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Home , Eugenol, Hydrogen peroxide

Science, Technology and Development ISSN : 0950-0707

Evaluation of in vitroAntioxidant potential of

Nikhat Parween*, Amber Jabeen, Birendra Prasad

Microbial & Molecular Genetics Lab., Department of Botany, Patna University, Patna-800005, India

*Department of Biochemistry, Patna University, Patna-800005, India

Abstract Eugenol (4-allyl-2-methoxyphenol) is a major volatile polyphenol of and several medicinal plants like- Ocimum sanctum, Origanium vulgare, Zingiber officinale and many more. The polyphenols present in various plant resources act as to curb the degradations brought about by free radicals to certain extent. They are frequently used in drug formulation due to its wide range of therapeutic properties that helps in maintenance of overall good health, provides protection and cure diseases. Therefore, we intend to evaluate the in vitro free scavenging activity of eugenol by determining its scavenging activity (SRSA), ferric reducing antioxidant power (FRAP) assay, scavenging activity and reducing power assay. Eugenol (50μM) showed almost 2.35 folds, 5.1 folds and 3.6 folds increase in superoxide scavenging ability, reducing ability, reducing power as compared to standard alpha-tocopherol and ascorbic . The graph was statistically analyzed using one-way ANOVA and t-test using Graphpad Prism 8.2.0. Strong antioxidative property of eugenol confers reduction of which in turn helps in maintaining good health because oxidative stress is the hallmark of many diseases. So, this makes eugenol convenient source of natural antioxidant in pharmaceutical preparation.

Keywords: Eugenol, antioxidant, free radical, oxidative stress.

I. INTRODUCTION Oxidative stress is the hallmark of many diseases and it results due to imbalance between free radical production and antioxidant defenses in our body which results in cellular damages. Free radicals are containing having odd number of electrons. Due to odd number of electrons, it is highly reactive and its accumulation damage biomolecules and causes oxidative stress condition [1]. Antioxidant neutralizes or makes free radicals less reactive by donating its electrons. It is natural, man-made (exogenous) and enzymatic (, , ) or non-enzymatic (glutathione, beta-carotene, alpha-tocopherols) which balances the oxidative status of an organisms by scavenging free radicals and prevents cells from detrimental damages caused by oxidation. It has been suggested that polyphenols exhibiting strong antioxidant activity can break a vicious cycle in progression of the diseases [2,3]. The polyphenol and flavonoid molecules present in various plant resources act as antioxidant to curb the degradations brought about by free radicals to certain extent. They are frequently used in drug formulation due to its wide range of therapeutic properties that helps in maintenance of overall good health, provides protection and cure diseases [4,5]. Strong antioxidative property of polyphenols confers reduction of oxidative stress that helps in maintaining good health. Eugenol (4-allyl-2-methoxyphenol) is one such interesting polyphenol which has opened broad range of research. The presence of phenolic group confers strong antioxidant activity (Fig. 1) [6]. It is present in most medicinal plants and possesses remarkable spectrum of pharmacological activities like antipyretic, , anti-inflammatory, , anticancer, central and neuro protective action and effects (Fig. 2). Therefore, we designed to evaluate their antioxidant activity in vitro, by determining superoxide scavenging activity (SRSA), scavenging activity, reducing power assay and ferric reducing antioxidant power (FRAP) assay, as these biochemical assays are reliable methods to assess the total antioxidant power of any compound.

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Figure 1. Structure of eugenol

Figure 2.Antioxidant potential of Eugenol

II. EXPERIMENTAL METHODS

2.1. Preparation of eugenol doses:

Eugenol and other reagents and chemicals were all purchased from Sigma-Aldrich. Eugenol, concentrations of 1µM, 10µM and 50µM in were made.

2.2. Measurement of in vitro Antioxidant activity:

Superoxide radical and Hydrogen Peroxide Scavenging Activity: The superoxide radical scavenging activity (SRSA) of eugenol was assessed by Pyragallol Method [7]. Reaction system was started by adding 4.5ml of 50mmol/L Tris-HCl (pH, 8.2, pre heated to 25°C in a bath for 20mins), 0.5 ml of 2 mmol/L pyragallol and 0.1ml of the sample. Reaction was carried out at 25°C in a water bath for 5mins and reaction was stopped by adding 1ml of 8mol/L HCl solution. Finally, absorbance was measured at 325 nm of reaction mixture. The ability of eugenol to scavenge hydrogen peroxide was assessed by the method of Binti Zaaba et al., 2016 [8]. H2O2 (40mM) was prepared in phosphate buffer and different concentration of eugenol

