These compounds have many notable medicinal properties as antidiabetic¹¹, hepatoprotective¹², antioxidant¹³, antimalarial¹⁴, immunomodulatory¹⁵ and antineoplastic properties¹⁶.

Therefore, we have undertaken this present investigation to evaluate the antihyperlipidemic effect of Amritarishta prepared by traditional and modern methods as Amritarishta-T and Amritarishta-M respectivelly and their marketed formulation.

2. MATERIALS AND METHODS:

2.1 Preparation of Amritarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I³. All the ingredients of Amritarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Amritarishta was done by Dr. G. D. Bagchi, Scientist, Department of Taxonomy and Pharmacognosy, Central Institute of Medicinal and Aromatic Plants, Lucknow. Prepared herbarium has been deposited in the Central Institute of Medicinal and Aromatic Plants, Lucknow for future reference. Identification of all the individual plant material was done as per The Ayurvedic Pharmacopoeia of India. Quantity of ingredients taken for the preparation of batch size 3.072 l of Amritarishta has been calculated according to the formula as given in The Ayurvedic Formulary of India, Part-I, 2000.

According to this method, coarsely powdered stems of guduchi (Tinospora cordifolia) with prescribed ingredients as Aegle marmelos (stem bark), Oroxylum indicum (roots), Gmelina arborea (stem bark), Stereospermum suaveolns (stem bark), Premna integrifolia (stem bark), Hedysarum gangeticum (entire plant), whole plant of Paederia foetida, entire plant of Solanum indicum, entire plant of Solanum xanthocarpum and Tribulus terrestris were placed in polished vessel of brass along with prescribed quantity of water (12.288l) and allowed to steep. After 12 h of steeping, this material was warmed at medium flame until the water for decoction reduced to one fourth of the prescribed quantity(3.072 l) , then the heating was stopped and it was filtered in cleaned vessel and after that jaggery was added and mixed properly. Then, prakshepa dravyas as svet jiraka, raktapuspaka, saptaparni, sunthi, marica, pippali, nagakesara, mustaka, katuka, ativisa and indravaruni in fine powdered form were added and this sweet filtered material was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days completion of fermentation was confirmed by standard tests¹⁷.The fermented preparation was filtered with cotton cloth and kept in clean covered vessel for further next seven days. Then, when the fine suspended particles settled down, it is strained again and poured in amber colored glass bottles previously rinsed with ethyl alcohol, packed and properly labelled.

2.2 Preparation of Amritarishta-M:

Method of preparation of Amritarishta-M was same as followed for Amritarishta-T only in addition to jaggery, yeast was also added for inducing fermentation¹⁸.

2.3 Animals:

Adult wistar albino rats, weighing between 200-220g of either sex were acclimatized to normal environmental conditions in the animal house for one week. The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22ºC±2ºC) and humidity (55±5%) with 12:12 hour light and dark cycle. All the animals were given a standard chow diet (Hindustan Lever Limited) and water ad libitum. The guidelines of the Committee for the Purpose of Control and Supervision of Experimentals on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animals Ethics Committee of Shri Sarvajanik College of Pharmacy, Mehsana, Gujarat (CPCSEA No. 07/09).

2.4 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.

2.5 Assay of lipid per oxidation:

The extent of lipid per-oxidation in goat liver homogenate was measured in vitro in terms of formation of thiobarbituric acid reactive substances (TBARS) by using standard method¹⁹ 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 2000 rpm 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 method²⁰. To study the comparative response, the experiment was divided into five groups. Liver homogenate (5%, 3ml) was aliquoted to different glass petri dishes. The first two groups were treated as control and standard where buffer and Vitamin E was added respectively. In the third to fifth group, different concentration (100, 150, 200, 250 and 300 µg/ml) of Amritarishta-T, Amritarishta-M and marketed Amritarishta were added. Lipid per oxidation 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% trichloroacetic acid. After 10 min, tubes were centrifuged and supernatant was separated and mixed with 1.5ml of 0.67% thio-barbituric acid. The mixture was heated on a water bath at 85⁰C for 30 min, and then on 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) as-

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

2.6 Determination of Antihyperlipidemic Activity:

Experimental design:

All the animals were randomly divided into the six groups with six animals in each group.

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

Group II (+ve Control): High Fat Diet (HFD)

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

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

Group V: HFD + marketed Amritarishta (2.0 ml/kg/day p.o)

Group VI: HFD +Atorvastatin (1.2 mg/kg/day p.o)

The composition of the two diets was 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 normal saline along with normal diet. Group II, III, IV, V and VI received high fat diet plus cholesterol for induction of hyperlipidemia. In addition to this, group III, IV and V were administered with Amritarishta-T, Amritarishta-M and marketed Amritarishta (2ml/kg/day p.o) respectively while group VI received Atorvastatin (1.2 mg/kg/day p.o) for nine weeks²¹.

Body weight of each animal was noted at the beginning and at the end of the experiment. During the whole period, free access to food and water was provided to the animals. Twenty hours prior to the end of the experiment, food was withdrawn and blood samples were taken by retro-orbital plexus. The blood samples were centrifuged for 12 min at 1600 rpm for the separation of serum. Serum total cholesterol²², serum HDL²³, serum LDL²³, serum VLDL²⁴and serum triglycerides²⁴ 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 formulae²³--

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

Atherogenic index = (LDL+VLDL)/HDL

2.7 Statistical analysis:

The results are expressed as mean ± SEM. Statistical analysis of data among the various groups was performed by using one way analysis of variance (ANOVA) followed by the Tukey’s test using Graph Pad Prism software of statistics. Significance value (P<0.05) was considered statistically significant as compared to control group.

