Evaluation of Antihyperglycemic and Antihyperlipidemic Activity of Leaf Extracts of Breynia vitis-idaea in Alloxan Induced Diabetic Rats

Aim: The present study was carried out to evaluate the hypoglycemic and hypolipidemic activity of alcohol and aqueous leaf extracts of Breynia vitis-idaea (Burm. F.) Fischer in alloxan induced diabetic rats. Methods: Diabetes was induced into albino Wistar rats by intraperitonial administration of alloxan (120 mg/kg). Normal and diabetic rats divided into different groups of six each. Alcohol and aqueous leaf extracts (300 mg/kg and 600 mg/kg) and standard drug (Glibenclamide 500 μg/kg) was administered orally for 21 days. Blood samples were collected from overnight fasted normal and diabetic rats on 0th, 7th, 14th and 21st days of treatment. Hypoglycemic activity was evaluated by measuring serum glucose level and glycosylated haemoglobin level after dosing with aqueous and alcohol extracts. Hypolipidemic activity was evaluated by measuring various biochemical parameters like total cholesterol, triglycerides, low density lipoprotein, very low density lipoprotein, high density lipoprotein and phospholipids. Results: Both the extracts significantly (P<0.001, p<0.01) reduced fasting blood glucose of alloxan diabetic rats in a dose-related manner, when compared to control and standard. They also have a significant recovery in the levels of parameters measured in lipid profile, when compared to control and standard group. Conclusion: The present investigation established pharmacological evidence to support the folklore claim that it is used as hypoglycemic and hypolipidemic agent.


INTRODUCTION
Diabetes mellitus (DM) is a multifactorial disease which is character ized by hyperglycemia, lipoprotein abnormalities, raised basal meta bolic rate, defect in reactive oxygen species scavenging enzymes and altered intermediary metabolism of major food substances.Diabetes is a major degenerative disease having complications which include hypertension, atherosclerosis and microcirculatory disorders. 1The in creasing incidence of the disease worldwide may be due to sedentary life style, unhealthy diet, obesity and other predisposing risk factors. 2 It is projected to become of the world's main disablers and killers, as the number of people with diabetes multiplies worldwide.The disease has taken an ever increasing share of national and international healthcare budgets. 3he World Health Organization has also recommended the evaluation of the effective use of plants, because of the modern drugs are not safe.The synthetic hypoglycemic agents used in clinical practices have serious side effects like haematological effects, coma, disturbances of liver and kidney. 4It is widely accepted that the most challenging goal in the management of diabetes is to achieve blood glucose level as close to normal as possible.However, in the indigenous system of medicine good numbers of plants are mentioned for the cure of diabetes and some of them have been experimentally evaluated and active principles were isolated.In the present scenario, the demand for herbal products is growing expo nentially throughout the world and major pharmaceutical companies are currently conducting extensive research on plant material for their potential medicinal value. 5any medicinal plants used in ethnomedical practices in India are known or little known to scientific world.The main objective of the study was to assess the hypoglycemic and hypolipidemic potential of leaves of Coral berry tree Breynia vitis-idaea belonging to the family Euphor biaceae.It is known as "Surasaruni" in Hindi. 6reynia vitis-idaea (Burm.f.) is an evergreen 1.55 m tall glabrous tree or large erect shrub with horizontal branches found in the Gangetic plain, Western Peninsula, China, Malay Peninsula and Sri Lanka.These plants are planted as ornamental hedge in garden.Leaves are 13 cm long, elliptic to ellipticovate, alternate dark brown or black when dry. Bark is yellowish grey, flowers are small, greenish yellow or pink.The fruits are fleshy, pink to red which turns black when ripe and measures 23 mm in diameter.The seeds are black and have a very hard seed coat.Root contains βsitosterol.Leaves contain triacontane, ceryl alcohol, lanosterol, pentatriacontanoic acid.A new sulphur containing spiroketal glycoside, breynin I and a new terpenic glycoside, breyniaionoside E together with 10 known compounds were isolated from the aerial parts of Breynia vitis-idaea.A decoction of the roots is employed as mouthwash for toothache.Leaves applied as poultice to hasten suppuration.Leaf juice given after parturition to prevent haemorrhage.Dried leaves smoked like tobacco to relief in tonsilitis.Astringent bark used to guard against haemorrhage. 79Aqueous, ethanol extracts of plant proves the anticancer action in HEPG2 cell lines against carbon tetra chloride induced toxicity in cell line. 10Leaves warmed along with leaves of Dodonaea viscosa, Dalbergia paniculata and applied for 23 days regularly to cure swelling of legs and testis. 11In the current literature, there is not much data con cerning the effect of Breynia vitis-idaea on the blood glucose level and parameters used in lipid profile.Therefore, the present study has been planned to investigate the effect of extracts in alloxan induced diabetic rats and to compare it with diabetic untreated and glibenclamide as a reference standard.

