1. 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 which causes hypertension and finally 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.

Balarishta is a polyherbal hydroalcoholic Ayurvedic preparation and is used as antioxidant and advised as a choice of remedy in mostly all types of fevers³.

The chief ingredient of Balarishta is guduchi, dried stem of Tinospora cordifolia. The chemical constituents reported from stems of Tinospora cordifolia belong to different classes such as alkaloids as tinosporin^4-5, glycosides as cordifoliosides-A and cordifolioside-B^6-7, steroids as β- sitosterol⁸, sesquiterpenoid as tinocordifolin⁹ and a large amount of phenolic compounds as gallic aciod, ellagic acid, catechin and epicatechin¹⁰. 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 Balarishta prepared by traditional and modern methods as Balarishta-T and Balarishta-M respectively and their marketed formulation.

2. MATERIALS AND METHODS:

2.1 Preparation of Balarishta-T:

This was prepared by the method as given in The Ayurvedic Formulary of India, Part-I³. All the ingredients of Balarishta were procured from local market, Jamnagar while jaggery was procured from local market, Mehsana. Authentication of all the ingredients of Balarishta 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.

According to this method, dried roots of Sida cordifolia and Withania somnifera were coarsely powdered and then 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, dhataki flowers (Woodfordia floribunda) and prescribed quantity of coarsely powdered prakshepa dravyas as Ipomoea digitata (roots), Ricinus communis (roots), Alpinia galangal (roots), Eletteria cardamomum (seeds), Ipomoea tridentate (entire plant), Eugenia caryophyllus (flower bud), Andropogon muricatus (roots) and Tribulus terrestris (fruits) were added and this sweet filtered fluid was placed for fermentation in incubator for fifteen days at 33±1°C. After 15 days, completion of fermentation was confirmed by standard tests¹⁰. 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 labeled.

2.2 Preparation of Balarishta-M:

Method of preparation of Balarishta-M was same as followed with Balarishta-T only dhataki flowers were replaced with yeast for inducing fermentation¹¹.

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 Experiments on Animals (CPCSEA) of the Government of India were followed and prior permission was granted from the Institutional Animals Ethics Committee of Shri Sarvajanik Pharmacy College, 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 method¹² 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 method¹³. 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 Balarishta-T, Balarishta-M and marketed Balarishta 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 (+veControl): High Fat Diet (HFD)

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

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

Group V: HFD + marketed Balarishta (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 were fed with high fat diet plus cholesterol for induction of hyperlipidemia. In addition to this, group III, IV and V were administered with Balarishta-T, Balarishta-M and marketed Balarishta (2ml/kg/day p.o) respectively while group VI received Atorvastatin (1.2 mg/kg/day p.o) for nine weeks¹⁴.

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 at 1600 rpm for 12 minutes for the separation of serum. Serum total cholesterol¹⁵, serum HDL¹⁶, serum LDL¹⁶, serum VLDL¹⁷and serum triglycerides¹⁷ 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 formulae¹⁶--

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 Balarishta-T, Balarishta-M and its marketed formulation, inhibited ferrous sulphate induced lipid per oxidation in a dose dependent manner. Balarishta-T and Balarishta-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 196.61, 201.72 and 206.09 µg/ml with Balarishta-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 thereof¹².

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

More than hundred percent increase in serum total cholesterol was noticed in rats fed with high fat diet as compared to rats fed with normal diet. Administration of Balarishta-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 Balarishta as Balarishta-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 Balarishta.

Table 1. Effect of Balarishta-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+Balarishta-T	215.94±1.73	222.26±2.43^b
4.	HFD+Balarishta-M	215.84±2.14	221.45±3.16^b
5.	HFD+Marketed Balarishta	216.27±3.17	222.12±1.96^b
6.	HFD+Atorvastatin(Std)	214.92±1.48	220.94±2.14^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

Fig.1. Effect of Balarishta-T, M and its marketed formulation on lipid per oxidation model

All values are shown as mean ± SEM of three replicates

Table 2. Effect of Balarishta-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

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

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

Balarishta-T

HFD

2.0 ml/kg

125.12±

0.84^b

52.54±

0.18^b

52.16±

0.27^b

20.14±

0.42^b

100.70±

0.28^b

1.376±

0.0041^b

Balarishta-M

HFD

2.0 ml/kg

127.54±

0.63^b

52.16±

0.12^b

54.65±

0.32^b

20.94±

0.69^b

104.70±

0.39^b

1.449±

0.0074^b

Marketed Balarishta

HFD

2.0 ml/kg

128.14±

1.14^b

51.98±

0.21^b

56.72±

0.19^b

21.12±

0.54^b

105.60±

0.42^b

1.497±

0.0063^b

Atorvastatin

(Standard)

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

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 Balarishta-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 process¹⁸.

In the evaluation of hypolipidemic activity significant reduction in body weight was observed in Balarishta 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 triglycerides¹⁹.

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 arteries²⁰.

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 therapy²¹.

In the present study, Balarishta-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 Balarishta treated groups is an important finding of this experiment. Most of the hypolipidemic drugs do not decrease serum triglycerides level but both types of Balarishta as Balarishta–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.

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Received on 14.10.2013 Accepted on 22.11.2013

Asian J. Res. Pharm. Sci. 4(1): Jan.-Mar. 2014; Page 07-11