Biochemical Assays for Antioxidant Activity Assessment

Antioxidant activity may also be measured in biological system, i.e., in vivo and in vitro models. These include measurement of oxidative stress marker of the adduct or end product of ROS with the molecules, such as lipid, protein, DNA, and other molecules. These methods include thiobarbituric acid reactive substances (TBARS), SOD, CAT, GPx, GSH, and ferrous oxidation-xylenol orange (FOX) assay. These assays may be carried out in blood, urine, breath and tissues. Some of the examples are described below:

6.5.2.1 TBARS

TBARS method determines the extent of lipid peroxydation in sample. TBARS is the reaction product of thiobarbituric acid (TBA) and malondialdehyde (MDA) which results from the decomposition of lipid hydroperoxide in the sample which is read spectrophotometrically at 532 nm (Ohkawa et al. 1979).

6.5.2.2 Protein Carbonyl

Protein carbonyl content results from the oxidative cleavage of protein. In this case, 2,4-dinitrophenylhydrazine (DNPH) reacts with protein carbonyl and forms a Schiff base to produce corresponding hydrazone. The amount of protein hydrazone produced is quantified spectrophotometrically at an absorbance between 360 and 380 nm (Levine et al. 1990).

6.5.2.3 FOX

Hydroperoxide content of the lipid can be determined from its ability to oxidize ferric (Fe2+) to ferrous (Fe3+). Ferrous (Fe3+) formed a complex with xylenol orange reagent (bluish-purple color) which is measured at 560 nm (Nourooz-Zadeh et al. 1994).

6.5.2.4 CAT

Catalase activity can be measured by using H2O2 as a substrate according to the method of Aebi (1984).

6.5.2.5 SOD

SOD is measured using the method of Kakkar et al. (1984) where nicotinamide adenine dinucleotide (NADH) is used as a substrate. The color intensity of the chromogen (purple color) in butanol layer is measured against butanol (blank) on spectrophotometer at 560 nm (Kakkar et al. 1984).

6.5.2.6 ROS

In this assay, 2′,7′-dichlorofluorescein diacetate (DCFDA) is used to measure ROS level. It undergoes cellular oxidation by ROS and gets converted into fluorescent dichlorofluorescein (DCF) which is highly fluorescent at 530 nm (Moein and Moein 2012).

Instrumental Technique (Antioxidant Analyzer)

Recently, an instrument named PHOTOCHEM Antioxidant Analyzer developed by Analytik Jena UK is being used for the measurement of antioxidant property of different products. It is capable of measuring both water-soluble and lipid-soluble antioxidants in a single system. It is based on the principle of photochemiluminescence with luminometric detection. It can measure the antioxidant capacity of lyophilized vegetables, fruits juices, beer, and water; lipid-soluble antioxidative capacity in baker's yeast, cheese, tea, and coffee; and lipid-soluble antioxidative capacity in edible oilandsalamiextracts(selectscience.net/product-news/rapid-and- accurate-antioxidant-measurement-in-foods).

 
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