In Vitro Evaluation of Antioxidant Potential of Ayurvedic Preparations Lauha Bhasma and Mandura Bhasma

 

Mrunali Potbhare¹*, Deepak Khobragade²

¹Shri Indu Institute of Pharmacy, Hyderabad

²Vijaya College of Pharmacy, Hayathnagar, Hyderabad

 

ABSTRACT:

The changing environmental conditions and food habits have evoked a great deal of attention toward the study of free radicals. Free radicals are introduced in our body by various endogenous systems, exposure to different physiochemical conditions or some pathological states. Free radicals are exposure to body leads to condition called oxidative stress. When body is unable to manage increased oxidative stress, we need to supply a source of antioxidant to the body. An antioxidant can be broadly defined as a substance inhibits oxidative damage to a target molecule mainly caused by free radicals. Antioxidants are very important in treating many disorders or are used as supportive treatment for various disorders which are caused or aggravated by oxidative stress. Lauha bhasma and Mandura bhasma are well known ayurvedic preparations traditionally used as a haematinic for treating various disorder specially blood disorders. Antioxidants and antioxidant potential are very important in treating many blood disorders and are very supportive in the haematinic action. The present study evaluates the antioxidant potential of Lauha bhasma and Mandura bhasma. The antioxidant activity was evaluated by DPPH scavenging activity and Hydrogen peroxide scavenging activity, Ascorbic acid was used as standard antioxidant agent for comparative evaluation. The results show that both the bhasma have significant antioxidant activity i.e. more than 80% of that of standard ascorbic acid. Mandura bhasm being having comparatively more antioxidant potential than Lauha bhasma.

 

 

 

1. INTRODUCTION:

In modern life we are constantly exposed free radicals, which can damage any cell in the body with which they come into contact and cause hazardous effects.^1,2 The free radicals cause a state of increased oxidative stress which is mainly responsible for all the     damage done to the body by free radicals^3,4. Though the body has its own mechanism to combat oxidative stress, when exposure is more than body’s capacity, the problem aggravates^5,6,7.

 

The harmful effects of free radicals can be prevented by use of substances called antioxidants^8,9. Antioxidants are a group of compounds that neutralise or prevent free radicals or reactive species and avoid cell or tissue damage caused by oxidative potential of free radicals^10,11,12. Many natural substances have been explored for their antioxidant activities^,13-16. There is still need to evaluate new substances for their antioxidant potential to find better alternative sources for treatment of conditions related to oxidative stress caused by free radicals. Lauha bhasma and Mandura bhasma are very popular ayurvedic preparations generally used for treating weakness and other disorders^17,18,19. Traditionally these bhasmas are very effectively used for treating anaemia specially iron deficiency anaemia. Studies have demonstrated that iron deficiency anaemia significantly decreases mineral-dependent antioxidant enzymes glutathione peroxidase activity in red blood cells of rats ^20,21 and humans^22,23and also in the liver, kidney, heart, and brain of rats²².  Since these bhasma have anti anaemic activity and are used for treating iron deficiency anaemia, they may have antioxidant activity also. Depending on the available traditional information regarding the uses of lauha bhasma and mandura bhasma, the present investigation was undertaken to investigate and confirm the antioxidant effects of these bhasma using in-vitro antioxidant activity evaluation. DPPH and Hydrogen Peroxide free radical scavenging methods were used for evaluating antioxidant activities. Ascorbic acid was used as standard antioxidant substance for comparative evaluation.

 

2. MATERIAL AND METHODS:

2.1 Materials

Marketed preparations of Lauha bhasma and Mandura bhasma were purchased from local ayurvedic medical market. Ascorbic acid, hydrogen peroxide and DPPH were purchased from SD Fine Chemicals, Mumbai, India. All other chemicals used were of AR grade and used as received.

 

2.2. Evaluation of In Vitro Antioxidant Activity

2.2.1.: Determination of 1,1- Diphenyl-2-Picrylhydrazyl (DPPH) Radical Scavenging Activity

Solutions of ascorbic acid, Lauha bhasm and Mandura bhasma were prepared by dissolving/dispersing bhasma in distilled water and preparing dilutions ranging from 5 mcg/ml to 250 mcg /ml. Total seven dilutions were prepared for evaluation of antioxidant activity in this study. DPPH (1,1- diphenyl-2-picrylhydrazyl )0.004% w/v solution was prepared by dissolving 4 mg of DPPH in 100 ml of ethanol. The solution was kept overnight in dark place for the generation of DPPH radical.

