Evaluation of in vitro antioxidant capacity and reducing potential of polyherbal drug- Bhāraṅgyādi.

BACKGROUND: Present work was designed to investigate antioxidant activity of polyherbal formulation in search for new, safe and inexpensive antioxidant. Clerodendrum serratum, Hedychium spicatum and Inula racemosa, were extensively used in ayurvedic medicine and were investigated together in the form of polyherbal compound (Bhāraṅgyādi) for their antioxidant potential.
MATERIALS AND METHODS: Hydroalcoholic extract was prepared from the above samples and was tested for total reducing power and in vitro antioxidant activity by ABTS(+) assay, Superoxide anion scavenging activity assay and lipid per-oxidation assay. RESULT: Reducing power shows dose depended increase in concentration maximum absorption of 0.677 ± 0.017 at 1000 μg/ml compared with standard Quercetin 0.856±0.020. ABTS(+) assay shows maximum inhibition of 64.2 ± 0.86 with EC50 675.31 ± 4.24. Superoxide free radical shows maximum scavenging activity of 62.45 ± 1.86 with EC50 774.70 ± 5.45. Anti-lipidperoxidation free radicals scavenge maximum absorption of 67.25± 1.89 with EC50 is 700.08 ± 6.81. Ascorbic acid was used as standard with IC50 value is 4.6 μg/ml. The result suggests polyherbal formulation to be a good potential for antioxidant activity. Oxidative stress results from imbalance between free radical-generation and radical scavenging systems. This will lead to tissue damage and oxidative stress.
CONCLUSION: In conclusion, we strongly suggest that Polyherbal compounds are source of potential antioxidant for radical scavenging. The highly positive correlation of antiradical scavenging activity and total polyphenolic content in Polyherbal compounds indicates that polyphenols are important components which could be used for the free radical scavenging activity. Further study is needed for isolation and characterization of the active moiety responsible for biological activity and to treat in various stress condition.

INTRODUCTION

Free radicals are atoms, molecules or ions with one or more unpaired electrons on an open shell configuration. This electron imbalance causes high reactivity, creating other free radicals by chain reactions. Presence of these free radicals affect human health causing several diseases like cancer, diabetes, hypertension, heart attack and degenerative diseases. The majority of free radicals that damage biological systems are oxygen radicals and other reactive oxygen species (ROS),which are byproducts formed in the cells of aerobic organisms and have important roles in cell signaling.[1] The production of free radicals in cells can happen both accidentally and deliberately. An example of deliberate reaction is the superoxide generated by activated phagocytes and in catalytic reaction by ribonucleotide reductase.[2] An example of accidental generation of free radicals would be the leakage of O2¯•, H2O2 and other ROS at the interface of the bacterium and the activated phagocyte.[3] However, the major source of free radicals under normal circumstances is the electron leakage that happens from electron transport chain as seen in the mitochondria, generating O2¯• from O2.[4] These free radicles may oxidized nucleic acid, protein, DNA and can initiate degenerative disease. Natural exogenous antioxidant in diet plays a potential role in interfering with oxidation process in biological system.[5] Hence there arises a need for natural antioxidants rather than synthetically manufactured ones which cause in vivo side effects,[6] maintaining a critical balance between radical generation and defense. Liver toxicity and carcinogenesis have been reported by accumulation of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).[7] As the body cannot synthesize these essential antioxidants these must be consumed from natural antioxidants sources.

Bhāraṅgyādi polyherbal composed of three herbal drugs namely Clerodendrum serratum, Hedychium spicatum and Inula racemosa, which are extensively used in ayurvedic system of medicine mainly for the treatment of Bronchial Asthma. Clerodendrum serratum known as Bhāraṅgī is found to have anti-inflammatory,[8] antihistaminic, antiallergic, antioxidant and hepatoprotective properties[9] mainly used in respiratory tract diseases. Hedychium spicatum known as Sati is found to possess hypotensive, anti-inflammatory, vasodilatory, tranquillizing, antimicrobial, CNS-depressant, hypothermic, spasmolytic and analgesic effects.[1011] Inula racemosa known as Puṣkaramūla is beneficial for cardiovascular system, angina and dyspnoea.[12]

We have recently reported the evaluation of the phenol and flavonoid content of polyherbal drugs Bhāraṅgyādi.[13] The objective of this study was to carry out the total antioxidant activity of polyherbal compounds by using different standard tests.

