Effect of Ethanolic Extract of Embelia Ribes on Dyslipidemia in Diabetic Rats

Diabetes mellitus has been treated orally with herbal remedies based on folk medicine since ancient times. Embelia ribes burm (Myrsinaceae), known commonly as vidanga, was used in Ayurveda for its anthelmintic activity. Ayurveda describes vidanga as pungent, causes increase in digestive fire, and cures flatulence and colic. A single study reported the antihyperglycemic activity of decoction of E. ribes in glucose-induced hyperglycemic albino rabbits. In the present study, the lipid-lowering and antioxidant potential of ethanolic extract of E. ribes burm was investigated in streptozotocin (40 mg/kg, IV, single injection)-induced diabetes in rats. Twenty days of orally feeding the extract (200 mg/kg) to diabetic rats resulted in significant (P < 0.01) decrease in blood glucose, serum total cholesterol, and triglycerides, and increase in HDLcholesterol levels when compared to pathogenic diabetic rats. Further, the extract also lowered the liver and pancreas thiobarbituric acid–reactive substances (TBARSs) values (P < 0.01) when compared to TBARS values of liver and pancreas of pathogenic diabetic rats. The results of test drug were comparable to gliclazide (25 mg/kg, orally), a standard antihyperglycemic agent. This is the first pilot study to provide biochemical evidence of potential of E. ribes in diabetic dyslipidemia.

Improved control of hyperglycemia does moderate diabetesassociated dyslipidemia; therefore, lipid-modifying treatment is warranted in many diabetic patients. There is also considerable evidence that oxidative damage is increased in diabetes, though the mechanisms are not clear [2,3].
Efforts continue in the field of medicine to find insulin substitutes from synthetic or plant sources for the treatment of diabetes. In traditional medicine, several medicinal plants or their extracts have been used to treat diabetes [4].
Embelia ribes burm (family, Myrsinaceae), known commonly as vidanga, is used in Ayurveda as anthelmintic [5]. Ayurveda describes vidanga as pungent, causes increase in digestive fire, and cures flatulence and colic. One Ayurvedic formulation, vidangadya curna (powder of vidanga), containing vidanga as main ingredient is taken with honey to alleviate obesity [6]. In a preliminary study, Tripathi [7] reported the antihyperglycemic activity of decoction of the E. ribes fruits in glucose-fed albino rabbits. The present study was undertaken to investigate the effect of ethanolic extract of E. ribes on diabetic dyslipidemia induced by streptozotocin (STZ) in wistar rats.

Preparation of the Extract
Dried E. ribes fruits, 200 g, were purchased locally from a grocery shop in New Delhi, India (in India, it is commonly available) and authenticated by a pharmacognosist, Prof. Mohd. Ali in our institute. A voucher specimen was retained in the department (UB # 04).
The fruits were soxhlet extracted with 90% ethanol in a soxhlet apparatus for 72 hours. The solvent was removed under reduced pressure to give a dry extract, 7% yield w/w (with respect 160 U. BHANDARI ET AL. to the crude material) and dose equivalent to 200 mg of the crude drug per kilogram body weight was calculated, and suspended in 2% v/v Tween 80 solution for the experiment.

Experimental Induction of Diabetes in Rats
Experiments on animals were conducted after obtaining approval from Hamdard University Animal Ethics Committee, which is registered with Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India, India (Registration no. 173/CPCSEA, dated 28 January, 2000).
Wistar rats of either sex (150 to 200 g) were obtained from the central animal house facility of Hamdard University of Delhi. They were acclimatized in an air-conditioned room at 22 • C ± 2 • C for 7 days and provided with free access to food (Gold Mohur rat pellet diet, Lipton India, Bangalore, India) and water.
After fasting for 18 hours, the rats were injected intravenously through tail vein with a single dose of 40 mg/kg STZ (Sigma, St. Louis, MO, USA), freshly dissolved in citrate buffer (pH 4.5). After injection, the rats had free access to food and water and were given 5% glucose solution to drink overnight to counter hypoglycemic shock. Diabetes in rats was identified by moderate polydipsia and marked polyuria.
After 3 days, the fasting blood glucose levels were determined by ortho-toluidine method [8,9]. The rats showing fasting blood glucose more than 200 mg/100 dL were considered diabetic and selected for the experimentation [10,11].

