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Molecules 2020, 25, 3191 6 of 24 Scheme 3. Reaction of N,N-dimethyl-p-phenylenediamine with hydroxyl radical [55]. The FC (Folin-Ciocalteau) assay was initially developed for proteins analysis [56], but later this method was extended by Singleton [57] for the analysis of total phenolic compounds in wine. From chemical point of view, this method is based on the oxidation of phenolic compounds. Folin-Ciocalteau phenol reagent consists of a mixture of phosphomolybdic acid and phosphotungstic acid, in which the molybdenum and tungsten are in the oxidation state 6+ and are reduced to the oxidation state of 5+ during the reaction. This is accompanied by a color change from yellow original color of the solution of Na2WO4/Na2MO4 to blue because of the formation of complexes with phenols (Phenol-MoW11O404−). The absorbance is usually measured in the range from 750–765 nm [43,44]. This method is mostly used for the determination of total phenolic compounds in various samples (food products, plants, plant extracts), but it can also react in alkaline conditions with non-phenolic compounds, such as amino acids, aromatic amines, ascorbic acid, sulphur dioxide, Cu+ ions, and others [43]. The FRAP (ferric reducing antioxidant power) assay is based on reduction of colorless Fe3+-2,4,6-tripyridyl-s-triazine complex to the intensively blue Fe2+-2,4,6-tripyridyl-s-triazine complex in acidic medium (Scheme 4). FRAP values are calculated from increasing absorbances measured at 593 nm [39,42,43]. The FRAP methods has several limitations. Compounds (even without antioxidant properties) with redox potential lower than the redox potential of Fe3+/Fe2+ pair may reduce Fe3+ to Fe2+ and can increase the FRAP value to obtain false high results. But, on the other hand, not all antioxidants reduce Fe3+ fast enough to measure [58]. Ferric reducing antioxidant power assay cannot be used for antioxidant acting as radical quench (with transfer of hydrogen) and for compounds absorbing at the wavelength of the determination, as the signals can interfere then [43,58,59]. Scheme 4. Reduction of Fe3+ (FRAP assay) [52]. Oxygen radical absorbance capacity assay (ORAC) is the method for measuring the scavenging activity against peroxyl radicals. The generation of peroxyl radicals have to be induced by AAPH (2,2′-azobis-(2-amidino-propane) dihydrochloride). The antioxidant activity is measured by determining the loss of fluorescence and fluorescein is usually used as a probe [59,60]. This method is popular mainly in the determination of antioxidant ability of foods and is limited by low reactivity of fluorescein and with peroxyl radicals [61].PDF Image | Antioxidant Activity Determination of Silver Nanoparticles
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