INTRODUCTION:

Ayurvedic medicine or Ayurveda is one of the world’s oldest medical systems. It originated in India and has evolved here over thousands of years. The term “Ayurveda” combines the Sanskrit words ayur (life) and veda (science or knowledge) which means “The science of life”. Ayurveda was the first holistic system of diagnosis and treatment integrating nutrition, hygiene, rejuvenation and herbal medicine. ⁽¹⁾Ayurveda contains 8 branches of sciences and 10 different diagnostic tools based on the Tridosha theory (three humours of the body). Ayurveda comprises of various types of medicines including the formulations forms namely Arishta (Fermented decoction) Asava (fermented infusions) these are regarded as valuable therapeutics due to their efficacy and desirable features. ^{(2, 3)} Arishta is prepared with decoction of herb in boiling water while Asava are prepared by directly using fresh herbal juice. ^{(4, 5)}

Balarishta is a poly herbal hydro alcoholic preparation and used to cure gastric problems (vata vyadhi) as restorative and as prime tool for the treatment of Arthritis, Rheumatism and all other auto immune disorders. The major components of Balarishta are dried roots of Sida cordifolia reported that they contain the chief constituents are alkaloids: ephedrine, vasicine, vasicinone, phytosterols, fatty acids, a rich quantity of phenolic substances and flavanoids as like rutin, flavones as well as saponins like keampferol and sitoindosides. Roots of Withania somnifera have been found to contain withanolides, a group of steroidal lactones which is known for its usefulness in the treatment of hypercholesterolemia, arthritis in combination with other drugs which is also credited to be hypoglycemic and diuretic. All these compounds have many favorable effects on human health such as lowering of human low density lipoproteins, treating of heart disease and cancer because of their anti oxidant property. ^(6-8)

Balarishta was a fermented liquid preparation, made with the composition of ingredients in the formulation given below. It contains not more than 10 per cent, and not less than 5 per cent of alcohol that is self generated in the preparation over a period of time, as prescribed in Ayurvedic Formulary of India, Part II (AFI).

Name of the ingredient/herbs used in the preparation of Balarishta^{(9, 10)}

S. NO	Name of the ingredients or Plants	Botanical name	Part used	Amount used
1	Bala	Sida cordifolia Linn	Root	4800g
2	Aswagandha	Withania somnifera	Root	4800g
3	Water (for decoction reduced to)	-	-	49.152L (12.288L)
4	Guda	Saccharum officinarum	-	14400g
5	Dhataki	Woodfordia fruicosa Kurz	Flower	768g
6	Payasa	Fritillaria volylei H.	Root	96g
7	Panchangula	Ricinus communis Linn.	Root	96g
8	Rasna	Pluchea lanceolata	Leaf or root	48g
9	Ela	Elettaria cardamom	Seed	48g
10	Prasarani	Paedereria foetida Linn	Leaf	48g
11	Devapuspa	Syzigium aromantic	Flower bud	48g
12	Usira	Vetiveria zizaniodes Linn.	Root	48g
13	Svadamstra	Tribulus terrestris Linn	Fruit	48g

MATERIALS AND METHODS:

Collection of Balarishta:

Three brands of Balarishta (B1, B2, and B3), were selected of different manufactures from local Authenticated Ayurvedic store and used further for Preliminary Phytochemical Screening^{(11, 12)} and Anti oxidant assay.

ANTIOXIDANT ACTIVITY OF BALARISTHA FORMULATIONS

Reducing Power Assay^(13-15)

Reagents Preparation:

Phosphate buffer: Phosphate buffer prepared by dissolving 2.38 g of disodium hydrogen phosphate (ortho), 0.19 g of potassium dihydrogen phosphate, and 8 g of sodium carbonate in 1000 ml of deionized water. Adjust the pH, if necessary.

Potassium ferricynade (1% w/v): Potassium ferricynade prepared by adding 1 g of Potassium ferricynade in 100 ml of distilled water.

