INTRODUCTION:

Diabetics have accelerated levels of oxidative stress and this contributes massively to most cardiovascular, neurological, retinal and renal diabetic complications ¹. Diabetes mellitus is a heterogeneous metabolic disorder as old as mankind and its incidence is considered to be high all over the world ².It is characterized by hyperglycemia. Hyperglycemia significantly diminishes glutathione levels lowering defenses against oxidative stress³.

A multitude of herbs, spices and other plant material has been described for the treatment of diabetes throughout the world ⁴. Furthermore, after the recommendations made by WHO (world health organisation) on diabetes mellitus, investigations on hypoglycemic agents from medicinal plants have become more important. The levels of serum lipids are usually elevated in diabetes mellitus and such an elevation represents the risk factor for coronary heart disease. Moreover, Diabetic patients experience a two to three fold increase in cardiovascular morbidity and mortality in comparison to non diabetics ⁵.

Arjunarishta, also named as Parthadhyarishta, is a polyherbal hydro-alcoholic preparation and is advised as a choice of remedy in cardiac insufficiency, chronic ailments in lungs, bilious affection, blood dysentery, inflammatory conditions and in the fracture of bones ⁶. It is very safe and natural and gives excellent results in heart and respiratory problems undoubtedly.

The chief ingredient of Arjunarishta is stem bark of Terminalia arjuna Roxb. It contains hydrolysable tannins, triterpenoid glycosides (arjunglucoside-1, arjunglucoside-2), sapogenins as arjunic acid, arjunolic acid, cardenolides , flavonoids, phenolics, phyto-sterols, mineral salt and sugar ⁷ .

Therefore we undertook the present investigation to evaluate the anti-diabetic effect of Arjunarishta–T and Arjunarishta-M prepared by traditional and modern methods, respectively, in Alloxan induced hyperglycemic animals.

MATERIAL AND METHOD:

Preparation of Arjunarishta

Arjunarishta-T:

This was prepared by method given in Bhasajya kalpana vigyan ⁶, according to this, Coarsely powdered Arjuna bark (Terminalia arjuna), fruits of Draksha (Vitis vinifera) and madhuca (Bassia longifolia) flowers were placed in polished vessel of brass along with prescribed quantity of water (26L), and allowed to steep. After 12h this material was warmed at low temperature till one fourth of water remained (6.5 L), then the heating was

Table-1.The anti-hyperglycemic effect of Arjunarishta-T and Arjunarishta-M on fasting blood sugar level (mg/dl) and blood glutathione level (mg/dl) of diabetic rats.

GR OUP	DOSE (ml/ kg bw)	ASTING BLOOD GLUCOSE and BLOOD GLUTATHIONE LEVEL (mg/dl)
		Day1		Day7		Day14		Day21
		FBS	GSH	FBS	GSH	FBS	GSH	FBS	GSH
I	2.0	66.8±2.1	27.71±0.7	72.01±2.2	24.25±0.67	73.5±2.8	25.0±0.49	75.6±2.3	24.5±0..39
II	2.0	205.15±3.15	14.17±0.71	267.72±2.95	14.01±2.1	285.79±4.85	13.7±2.7	291.70±6.5	12.79±2.03
III	10mg/kg	202.15±3.0	17.8±1.0	180.63±6.1	20.48±1.8	136.16±2.3	22.73±1.2	116.72±4.2***	25.63±2.6***
IV	2.0	201.17±2.95	15.17±2.2	198.17±2.45	15.97±1.95	180.7±1.9	16.17±1.8	164.0±1.85***	16.75±1.5***
V	2.0	202.49±1.0	16.75±2.15	195.27±2.0	17.0±2.0	176.45±1.5	17.25±1.75	149.0±1.01***	17.49±1.63***

All value are expressed as mean ± SEM, n=6, ***P<0.001,

Table2.Effect of oral administration of Arunarishta-T and M on body weight of Alloxan induced diabetic rats.

