Development and Validation of a Microplate-based .BETA.-carotene Bleaching Assay and Comparison of Antioxidant Activity (AOA) in Several Crops Measured by .BETA.-carotene Bleaching, DPPH and ORAC Assays

Abstract

The reproducibility and reliability of the micro-β-carotene-linoleic acid bleaching (BCB) assay have been improved, enabling comparison to the antioxidant activity (AOA) of extracts from eleven kinds of crops evaluated by the oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. All assays were conducted using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator, and AOAs were expressed as micromoles of Trolox equivalent per gram of weight. The coefficient variation (CV) of the BCB assay using a microplate was 14.40%, which was comparable to that of the ORAC assay (H-ORAC (14.10%), L-ORAC (18.76 %)).Keywords: AOA, β-carotene bleaching assay, DPPH assay, ORAC assay, Trolox, plate reader, tocopherol *To whom correspondence should be addressed. E-mail: ichiho@affrc.go.jp IntroductionAntioxidants are believed to play an important role in preventing chronic illnesses such as heart disease, stroke, cancer, Alzheimer's disease, rheumatoid arthritis, and cataracts. As people have become more health-conscious, research on antioxidants has increased. Recently, reviews of AOA and measuring assays have been published (Koleva et al., 2002;Prior et al., 2005;Roginsky and Lissi 2004). These studies compared several commonly used assays for measuring AOA, and summarized the reaction mechanisms. Multiple reaction characteristics and mechanisms, as well as different phase localizations, were usually involved. Thus, no single assay accurately reflects all the radical sources or all the antioxidants in a system . In addition, antioxidants may respond differently to different radical or oxidant sources. Therefore, it is important at the outset to understand that AOA cannot be measured accurately and quantitatively by any simple universal assay .Antioxidants can deactivate radicals by two major mechanisms: hydrogen atom transfer (HAT), and single electron transfer (SET) (Huang et al., 2005). Therefore, we used the oxygen radical absorbance capacity (ORAC) assay (a HATbased method) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay (a SET-based method) to evaluate the AOA of crop extracts from Ibaraki Prefecture, Japan. We used the β-carotene bleaching (BCB) assay to evaluate the AOA of crop extracts and compared it to the AOA measured by the ORAC and DPPH assays. The BCB assay is based on the competitive bleaching of β-carotene during the antioxidation of linoleic acid in aqueous emulsion, and is monitored as decay of absorbance in the visible region (Miller, 1971). However, this assay is poorly quantified since the AOA is only given as an inhibition percentage and it is difficult to obtain repeatable data (Roginsky and Lissi, 2004). We therefore, for the first time, tried to express the AOA of the BCB assay as the Trolox equivalent. All three assays were conducted using the Trolox calibrator (positive control), and AOA was expressed as micromoles of the Trolox equivalent per gram of weight. In this study, we also ascertained the reproducibility and reliab...

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