3.3. Ferric Reducing Antioxidant Power (FRAP) Assay

This method is based on the ability of antioxidants to reduce Fe³⁺ to Fe²⁺ ions in the presence of 2,4,6-tripyridyl-s-triazine (TPTZ). Within the reaction, the colourless Fe(III)-TPTZ complex is converted into the blue-coloured Fe(II)-TPTZ complex and the change in the absorbance at 593 nm, that directly reflects the TAC of sample, is measured spectrophotometrically. Ferric reducing power could be expressed as IC₅₀ value, in % or reported as Fe²⁺ equivalents. The FRAP reaction is carried out at acidic solution (pH 3.6) to maintain iron solubility. In such environment the ionization potential that drives hydrogen atom transfer decreases and increases the redox potential, which is the dominant reaction mechanism. Because the reaction detects compounds with redox potentials of <700 mV, which is comparable with that of ABTS^•+ (680 mV), similar compounds react in both the ABTS and FRAP assays [68]. FRAP cannot detect compounds that act by radical quenching (hydrogen transfer), particularly thiols (as glutathione).

However, FRAP is simple, rapid (generally 4–6 min), inexpensive, and can be performed using semiautomatic or automated protocols. Mathew et al. [71] used this approach to test reducing potential of sixteen compounds including BA and CFA. They found very low reducing power of BA, whereas CFA showed a strong reducing potential. Jitareanu et al. [95] positioned the reducing potential of the NCA compounds in the following order: CFA > p-CA > CinA. Similar results were obtained by Masek et al. [84] after comparing the reduction activity of p-CA (4.6%) and CFA (30.8%) at their equivalent concentration of 30 µg/mL. They also reported that increasing concentration of the tested compounds resulted in their higher reduction activity. The effect of the dose on the reducing power of CFA and ChA was also observed by Liu et al. [80]. Their results showed that the FRAP value of CFA was significantly lower than that of ChA at low concentrations (10–100 µmol/L); however, its reducing power was higher than ChA at concentrations in the range of 250–500 µmol/L. The above results demonstrate that ferric reducing power of antioxidants depends on the concentration of compound [80,84] the degree of hydroxylation, and the extent of conjugation of antioxidant [68]. When comparing RA, CinA, p-CA, and CFA, the presence of an additional hydroxyl group increases reducing activity.