INTRODUCTION:

The association of raised serum cholesterol and Triglycerides with cardiovascular disease is well known. Hypolipidemic drugs are those, which lower the level of lipids and lipoproteins in blood¹. The hypolipidemic drugs have attracted considerable attention because of their potential to prevent cardiovascular disease by retarding the accelerated atherosclerosis in hyperlipidemic individuals and accelerated atherosclerosis cause hypertension, which can cause heart attack. This is the second leading cause of death in the world. Heart attack can occur in any person, manifests itself in various ways- as a sudden episode of weakness of half of the body, confusion, slurring of speech, visual disturbances, headache, vertigo, altered consciousness, usually happening altogether².

Ashwagandharishta is a polyherbal hydro-alcoholic ayurvedic preparation and is used as rasayana³. Rasayanas are used to promote health and longevity by increasing defence against disease, arresting the ageing process and revitalizing the body in debilitated conditions⁴.

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 diuretic⁵.The pharmacological effect of the roots of Withania somnifera is attributed to Withanolides, a group of steroidal lactones⁶.

Besides Withania roots,all the other ingredients of Ashwagandharishta as Arjuna ( Bark of Terminalia arjuna), Liquorice (roots of Glycyrrhiza glabra), Majith ( roots of Rubia cordifolia), Rasna ( roots of Alpinia chinensis), Taj ( inner bark of Cinnamomum zeyleynicum), Nagarmotha ( rhizomes of Cyperus rotundus), haritaki ( fruits of Terminalia chebula), turmeric( rhizomes of Curcuma longa), Nagakesara ( stamens of Messua ferrea) etc. contain a rich quantity of phenolic compounds and flavonoids and possess significant antioxidant activity^7-12. Therefore, we undertook the present investigation to evaluate the hypolipidemic effect of Ashwagandharishta prepared by traditional and modern methods, Ashwagandharishta-T and Ashwagandharishta-M respectively in high fat diet induced hyperlipidemic wistar albino rats.

MATERIAL AND METHOD:

Preparation of Ashwagandharishta:

Ashwagandharishta-T:

This was prepared by method as given in Ayurvedic Formulary of India³.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. Authentication 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 tests¹³.The fermented preparation was filtered with cotton cloth and kept in cleaned covered vessel for further next seven days. Then, the preparation 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 fermentation¹⁴.

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 and given a standard chow diet (Hindustan Lever Ltd) and water ad libitum.

Chemicals:

Thiobarbituric acid was obtained from Loba Chemie, India. Ferrous sulphate, trichloro acetic acid, Potassium dihydrogen phosphate, Potassium hydroxide, were of analytical grade and obtained from Ranbaxy fine chemicals.

Assay of lipid per oxidation:

The extent of lipid peroxidation in goat liver homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS) by using standard method¹⁵ with the help of spectrophotometer. Goat liver was purchased from local slaughter house. Its lobes were dried between blotting paper (to remove excess blood) and were cut into small pieces with a heavy-duty blade. They were then homogenized in glass-Teflon homogenizing tubes in cold phosphate buffer saline (pH 7.4). It was centrifuged at 2000rpm for 10 min, and supernatant was diluted with phosphate buffer saline up to final concentration of protein 0.8-1.5 mg/0.1ml. Protein concentration was measured by using standard method¹⁶. To study the comparative response, the experiment was divided into five groups. Liver homogenates (5%, 3ml) was aliquoted to seven different glass Petri dishes. The first two groups were treated as control and standard where buffer and Vit. E was added. In the third to seventh group, different concentration (100, 150, 200, 250 and 300 µg/ml) of Ashwagandharishta-T and Ashwagandharishta-M were added. Lipid peroxidation was initiated by adding 100µl of 15mM ferrous sulphate solution to 3 ml of liver homogenate. After 30 min, 100µl of this reaction mixture was taken in a tube containing 1.5ml of 10% trichloro acetic acid. After 10 min, tubes were centrifuged and supernatant was separated and mixed with 1.5ml of 0.67% thiobarbituric acid. The mixture was heated in a water bath at 85°C for 30 min, and in boiling water bath to complete the reaction. The intensity of pink colored complex formed was measured at 535 nm.

The percentage of inhibition of lipid per-oxidation was calculated by comprise the results of the test with those of controls as per the following formula i.e. Eq. (1);

Percentage Inhibition = (Control Absorbance- Test Absorbance) X 100/Control absorbance.

Determination of hypolipidaemic activity:

Experimental design:

Thirty wistar albino rats were divided in following five groups of six each.