Group	Dose (ml/kg bw)	Average body weight
Group	Dose (ml/kg bw)	Day1	Day7	Day14	Day21
I	2.0	200.2±3.4	203.4±2.6	204.12±4.2	206.19±4.5
II	2.0	200.5±4.6	169.43±3.4	160.47±4.2	142.7±2.4
III	10mg/kg	208.4±2.4	194.27±1.6	190.8±2.7	187.37±3.4***
IV	2.0	208.5±3.2	191.25±4.2	183.15±2.4	182.34±2.6***
V	2.0	209.4±2.5	190.26±3.2	182.95±2.6	181.92±3.4***

All value are expressed as mean ± SEM, n=6.***P<0.001.

Table3.Effect of oral administration of Arjunarishta-T and M, on serum lipid profile in Alloxan induced diabetic rats after three weeks of treatment.

Group	Dose (ml/kg bw)	Serum cholesterol	Serum triglyceride	Serum HDL	Serum LDL	Serum creatinine	Serum urea	ALP
I	2.0	135.0±6.2	84.65±5.2	41.46±1.3	91.5±4.1	0.49±0.79	25.7±1.2	118.72±2.2
II	2.0	275.35±17.5	200.0±11.7	27.17±0.31	195.47±10.5	1.72±0.32	64.85±1.7	318.42±5.9
III	10 mg/kg	146.13±6.0*	109.1±4.8*	59.17±1.71*	84.17±3.2*	0.52±.21*	29.06±2.05*	122.63±3.92*
IV	2.0	155.14±5.5*	111.17±3.3*	42.06±0.2*	99.16±4.6*	0.61±0.4*	33.17±3.17*	144.47±6.73*
V	2.0	153.42±6.2*	113.18±3.2*	42.85±0.39*	98.91±5.7*	0.63±0.49*	34.0±4.0*	145.06±5.6*

All values are expressed as mean ± SEM , n=6, *P<0.05

stopped and filtered in a cleaned vessel and immediately 2.5kg of Jaggery (juice of Saccharum officinarum) was added. This sweet filtered material was placed in incubator for 15 days at 33°C ± 1°C after adding Dhataki flowers (Woodfordia floribunda). After 15 days, completion of fermentation was confirmed. The fermented preparation was filtered with cloth and kept in cleaned covered vessel for further next seven days. Then, the liquid was poured in ambered glass bottles.

Arjunarishta-M: Method of preparation was same as followed with Arjunarishta-T, only Dhataki flowers were replaced by Yeast for inducing fermentation⁸.

Animals:

Adult Wistar albino rats, weighing between 180-210g of either sex were acclimatized to normal environmental conditions in the laboratory for one week, given a standard chow diet (Hindustan Lever Ltd), and water ad libitum.

Induction of diabetes:

The animals were fasted for 18 h and made diabetic by injecting Alloxan monohydrate (150mg/kg, i.p) dissolved in saline normal saline ⁹. The diabetic state was confirmed when the blood sugar value was greater than 180mg/dl.

Experimental design:

All the animals were randomly divided into the five groups with six animals in each group. Group I, II and III were served as normal, diabetic and standard (glibenclamide 10mg/kg, p.o), respectively ¹⁰.Group IV and V were treated with Arjunarishta-T and Arjunarishta-M, respectively at the dose of 2ml/kg, p.o. per day.

Diabetic animal models:

Rats were made diabetic by a single intraperitoneal injection of Alloxan monohydrate (Loba chemie, Mumbai). Alloxan was first weighed individually for each animal according to the weight and then solubilized with 0.2ml saline just prior to injection. Two days after Alloxan injection rats with plasma glucose level of greater than 180mg/dl were selected for the present study. Treatment with Arjunarishta-T and M was started 48h after Alloxan injection. Blood samples were drawn at weekly interval until the end of study i.e three weeks. Fasting blood glucose, blood glutathione estimation and body weight measurement were done on day 1, 7, 14 and 21^st of the study. On 21^st day blood was collected by retro orbital puncture under mild ether anesthesia and fasting blood sugar was estimated ¹¹. serum was separated and analyzed for serum cholesterol ¹², serum triglycerides ¹³, serum HDL ¹⁴, serum LDL ¹⁵, serum creatinine ¹⁶, serum urea ¹⁷, serum alkaline phosphatase ¹⁸.