Drugs and chemicals
The following drugs and chemicals were used in the experiment: Alloxan monohydrate was purchased from Central Drug house, New Delhi, India and Glibenclamide was purchased from Bal Pharma, Bangalore, India.Diagnostic kits of Glycosylated haemoglobin was purchased from Coral Clinical System, Goa, India and diagnostic kits of cholesterol, phospho lipids, triglycerides, HDL, VLDL and LDL were purchased from Span diagnostics Ltd., Surat, India and rest all other reagents and chemicals were of analytical grade.

Plant material
The leaves of Breynia vitis-idaea were collected from the vicinity of Tirunnelveli (Tamil Nadu, India).Taxonomic identification was car ried out by V. Chelladurai, Research OfficerBotany (Retired scientist CCRAS).A voucher specimen (JC Nagar) was deposited in the her barium of the department of Pharmacognosy in the college for future reference.The collected leaves were washed thoroughly in tap water to remove any unwanted matter and then dried under shade for one week.After complete drying, leaves were pulverized into coarse powder.The powder stored in airtight container in cool and dark place to prevent deteriora tion by elevated temperature, light and moisture.

Preparation of crude extracts
Coarsely powdered, shade dried leaves of Breynia vitis-idaea was charged into a soxhlet apparatus and successive hot extraction was carried out using ethanol (70% v/v) for 24 h.The liquid extract was concentrated in rotary flash evaporator at a temper ature not exceeding 50ºC (yield 12.65% w/w).The alcohol extract was formulated as a suspension in distilled water using 2% v/v Tween80 as suspending agent for animal studies.The aqueous extract was prepared by maceration method.The coarsely powder of leaves kept with chloroform water for 24 h.The macerate was filtered and filtrate concentrated in rotary flash evaporator (yield 15.3% w/w).Aqueous extract was formulated by dissolving in distilled water for animal studies.The extracts were preserved in desiccators for further experiments.

Animals used
Swiss albino mice weighing 20-30 g and albino rats (Wistar strain) weighing 170 ± 10 g of either sex were used for the study.The animals were procured and housed in the animal house at least 2 weeks prior to the study, for acclimatization.Animal house was well maintained under standard hygienic conditions, at a temperature (15-20 ± 5°C), room humidity (60% ± 10%) with 12 h day and night cycle with food and water ad libitum.All the pharmacological experiments were as per CPCSEA (Committee for the Purpose of Control and Supervision on Experiments on Animals) norms after obtaining approval of the Institutional Animal Ethics Committee (Reg.No. 870/ac/08/CPCSEA).

Acute toxicity studies
These studies were carried out to study the acute toxic effects and deter mine minimum lethal dose of the drug extracts.Swiss albino mice of either sex weighing between 2030 g fasted overnight, were used for the study.Each extract was orally administered at doses of 30, 100, 300, 1000 and 3000 mg/kg body weight to separate groups of mice.Subsequent to administration of drug extracts, the animals were observed closely for the first three hours, for any toxic manifestations, like increased motor activity, salivation, clonic convulsions, coma and death.Subsequently observations were made at regular intervals for 24 h.The animals were under further investigation up to a period of 1 week. 12