Group I (-ve Control): Normal diet (Standard chow diet)

Group II (+veControl): High Fat Diet (HFD)

Group III: HFD+ Ashwagandharishta-T (2.0 ml/kg bw/day p.o)

Group IV: HFD+ Ashwagandharishta-M (2.0 ml/kg bw/day p.o)

Group V: HFD+Atorvastatin (1.2 mg/Kg b.w./day p.o)

The compositions of the two diets were as follows:

Control Diet(Normal):

Wheat flour 100g

Sucrose 50g

Hydrogenated vegetable oil 5ml

Casein 20g

Cellulose 4g

Salt mixture (NaCl, KCl, CaCl₂) 1.5g

Citric acid 0.5ml

Vitamin B complex composition

High fat Diet

Wheat flour 100g

Sucrose 50 g

Hydrogenated vegetable oil 10ml

Casein 20g

Butter 10g

Cellulose 4g

Salt mixture (NaCl, KCl, CaCl2): 1.5g

Cholesterol (dried egg yolk) 0.5g

Citric acid 0.5ml

Vitamin B complex composition.

Procedure:

Group I served as normal control and was given water 2.0ml/kg bwt orally along with normal diet. Group II, III, IV and V received high fat diet plus cholesterol for induction of hyperlipidemia. In addition to this, group III and IV were given Ashwagandharishta –T and Ashwagandharishta-M (2 ml/kg b.w. /day p.o) while group V received Atorvastatin (1.2 mg/kg b.w. /day p.o) for nine weeks ¹⁷.

Body weight of each animal was noted at the beginning and at the end of the experiment. During the whole period, the animals have free access to food and water. Twenty hours prior the end of the experiment, food was withdrawn and blood samples were taken by retro-orbital plexus. The blood samples were centrifuged for 12 minutes at 16 00 rpm. Serum Total Cholesterol¹⁸, Serum HDL¹⁹, Serum LDL¹⁹, Serum VLDL²⁰, Serum Triglycerides²⁰, were determined in each blood sample.

These parameters were estimated by using Span Diagnostic and Erba Diagnostic Kits.

The LDL, VLDL and Atherogenic index were calculated by using the following Friedewald formulae¹⁹

LDL = TC – HDL – VLDL (where VLDL = TG/5)

Atherogenic index = (LDL+VLDL)/HDL

Statistical analysis

All data are expressed as mean ±SEM. For comparison amongst different groups, one-way ANOVA was performed. A significance value (p< 0.001) was considered statistically significant.

Table-1 Effect of Ashwagandharishta- T and M on Lipid Per-oxidation model

Concentration ( µg/ml)	Percentage Inhibition
Concentration ( µg/ml)	Ashwagandharishta-T	Ashwagandharishta-M
300	63.48±0.62	62.15±0.67
250	60.15±0.58	59.24±0.62
200	58.27±0.63	57.32±0.54
150	45.16±0.57	43.78±0.59
100	35.74±0.48	33.97±0.51
Vitamin E ( 5mM)	68.32±0.42
IC₅₀ (µg/ml)	181.88	191.05

All values are expressed as Mean ± SEM of Replicates of three

RESULTS:

Assay of lipid peroxidation:

The results presented in Table-1, showed that both the Ashwagandharishta-T and M, inhibited ferrous sulphate induced lipid per-oxidation in a dose dependent manner. The Ashwagandharishta T and M at 300µg/ml exhibited maximum inhibition (63.48% and 62.15% respectively), which is nearly equal to the inhibition produced by Vit. E (5mM). The IC₅₀ value was found to be 181.88 and 191.05 µg/ml with Ashwagandharishta-T and M respectively. The inhibition could be caused by the absence of ferryl-perferryl complex or by changing the ratio of ferric to ferrous or by reducing the rate of conversion of ferrous to ferric or by changing the iron itself or combination thereof¹⁵ .

Hypolipidemic activity:-

A significant reduction in body weight of rats was observed in Ashwagandharishta-T and M treated groups III and IV, respectively in comparison to control group as shown in Table-2.