MATERIALS AND METHODS

Plant collection

Clerodendrum serratum, Hedychium spicatum and Inula racemosa (Ingredients of Bhāraṅgyādi compound) were collected from local market of Varanasi (India). The identification of the drugs was done by Prof. A. K. Singh, Department of Dravyaguṇa, S.S.U., Varanasi (Identification number DG/AKS/604). The plants were dried in open air and then kept in an oven at 60°C for some time, then grinded into a fine powder by using electric blender and stored in clean labeled airtight bottles.

Preparation of the plant extract

Hundred grams of plant powder was subjected to hydroalcoholic extraction (Distilled water: Ethanol = 2:1) by hot percolation method through soxhlet apparatus. Thereafter extract was dried using rotary evaporator and dried extract was put through a process of standardization. The percentage yield of the extract was determined and was found to be 12%..

Experimental design

Total reducing potential

The reducing power of the hydroalcoholic extract of Bhāraṅgī extract was determined according to the developed method.[14] The 2.5 ml of the solution of extract (100-800 µg/ml) was mixed with equal volume of phosphate buffer (0.2 M, pH 6.6) and 1% potassium fericyanide and placed in water bath at 50°C for 20 min. It was then cooled rapidly and 2.5 ml of 10% trichloroacetic acid was added and vortexed. This incubation mixture was centrifuged at 3,000 rpm for 10 min and its 5 ml supernatant was mixed with equal volume of distilled water and 1 ml of 0.1% ferric chloride. It was further incubated at room temperature for 10 min and absorbance was read at 700 nm. The reducing property of test sample was standardized against Quercetin and expressed as difference in O.D. from control, as well as test as 0.1, and expressed as µg/ml. A higher degree of absorbance indicates the stronger reducing power.

ABTS assay/Total antioxidant capacity

ABTS*+ radical-scavenging activity of polyherbal extracts was determined according to Re et al. 1999. ABTS*+ radicals were pre-generated by adding 5 ml of a 4.9 mM potassium persulfate solution to 5 ml of a 14 mM ABTS solution and kept for 16 h in the dark. This solution was suitably diluted with distilled water to yield an absorbance of 0.70 at 734 nm and then used for antioxidant assay. Solution of vitamin C (50 µg/ml) was used as standard. It 50 µl was added to 950 µl of ABTS solution, vortexed for 10 s and after 6 min and then reduction in absorbance was recorded at 734 nm, using distilled water as a blank, on ELICO (SL-150) UV-vis spectrophotometer. Same volume of test solutions of each extract was also taken in similar manner.

Superoxide radical-scavenging activity

This assay was based on the capacity of the extract to inhibit the photochemical reduction of nitro blue tetrazolium (NBT).[15] In brief, each 3 ml reaction mixture contained 0.01 M phosphate buffer (pH 7.8) (PBS), 130 mM methionine, 60 mM riboflavin, 0.5 mM EDTA, NBT (0.75 mM) and 0.5 ml of test sample solution/standard solution. The tubes were kept in front of a fluorescent light and absorbance was taken after 6 min at 560 nm against blank (0.01 M PBS). Identical tubes were kept in dark, which served as controls. The percentage inhibition of superoxide generation was measured by comparing the absorbance of the control tube with that of test material/standard containing tubes.

