The fluorescence signals were measured in the microplate reader at 528 ± 20 nm for emission and TSA HDAC cell line 485 ± 20 nm for excitation. The fluorescence signal was measured immediately after HOCl addition. Cysteine was used as positive control (IC50 = 0.07 μg/ml). The ONOO− scavenging capacity was measured by monitoring the ONOO−-induced
oxidation of non-fluorescent DHR to fluorescent rhodamine (Gomes et al., 2007). ONOO− was synthesized as previously described by Gomes, Costa, Lima, and Fernandes (2006). Reaction mixtures contained the following reactants at the indicated final concentrations (final volume of 300 μl): DHR (5 μM), ONOO− (600 nM) and aqueous solutions of antioxidant microcapsules or trolox (five concentrations). The fluorescence signal was measured in the microplate reader after 5 min incubation, with wavelengths of emission at 528 ± 20 nm and excitation at 485 ± 20 nm. In a parallel set of experiments, the assays were performed in the presence of 25 mM NaHCO3 in order to simulate the physiological CO2 concentration. This evaluation is important because, under physiological conditions, the reaction between ONOO− and bicarbonate is predominant (k = 3–5.8 × 104 M−1 s−1), generating
nitrogen dioxide ( NO2) and carbonate radical anion (CO3 −). Ascorbic acid was used as positive control (IC50 = 0.22 μg/ml PD98059 cell line and IC50 = 0.31 μg/ml in the absence and presence of NaHCO3, respectively). Protein content was determined according to the Kjeldahl method (AOAC, 1997), using the conversion factor of 6.25. The amino acid analysis was carried out according to White, Hart, and Kry (1986). Both analyses were performed in duplicate. Two approaches were used to present and discuss the capacity of GA and MD microcapsules to scavenge ROS and RNS. The first one aimed to compare the antioxidant capacity of the microcapsules as a whole, regardless the fact that they do not have the same antioxidant concentration (Table 1). The second approach discusses the effects of the addition of 1 μmol of antioxidant molecule
per gramme of biopolymer (GA or MD) in comparison to the biopolymer alone (empty microcapsule) (Table 2). Except for trolox, it is not possible to compare the microencapsulated antioxidants with the correspondent not microencapsulated ones since carotenoids Cyclin-dependent kinase 3 and tocopherol are lipophilic, thus they are not soluble in the solvents used in the methods. Microencapsulation, both using GA and MD as wall material, resulted in suppression of trolox scavenging capacities against HO and ONOO− (Table 3). However, microencapsulation of trolox with GA improved the ROO , H2O2 and HOCl scavenging capacity as compared to trolox alone, being about 2-, 57- and 96-fold more potent, respectively (Table 3). Both empty microcapsules presented capacity to scavenge ROO , although GA was more potent than MD (Fig. 1).