Induction of diabetes mellitus
In the present work alloxan monohydrate was used to induce hypergly cemia in animals at the dose of 120 mg/kg body weight by intraperitonial injection. 13The fasting blood glucose levels were determined after 72 h of alloxan administration.Rats having blood glucose level above 200 mg/dl were selected for the study.Diabetic rats were divided in six groups; each group comprised of six rats. 14roup 1Normal control.Group 2-Positive controlUntreated alloxan diabetic rats.Group 3-StandardAlloxan diabetic rats treated with glibenclamide (500 µg/kg, p.o.) Group 4Alloxan diabetic rats treated with aqueous extract (300 mg/kg, p.o) Group 5Alloxan diabetic rats treated with aqueous extract (600 mg/kg, p.o) Group 6Alloxan diabetic rats treated with alcohol extract (300 mg/kg, p.o) Group 7Alloxan diabetic rats treated with alcohol extract (600 mg/kg, p.o) Doses of aqueous extract, alcohol extract, standard drug and normal saline were calculated according to the body weight of each animal.Suspension of extracts, standard drug and normal saline were adminis tered orally to each animal using stainless steel feeding needle fitted on a plastic syringe.The treatment schedule was once daily for 21 days and animals were fed on laboratory diet of pellet chow and water ad libitum.They were fasted for 18 h prior to blood withdrawal.

Determination of hypoglycemic activity
Blood samples were collected by orbital sinus puncture under mild ether anaesthesia.Serum was separated by centrifuging blood at 6000 rpm for 15 min.Serum glucose estimation was performed on 0 th , 7 th , 14 th and 21 st day by end point method using Autochem Nexgen semi autoanalyzer (Span diagnostics, Surat, India) with the help of glucometer (Glucochek, Surat, India).On 21 st day estimation of glycosylated hemoglobin was also performed using UV visible spectrophotometer (Systronic 2203) with the help of Glycohemoglobin reagent kit.

Determination of hypolipidemic activity
Blood samples were collected by orbital sinus puncture under mild ether anaesthesia on 21 st day from the start of treatment.Serum was separated and analyzed for various biochemical parameters-Cholesterol, Triglyc erides, HDL, LDL, VLDL and Phospholipids by using various kits.

Statistical analysis
The data obtained were statistically analyzed by one way analysis of variance (ANOVA) and expressed as mean ± S.E.M. followed by Tukey Kramer Multiple Comparison Test using instat software.

Acute toxicity studies
Acute toxicity study revealed the nontoxic nature for both the extracts.There was no mortality and no toxic reactions found at any of the doses tested until the end of the study period.As per OECD guidelines, therapeutic range was considered between 1/10 to 1/5 times of LD 50 .Accordingly, 300 mg/kg and 600 mg/kg BW doses for both the extracts were selected for determination of pharmacological studies.

Hypoglycemic activity
Hypoglycemic activity of aqueous and alcohol extracts of Breynia vitis-idaea were evaluated in alloxan induced diabetic rats.Administration of alloxan increases the serum glucose level in normal rats.The effects of extracts and glibenclamide on serum glucose level in diabetic rats are depicted in Table 1.The fall in serum glucose levels of the extracts and glibenclamide treated groups were compared with that of positive control (diabetic untreated) group.Both aqueous and alcohol extracts showed significant hypoglycemic effect in comparison with positive control group on 7 th day itself.The continuous treatment for three weeks leads to a dose dependent fall in serum glucose level.The dose of 600 mg of both the extracts decreases the serum glucose level towards normal level.The concentrations of serum glycosylated haemoglobin level in diabetic rats are depicted in Table 2.The concentration of serum glycosylated haemoglobin level also found significant when compared to positive control group.Administration of 300 mg/kg of aqueous extract showed 13.61%, 41.94%, 60.89% decline in glucose levels of experimental animals on day 7 th , 14 th , 21 st respectively.The glycosylated haemoglobin level was found 52.25% less, when compared to diabetic control group.Administration of 600 mg/kg of aqueous extract showed 20.04%, 47.83%, 66.62% decline in glucose levels of experimental animals on day 7 th , 14 th , 21 st respectively.The glycosylated haemoglobin level was found 59.25% less, when compared to diabetic control group.Administration of 300 mg/kg of alcohol extract showed 17.47%, 44.37%, 63.37% decline in glucose levels of experimental animals on day 7 th , 14 th , 21 st respectively.The glycosylated haemoglobin level was found 52.75% less, when compared to diabetic control group.Administration of 600 mg/kg of alcohol extract showed 19.34%, 45.44%, 63.67% decline in glucose levels of experimental animals on day 7 th , 14 th , 21 st respectively.The glycosylated haemoglobin level was found 59% less, when compared to diabetic control group.