Lipid per oxidation assay

A modified thiobarbituric acid-reactive specie (TBARS) assay[16] was used to measure the lipid peroxide.[17] Here, Malondialdehyde (MDA), a secondary product of the oxidation of polyunsaturated fatty acids, reacts with two molecules of thiobarbituric acid (TBA), yielding a pinkish red chromogen with an absorbance maximum at 532 nm. The egg yolk homogenate was prepared in distilled water, (10%, v/v) and 0.1 ml of extract was mixed in a test tube and the volume was made up to 1 ml, by adding distilled water. Finally, 0.05 ml FeSO4 (0.07 M) was added and incubate it for 30 min, to induce lipid peroxidation. Thereafter, 1.5 ml of 20% acetic acid (pH adjusted to 3.5 with NaOH) and 1.5 ml of 0.8% TBA (w/v) (prepared in 1.1% sodium dodecyl sulphate) and 0.05 ml 20% TCA were added, vortexed and then heated in a boiling water bath for 60 min. After cooling, 5.0 ml of 1-butanol was added to each tube, vortexed thoroughly and centrifuged at 3000 rpm for 10 min. The absorbance of the organic upper layer was measured at 532 nm against blank, where 0.1 ml of distilled water was used in place of the extract.

Statistical analysis

The data were subjected to statistical analysis software SigmaStat version 3.1 for correlation coefficient. The correlation of the data was determined by Pearson's test. P <0.05 was considered as statistically significant.

RESULTS

Several concentrations, ranging from 100-1000 µg/ml of the hydroalcoholic extract of Bhāraṅgyādi were tested for their antioxidant activity in different in vitro models. The percentage of inhibition was observed that free radicals were scavenged by the test compounds in a concentration dependent manner up to the given concentration in all the models.

Reducing potential of polyherbal drug shown in Figure 1, as a function of concentration dose dependent increase of Fe3+ to Fe2+ was observed. Amongst different concentration of extracts tested, 100, 200, 400, 600, 800, 1000 µg/ml shows maximum absorption of 0.677 ± 0.017 at 1000 µg/ml compared with standard Quercetin 0.856 ± 0.020.

Figure 1

Reducing power of polyherbal Bhāraṅgyādi compounds in hydroalcoholic extract and comparison with quercetin

Bhāraṅgyādi extract in different concentration effectively scavenge the ABTS + radicals showed percentage of inhibition in different concentration of 50, 100, 200, 400, 600, 800, 1000 µg/ml as 12.49 ± 1.14, 21.96 ± 1.14, 28.31 ± 1.6, 37.66 ± 1.27, 47.29 ± 0.84, 56 ± 0.85 and 64.2 ± 0.86 with EC50 675.31 ± 4.24. Superoxide free radicals were scavenged in different concentration of Bhāraṅgyādi extract showed superoxide scavenging activity as (% of inhibition) 7.9 ± 1.24, 16.98 ± 1.57, 26.64 ± 1.84, 40.45 ± 0.84, 52.12 ± 1.26 and 62.45 ± 1.86, respectively. An EC50 value for Bhāraṅgyādi extracts is 774.70 ± 5.45 proving again the good antioxidant activity of Bhāraṅgyādi compound.

Anti-lipid peroxidation free radicals were scavenged in different concentration of Bharangyadi extract in different concentration showed the lipid peroxide radical scavenging activity as 8.36 ± 1.86, 17.02 ± 1.86, 31.64 ± 0.88, 46.31 ± 1.22, 56.6 ± 1.96, 67.25 ± 1.89 with EC50 is 700.08 ± 6.81. Ascorbic acid was used as a reference compound and its IC50 value is 4.6 µg/ml.

