Assay Dilution Factors Confound Measures of Total Antioxidant Capacity in Polyphenol‐Rich Juices

Abstract

The extent to which sample dilution factor (DF) affects total antioxidant capacity (TAC) values is poorly understood. Thus, we examined the impact of DF on the ORAC, FRAP, DPPH, and total phenols (TP) assays using pomegranate juice (PJ), grape juice (GJ), selected flavonoids, ascorbic acid, and ellagic acid. For ORAC, GJ was comparable to PJ at DF 750, but at DF 2000, the ORAC value of GJ was 40% more than PJ. Increasing DF increased GJ and PJ, DPPH, TP, and FRAP values 11% and 14%, respectively. Increased test concentrations of quercetin and catechin resulted in 51% and 126% greater ORAC values, but decreased naringenin by 68%. Flavonoids, but not ellagic acid or ascorbic acid, may contribute to the dilution effect on the variation of final TAC values. Thus, reporting TAC or TP using a single DF may introduce uncertainty about the confidence of TAC assay values, especially when comparing different juices. These results underscore the importance of using compatible test standards for reporting TAC values.

Measurement of total phenolic content in wine using an automatic Folin–Ciocalteu assay method

“…The reduced responses at one and twofold dilutions did not appear to be directly related to saturation of the reagent in the system because the peak areas of the gallic acid standards could be linearly increased beyond the peak areas of the undiluted wine samples. As individual or combinations of compounds or dilutions of samples have shown to influence the determination of the total antioxidant activity (Parker et al ., ; Bolling et al ., ; Sipel et al ., ), it was surprising to see such differences in our results. We speculated that differential reaction responses of the various compounds in wines existed, some with rapid and some with slow reactions with the F‐C assay, as we examined in a test below.…”

“…In general, more complex combinations had significantly higher ORAC values than less complex combinations, with most having ORAC values that were additive. The sample dilution factor (DF) has affected total antioxidant capacity values in various phenolic compounds or in grape juice or pomegranate juice when the final flavonoid AOA values were expressed as GAE (Bolling et al ., ). Similarly, effects of sample dilution on the measured (and recalculated) antioxidant capacity by four different methods were observed, with differences up to 28% between dilutions (Sipel et al ., ).…”

Solvent effect on total phenolic contents, antioxidant, and antibacterial activities of Matricaria pubescens

“…AAPH, FL, and trolox solutions were prepared in 75 mM phosphate buffer saline (pH 7.4). The ORAC values were calculated by a regression equation between the trolox concentration and the net area under the FL decay curve (Bolling et al, 2012). The ORAC capacities were expressed as mmol of trolox equivalents (TE) per 100 g of dry weight (DW).…”

Contributions of phenolics and added vitamin C to the antioxidant capacity of pomegranate and grape juices: synergism and antagonism among constituents

“…Fractions were reconstituted or diluted with the appropriate assay buffers or solvents prior to analysis. For total phenol and TAC analyses, dilutions were adjusted to enable raw assay values to fall approximately midpoint of the linear assay ranges as dilution is known to affect final TAC and total phenol values (Bolling et al, 2012).…”

“…Total antioxidant capacity assays 2,2-Diphenyl-1-picrylhydrazyl radical scavenging was assessed as previously described (Benzie & Strain, 1996;Bolling et al, 2012). Briefly, methanol-diluted juice, fractions or standards were added to 10 lM DPPH (final concentration) in methanol.…”