate was evaporated in vacuum to give a residue (yield: 8.2% w/w).

 

2.4. Biochemical parameters

Blood glucose, total cholesterol, HDL-cholesterol and triglyceride levels in serum were measured spectrophotometrically by methods prescribed by the manufacturer²¹. Urine sugar was detected by reagent-based uristrix from Bayer.

 

2.5. Induction of experimental diabetes

A freshly prepared solution of STZ (60 mg/kg) in 0.1M citrate buffer, pH 4.5 was intraperitoneal injected to overnight fasted rats ²². FBG level was estimated at the time of induction of diabetes and postprandial blood glucose (PPG) was checked regularly up to stable hyperglycemia, usually after one week with STZ.

Depending on their glucose levels, the animals were divided in three groups:

Group I Sub-diabetic animals with nearly normal FBG of 80-120 mg/dl but showing abnormal glucose tolerance.

Group II Mild-diabetic animals with FBG of 120-250 mg/dl.

Group III Severely-diabetic animals showing FBG above 250 mg/dl.

 

2.6. Experimental design

Initial screening of the extract for the hypoglycemic activity was done in normal healthy rats by conducting glucose tolerance test (GTT). The antidiabetic effect was assessed in diabetic animals by two methods: (i) by studying the effect of different doses of the ethanolic extract on blood glucose levels of sub-diabetic and mild-diabetic rats during GTT. The most effective dose was again tested on STZ-induced sub-diabetic rats for glucose tolerance, and (ii) by giving the extract (350 mg/kg) daily for 14 days to STZ-diabetic rats and studying their effects on FBG, postprandial blood glucose (PPG) level and urine sugar. Total lipid profile was estimated to assess the effect of ethanolic extract on abnormal lipid profile seen in severely-diabetic rats. An attempt was made to determine the LD₅₀.

 

2.6.1. Assessment of hypoglycemic activity in normal healthy rats

Five groups of six rats in each were used in the experiment. Group 1 served as untreated control and animals of groups 2, 3, 4 and 5 received different doses of plant extract (200, 300, 350 and 400 mg/kg, respectively). In overnight fasted rats, initial fasting blood samples were taken and then different doses (200, 300, 350, 400 mg/kg) of the ethanol extract were given orally to different groups of animals and their effect on FBG was studied hourly up to 2 h. The animals were then orally administered 2 g/kg of glucose solution and glucose tolerance was studied at 1 h intervals for another 3 h. Thus, the total period of blood collection was up to 5 h.

 

2.6.2. Assessment of antidiabetic activity by GTT in diabetic rats

Different doses (200, 300, 350 and 400 mg/kg) of extract and the standard drug metformin (500 mg/kg) were assessed to find out the effective dose. GTT-based activity testing was carried out on sub-diabetic and mild-diabetic rats. Five groups of six rats each were used in the experiment. In overnight-fasted diabetic rats, the initial fasting blood samples were drawn from orbital plexuses (for FBG) and then water was given orally. After 90 min, blood was drawn again that gives the ‘0 h’ value for blank GTT. The animals were then given a glucose solution (2 g/kg) orally and blood samples were drawn at 1, 2 and 3 h after glucose administration to get the GTT pattern of the untreated diabetic rats (control). After a week, same animals were again fasted overnight. Fasting blood samples were drawn and different graded doses (200, 300, 350 and 400 mg/kg) of plant extract and metformin were administered to different groups.

Table 1

Effect of different doses of Dioscorea japonica leaves ethanol extract on the fasting blood glucose and glucose tolerance in normal healthy rats (mean± S.D.)

 2 g/kg of glucose solution given orally.

 p < 0.01 when compared with control group.

 

Table 2

Effect of 14-day treatment with ethanol extract of Dioscorea japonica leaves on the fasting blood glucose (FBG), postprandial glucose (PPG) and urine sugar (US) of stz-diabetic rats.

n = 5, p < 0.001 when compared with initial (before treatment) values.

 

Table 3

Effect of 14-day treatment with ethanol extract of Dioscorea japonica leaves on the fasting  lipid profile of STZ induced severely-diabetic rats

n = 6 in each group; values are presented as mean±S.D.

p < 0.001 when compared with initial (before treatment) values.

