Original article
Comparison of ABTS/DPPH assays to measure antioxidant capacity in popular antioxidant-rich US foods

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Abstract

To evaluate the comparability of the two most common radical scavenging assays using 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the 50 most popular antioxidant-rich fruits, vegetables and beverages in the US diet were identified and analyzed for their antioxidant capacities, total phenolics and flavonoids content. Spearmans–Rho correlation coefficients were calculated in order to characterize the relationship between antioxidant capacities, total phenolics and flavonoids content. Antioxidant capacity showed a strong positive relationship comparing both assays (ρ = 0.949, p < 0.001). Antioxidant capacity detected by ABTS assay was stronger positively associated with the oxygen radical absorbance capacity (ORAC) from USDA database (for ABTS: ρ = 0.593, p < 0.001; for DPPH: ρ = 0.539, p < 0.001, respectively), phenolics (for ABTS: ρ = 0.946, p < 0.001; for DPPH: ρ = 0.897, p < 0.001, respectively) and flavonoids content (for ABTS: ρ = 0.718, p < 0.001; for DPPH: ρ = 0.708, p < 0.001, respectively). Antioxidant capacity detected by ABTS assay was significantly higher for fruits, vegetables and beverages compared to that by DPPH assay. The high-pigmented and hydrophilic antioxidants were better reflected by ABTS assay than DPPH assay. These data suggest that ABTS assay may be more useful than DPPH assay for detecting antioxidant capacity in a variety of foods.

Introduction

Different assays have been introduced to measure antioxidant capacity of foods and biological samples. The concept of antioxidant capacity first originated from chemistry and was later adapted to biology, medicine, epidemiology and nutrition (Cao and Prior, 1998, Floegel et al., 2010, Pellegrini et al., 2003). It describes the ability of redox molecules in foods and biological systems to scavenge free radicals. This concept provides a broader picture of the antioxidants present in a biological sample as it considers the additive and synergistic effects of all antioxidants rather than the effect of single compounds, and may, therefore, be useful to study the potential health benefits of antioxidants on oxidative stress-mediated diseases (Brighenti et al., 2005, Puchau et al., 2009).

In recent years, a wide range of spectrophotometric assays has been adopted to measure antioxidant capacity of foods, the most popular being 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, among others such as oxygen radical absorbance capacity (ORAC) and ferric reducing ability of plasma (FRAP) assay (Brand-Williams et al., 1995, Kim et al., 2002, Ou et al., 2002, Re et al., 1999, Thaipong et al., 2006, van den Berg et al., 1999, van den Berg et al., 2001). Most of the assays employ the same principle: a synthetic colored radical or redox-active compound is generated; and the ability of a biological sample to scavenge the radical or to reduce the redox-active compound is monitored by spectrophotometer, applying an appropriate standard to quantify antioxidant capacity, e.g. as Trolox equivalent antioxidant capacity (TEAC) or vitamin C equivalent antioxidant capacity (VCEAC). Furthermore, there are two types of assays. One approach is based on an electron transfer and involves reduction of a colored oxidant, e.g. in ABTS, DPPH and FRAP assay. The other approach involves a hydrogen atom transfer, like ORAC assay, in which antioxidants and substrate compete for thermally generated peroxyl radicals (Dudonné et al., 2009, Rodriguez-Amaya, 2010). In specification, the ABTS assay is based on the generation of a blue/green ABTS·+ that can be reduced by antioxidants; whereas the DPPH assay is based on the reduction of the purple DPPH· to 1,1-diphenyl-2-picryl hydrazine. Both assays are convenient in their application and thus most popular; nevertheless they are limited as they use non physiological radicals. In contrast, the ORAC assay detects chemical change in a fluorescent molecule caused by a free radical attack. It is based on peroxyl radicals that reflect physiological relevant perturbations. The FRAP assay is different from the others as there are no free radicals involved but the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) is monitored. When applied to food analysis, the antioxidant capacity measurements may be different depending on the assay used. Many studies for the analysis of assay comparability previously focused on only few, selected or exotic foods (Gorinstein et al., 2010, Kim et al., 2003, Pellegrini et al., 2003, Samaniego Sanchez et al., 2007). Food analyses that based their sample selection on population intake data are currently rare. Thus, information on the comparability of different antioxidant capacity assays is not readily available for a variety of foods commonly consumed in the United States (US). In this population not only fruits and vegetables, but also beverages contribute strongly to antioxidant intake.

Thus, the purpose of this study was to evaluate the comparability of antioxidant capacity measurements obtained by ABTS and DPPH assay, when applied to a large number of food samples containing a variety of fruits, vegetables and beverages that are major contributors to antioxidant intake in the US diet.

Section snippets

Reagents and standards

ABTS, (+)-catechin, DPPH, Folin–Ciocalteu's phenol reagent, and gallic acid were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ascorbic acid was obtained from Fisher Scientific (Fair Lawn, NJ, USA). 2,2-Azobis(2-amidinopropane) dihydrochloride was purchased from Wako Chemicals Inc. (Richmond, VA, USA). All chemicals used were of analytical grade. Standard solutions were prepared with distilled deionized water.

Selection of food samples

To cover a variety of antioxidant-rich foods that are widely consumed in the

Results

Among the 50 most popular foods in the US diet with high antioxidant capacity were 18 fruits, 13 vegetables and 19 beverages. The antioxidant capacities of foods are summarized in Table 1. The highest antioxidant capacities were detected for strawberry by DPPH assay (520.7 ± 39.3 mg VCE/100 g) and for blueberry by ABTS assay (476.6 ± 28.9 mg VCE/100 g). Although their ranking differed slightly, both assays identified the top ten foods according to their antioxidant capacities as blueberry, black plum,

Discussion

The present study showed that relative to DPPH assay, the ABTS assay was more strongly correlated with ORAC from USDA database, phenolics and flavonoids content of the 50 most popular antioxidant-rich foods in the US diet. The results suggest that ABTS assay better reflects the antioxidant contents in a variety of foods than DPPH assay.

It has been previously reported that antioxidant capacity determined by in vitro assays differs (Ou et al., 2002, Wootton-Bearda et al., 2010, Wu et al., 2004).

Conclusions

The findings of the present study based on the analysis of a large number of food samples rich in antioxidants strongly suggest that compared to DPPH assay the ABTS assay better estimates the antioxidant capacity of foods, particularly fruits, vegetables and beverages. The data show that the antioxidant capacities measured by ABTS assay are strongly correlated with the ORAC from USDA database, phenolics and flavonoids content in the 50 most popular antioxidant-rich foods of the US diet. These

Acknowledgments

This study was supported by the University of Connecticut Hatch Project No. CONS00846 from the United States Department of Agriculture.

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    Presented in part at the 2010 Experimental Biology Conference in Anaheim, CA, April 2010.

    1

    Present address: Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany.

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