(468) Comparison of Methods to Determine Antioxidant Levels in Fruit

Abstract

In preliminary studies, we found that relative and absolute antioxidant (AO) levels varied within and among small fruit types. AO levels were affected by assay method used, time of reaction, volume of sample, and the ratio of reactants to total AO activity. To identify the physicochemical parameters that affect accuracy and reproducibility, a series of experiments were conducted to test the roles of AO assay, different AOs, and AO concentration on measured AO content and reaction kinetics. Three assays (DPPH, FRAP, ABTS) were used to evaluate AO capacity of seven fruit types (black and red raspberry, blackberry, strawberry, grape, elderberry, and cranberry) and nine purified AOs (ascorbic, caffeic, chlorogenic, gallic, and ellagic acids, α-tocopherol, trolox, cyanidin-3-glucoside, and quercetin). Ascorbic acid, trolox, caffeic acid, chlorogenic acid, and α-tocopherol exhibited simple reaction kinetics and reached endpoints quickly, regardless of assay. Gallic and ellagic acids, quercetin, cyanidin-3-glucoside, and all fruit extracts exhibited more complex kinetics and long reaction times (>70 min) to reach an endpoint. Moreover, the latter four AOs had the highest AO capacity among the compounds tested. We observed differences in reactivity between assays, compounds and fruit extracts, but relative AO activity was comparable, although the absolute values differed. Since AO capacity of fruit extracts is a composite of the individual AOs present, it is important that reactions progress to near steady state, assay reactants are in excess of (30–50×) the AO capacity being measured, more than one assay is used to describe the total AO activity of fruit samples. Thus, there may not be a single AO assay method that completely defines the AO activity of a given fruit.