Trichloroacetic acid (10% w/v): Trichloroacetic acid prepared by adding 10 g of TCA in 100 ml of distilled water.

Iron (III) chloride (0.1% w/v): Iron (III) chloride prepared by adding 100 mg of ferric chloride in 100 ml of distilled water.

Procedure

The reducing power of sample was determined by the method of Yen and Duh. Different concentrations of arishta (20 – 100 μg/ml) were mixed with 2.5 ml of phosphate buffer (200 mM, pH 6.6) and 2.5 ml of 1 % potassium ferricyanide. The mixtures were incubated at 50°C for 20 min. After incubation, 2.5 ml of 10% TCA were added to the mixtures, followed by centrifugation for 10 min. The upper layer (5 ml) was mixed with 5 ml of distilled water; 1 ml of 0.1 % ferric chloride and the absorbance of the resultant solution were measured at 700 nm.

Hydrogen Peroxide Scavenging Assay ⁽¹⁶⁾

Reagents Preparation:

Phosphate buffer saline (pH 7.4): Phosphate buffer saline prepared by dissolving 2.38 g of disodium hydrogen phosphate (ortho), 0.19 g of potassium dihydrogen phosphate, and 8 g of sodium chloride in 1000 ml of deionized water. Adjust the pH, if necessary.

Procedure

A solution of hydrogen peroxide (40mM) was prepared in phosphate buffer, pH 7.4. The concentration of hydrogen peroxide was determined by absorption at 230 nm using a spectrophotometer. 20-100 μg/ml concentrations of arishta in distilled water were added to a hydrogen peroxide solution and absorbance at 230 nm was determined after ten minutes against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid was used as the standard.

Percent inhibition= (A0-A1) /A0×100

Where,

A0 absorbance of the control reaction

A1 absorbance in the presence of the sample

Nitric Oxide Scavenging Assay ^(17-19)

Nitric Oxide Scavenging Assay determines nitric oxide based on the enzymatic conversion of nitrate to nitrite by nitrate reductase in mammalian body. Since most of the NO was oxidized to nitrite (NO₂) and nitrate (NO₃^-), the concentrations of these anions have been used as a quantitative measure of NO production.

Reagents Preparation:

1. Griess reagent A (1% Sulphanilamide in 2% phosphoric acid): The reagent was prepared by dissolving 0.25 g Sulphanilamide in 25 ml of 2% phosphoric acid solution (2 ml phosphoric acid in 100 ml deionized water) and stored at cool temperature not allow to freeze, protected from light.

2. Griess reagent B (0.1% N-naphthyl-ethylene diamine dihydrochloride): The reagent was prepared by dissolving 0.025 g N-naphthyl-ethylene diamine dihydrochloride in 25 ml deionized water and stored at cool temp, protected from light.

Procedure

The chemical source of NO was sodium nitroprusside (10 mm) in 0.5 m phosphate buffer, pH 7.4, which spontaneously produced NO in an aqueous solution. NO interacted with oxygen to produce stable products, leading to the production of nitrites. After incubation for 60 min at 37°C, Griess reagent (mixture of reagent A and B) was added. The same reaction mixture without the extract of sample but with equivalent amount of distilled water served as control. Ascorbic acid was used as positive control. The percent inhibition was calculated as per the equation mentioned in the Hydrogen peroxide scavenging assay.

Statistical analysis

Tests were carried out in duplicate and represented using Ms Windows based graph pad Prism (version 6) software. Results were expressed as graphically / mean ± standard deviation.

RESULTS AND DISCUSSION:

Phytochemical screening of Balarishta formulations:

Balarishta formulations (B1, B2, B3) were showed the positive results for carbohydrates, steroids, tannins, phenols, flavonoids and glycosides tests.