3. RESULTS:

The results presented in Figure. 1, showed that Amritarishta-T, Amritarishta-M and its marketed formulation, inhibited ferrous sulphate induced lipid per oxidation in a dose dependent manner. Amritarishta-T and Amritarishta-M at 300 µg/ml exhibited maximum inhibition, which was nearly equal to the inhibition produced by Vitamin E (5mM). The IC₅₀value was found to be 236.84, 243.66 and 247.98 µg/ml with Amritarishta-T, M and its marketed formulation 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 thereof¹⁹ .

A significant reduction in the body weight of rats was observed in Amritarishta-T, Amritarishta-M and its marketed formulation treated groups as compared to high fat died fed control group as shown in Table 1.

A one hundred seven (107%) increase in serum total cholesterol was noticed in rats fed with high fat diet as compared to rats fed with normal diet. Administration of Amritarishta-T, M and its marketed formulation showed significant reduction in serum cholesterol, serum LDL, serum triglycerides while showed significant rise in serum HDL as compared to high fat diet fed control group as shown in Table 2.

All the test formulations of Amritarishta as Amritarishta-T, M and its marketed formulation also showed significant decrease in atherogenic index as compared to high fat diet control group as shown in Table 2, which strongly supports anti-atherosclerotic property of Amritarishta

Table 1. Effect of Amritarishta-T, M and its marketed formulation on body weight of high fat diet induced hyperlipidemic rats

S. No	Treatment Groups	Initial Body Weight ( g)	Final Body weight (g)
1.	Normal	213.72±2.27	215.46±1.84
2.	HFD Control	216.15±2.14	232.14±2.41^a
3.	HFD+Amritarishta-T	215.92±1.98	223.24±1.62^b
4.	HFD+Amritarishta-M	216.24±2.37	224.56±1.94^b
5.	HFD+Marketed Amritarishta	216.73±2.95	224.13±2.48^b
6.	HFD+Atorvastatin(Std)	214.2±1.92	216.0±1.84^b

All values are expressed as mean ±SEM (n = 6); HFD, High fat diet

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to HFD control

4. DISCUSSION:

Lipids are widely involved in oxidative 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, anaemia, 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 Amritarishta-T and M were evaluated for free radical scavenging activity and showed potent anti-oxidant activity and evidenced that free radical scavenging potential helps in ameliorating disease process²⁵.

In the evaluation of hypolipidemic activity significant reduction in body weight was observed in Amritarishta treated groups as compared to high fat diet fed control group which suggests that certain enzymes are secreted in quantity involved in bile acid synthesis and its excretion and this may cause decrease in serum cholesterol and serum triglycerides²⁶.

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 arteries²⁷.

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-atherosclerotic 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 with lipid lowering therapy²⁸.

In the present study, Amritarishta-T and M showed significant reduction in total cholesterol and LDL cholesterol level as compared to high fat diet fed control group. 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 disease. The decrease in serum triglyceride level and reduction in atherogenic index in Amritarishta treated groups is an important finding of this experiment. Most of the hypolipidemic drugs do not decrease serum triglycerides level but both types of Amritarishta as Amritarishta–T and M reduced the elevated serum triglyceride level significantly. Thus, both of these preparations maintained the serum parameters near to the normal level significantly. Reverse back of atherogenic index provides strong additional benefits in the prevention and treatment of atherosclerosis.

Table 2. Effect of Amritarishta-T, M and its marketed formulation on serum lipid profile in high fat diet induced hyperlipidemic 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	0.944±0.0031
II	Control	HFD	2.0 ml/kg water	224.12±0.42^a	44.15±0.09^a	134.16±0.28^a	31.15±0.63^a	155.75±0.57^a	3.743±0.0046^a
III	Amritarishta-T	HFD	2.0 ml/kg	138.12±1.04^b	49.12±0.14^b	66.42± 0.46^b	23.42±0.36^b	117.10±0.59^b	1.829±0.0015^b
IV	Amritarishta-M	HFD	2.0 ml/kg	141.54±0.96^b	49.06±0.23^b	68.25± 0.59^b	25.14±0.47^b	125.70±0.68^b	1.904±0.0027^b
V	Marketed Amritarishta	HFD	2.0 ml/kg	140.62±0.59^b	48.98±0.41^b	69.17± 0.63^b	24.96±0.78^b	124.80±0.82^b	1.922±0.0048^b
V	Atorvastatin (Stan-dard)	HFD	1.2 mg/kg	115.70±0.73^b	54.52±0.12^b	42.15± 0.54^b	19.28±0.34^b	96.40±0.81^b	1.127±0.0032^b

All values are expressed as mean ±SEM (n = 6); HFD, High fat diet

a P<0.001 significant as compared to normal

b P<0.001 significant as compared to HFD control

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Received on 01.12.2013

Modified on 12.12.2013

Accepted on 14.12.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(6): November –December 2013, 315-319