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J. Agric. Food Chem. 2004, 52, 48−54
Extending Applicability of the Oxygen Radical Absorbance Capacity (ORAC−Fluorescein) Assay ALBERTO DAÄ VALOS, CARMEN GOÄ MEZ-CORDOVEÄ S,
AND
BEGON˜ A BARTOLOMEÄ *
Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
The ORAC-fluorescein (ORAC-FL) method recently validated using automatic liquid handling systems has now been adapted to manual handling and using a conventional fluorescence microplate reader. As calculated for Trolox, the precision of the method was 1 > 3 > 2 > 5, that seemed not to be related with their composition (see Materials and Methods). The same supplements assayed for their antiradical activity against a synthetic radical (2,2-diphenyl-1-picrylhydrazyl, DPPH•) followed a different relative order: 7 g 4 > 1 > 6 g 3 > 2 > 5 (31). The nature of the radical clearly conditioned the response of the antioxidant/s present in each supplement. In our opinion, the ORAC procedure is more convenient for evaluating antioxidant activity of dietary supplements because it involves peroxyl radicals, which are the most abundant radicals in biological systems. Final Remarks. The increasing interest in the evaluation of the antioxidant activity in complex matrixes has been aimed at the development of new methodologies and/or the improvement of the known ones. The ORAC method takes into account both inhibition percentage and inhibition time of the peroxyl radical action by antioxidants. Deficiencies of the ORAC assay associated with the use of β-phycoerythrin as fluorescent have been overcome by the use of fluorescein (FL). This paper proves suitability of manual handling and using a conventional fluorescence microplate reader for performing the ORAC-FL assay on pure compounds and food samples. Useful ORACFL data for benzoic and cinnamic acids and aldehydes, flavonoids, BHA, wines, and dietary antioxidant supplements are given. The results confirm that wine antioxidant activity is influenced by grape variety and enological practices. Oak aging
Extending Applicability of ORAC−Fluorescein
J. Agric. Food Chem., Vol. 52, No. 1, 2004
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Figure 4. ANOVA analysis of the ORAC value (a, c) and the total polyphenol content (b, d) corresponding to red oak-aged wines from Tempranillo (n
) 12) and Cabernet Sauvignon (n ) 10) varieties from different vintages (1989−2002). Error bars represent the 95% confidence intervals for mean.
Figure 5. Time course of the reaction of (a) fluorescein (FL) with AAPH in the presence of commercial antioxidant supplement #4 (20 µg/mL), and of
(b) β-phycoerythrin (PE) with AAPH in the presence commercial antioxidant supplement #4 (65 µg/mL). Table 5. Trolox Equivalents and Linear Ranges (Net AUC vs
Concentration) of the Commercial Dietary Antioxidant Supplements Studied
evaluating the antioxidant activity of both wine and antioxidant dietary supplement samples. ACKNOWLEDGMENT
supplement
Trolox-equivsa,b
conc range (µg/mL)
slope
intercept
r2
1 2 3 4 5 6 7
1.47 ± 0.08 0.079 ± 0.003 0.704 ± 0.035 3.18 ± 0.08 0.018 ± 0.001 2.12 ± 0.08 2.27 ± 0.08
16.7−66.7 166−1000 26.7−114 6.66−28.6 1000−4000 8.30−40.0 10.0−40.0
0.5712 0.0364 0.2936 1.1571 0.0085 0.9008 0.8578
8.4502 3.7752 4.1812 5.7536 2.4959 3.2659 5.0344
0.9942 0.9966 0.9959 0.9954 0.9926 0.9920 0.9921
a Expressed as µmol of Trolox equivalent/mg of dietary supplement. b Results are presented as the mean (n ) 3) ± SD.
increases the antioxidant activity of wines, but grape annual characteristics as well as bottle-aging effects can induce differences among vintages. The antioxidant capacity of the dietary antioxidant supplements studied is variable, ranging from 0.018 to 3.18 µmol of Trolox equivalent/mg of supplement, which, in any case, is considerably lower than the values found for pure flavonoids such as (+)-catechin (51.3 µmol) and quercetin (34.6 µmol). Based on the results, we propose the ORAC-FL assay as a simple and sensitive method for
We thank Dr. Howard (Food Science and Horticulture Department, University of Arkansas, AR) for helpful discussion about the methodology, Dr. Rial (Instituto de Investigaciones Biolo´gicas, CSIC, Madrid, Spain) for facilities with the fluorescence plate reader, and Mr. Suberviola (Viticulture and Enology Station of Navarra, EVENA, Olite, Navarra, Spain) for providing us with wine samples. LITERATURE CITED (1) Glazer, N. Fluorescence-based assay for reactive oxygen species: a protective role for creatinine. FASEB J. 1988, 2, 24872491. (2) Glazer, A. N. Phycoerythrin fluorescence-based assay for reactive oxygen species. Method Enzymol. 1990, 186, 161-168. (3) Cao, G.; Alessio, H. M.; Cutler, R. G. Oxygen-radical absorbance capacity assay for antioxidants. Free Radic. Biol. Med.1993, 14, 303-311. (4) Cao, G.; Verdon, C. P.; Wu, A. H. B.; Wang, H.; Prior, R. L. Automated assay of oxygen radical absorbance capacity with the COBAS FARA II. Clin. Chem. 1995, 41, 1738-1744.
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