 

Table 4

Effect of the ethanolic extract of Dioscorea japonica leaves (200-400 mg/kg) and metformin (500 mg/kg) on the glucose tolerance in sub-diabetic and mild-diabetic rats

Values are presented as mean±S.D. FBG: Fasting blood glucose.

p < 0.001 compared with their control.

p < 0. 01 compared with their control.

Blood sugar level (from 255±5.8 mg/dl to 68±9.6 mg/dl)

 

 

After 90 min, blood samples were drawn again. This serves as ‘0 h’ sample of the treated-diabetic rats. The animals were given glucose solution (2 g/kg) orally and blood samples were drawn at 1, 2 and 3 h after glucose administration to get the GTT pattern of the treated-diabetic rats (same animals). Glucose tolerance studies were also carried out with a dose of 350 mg/kg of the extract in STZ-induced diabetic rats.

 

 

2.6.3. Treatment of diabetic rats

Three groups of five rats each were used in the experiment. Group 1 served as normal healthy control group and Group 2 as diabetic-untreated control.  Group 3 was treated daily with a dose of 350 mg/kg extract for 14 days.  At the beginning and end of the experiment, FBG and urine sugar (US) were estimated. Postprandial blood glucose (PPG) was estimated daily during the treatment period.

 

2.7. LD₅₀ experiment

Four groups of rats of both sex (6 animals per group, 3 females and 3 males), weighing about 150-180 g were orally administered by a single dose of 875 mg/kg, 1.750, 3.5 and 5.250 g/kg of ethanolic extract of Dioscorea japonica leaves. Then rats were observed for gross behavioural, neurologic, autonomic, and toxic effects continuously. Food consumption, faeces and urine were also examined at 2 h and then at 6 h intervals for 24 h.

 

2.8. Statistical calculations

Data were expressed as mean± S. D. for all experiments and significant differences between groups were calculated according to Student’s two-tailed t-test. Values corresponding to p< 0.05 were considered statistically significant.

 

3. RESULTS

3.1. Effect in normal healthy rats

In order to know the optimum effective dose of the ethanolic extract of Dioscorea japonica on FBG and glucose tolerance, different doses of the ethanolic extract were given to overnight-fasted healthy rats. The extract at doses of 200 and 300 mg/kg did not had any appreciable effect on the FBG, but there was a slight improvement in glucose tolerance of about 10.6 and 13.8% at 1 h peak value (3 h in Table 1), respectively. A dose of 350 mg/kg ethanol extract brought about a slight fall (6.0%) in FBG after 1 h of the extract administration, and improved glucose tolerance by 17.1% at the 1 h peak value (3 h in Table 1) when compared to control rats which received equal volume of water instead of extract. The higher dose of 400 mg/kg had more or less the same effect as that of 350 mg/kg. It therefore appears that 350 mg/kg of the ethanol extract of Dioscorea japonica leaves is the most effective dose on FBG and GTT of healthy rats. One of the reasons why even with the optimal dose of 350 mg/kg, the fall was only 17.1% could be that normal regulatory mechanisms operate to prevent hypoglycemia in normal animals.

 

The maximum fall in FBG was after 1 h and maintained up to 2 h during fasting. So in further experiments, effect of the extract on FBG was observed after 90 min of the extract administration considering that it takes a minimum of 1 h to act and then GTT was performed by giving glucose solution.

 

3.2. Effect on diabetic rats during GTT

In order to choose the optimum dose for the diabetic animals, different doses of ethanolic extract (200, 300, 350 and 400 mg/kg) were evaluated on glucose tolerance in diabetic rats along with the standard drug metformin (500 mg/kg). The rats were treated with the extract and improvement in GTT was assessed by comparing the blood glucose level (BGL) before and after the treatment. A dose of 200 and 300 mg/kg of ethanolic extract reduced FBG by 9.8 and 18.2% (Table 4) within 90 min of the extract administration (0 h of GTT), while after 1 h of glucose administration (1 h of GTT); the BGL was reduced by 14.5 and 31.2%, respectively. A reduction of 16.4% was observed in 2 h of GTT with 200 mg/kg, while this fall increased further to 28.3% with 300 mg/kg of extract. A dose of 350 mg/kg significantly (p < 0.001) reduced FBG by 26.8% at 0 h and 37.2 and 60.6% at 1 and 2 h, respectively (Table 4). The higher dose of 400 mg/kg had about the same effect as that of 350 mg/kg. It therefore appears that 350 mg/kg of the ethanol extract of Dioscorea japonica is the effective dose on FBG and GTT of STZ-induced diabetic rats.

