Chances of Reduction in Cardiovascular Risk by Ashwagandharishta in Induced Diabetic Condition.

Tiwari Preeti¹* and Patel RK²

¹Department of Pharmacognosy, Shri Sarvajanik Pharmacy College, Mehsana-384 001(N. Gujarat).

²Shri. S.K. Patel College of Pharmaceutical Education and Research, Kherva- 382711, (N. Gujarat)

ABSTRACT:

The objective of the study was to evaluate the chances of reduction in cardiovascular risk factors associated with diabetic conditions. Both types of Ashwagandharishta-T and Ashwagandharishta-M, were prepared by traditional and modern methods, respectively, and evaluated for fasting blood sugar , blood glutathione levels and serum biochemical parameters in alloxan induced diabetic rats. Both the Ashwagandharishta preparations were able to maintain the tested parameters near to normal level significantly.

KEYWORDS: Cardiovascular risk , Antidiabetic, Ashwagandharishta, Glutathione

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 Organization) 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.

Ashwagandharishta is a polyherbal hydro-alcoholic preparation and is used as rasayana⁶. Rasayanas are used to promote health and longevity by increasing defence against disease, arresting the ageing process and revitalizing the body in debilitated conditions⁷. The chief ingredient of Ashwagandharishta is roots of Ashwagandha, Withania somnifera, commonly known for its usefulness in the treatment of hypercholesterolemia, arthritis in combination with other drugs, is also credited to be hypoglycemic and diuretic⁸.The pharmacological effect of the roots of Withania somnifera is attributed to Withanolides, a group of steroidal lactones⁹.

Therefore we undertook the present investigation to evaluate the anti-diabetic effect of Ashwagandharishta-T and Ashwagandharishta-M prepared by traditional and modern methods, respectively in alloxan induced hyperglycemic rats.

MATERIAL AND METHOD:

Preparation of Ashwagandharishta:

Ashwagandharishta-T:

This was prepared by method as given in Ayurvedic Formulary of India ⁶. Formula of Ashwagandharishta has been given in the Table-1. The ingredients of Ashwagandharishta were procured from Local market, Jamnagar. Identification of all the individual plant material was done as per Ayurvedic Pharmacopoeia of India. Authentification of all these ingredients was done in the Botany Department of Central Institute of Medicinal and Aromatic plants ( CIMAP), Lucknow. Prepared herbarium has been deposited in the CIMAP for future reference.

According to this method, coarsely powdered Ashwagandha roots ( Withania somnifera) with prescribed ingredients were placed in polished vessel of brass along with prescribed quantity of water( 24.576L), and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one eighths of the prescribed quantity ( 3.072 L) , then the heating was stopped and it was filtered in cleaned vessel and after that honey was added. Then, Dhataki flowers ( Woodfordia floribunda), and Prakshepa dravyas as Sonth, marich, pippali, tvak, Tejpatra, priyangu and nagakesara were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33^oC±1^oC. After 15 days, completion of fermentation was confirmed by standard tests¹⁰.The fermented preparation was filtered with cotton cloth and kept in cleaned covered vessel for further next seven days. Then, the liquid was poured in amber colored glass bottles, packed and properly labeled.

Ashwagandharishta-M:

Method of preparation was same as followed with Ashwagandharishta-T, only Dhataki flowers were replaced with Yeast for inducing fermentation¹¹.

Animals:

As per the guidelines of CPCSEA, adult Wistar albino rats, weighing between 200-220g 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 sterile normal saline. In order to stave off the hypoglycemia during the first day, 5% w/v glucose solution was given orally to diabetic rats four to six hours after alloxan administration .The diabetic state was confirmed when the blood sugar level was greater than 180mg/dl ¹².

Experimental design:

All the animals were randomly divided into the five groups with six animals in each group. Group I ( Normal rats), Group II Control ( Diabetic untreated rats), Group III ( Diabetic rats treated with Glibenclamide10 mg/Kgbwt p.o. per day)¹³, Group IV ( Diabetic rats treated with Ashwagandhrishta-T 2.0ml/Kgbwt. p.o.Per day) and Group V (Diabetic rats treated with Ashwagandhrishta-M 2.0ml/Kgbwt p.o.Per day).Rats in Group I and II were fed with normal saline as vehicle.