DISCUSSION

In living systems, free radicals are constantly generated and they can cause extensive damage to tissues and biological molecules leading to various diseases.[18] Many synthetic drugs protect against oxidative damage but, because of their adverse side effects an alternative solution to this problem is to consume natural antioxidants through food supplements and traditional medicines.[19] Recently, many plant extracts have been shown to be potent free radical scavengers. Asthma is one among those diseases caused by oxidative stress. Though contemporary medicines are useful in the prevention and management of diseases but wide range of toxic side effects and resistance to antibiotics compel us to search some new alternatives. The present study aim to evaluate the antioxidant effect of polyherbal compound namely Bhāraṅgyādi. These drugs have been extensively used for the management of respiratory tract diseases since a long time. The efficacy of these drugs is time tested and clinically proved but validation on scientific parameters is still lacking. Hydroethanolic extract was prepared and phytochemical analysis had been done. Phytochemical screening of the drugs showed that Bhāraṅgyādi compound has good content of phenolic and flavonoid content. Both these classes of compounds have good antioxidant potential and their effects on human nutrition and health are considerable.[2021] Natural phenolics exert their beneficial health effects mainly through their antioxidant activity by decreasing oxygen concentration, intercepting singlet oxygen, preventing 1st chain initiation by scavenging initial radicals such as hydroxyl radicals, binding metal ion catalysts, decomposing primary products of oxidation to non-radical species, and breaking chains to prevent continued hydrogen abstraction from substances.[22]

Previous studies suggested that the total polyphenolic content of the plant extracts will be positively correlated to the scavenging activities. The reducing potential of plant is mostly associated with the presence of reductones, which exert their mechanism of action by breaking the free radical chain by donating a hydrogen atom.

ABTS+ is a blue chromophore produced by the reaction between ABTS and potassium persulfate.[23] Addition of polyherbal extracts to this pre-formed radical cation reduced it to ABTS in a concentration dependent manner [Figure 2]. These results were compared with those obtained with gallic acid, which indicates that the extract is a potent antioxidant. Similar results were seen with lipid peroxide scavenging by extracts [Figure 3]. Similar trend of scavenging superoxide anion indicates that polyherbal extracts are potent superoxide scavenger [Figure 4]. Superoxide dismutase catalyses the dismutation of the highly reactive superoxide anion to oxygen and hydrogen peroxide.[24] Superoxide anion is the first reduction product of oxygen.[25] This is measured in terms of inhibition of generation of O2. Antioxidants may offer resistance against the oxidative stress by scavenging the free radicals, inhibiting the lipid per oxidation and by many other mechanisms and thus prevent disease.[26]

Figure 2

ABTS+ radical scavenging activity (%). ABTS scavenging activity with different concentrations of Bhāraṅgyādi hydroethanolic extract. The absorbance of only ABTS + solution at 734 nm- was 0.712 ± 0.032 (Experimental control). Trapping potential of ascorbic acid- EC50 33.81 ± 2.40

Figure 3

Lipid peroxide scavenging activity (%). Lipid peroxide scavenging activity with different concentrations of Bhāraṅgyādi hydroethanolic extract. Result was expressed in terms of % reduction in lipid per oxidation in comparison to control (reaction mixture without antioxidant having absorbance 0.672 ± 0.033 at 532 nm)

Figure 4

Superoxide radical scavenging activity (%) with different concentrations of Bhāraṅgyādi hydroethanolic extract. Trapping potential of Copper sulphate. EC50 44.90 ± 1.40.

The peroxidation of membrane lipids initiated by oxygen radicals may lead to cell injury. Initiation of lipid per oxidation by ferrous sulphate takes place either through ferryl-perferryl complex[27] or through –OH radicals by Fenton reaction[28] thereby initiating a cascade of oxidative reactions. The results obtained in the present studies may be attributed to several reasons viz, the inhibition of ferryl- perferyyl complex formation; scavenging of OH or superoxide radicals or by changing the ratio of Fe3+/Fe2+, reducing the rate of conversions of ferrous to ferric or by chelating of the iron itself. The moderate activity of the extract may probably be due to the rapid and extensive degradation of the antioxidant principles in an ex vivo state. It is also known that the –OH radical which initiates lipid peroxidation has a difficult to investigate by conventional methods.[29]