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was added to 0.6ml of H2O2 solution and final volume was made to 3ml. The absorbance of the reaction mixture was measured at 230nm. The SRSA and scavenging of hydrogen peroxide (%) for eugenol was calculated as follows:

Ab−As Inhibition Rate (%) = *100 Ab

Where; Ab= Absorbance values of the blank control

As= Absorbance values of the sample

Reducing Power and FRAP (Ferric reducing antioxidant power) Assay: Fe3+ reducing power of eugenol was determined by R-power assay. Sample was mixed with PBS (0.2M, pH 6.6), 1% potassium ferricyanide solution and vortexed for 5mins. Reaction mixture was incubated at 50°C for 20mins and then 10% TCA was added and centrifuged at 3000rpm for 10mins. Supernatant was collected and finally mixed with distilled water and 1% ferric chloride. The absorbance of the reaction mixture was measured at 700nm against blank. Ascorbic acid was used as reference standard. For FRAP assay, reaction solution was freshly prepared by mixing 10ml of 300mM acetate buffer (pH 3.6), 1ml of 10mM TPTZ in 40mM HCl and 1ml of 20mM ferric chloride. The solution was pre-warmed at 37°C and then sample was added in 9:1 ratio. After 4mins incubation at 37°C absorbance was recorded at 593nm. Ascorbic acid was used as positive control. FRAP value of sample (μM)= Change in O.D of sample from 0 to 4 mins × FRAP value of standard (1000 μM) Change in O.D of sample from 0 to 4 mins

III. RESULTS

3.1 Antioxidant activity of eugenol under in vitro condition: Increased free radical production results in oxidative stress which in turn causes detrimental damages of biomolecules such as DNA, , lipids, carbohydrates and excessive production of it results in oxidative stress condition. Natural and polyphenols inhibit or limit the production of free radicals. The antioxidant activity of phenolic compound is mainly due to their properties, due to which they act as hydrogen donators, quenchers and reducing agents [9]. Therefore, we analyzed the free radical scavenging activity of eugenol in vitro by superoxide scavenging activity (SRSA), ferric reducing antioxidant power (FRAP) assay, hydrogen peroxide scavenging activity and reducing power assay. These biochemical assays are reliable methods to assess the total antioxidant power of any compound. These various antioxidant activities were compared with standard antioxidants such as ascorbic acid, butylated hydroxytoluenes (BHT) and α-tocopherol. SRSA and FRAP assay: Superoxide is a potent inducer of oxidative stress. It causes biological by inactivating iron sulphur cluster containing , thereby generating free iron in the which ultimately generates highly reactive . The superoxide radical scavenging activity (SRSA) of eugenol was assessed by Pyragallol Autoxidation Method [7]. and the radical scavenging ability of eugenol was expressed in terms of percentage of scavenged. Eugenol showed maximum superoxide scavenging activity (52.34±1.25%) at higher concentration (50µM) in comparison to lower concentration of 1µM (28.89±2.17%) followed by 10µM (38.55±1.09%), respectively (***p<0.001, ANOVA, Table-1). Percentage superoxide scavenging activity was maximum at higher dose of eugenol. 50μM of eugenol showed almost 2.35 folds increase in scavenging ability as compared to standard alpha-tocopherol having 22.18±3.2% superoxide scavenging activity (Fig. 3A). While, FRAP (ferric reducing antioxidant power) assay measures the reducing ability of any compound to reduce Fe3+ to Fe2+. This method depends upon reduction of ferric tripyridyltriazine (TPTZ) complex to ferrous tripyridyltriazine at low pH. Reducing ability describes the electron donating ability of compound, which confers its antioxidant ability. Results demonstrated that eugenol at higher dose exhibited strong iron reducing ability as compared to lower dose. Eugenol showed almost 5.1 folds increase in reducing ability as compared to standard