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 Ashwagandharishta-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 sugar, 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 plexus under mild ether anesthesia and fasting blood sugar was estimated ¹⁴. Serum was separated and analyzed for serum cholesterol¹⁵, serum triglycerides¹⁶, serum HDL¹⁷, serum LDL¹⁷, serum creatinine¹⁸, serum urea¹⁹, serum alkaline phosphatase²⁰ by using Span and Erba diagnostic kits.

Determination of blood glutathione:

Blood glutathione was estimated by the method of Beutler et al²¹.

Statistical analysis:

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

RESULTS AND DISCUSSION:

The anti-hyperglycemic effect of Ashwagandharishta-T and Ashwagandharishta-M on the fasting blood sugar level and blood glutathione level of diabetic rats is shown in Table-2.

Administration of alloxan (150mg/kgi.p.) led to more than 1.5 fold elevation of fasting blood sugar level, which was maintained over a period of 3 weeks. Three week of daily treatment of Ashwagandharishta-T and Ashwagandharishta-M (2 ml/kg p.o.) caused significant fall in blood sugar level by 42% and 47%, respectively.

GSH, being the most important bio-molecule against chemically induced toxicity can participate in the elimination of reactive intermediates by reduction of hydro-per-oxidase in the presence of glutathione per-oxidase. The most important mechanism implicated in 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 diabetes²².

In our study, in alloxan treated diabetic rats the GSH ( blood glutathione level) decreased significantly(p<0.001). Glibenclamide (10mg/kg p.o) treated rats showed increase in GSH levels on both 14^th and 21^st day of treatment. Similarly, Ashwagandharishta-T and Ashwagandharishta- M (2.0 ml/kg p.o) treated rats also showed significant increase in GSH level (p< 0.001).

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 ²³.

Table1 Prescribed Formula of Ashwagandharishta as per Ayurvedic Formulary of India for Batch Size 12.288L and used for Batch size 3.072 L

S. No.	Vernacular name of Drugs	Botanical name	Plant part	Prescribed Quantity for Batch size 12.288 L	Quantity Taken for Batch size 3.072 L
1.	Ashwagandha	Withania somnifera	Rt.	2.4 Kg	600 g
2.	Kali musali	Curculigo orchioides	Rt.	960 g	240 g
3.	Manjistha	Rubia cordifolia	Rt.	480 g	120 g
4.	Haritaki	Terminalia chebula	Fr.	480 g	120 g
5.	Haridra	Curcuma longa	Rz.	480 g	120 g
6.	Daruharidra	Berberis aristata	St.	480 g	120 g
7.	Yasti	Glycyrrhiza glabra	Rt.	480 g	120 g
8.	Rasna	Alpinia chinensis	Rt.	480 g	120 g
9.	Vidari	Pueraria tuberosa	Rt.	480 g	120 g
10	Arjuna	Terminalia arjuna	StBk	480 g	120 g
11	Mustaka	Cyperus rotundus	Rz.	480 g	120 g
12	Trivrt	Ipomoea turpethum	Rt.	480 g	120 g
13.	Svet Sariva	Hemidesmus indicus	Rt.	384 g	96 g
14.	Krisna sariva	Ichnocarpus frutescens	Rt.	384 g	96 g
15.	Svet Chandan	Santalum album	Htwd	384 g	96 g
16.	Rakta Chandan	Pterocarpus santalinus	Htwd	384 g	96 g
17.	Vacha	Acorus calamus	Rz.	384 g	96 g
18.	Chitraka	Plumbago zeyleynica	Rt.	384 g	96 g
19.	Water for decoction Water reduced to	-	-	98.304 L 12.288 L	24.576 L 3.072 L
Prakshepa Dravyas
20.	Madhu ( Honey)	-	-	14.4 Kg	3.6 Kg
21.	Dhataki	Woodfordia floribunda	Fl.	768 g	192 g
22.	Sonth	Zingiber officinalis	Rz.	96 g	24 g
23.	Marica	Piper nigrum	Fr.	96 g	24 g
24.	Pippali	Piper longum	Fr.	96 g	24 g
25.	Tvak	Cinnamomum zeyleynicum	StBk	192 g	48 g
26.	Elaichi ( Chhoti)	Eletteria cardamomum	Sd.	192 g	48 g
27.	Tejpatra	Cinnamomum tamala	Lf.	192 g	48 g
28.	Priyangu	Callicarpa microphylla	Fl.	192 g	48 g
29.	Nagakesara	Mesua ferrea	Stmn	96 g	124 g

