New approach for evaluation of the antioxidant capacity based on scavenging DPPH free radical in micelle systems
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 (ABTS+) 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 (DPPH) assay (Bortolomeazzi et al., 2010, Cheng et al., 2006, Katsube et al., 2004). Among the methods mentioned above, the DPPH 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 DPPH assay method is based on the scavenging of the stable DPPH by an antioxidant. The absorbance of the radical in the range of 515–520 nm is monitored. The DPPH method was first reported by Blois (1958), who observed that the DPPH 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 DPPH scavenging test became a popular method to estimate the antioxidant capacity. For the DPPH method, factors influenced the scavenging activity are solvent, pH, sample concentration and reaction time. The fact that dissolution of DPPH 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 DPPH in aqueous solution. Chen et al. (2009) reported a colloidal synthesis of stable, water-soluble DPPH nanoparticles which were used as a new type of electron paramagnetic resonance (EPR) standard. Moreover, Sharma and Bhat (2009) reported the scavenging of DPPH free radical in buffered methanol. The free radical scavenging by butylated hydroxytoluene (BHT) was markedly influenced by the reaction medium. The DPPH 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 DPPH has been mostly performed in organic solvents. Thus, in this work, we attempted to develop a DPPH 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 DPPH 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 (DPPH), 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
DPPH is well known as a stable organic free radical which has been used for estimation of the antioxidant capacity. However, the solubility of DPPH in water limits its applications in aqueous solution. To overcome this limitation, the incorporation of DPPH into the micelle solution was used to improve the solubility of DPPH 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 DPPH 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 DPPH 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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