New approach for evaluation of the antioxidant capacity based on scavenging DPPH free radical in micelle systems

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Abstract

A simple and sensitive method was developed to evaluate the antioxidant capacity using a 2,2-diphenyl-1-picrylhydrazyl radical (DPPHradical dot) incorporated in surfactants. Parameters possibly affected the DPPHradical dot scavenging activity were investigated including the type of surfactant, concentration of surfactant, solution pH and concentration of buffer. The optimum micelle system for determining the antioxidant capacity was 2 mM CTAB in 0.1 M acetate buffer (pH 4.6). The IC50 values of various antioxidants were calculated and compared to those prepared in methanol. Furthermore, the rate of the reaction between DPPHradical dot and antioxidants was investigated. The rate constants observed in the micelle system were significantly faster than those in methanol, and this allowed shorter analysis time. This method was validated through linearity, limit of detection and precision by comparing with conventional methanolic assay. The purposed method was applied to evaluate the antioxidant capacity from aqueous extracted plant samples with satisfactory results.

Introduction

Antioxidants play a very important role in the body defense system against reactive oxygen species (ROS). The ROS are the harmful byproducts generated during normal cell aerobic respiration (Gutteridge & Halliwell, 2000). In addition, different environmental stress factors such as pollution, drought, temperature, excessive light intensities and nutritional limitation are able to increase the production of ROS (Arora et al., 2002, Ehling-Schulz and Scherer, 1999, Rijstenbil, 2002). Since ROS are considered responsible for a number of diseases such as cardiovascular disease, some forms of cancer, cataract, age-related muscular degeneration, and rheumatoid arthritis (Diplock, 1994, Esterbauer et al., 1992, Luis and Navab, 1993). Therefore, numerous studies have been conducted in order to evaluate the antioxidant capacity of certain compounds or plant materials. A wide variety of methods have been developed for the investigation of antioxidant capacity (Prior, Wu, & Schaich, 2005) including the total radical trapping parameter (TRAP) assay (Mulholland & Strain, 1991), the 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonate) radical cation (ABTSradical dot+) assay (Hung et al., 2009, Miller et al., 1993), the ferric reducing antioxidant power (FRAP) assay (Benzie and Strain, 1996, Pérez-Jiménez et al., 2008), the oxygen radical absorbance capacity (ORAC) assay (Cao et al., 1995, Mesa et al., 2008), the cupric reducing antioxidant capacity (CUPRAC) assay (Apak, Güçlü, Özyürek, & Karademir, 2004), the electrochemical estimation of total reducing capacity (Chevion, Roberts, & Chevion, 2000) and the 2,2-diphenyl-1-picrylhydrazyl radical (DPPHradical dot) assay (Bortolomeazzi et al., 2010, Cheng et al., 2006, Katsube et al., 2004). Among the methods mentioned above, the DPPHradical dot method is widely used due to its stability, simplicity and its simple reaction system which involves only the direct reaction between the radical and an antioxidant.

The DPPHradical dot assay method is based on the scavenging of the stable DPPHradical dot by an antioxidant. The absorbance of the radical in the range of 515–520 nm is monitored. The DPPHradical dot method was first reported by Blois (1958), who observed that the DPPHradical dot was reduced by thiol-containing amino acid cysteine and other active compounds. Later, Brand-Williams, Cuvelier, and Berset (1995) revised the original method and the DPPHradical dot scavenging test became a popular method to estimate the antioxidant capacity. For the DPPHradical dot method, factors influenced the scavenging activity are solvent, pH, sample concentration and reaction time. The fact that dissolution of DPPHradical dot must be done in organic solvents (for examples, methanol and ethanol) limited the use of this radical in aqueous solution. To date, many researchers have developed methods to employ DPPHradical dot in aqueous solution. Chen et al. (2009) reported a colloidal synthesis of stable, water-soluble DPPHradical dot nanoparticles which were used as a new type of electron paramagnetic resonance (EPR) standard. Moreover, Sharma and Bhat (2009) reported the scavenging of DPPHradical dot free radical in buffered methanol. The free radical scavenging by butylated hydroxytoluene (BHT) was markedly influenced by the reaction medium. The DPPHradical dot scavenging capacity of BHT in buffered methanol was better than in the methanol solution with IC50 values of 9.7 μM and 60.0 μM, respectively. However, this method suffered from the use of a large volume of organic solvents.

Until now, the assay based DPPHradical dot has been mostly performed in organic solvents. Thus, in this work, we attempted to develop a DPPHradical dot method for evaluation of the capacity of antioxidants in aqueous solution using surfactant aggregates or micelles. When surfactants are added to aqueous solution at the concentration higher than the critical micelle concentration (cmc), they spontaneously form micelles with the hydrophobic tails pointed toward the center and the hydrophilic head groups pointed at the surface of the micelle. Parameters that can affect the estimation of the antioxidant capacity including the type of surfactant, concentration of surfactant, solution pH and concentration of buffer were explored. Reaction rates of DPPHradical dot and antioxidants in the micelle system were also evaluated. This new method demonstrated to run well in aqueous solution and can be used to determine the antioxidant capacity of both hydrophilic and hydrophobic antioxidants.

Section snippets

Chemicals and instruments

All chemicals used were of analytical grade and were used without further purification. 2,2-Diphenyl-1-picrylhydrazyl (DPPHradical dot), α-tocopherol and ferulic acid were obtained from Sigma (Sigma-Aldrich Group, USA). Sodium dodecyl sulphate (SDS) was purchased from BDH (BDH-Prolabo, England). Triton-X 100, cethyltrimethylammonium bromide (CTAB), syringic acid, guaiacol, p-coumaric acid, p-cresol and o-cresol were obtained from Fluka (Sigma-Aldrich Group, USA). Gallic acid, protocatechuic acid, caffeic

Results and discussion

DPPHradical dot is well known as a stable organic free radical which has been used for estimation of the antioxidant capacity. However, the solubility of DPPHradical dot in water limits its applications in aqueous solution. To overcome this limitation, the incorporation of DPPHradical dot into the micelle solution was used to improve the solubility of DPPHradical dot in aqueous solution. Estimation of the antioxidant capacity in aqueous solution should give more benefit to the biological system than the evaluation in methanolic solution.

Conclusion

We have successfully developed the modified DPPHradical dot assay to evaluate the antioxidant capacity of both hydrophilic and lipophilic antioxidants by the assistance of the surfactant. Parameters affected the performance of the assay such as the CTAB concentration, buffer pH and concentration were optimized. Common antioxidants (12 compounds) were used to evaluate the feasibility of the assay. The modified DPPHradical dot method was relatively faster than the conventional methanolic assay and did not require the

Acknowledgements

This research was financially supported by the National Research University Project, Khon Kaen University, the Thailand Research Fund (RTA5380003) and the Center for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education.

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