Table-2. The anti-hyperglycemic effect of Ashwagandharishta-T and Ashwagandharishta-M on fasting blood sugar level (mg/dl) and blood glutathione level (mg/dl) of diabetic rats.

GROUP	DOSE (ml/kg bw)	FASTING BLOOD SUGAR 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	201.35±1.94	15.26±2.15	198.29±3.2	16.14±2.95	180.91±1.75	16.25±1.28	168.0±1.29***	16.92±1.29***
V	2.0	202.63±1.25	16.79±1.28	195.48±1.25	16.82±2.06	177.12±2.13	16.85±1.72	154.12±2.05***	17.15±2.61***

All values are expressed as mean ± SEM, n=6. ***P<0.001 vs control group

Table-3: Effect of Oral administration of Ashwagandharishta-T and M on Body weight of Alloxan induced diabetic rats

Group	Dose (ml/kg bw)	Average Body Weight ( g)
		Day 1	Day 7	Day 14	Day 21
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	10 mg/kg	208.4±2.4	194.27±1.6	190.8±2.7	187.37±3.4***
IV	2.0	208.7±2.5	192.27±3.4	184.64±1.6	183.52±1.27***
V	2.0	209.3±3.6	191.16±4.2	183.92±2.4	182.14±3.2***

All values are expressed as Mean ± SEM, n=6. ***p < 0.001 vs control group

Table 4: Effect of oral administration of Ashwagandharishta –T and M, on serum lipid profile in Alloxan induced diabetic rats after three weeks of treatment.

Group	Dose (ml/kg bwt/dayp.o.)	Serum cholesterol (mg/dL)	Serum triglyceride ( mg/dL)	Serum HDL (mg/dL)	Serum LDL (mg/dL)	Serum creatinine ( mg/dL)	Serum urea (mg/dL)	ALP (IU/L)
I	2.0	155.0± 6.2	84.65± 5.2	47.46± 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***	39.17± 1.71***	84.17± 3.2***	0.52± 0.21***	29.06± 2.05***	122.63± 3.92***
IV	2.0	134.9± 2.6***	110.85± 1.8***	37.94± 0.84***	93.89± 2.2***	0.58± 0.25***	31.75± 0.92***	139.5± 2.4***
V	2.0	135.2± 2.4***	111.19± 1.5***	37.25± 1.2***	93.92± 1.81***	0.59± 0.23***	32.12± 1.4***	140.7± 1.92***

All values are expressed as mean ± SEM , n=6, ***P<0.001 vs control group

The standard anti-diabetic drug taken was Glibenclamide and the test preparations were Ashwagandharishta-T and Ashwagandharishta-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-3).

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

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

Ashwagandhrishta- T and Ashwagandharishta-M at the dose of 2 ml/kg body weight orally significantly reduced total serum cholesterol (51% and 51%), serum LDL (52% and 52%) and triglycerides ( 44% and 44%) when compared to diabetic control. The increase in HDL was 40% and 37% with Ashwagandharishta-T and Ashwagandharishta-M respectively .Ashwagandharishta-T and Ashwagandharishta-M also significantly reduced serum creatinine ( 66% and 66%), Serum urea (51% and 50%) and serum alkaline phosphtase levels ( 56% and 56%) (Table-4 ).

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

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Received on 05.01.2010

Accepted on 20.03.2010

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Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 171-174