EFFECT OF ANTIOXIDANT ACTIVITY ON BALARISTHAFORMULATIONS

Reducing Power Assay

Compounds with reducing power indicate that they are electron donors and can reduce the oxidized intermediates of lipid peroxidation processes, so that they can act as primary and secondary antioxidants. Reducing power is associated with antioxidant activity and serves as a significant reflection of the antioxidant activity. In this assay, the yellow colour of the test solution changes to various shades of green and blue depending on the reducing power of each formulation. Presence of reducers causes the conversion of the Fe3+/ferricyanide complex used in this method. By measuring the formation of Pearl’s Prussian blue at 700nm, the concentration of Fe³⁺ ion was determined. Higher absorbance indicated greater reducing ability of the test solution.

Fig. no.1: Reducing power activities of Balarishta formulations in comparison with a standard Ascorbic acid

The present investigation, concentration dependent reducing ability of the formulations were studied and Balarishta formulations showed antioxidant potential in a dose dependent manner as shown in figure 1. B3 showed more reducing ability compared to B1 and B2 but formulation reducing ability was very less in comparison to standard Ascorbic acid.

Hydrogen Peroxide Scavenging Assay

Hydrogen peroxide was a reactive oxygen species that accumulates during oxidative stress and toxic to cells. Excessive accumulation leads in damage of DNA, proteins and lipids. Oxidative damage can leads to ageing, asthma, arthritis, cardiovascular disease, atherosclerosis, Down’s syndrome and neurodegenerative diseases. Recent studies indicated that antibodies have capacity to convert molecular oxygen into hydrogen peroxide it contributes to the normal recognition and destruction processes of the immune system. Measurement of this reactive species may help to determine how oxidative stress modulates varied intracellular pathways.

Fig. no 2: Hydrogen peroxide scavenging activity of Balarishta formulations in comparison with a standard Ascorbic acid

Percent inhibition of H₂O₂of Balarishta formulations B1, B2, B3 were 88.59 ± 0.009, 86.95 ± 0.039 and 87.26 ± 0.040 respectively at a concentration of 100 µg/ml. The Balarishta formulations B1 showed good scavenging activity of hydrogen peroxide which has been compared with Ascorbic acid. B1 formulation was the better than the B2 and B3.

Nitric Oxide Scavenging Assay

Nitric oxide or reactive nitrogen species in mammalian cells generally formed when their reaction occurs with oxygen or super oxides such as NO₂, N₂O₄, N₃O₄, NO₃^- and NO₂^- which are highly reactive in nature. Excessive production of NO leads to the harmful effects and can cause detrimental to human body so that it can lead to disorders like inflammation, cancer and other pathological complications.

Fig. no 3: Nitric Oxide Scavenging Assay of Balarishta formulations in comparison with a standard Ascorbic acid

Percent inhibition of NO^- of Balarishta formulations B1, B2, B3 were 50.58 ± 0.048, 30.79 ± 0.048 and 54.20 ± 0.097 respectively at a concentration of 100 µg/ml. B3 formulation showed good activity then the B1 and B3.

CONCLUSION:

In current research work the Balarishta formulations (B1, B2, and B3) were screened for the Antioxidant activity using Reducing Power Assay, Hydrogen Peroxide Scavenging Assay, Nitric Oxide Scavenging Assay methods. Ascorbic acid has been used as standard Antioxidant drug. Results conclusively state that the Balarishta formulations have shown the presence of the Alkaloids, Steroids, Phenols, Flavonoids, Tannins and Carbohydrates. Balarishta has well anti oxidant activity comparatively with ascorbic acid in all the three models in dose dependent manner.

ACKNOWLEDGEMENT:

The author acknowledges the Department of Pharmacognosy and Phytochemistry, Centre for Pharmaceutical Sciences JNTUH for providing necessary facilities.

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Received on 21.04.2015 Modified on 18.05.2015

Res. J. Pharmacognosy & Phytochem. 7(2): April-June 2015; Page 107-110

DOI: 10.5958/0975-4385.2015.00018.7