Table-2 Effect on Body Weight of rats of all groups

S. No.	Treatment Groups	Initial Body Weight ( g)	Final Body weight (g)
1.	Group I	213.72±2.27	215.46±1.84
2.	Group II	216.15±2.14	232.14±2.41
3.	Group III	215.56±1.87	217.25±2.34***
4.	Group IV	215.17±2.32	219.27±1.73***
5.	Group V	214.2±1.92	216.0±1.84***

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

A one hundred seven percent (107%) increase in serum total cholesterol (TC) was noticed in rats fed with high fat diet and cholesterol (group II) in compare to rats fed normal diet (group I). Administration of Ashwagandharishta –T and M lowered the serum total cholesterol by 47% and 46% in group III and group IV, respectively (Table-3). A significant reduction in serum LDL cholesterol was observed in Ashwagandharishta –T and M treated groups and lowering percentage was 66% and 65 % respectively. The HDL cholesterol level was significantly reduced in group II due to high fat diet, which became normal in group-III and IV, with the effect of Ashwagandharishta- T and M .Ashwagandharishta-T and M treated groups showed reduction in atherogenic index in comparison to control.s Both of these preparations as Ashwagandharishta- T and M showed significant decrease in serum triglycerides that was 35 and 34 percent respectively.

DISCUSSION:

Lipids are widely involved in oxidation reactions and these reactions, can be induced by free radicals called Reactive Oxygen Species (ROS). Oxidative stress caused by ROS in the living cell is associated with numerous diseases, like coronary heart disease, atherosclerosis, inflammation, cancer, anemia, and age related muscular degeneration and ageing. Use of anti oxidants (substances that when present in low concentrations with those of an oxidizable substrate, significantly retard oxidation of that substance) can postpone problems caused by ROS and they retard oxidation process. Enzyme modifying actions of anti-oxidants could account for their pharmacological activities. In our present study Ashwagandharishta-T and M was evaluated for free radical scavenging activity and showed potent anti-oxidant activity and evidenced that free radical scavenging potential helps in ameliorating disease process²¹.

Table-3: Effect of Ashwagandharishta-T and M on Various Serum Levels in Hyperlipidaemic Rats.

Groups	Treatment	Diet	Dose ml or mg/kg b.wt/day p.o	Total Cholesterol mg/dl	HDL mg/dl	LDL mg/dl	VLDL mg/dl	Triglycerides mg/dl	Atherogenic index
I	Normal	Normal diet	2.0 ml/kg water	108.25±0.34	56.45± 0.11	36.92 ±0.33	16.26± 0.58	81.30± 0.74	0.942
II	Control	High fatty diet	2.0 ml/kg water	224.12±0.42	44.15± 0.09	134.16± 0.28	31.15± 0.63	155.75± 0.57	3.744
III	Ashwagandharishta-T	High fatty diet	2.0 ml/kg	118.46±0.54 ***	52.53± 0.14***	45.53± 0.43***	20.12± 0.42***	100.60± 0.27***	1.249
IV	Ashwagandharishta-M	High fatty diet	2.0 ml/kg	120.17±0.62***	52.45± 0.07***	47.16± 0.37***	20.51± 0.24***	102.55± 0.34***	1.290
V	Atorvastatin ( Standard)	High fatty diet	1.2 mg/kg	115.70±0.73***	54.52± 0.12***	42.15± 0.54***	19.28± 0.34***	96.40± 0.81***	1.126

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

A rise in LDL may cause deposition of cholesterol in the arteries and aorta and hence it is a direct risk factor for coronary heart disease. LDL carries cholesterol from the liver to the peripheral cells and smooth muscle cells of the arteries²².

HDL promotes the removal of cholesterol from peripheral cells and facilitates its delivery back to the liver. Therefore, increased levels of HDL are desirable. On the contrary, high levels of VLDL and LDL promote arteriosclerosis. LDL, especially in its oxidized form, is taken up by macrophages via a scavenger mechanism. Therefore, anti-arteriosclerotic drugs should reduce VLDL and LDL and/or elevate HDL.The search for hypolipidemic drugs follows that high level of serum cholesterol is associated with an increased incidence of coronary heart diseases. Reduction in LDL cholesterol and increase in HDL cholesterol concentration are significantly related to lipid lowering therapy²³.

In the present study, Ashwagandharishta- T and M resulted significant reduction in total cholesterol and LDL cholesterol level. A significant fall in HDL cholesterol to total cholesterol ratio was observed in Group II (high fat diet treated rats). Low level of HDL cholesterol is associated with high risk of coronary artery disease²⁴. The decrease in serum triglyceride level and reduction in atherogenic index in Ashwagandharishta treated groups is an important finding of this experiment. Most of the hypolipidemic drugs do not decrease serum triglycerides level but both types of Ashwagandharishta showed significant decrease in serum triglyceride. Reverse back of atherogenic index provides strong additional benefits in the prevention and treatment of atherosclerosis.

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Received on 05.01.2010 Modified on 10.02.2010

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 574-577