Hypolipidemic activity
The lipid profile of normal control, positive control, glibenclamide and extracts treated diabetic rats are depicted in Table 3 and 4. In alloxan induced diabetic rats there was a significant increase in total cholesterol, triglycerides, phospholipids, LDLcholesterol, VLDLcholesterol and significant decrease in HDLcholesterol in serum, when compared to normal control.The extracts and glibenclamide treated rats were signifi cantly decrease the total cholesterol, triglycerides, phospholipids, LDL cholesterol, VLDLcholesterol and increase the HDLcholesterol on day 21.Both the extracts showed almost same effect on serum glucose level.In the diabetic untreated rats the lipid profile levels remained higher without much change during the study period of 21 days.

DISCUSSION
Type I diabetes mellitus is a chronic disease characters by high blood glucose level due to an absolute or relative deficiency or circulating insulin levels.Various types of oral hypoglycemic agents are available along with insulin for treating diabetes mellitus.It is generally accepted that sufonylureas including glibenclamide, produce hypoglycaemia by stimu lating the pancreatic βcells to release more insulin.Reducing hepatic insulin clearance, stimulate the release of somatostatin and suppressing the secretion of glucagon.Sulfonylureas have also been shown to suppress hepatic gluconeogenisis. 1516required evidence that will demonstrate drug's safety and effectiveness for its proposed use, a carefully designed and progressive sequence of preclinical (animal) and clinical (human) studies are undertaken.This study indicated that both of the extracts of Breynia vitis-idaea have potential to decrease blood glucose level as well as improving hyperlipi daemia and to reduce the complications associated with experimental diabetes.This study also supports the folklore usefulness of this plant in the treatment of diabetes.It can be concluded that the roots of this plant could be further investigated for antidiabetic bioactive principles.
The present study focused the scientific explanation about the hypo glycemic and hypolipidemic activity for both the extracts of leaves of Breynia vitis-idaea for the management of alloxan induced diabetes.Experimental animals were made diabetic using alloxan.Alloxan is a toxic glucose analogue, which selectively destroys insulin producing cells in the pancreas when administered to rodents and many other animal species.This causes an insulin dependent diabetes mellitus in these animals, with characteristics similar to type1 diabetes in humans.In diabetic rats, alloxan led the elevation of fasting blood glucose level, which was maintained over a period of 23 weeks.Decrease in blood glucose level may be due to the regeneration of βcells of the pancreas which was destroyed by alloxan. 1718ipid play an important role in the pathogenesis of complications involved with diabetes mellitus.The elevated level of serum cholesterol, LDL, VLDL, triglycerides and reduced level of HDL possess to be a rises of factor for developing microvascular complication leading atherosclerosis and cardiovascular diseases like coronary heart disease.The abnormal high concentration of serum lipid in diabetic mainly due to increased mobilization of free fatty acids from peripheral fat depots, since insulin inhibits the hormone sensitive lipase, insulin deficiency or insulin resistance may be responsible for dislipidimia.The present studies provide the introductory approach for the evalua tion of its traditional preparations in order to scientifically validate the therapeutic use of Breynia vitis-idaea in the control of diabetes as well as maintenance of various biochemical parameters.

CONCLUSION
Screening of Ayurvedic drugs/plants for biological activity assumes prime importance to establish physiological action of the drug.To obtain

Table 2 : Effect of aqueous and alcohol extracts of leaves of Breynia vitis-idaea on glycosylated hemoglobin conc. on 21 st day Groups Glycosylated hemoglobin conc.
*Values expressed as Mean ± SEM.One way ANOVA (*** p<0.001).Tukey Kramer Multiple Comparison Test in comparison with diabetic control group.