 

3.3. Effect on BGL and urine sugar of diabetic rats

After 14-day treatment of diabetic rats with extract, there was a 73.3% fall in FBG. The animals returned to normal

To check the effect of extract on the postprandial glucose (PPG) level of diabetic rats, PPG was estimated regularly during the treatment period. A 42% fall in PPG was observed after the treatment and also no urine sugar was observed, while initially, it was 1.5 g/l (Table 2).

 

3.4. Effect on FBG, lipid profile and urine sugar of severely-diabetic rats

 

It was intended to assess the effect of long-term treatment on BGL, urine sugar and associated abnormal lipid profile in STZ-induced severely-diabetic rats. Rats were treated with 350 mg /kg of ethanol extract once a day in the morning for 15 days. At the end of the treatment, the animals were compared with their own initial values and significant reduction (p < 0.001) of 52.7, 53.1 and 75% in FBG, PPG and urine sugar (US), respectively (Table 2) was observed. The various parameters of blood lipid profile of severely-diabetic rats were tested before and after the treatment. The enhanced levels of TC, LDL, VLDL cholesterol and TG were brought down significantly (p < 0.001) after the treatment period. A 49.3% fall in TC, 71.9% decrease in LDL cholesterol and 28.7% fall in TG was observed in treated-diabetic rats. There was also an increase of 30.3% in HDL cholesterol in the treated-diabetic rats (Table 3). At the end of the experiment, food intake and body weight were normal.

 

 

3.5. LD₅₀

Experiment was carried out on normal healthy rats. The behaviour of the treated rats appeared normal. No toxic effect was reported at doses up to 10 and 15 times of effective dose of the ethanol extract and there was no death in any of these groups. The consumption of food was increased by 20% in 10 and 15 times doses treatment within 2 h and it became normal afterwards; body weight was also normal.

4. DISCUSSION

The present study for the first time reports the hypoglycemic and antidiabetic effects of an ethanolic extract of Dioscorea japonica leaves. The study reveals that the maximum hypoglycemic effect was produced within 1 h during GTT (Table 1). This indicates that it takes about 1 h for the active ingredient(s) or its (their) metabolites in the ethanol extract to enter into the circulation and target tissues to bring about hypoglycemic effect, which is maintained for at least 3 h. Different doses of the ethanol extract were assessed on BGL of alloxan-diabetic rabbits during GTT, and the most effective dose was found to be 350 mg/kg.

The effect of 350 mg/kg of ethanolic extract was better than the effect of the same dose of the standard drug metformin during GTT. Maximum improvement in glucose tolerance, as indicated by reduction in peak blood glucose levels was observed at 1 and 2 h during GTT. Treatment of STZ-induced diabetic rats for 14 days (Table 2) brought down the elevated blood glucose levels ranging from 250 to 350 mg/dl to nearly normal range.

 

STZ selectively destroys pancreatic insulin-secreting -cells ^{(23, 24)} causing diabetes close to type 2 diabetes of humans. The elevated blood glucose levels in the diabetic animals used by us were in the range of 150–350 mg/dl which resembles both type 2 diabetes (150 to about 250 mg/dl) with partially functional pancreas, as well as type 1 (above 300 mg/dl) with considerable pancreas damage. This shows that the ethanol extract of Dioscorea japonica might be useful in both type 1 and 2 diabetes. This is an additional advantage of the extract over the existing sulfonylurea drugs, which act only when there is functional pancreas ^{(25, 27)}. It is well known that in uncontrolled type 2 diabetes mellitus, there will be an increase in TC, LDL and VLDL cholesterol and triglyceride with decrease in HDL cholesterol which contribute to the coronary artery disease ^{(20, 26)}. From this point of view, it is encouraging that the 14-day treatment of ethanol extract brought down the elevated levels of TC, LDL and VLDL cholesterol and TG in diabetic animals to nearly normal level (Table 3). There was increase in HDL cholesterol also, which was a desirable feature. The LD50 of the extract is high (no death even with 15 times of effective dose) indicating high margin of safety. These results clearly indicate the possible usefulness of ethanolic extract of Dioscorea japonica leaves in diabetes mellitus.

 

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Received on 29.02.2012          Modified on 11.03.2012

Accepted on 05.04.2012         © RJPT All right reserved