A FRAP Assay at pH 7 unveils Extra Antioxidant Activity from Green, Black, White and Rooibos Tea but not Apple Tea

Abstract

Abstract Aim: Realization of a ferric reducing antioxidant power (FRAP) assay at neutral pH and re-evaluation of tea antioxidant activity for comparisons with the standard FRAP assay. Method: A FRAP assay at neutral pH utilized ferrozine (7.3-(2-Pyridyl)-5, 6-diphenyl-1, 2, 4-triazine-4’, 4’’-disulfonic acid;ferrozine) dye in conjunction with Tris-HCl buffer (0.1M. pH 7.0) with 280 µl of regent addition to 20 µl of tea infusions and absorbance measurements at 562 nm with a microplate reader. Results: The microplate ferrozine FRAP assay (mFzFRAP) gave linear calibrations for ascorbic acid, gallic acid, ammonium ferrous sulphate, (AFS), trolox, cysteine and glutathione (R2 = 0.998 -1.000) with molar absorptivity (measures of sensitivity) similar to literature values. The analytical precision was 5-7% and the minimum detectable concentrations (MDC) were 1.4- 2.8 µM (0.4-0.8 nanomoles). Discussion: Values for FRAP were higher at pH 7.0 compared to pH 4.0 for gallic acid, ascorbic acid, glutathione, and cysteine possibly due to their ionization at high pH. The assay sensitivity for AFS and trolox were unchanged at pH 4.0 and pH 7.0. When assayed at pH 7 the water infusions from green tea, black tea, white tea, and rooibos tea had 200-360% antioxidant activity normally observable at low pH. Conclusion: A FRAP assay at pH 7 unveils extra antioxidant activity for green, black, white and Rooibos teas compared to values from the standard TptzFRAP (pH 3.6) method. As a recommendation, the antioxidant activity of teas and other herbal preparations should be re-evaluated over a wide pH range.