Mechanism of Antioxidant Activity
There are mainly three types of mechanism known for the antioxidant activity, viz., chain breaking, preventive, and synergetic. Schematic representation of these mechanisms is given in Fig. 6.4a–c.
Techniques for Measurement of Antioxidant Activity
There are three major techniques mostly used for the measurement of antioxidant activity in various samples.
Chemical Assays for Antioxidant Activity
There are many chemical assays used for the assessment of antioxidant activity in the products (herbal, nutraceuticals, and food items). Some of the well-documented and most practiced methods are described below.
6.5.1.1 Oxygen Radical Absorption Capacity
Oxygen radical absorption capacity (ORAC) method uses dichlorofluorescein as the fluorescent probe and an azo-compounds, such as 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) as the radical generator. It measures the inhibition of the peroxyl radical induced oxidation initiated by thermal decomposition of AAPH. Over time, the free radical generated from the thermal decomposition of AAPH quenches the signal from the fluorescent probe fluorescein. The subsequent addition of an antioxidant produces a more stable fluorescence signal due to the inhibition of fluorescein decay by single antioxidant and/or complex mixture. Rate of decay of fluorescence measures the antioxidant's capacity (Číž et al. 2010).
Fig. 6.4 (a–c) showing schematic representation of mechanism of chain breaking, preventive and synergetic action of antioxidants respectively
6.5.1.2 Determination of Total Phenolic Content (TPC)
Total phenolic content of the extracts are determined using Folin–Ciocalteu (FC) reagent using spectrophotometer, measured at 725 nm. This method is based on reduction ability of phenolic functional group. Oxidation and reduction reaction of phenolate ion takes place at base condition. The reduction of phosphotungstate– phosphomolybdenum complex (Folin–Ciocalteu reagent) by phenolat ion will change its color to blue. The reduction of complex will increase when the extract contains more phenolic compounds. Thus the color will be darker and the absorbance will be higher, showing higher antioxidant activity (Prior et al. 2005).
6.5.1.3 1,1′-Diphenyl-2-Picrylhydrazyl
DPPH (1,1′-diphenyl-2-picrylhydrazyl) assay is carried out as per the reported method of Brand-Williams et al. (1995). DPPH− free radical is obtained by dissolving DPPH in methanol and is stable when placed under the dark at −20 °C until used. As DPPH− reacts with antioxidants present in the sample, color changes from violet to yellow and absorbance of the solution so obtained is measured spectrophotometrically at 515 nm (Brand-Williams et al. 1995).
6.5.1.4 Trolox Equivalent Antioxidant Capacity
In this assay, ABTS {2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)} is used to measure the antioxidant capacity of the substance (food stuffs). Trolox equivalent antioxidant capacity (TEAC) is also known as ABTS assay and the procedure is based on the reported method of Arnao et al. (2001). When ABTS reacts with potassium persulfate, it becomes a free radical (ABTS+) which gives blue color to the solution. The phenolics, thiols, or vitamin C present in the food stuffs scavenge this ABTS+ free radical and convert it into its neutral colorless form which is measured spectrophotometrically. ABTS+ absorbs light at 734 nm (Arnao et al. 2001).
6.5.1.5 Ferric Reducing Antioxidant Power
Ferric reducing antioxidant power (FRAP) assay is carried out using the earlier reported method as described by Benzie and Strain (1996). When ferric chloride reacts with 2,4,6-tripyridyl-s-triazine (TPTZ) at low pH, ferric is converted into ferrous causing formation of ferrous tripyridyl triazine complex. FRAP values are obtained by comparing the absorbance change at 593 nm in reaction mixture with those containing ferrous ions in known concentration (Benzie and Strain 1996).
6.5.1.6 Determination of Total Reducing Power (TRP)
TRP is determined following the method of Negi et al. (2005). It is measured spectrophotometrically in terms of their capacity to reduce the potassium ferricyanide (Fe3+) to the potassium ferrocyanide (Fe2+), depending upon the concentration of the antioxidant compounds present in the sample, which in turn reacts with ferric chloride to form ferric ferrous complex that has an absorption maximum at 700 nm (Negi et al. 2005).