Experimental Procedure
Normal and diabetic rats (n = 10 each) were randomly divided into 4 groups of 10 rats each: Group I, normal healthy control; group II, pathogenic diabetic control (STZ treated only); group III, STZ + ethanolic E. ribes extract treated (200 mg/kg); group IV, STZ + gliclazide treated (25 mg/kg), a standard control. The test and standard drug were fed orally for 20 days. Groups I and II rats received 2% Tween 80 solution orally once a day for 20 days.

Note.
Values are mean ± SEM (n = 10). *P < 0.01 when compared with group I; # P < 0.01 when compared with group II.

Blood Collection and Biochemical Estimations
On the 21st day, fasting blood samples were collected from tail vein of all the groups of rats. Whole blood was collected for estimation of blood glucose [8,9]. Serum was separated for the estimation of total serum cholesterol [12], high-density lipoprotein (HDL)-cholesterol [13], and triglycerides [14].

Measurement of Tissue Lipid Peroxidation
On the evening of the 21st day, all fasted rats were killed by decapitation under light ether anaesthesia. Liver and pancreas were removed immediately and washed with ice-cold normal saline. Ten-percent homogenate of above tissues were prepared separately at 10,000 rpm in cooling centrifuge. Supernatant thus obtained was used for measurement of thiobarbituric acidreactive substances (TBARSs), which can be measured by the formation of malondialdehyde (MDA) after the breakdown of polyunsaturated fatty acids [15]. Liver and pancreas protein contents were evaluated by the method of Lowry using bovine serum albumin (BSA) as standard [16].

Statistical Analysis
The results are presented as mean ± SEM using 1-way analysis of variance test (ANOVA) followed by Dunnett's t test. P < 0.01 was considered significant.

RESULTS
The mean blood glucose levels in rats fed on normal diet (group I) alone was stable throughout the experimental period. Conversely, in the STZ-treated group, group II, there was significant rise in blood glucose level, as compared to group I. Drug treatment in STZ-treated rats for 20 days significantly reduced (P < 0.01) blood glucose levels in groups III and IV when compared to pathogenic group II rats ( Table 1).
The ethanolic extract of E. ribes also significantly ( P < 0.01) reduced serum total cholesterol and triglycerides and increased

Note.
Values are mean ± SEM (n = 10). *P < 0.01 when compared with group I; # P < 0.01 when compared with group II.
the HDL-cholesterol levels as compared to pathogenic diabetic rats, i.e., Group II. Furthermore, the results of the test drug were comparable to gliclazide, a standard antihyperglycemic agent.
There was no significant change in food consumption during the administration of ethanolic extract of E. ribes in dose of 200 mg/kg. However, the experimental animals showed marked polyuria and moderate polydipsia. STZ treatment also induced a statistically significant increase in liver and pancreas lipid peroxide levels (P < 0.01) as compared to group I. E. ribes and gliclazide treatments lowered the liver and pancreas TBARS values (P < 0.01) as compared to group II diabetic rats ( Table 2).

DISCUSSION
Cardiovascular diseases constitute the main cause of morbidity and mortality in diabetes mellitus. Diabetic individuals have a 2-to 4-fold increased risk of clinical atherosclerotic disease [17]. Dyslipidemia has been proven to be the most important modifiable risk factor contributing to atherosclerosis in diabetes [18]. Furthermore, there is widespread acceptance of a possible role for reactive oxygen species, generated as a result of hyperglycemia, in causing many of the secondary complications of diabetes, such as nephropathy, retinopathy, and neuropathy [19].
The inability of the modern synthetic approach to provide a satisfactory answer has led to a shift in focus to alternative forms of therapy based on drugs derived from plants. The present study was an effort to investigate the effect of ethanolic extract of E. ribes on diabetic dyslipidemia induced by STZ in rats. The study revealed the significant antihyperglycemic activity (P < 0.01) of ethanolic extract of E. ribes. Furthermore, extract treatment also produced significant fall in serum total cholesterol and triglyceride levels, indicating profound lipid-lowering activity of the test drug. The study also indicated the presence of antioxidant principles in the extract.
In conclusion, the present study shows that increased oxidative stress is apparent in STZ-induced diabetic animals. The ethanolic extract of E. ribes can protect tissues from lipid peroxidation. The extract also exhibits a significant lipid-lowering activity in these rats. Further studies are being undertaken to explain more fully the mechanism(s) of the lipid-lowering and antioxidant effects of E. ribes.