Antioxidants radical trap methods

The limitations and pitfalls of using artificial azo initiators were discussed in Chapter 1. Unfortunately, the use of such artificial initiators to evaluate antioxidants is common (see Section E). The so-called radical trap methods (also known as antioxidant status or capacity) are dependent on the use of either water-soluble or lipid-soluble radical trapping agents. These methods measure the relative reactivity of antioxidants toward artificial radicals, but they provide... 

Table 9.19. Free radical trapping methods to evaluate natural antioxidants ...

The ORAC method was first proposed by Cao and co-workers in 1993. Like in the TRAP method, they used a fluorescent indicator. Determination of antioxidant activity by this method is based on measurement of decreasing fluorescence of the indicator caused by the radicals generated in the system. The reaction mixture in their proposal consisted of a fluorescent indicator p-phycoerythrin (p-PE), 2,2 -azobis(2-amidinopropane) dihydrochloride (AAPH) as a peroxyl radical generator and the analysed sample [42]. Attributing the low purity of p-phycoerythrin (approx. 30%) to the low reproducibility of fluorescence and the occurrence of different forms of phycoerythrin, Ou and co-workers [43] modified the method by replacing the indicator with fluorescein (3, 6 -dihydroxyspiro[isobenzofuran-l[3H],9 [9H]-xanthen]-3-one). 

Popular EPR-based assays of antioxidant activity include the DPPH assay, in which the ability of compounds to quench (by H-atom transfer) the 1,1-diphenyl-2-picrylhydrazyl radical is used to rank their antioxidant activity. This method has been applied widely to the analysis of dietary antioxidants and extracts from medicinal plants.213 219 Extensive use has also been made of assays based on the competition between spin traps and test compounds for reaction with enzymatically-generated 02 and, in the presence of a metal catalyst, the OH rad-... 

Total radical trapping parameter (TRAP) assay is widely used in investigations and has various modifications [45-48]. This method presumes antioxidants capability to react with peroxyl radical 2.2-azobis (2-amidinopropane) dihydrochloride (AAPH). TRAP modifications differ in methods of registering analytical signal. Most often the final stage of analysis include peroxyl radical AAPH reaction with luminescent (luminol), fluorescent (dichlorofluorescein-diace-tate, DCFH-DA) or other optically active substrate. Trolox is often used as a standard. 

Other measurements of antioxidant activity include FRAP (ferric reducing-antioxidant power) (123-126), TRAP (total radical-trapping antioxidant parameter) (123, 127), phycoerythrin assay (128, 129), and test of metal chelating capacity (130, 131), among others. Reviews on methods for testing antioxidant activity have been published (9, 12). 

The rate of initiation is generally measured by an induction period method using an antioxidant (AH) that has a known stoichiometric factor n, defined as the number of radicals trapped by each molecule of antioxidant. Because a-tocopherol is known to have an n value of 2, a known concentration is used to determine the induction period, T, during which the oxidation is inhibited. 

An overview of die free radicals and reactions thereof is presented. Free radicals are atoms or groups having an unpaired electron and hence are paramagnetic. Electron paramagnetic resonance spectroscopy (EPR) and trapping methods are used to analyze radicals. In lipids, radical reactions lead to autoxidation and hence flavor reversion. Reactive oxygen species are key components involved in such reactions. Finally, descriptions for phenolic, sequesterant and enzymatic antioxidants and their mode of action are provided. 

Trapping of radicals by scavengers belongs to the most important methods to prevent autoxidation reactions. Radical scavengers are also referred to as chain breaking acceptors. Further, they are classified as primary antioxidants. 

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