The ROO scavenging capacity was measured by monitoring the effect of the microcapsules on the fluorescence decay resulting from ROO -induced oxidation of fluorescein (Ou, Hampsch-Woodill, & Prior, 2001). ROO was generated by thermodecomposition of AAPH at 37 °C. Reaction mixtures in the Staurosporine price wells contained
the following reagents at the indicated final concentrations (final volume of 200 μl): fluorescein (61 nM), AAPH solution in phosphate buffer (19 mM) and microcapsules aqueous solutions (four concentrations). The mixture was preincubated in the microplate reader during 10 min before AAPH addition. The fluorescence signal was monitored every minute
for the emission wavelength at 528 ± 20 nm with Metabolism inhibitor excitation at 485 ± 20 nm, until 180 min. Trolox was used as positive control (Net area (64 μM) = 23). The H2O2 scavenging capacity was measured by monitoring the H2O2-induced oxidation of lucigenin (Gomes et al., 2007). Reaction mixtures contained the following reagents at final concentrations (final volume of 300 μl): 50 mM Tris–HCl buffer (pH 7.4), lucigenin solution in Tris–HCl buffer (0.8 mM), 1% (w/w) H2O2 and aqueous solutions of antioxidant microcapsules or trolox (five concentrations). The chemiluminescence signal was detected in the microplate reader after 5 min of incubation. Ascorbic acid was used as positive control (IC50 = 171 μg/ml). The HO scavenging capacity was measured by monitoring the HO -induced oxidation of luminol (Costa, Marques, Reis, Lima, & Fernandes, 2006). The HO was generated by a Fenton system (FeCl2–EDTA–H2O2). N-acetylglucosamine-1-phosphate transferase Reaction mixtures
contained the following reactants at the indicated final concentrations (final volume of 250 μl): luminol (20 mM), FeCl2–EDTA (25, 100 μM), H2O2 (3.5 mM) and aqueous solutions of antioxidant microcapsules or trolox (five concentrations). The chemiluminescence signal was detected in the microplate reader after 5 min of incubation. Gallic acid was used as positive control (IC50 = 0.11 μg/ml). The HOCl scavenging capacity was measured by monitoring the HOCl-induced oxidation of DHR to rhodamine 123 (Gomes et al., 2007). HOCl was prepared by adjusting the pH of a 1% (w/v) solution of NaOCl to 6.2, with 10% H2SO4 (v/v). The concentration of HOCl was determined spectrophotometrically at 235 nm using the molar absorption coefficient of 100 M−1 cm−1 and further dilutions were made in 100 mM phosphate buffer (pH 7.4).