Determination of blood glutathione:

Blood glutathione was estimated by the method of Beutler et al¹⁹.

Statistical analysis:

All the values of the body weight, fasting blood sugar and biochemical estimation were expressed as mean ±SEM and analyzed for ANOVA.

RESULTS AND DISCUSSION:

The antihyperglycemic effect of Arjunarishta-T and Arjunarishta-M on the fasting blood glucose levels and blood glutathione level of diabetic rats is shown in Table-1.

Administration of alloxan (150mg/kgi.p.) led to more than 1.5 fold elevation of fasting blood glucose level, which was maintained over a period of 3 weeks. Three week of daily treatment of Arjunarishta –T and Arjunarishta-M (2 ml/kg p.o.) caused fall in blood sugar level by 44% and 49%, respectively.

GSH, being the most important biomolecule against chemically induced toxicity can participate in the elimination of reactive intermediates by reduction of hydro-peroxidase in the presence of glutathione peroxidase. The most important mechanism implicatedin the diabetogenic action of alloxan is by increased generation of oxygen free radical which cause a decrease in plasma GSH concentration. Hence, drugs that could prevent the generation of these oxygen free radicals or increase the free radical scavenging enzymes may be effective in alloxan induced diabetes ²⁰.

In our study, alloxan treated diabetic rats the GSH ( blood glutathione level) decreased significantly(p<0.05). Glibenclamide (10mg/kgp.o) treated rats showed increase in GSH levelson both 14^th and 21^st day of treatment. Similarly, Arjunarishta –T and arjunarishta-M (2 ml/kg p.o) treated rats also showed significant increase in GSH levels (p< 0.5).

In the present study, the observed significant increase in blood glucose level and a decrease in blood glutathione levels in diabetic rats could be due to destruction of ß –cells by alloxan reinforcing the view that alloxan induced diabetes probably through the generation of oxygen free radicals ²¹.

The standard anti-diabetic drug taken was Glibenclamide and the test preparations were Arjunarishta-T and arjunarishta-M, this shown that they could prevent the development of Diabetes mellitus in albino rats, due to their antioxidant property since they shown a significant decrease in fasting blood sugar level (FBS) and increase in GSH ( blood glutathione level) after treatment.

Vehicle control animals were found to be stable in their body weight but diabetic rats showed significant reduction in body weight during 21 days (Table-2).

Alloxan caused body weight reduction, which is increased by standard drug (Glibenclamide) and test preparations (Arjunarishta-T and Arjunarishta-M) nearly equal to the normal.

This study indicates that arjunarishta-T and Arjunarishta-M have good anti-diabetic acticity. Both the Arjunarishta-T And Arjunarishta-M exhibited significant antihyperglycemic activity in alloxan induced hyperglycemic rats without causing significant change in body weight.

Arjunarishta_T and Arjunarishta-M at the dose of 2 ml/kg body weight orally significantly reduced total serum cholesterol (44% and 44%), serum LDL ( 49% and 49%) and triglycerides 9 44% and 43%) when compared to diabetic control. The increase in HDL was 35% and 37% with Arjunarishta-T and Arjunarishta-M respectively. Arjunarishta-T and Arjunarishta-M also significantly reduced serum creatinine (64% and 64%), Serum urea (49% and 48%) and serum alkaline phosphtaselevels (55% and 55%)(Table-3).

The obtained result suggests that presence of alcohol could be beneficial in the faster absorption of poly-phenolic compounds present in Arjunarishta that is responsible for showing scavenging of alloxan induced reactive oxygen species.

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Received on 06.06.2009 Modified on 15.07.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 332-335