3.3.3. H2O2 Scavenging

The hydrogen peroxide (H₂O₂) scavenging activity was determined using a method modified from Hazra et al. [39] and Jiang et al. [40]. The assay was carried out according to Vaario et al. [36]. An aliquot of 2 mM H₂O₂ (Merck KGaA, Darmstadt, Germany) was added to the reaction mixture with the sample and a mixture containing 2.56 mM ammonium ferrous (II) sulfate (BDH Prolabo) in 0.25 mM H₂SO₄ (Merck KGaA) and 27.8 µM xylenol orange disodium salt (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) in 4.4 mM sorbitol (D(-)-sorbitol, AppliChem GmbH). After 30 min incubation, the absorbance of violet-colored ferric-xylenol orange complexes at 560 nm was measured. The assay measures the ability of the sample to scavenge H₂O₂ and prevent the oxidation of Fe(II) to Fe(III), which is indicated by the formation of a ferric–xylenol orange complex. The inhibition of the oxidation is expressed as an inhibition % of the reaction and the samples with 100% inhibition activity will remain yellowish. Sodium pyruvate (Sigma-Aldrich) was used as a reference compound.