 

An aliquot of 3 ml of 0.004% DPPH solution in ethanol and 0.1 ml of test sample at various concentrations were mixed. The mixture was shaken vigorously and allowed to reach a steady state at room temperature for 30 min. De-colorization of DPPH was determined by measuring the absorbance at 517 nm. A control was prepared using 0.1 ml of distilled water in the place of test sample

The percentage inhibition of free radical by the test sample was calculated using the formula:

 

Where, A0 is the absorbance of control; A1 is the absorbance of test sample.

2.2.2: Determination of Hydrogen Peroxide Scavenging Activity

Same dilutions of test and standard were used for evaluating antioxidant activity of Lauha Bhasma and Mandura Bhasma by Hydrogen Peroxide Scavenging Activity.

 

Hydrogen peroxide (0.002%v/v) solution was prepared by transferring 100 μl of hydrogen peroxide (30%) to a volumetric flask and the volume was made up to 15 ml with distilled water. From this, 1 ml of solution was transferred to a volumetric flask and was made to 100 ml with distilled water.

 

Phosphate buffer (100 mM, pH 7.4) solution was freshly prepared during the study.

Solution A: 276 mg of NaH₂PO₄ was weighed, transferred to a volumetric flask and the volume was made up to 100 ml with distilled water.

 

Solution B: 568 mg of Na₂HPO₄ was weighed, transferred to a volumetric flask and the volume was made up to 100 ml with distilled water.

From the above solutions, 12 ml of solution A and 88 ml of solution B were mixed and pH was adjusted to 7.4. Phenol red (0.2 mg/ml) containing Peroxidase (0.1 mg/ml) solution was also freshly prepared during the free radical study. 2 mg of phenol red and 1 mg of horse radish peroxidase were dissolved in 10 ml of 100 mM phosphate buffer solution.

 

Each 100 μl of the standard and test solutions with different concentrations  were mixed with 100 μl of 0.002% H₂O₂ and 0.8 ml of phosphate buffer and were pre-incubated for 10 min at 37⁰C. 1 ml of phenol red dye containing horseradish peroxidase solution was added to this reaction mixture. After 15 min 50 μl of 1 M NaOH was added and absorbance was measured at 610 nm immediately. A control was prepared using 0.1 ml of distilled water in the place of test or standard solution.

 

The percentage inhibition of radical by the test sample was calculated using the same formula as that of DPPH scavenging evaluation previously mentioned.

 

3. RESULTS AND DISCUSSION:

3.1.:  DPPH free Radical Scavenging Activity

The results of evaluation of antioxidant activity of  Mandura bhasma and Lauha bhasma indicated significant antioxidant activity in both the bhasma. Mandura bhasma showed relatively superior antioxidant potential than lauha bhasma. Moreover the antioxidant activity of both the bhasma was found to be dose dependant and comparable to antioxidant activity of standard, ascorbic acid. Lauha bhasma showed about 80 % of antioxidant activity as that of ascorbic acid and mandura bhasma showed about 85% of antioxidant activity as that of ascorbic acid. The results are depicted in Fig. No. 1.

 

Fig No. 1: Antioxidant activity of Mandura Bhasma and Lauha Bhasma using DPPH scavenging

 

3.2.:  H₂O₂ free Radical Scavenging Activity :

To consolidate the findings of antioxidant activity, the potency of Mandura bhasma and lauha bhasma was compared with antioxidant potential of Hydrogen peroxide, the results are shown in Fig. No. 2. The results indicate that the Mandura bhasma and lauha bhasma  has significant antioxidant activity.  Both the bhasma have more than 80 % of antioxidant activity as compared to ascorbic acid in  hydrogen peroxide scavenging study.. The results also confirm that mandura bhasma is having relatively superior antioxidant potential than lauha bhasma.

 

 

Fig No. 2: Antioxidant activity of Mandura Bhasma and Lauha Bhasma using H₂O₂ scavenging

 

 

4. CONCLUSION:

The results of the study confirm that the traditionally used Lauha bhasma and Mandura bhasma have significant antioxidant activity. Moreover the activity is comparable with that of ascorbic acid  as both the bhasma shows more than 80% of antioxidant activity as that of ascorbic acid.. This antioxidant potential may be one of the reasons of their therapeutic benefits. These bhasma need to be evaluated for more activities which are affected by antioxidant potential like ant anaemic or haematinic or anti-diabetic or protective activities.

 

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Received on 18.04.2017       Accepted on 20.05.2017     

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