Total Antioxidant Capacity Assay Using Optimized Ferricyanide/Prussian Blue Method

Abstract

The existing ferricyanide/Prussian blue assay of reducing capacity measurement was optimized so as to obtain a more reproducible, linear and additive response from antioxidants. The modification involved the simultaneous use of ferricyanide and iron(III) to regulate more favorable redox conditions for a greater variety of antioxidants. Prussian blue precipitation was hindered with the addition of sodium dodecyl sulfate, and the optimal pH was adjusted to 1.7 to maintain the redox activity of ferric ion while preventing its hydrolysis. Incubation of the reaction mixture at room temperature for 30 min enabled more complete oxidations than observed in the conventional ferricyanide method. The order of trolox equivalent antioxidant capacities was quercetin > rosmarinic acid > gallic acid > ferulic acid ≥ catechin > caffeic acid ≥ rutin ≥ ascorbic acid ≈ trolox. Synthetic antioxidant mixtures gave the theoretically expected total antioxidant capacities conforming to Beer's law. The assay was nonresponsive to simple sugars and citric acid (which are not true antioxidants) but responsive to biologically important thiols which are not oxidized by other Fe(III)-based assays. The assay was used in real sample solutions by using the method of standard additions to green tea, nettle, and sage, and validated against other similar antioxidant assays.

Effect of different fractions of Hibiscus rosa sinensis leaf extract on islets of Langerhans and antioxidant activity in non-obese diabetic (NOD) mouse

“…CHP stained histological sections slides were photographed using Olympus B × 60 Microscope. Total antioxidant capacity was estimated based on the method of Berker et al, (2009). Briefly 0.1 ml of sample, 0.9 ml of EtOH (96%), 5 ml H 2 O, 1.5 ml of 1 M HCl, 1.5 ml of ferricyanide solution (1%), 0.5 ml of 1% sodium dodecyl sulfate (SDS), and 0.5 of FeCl 3 .6H 2 O (0.2%) were added and the volume was made up to 10 ml.…”

Measurement of antioxidant activity

“…Antioxidants can either reduce the Fe 3+ in the solution to Fe 2+ , which binds the ferricyanide to yield Prussian blue, or reduce the ferricyanide to ferrocyanide, which binds the free Fe 3+ in the solution and forms Prussian blue. The simplified scheme for these two reactions is given blow (Berker, Gueclue, Tor, Demirata, & Apak, 2010 One drawback of this ferricyanide FRAP assay is the tendency of the Prussian blue to precipitate, forming a suspension and staining the measuring cuvette. Therefore, time to add Fe 3+ (FeCl3) is essential and may introduce error to result interpretation.…”

“…Therefore, time to add Fe 3+ (FeCl3) is essential and may introduce error to result interpretation. In order to stabilize the Prussian blue against precipitation, Berker et al (2010) proposed addition of the surfactant sodium dodecyl sulphate, while adjusting the optimal pH to 1.7 to maintain the redox activity of ferric ion without hydrolysis. The authors suggested that this modification also allows evaluation of antioxidants whose redox potential do not exceed that of the Fe 3+ /Fe 2+ in conventional FRAP assay (i.e.…”

Methods of measurement and evaluation of natural antioxidant capacity/activity (IUPAC Technical Report)

“…The ferricyanide/Prussian blue assay of reducing capacity measurement was modified so as to obtain a more reproducible, linear, and additive response from antioxidants with respect to concentration [19]. The simultaneous use of ferricyanide and ferric ions as chromogenic oxidants supplied more favourable redox conditions for a greater variety of antioxidants.…”