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ascorbic acid (***p<0.001, ANOVA, Fig.3B). Increased absorbance at 593nm signified correlation between eugenol dose and it’s FRAP values. Hydrogen peroxide scavenging and reducing power assay: Oxidative stress results in an increase cellular concentration of (ROS) during metabolic processes, which ultimately get converted to hydrogen peroxide, potent inducers of oxidative stress. The ability of eugenol to scavenge hydrogen peroxide was assessed by the method of Binti Zaaba et al., 2016 [8]. Eugenol showed maximum hydrogen peroxide scavenging activities (28.84±3.23%) at higher concentration (50µM) as compared to lower concentration of 1µM (16.39±0.97%) and 10µM (22.06±2.6%), respectively (***p<0.001, ANOVA, Fig. 3C). Eugenol showed inhibition percentage (15-30%) at different concentrations. The scavenging activity of eugenol was effective in the order 1μM<10μM< 50μM. Reducing power ability serves as an indicator of the antioxidant potential of a compound and its ability to reduce the oxidized intermediates of lipid peroxidation by donating electron. Reducer in turn causes conversion of Fe3+/ferricyanide complex. It is measured by spectrophotometric detection at 700nm. Higher absorbance indicates increased reducing power of reaction mixture. During present investigation reducing power of varying doses of eugenol was measured. All the concentrations of eugenol exhibited higher reductive potential as compared to standard ascorbic acid. Eugenol (50µM) showed almost 3.6 folds higher reducing ability to that of ascorbic acid (***p<0.001, ANOVA, Fig. 3D). Increased absorbance indicated the higher reducing potential of the eugenol. The results of in vitro antioxidant study suggested the potential scavenging activity and reducing power of the eugenol.

Figure 3. In vitro antioxidant activity of eugenol: (A) Effect of eugenol on superoxide radical scavenging ability (B) FRAP (Ferric ion reducing ability) of eugenol (C) Hydrogen peroxide scavenging ability of eugenol (D) Reducing power ability of different doses of eugenol at 700nm. The graph was statistically analyzed using one-way

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ANOVA in GraphPad Prism 8.2.0 (*** signifies p-value<0.001). Error bar represents S.E.M (Standard error of mean).

Table 1: Free radical scavenging ability of eugenol

- Treatment % O2 Scavenging ability (Mean ±SEM)

Eugenol (1μM) 28.89 ± 1.04

Eugenol (10μM) 38.55 ± 0.91

Eugenol (50μM) 52.34 ± 1.712

IV. CONCLUSION Recent studies revealed a key role of antioxidant capacity of polyhenols contributing to health benefits. Our study clearly demonstrated that eugenol is a potent natural antioxidant compound that exerts its action by scavenging free radicals or by inhibiting generation of free radicals as shown by the in vitro assays. Strong antioxidative property of eugenol confers reduction of oxidative stress that helps in maintaining good health. So, this makes eugenol convenient source of natural antioxidant in pharmaceutical preparation.

REFERENCES

[1] J. R. Cypser and T. E. Johnson, “A Multiple stressors in Caenorhabditis elegans induce stress hormesis and extended longevity”, Journal of Gerontology A- Biological Science and Medical Science. 57: 109-14, 2002. [2] I. Gulcin, “Antioxidant activity of eugenol: a structure-activity relationship study”, J Med . 14(9): 975- 85, 2011. [3] I. Gulcin, M. Elmastas,Y. A. Hassan, “Antioxidant activity of clove oil”. Arabian Journal of Chemistry. 489- 499, 2012. [4] O. J.Owolabi, E. K. I. Omogbai and O.Obasuyi, “Antifungal and antibacterial activities of the ethanolic and aqueous extract of Kigeliaafricana (Bignoniaceae) stem bark”. African Journal of Biotechnology, Vol. 6 (14): 1677- 1680, 2007. [5] C. J. Bailey and C. Day,“Traditional Plant Medicines as Treatments for Diabetes”. Diabetes Care. 12(8): 553- 564,1989. [6] M. O. Gata, M. H. Oshi, S. U. Rano, T. E. Ndo, “ Antioxidant Activity of Eugenol and Related Monomeric and Dimeric Compounds”. 48:1467-1469,2000. [7] Q. A. Zhang, X. Wang, Y. Song , X. H. Fan, J. F. Garcia Martin, “Optimization Pyrogallol Autoxidation Conditions and Its Application in Evaluation of Superoxide Anion Radical Scavenging Capacity for Four Antioxidants”. J AOAC Int, 99(2):504-11, 2016. [8] N. A. A. Binti Zaaba, G.R. Gayathri, V.P. V. Vishnu Priya, “In vitro free radical scavenging activity of tea tree oil and clove oil”. Asian Journal of Pharmaceutical and Clinical Research. 9: 179-83, 2016. [9] D. M. Kasote, S. S. Katyare, M. V. Hegde and H. Bae, “Significance of Antioxidant Potential of Plants and its Relevance to Therapeutic Applications”. International Journal of Biological Sciences.11(8): 982-991, 2015.

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