2.11. In Vitro Determination of the Antioxidant Activity of EGCG-grafted-Chitosan

The same concentrations of EGCG and nanoparticles as those used in the bacterial growth inhibition tests were employed to determine antioxidant activity using ABTS, DPPH, and FRAP assays. The ABTS and DPPH tests were used to evaluate the radical-trapping activity, while the FRAP test was used to evaluate the reducing power of the developed materials [39].

ABTS radicals were propagated by oxidation of ABTS (7.0 mM) with potassium persulfate (K₂S₂O₈, 4.95 mM) in the dark (12 h) at 25 °C. Then, ABTS was diluted with PBS (0.2 M, pH 7.4) to reach an absorbance value of 0.7 measured at 734 nm. The absorbance value of a mixture of a 20 μL sample and 200 μL of the working solution was measured at 734 nm by a microplate reader (Veloskan™ LUX, Thermo Fisher Scientific, Waltham, MA, USA) after 30 min of reaction. The scavenging activity was reported as ABTS radical inhibition (%) and calculated according to Equation (3).

where the absorbance of water instead of the sample was the control (Abs₀); the absorbance of the sample with ABTS is Abs₁ and the absorbance of the water instead of ABTS is Abs₂. Trolox standard solution was prepared and assayed under the same conditions. Results are expressed in terms of TEAC, which represents the mmol/L Trolox equiv/g sample.

DPPH radical scavenging assays were performed according to Hu et al. [15]. Pure EGCG or the nanoparticles were homogeneously dispersed in water at the same concentrations as those used to evaluate bacterial growth inhibition. The samples (50 μL) and methanolic DPPH solution (200 μL) at 0.4 mM were added to a 96-well microplate. The reaction was carried out in the dark at 25 °C/30 min. The absorbance was measured at 515 nm by using a microplate reader (Veloskan™ LUX, Thermo Fisher Scientific, Waltham, MA, USA). A Trolox standard solution was prepared and assayed under the same conditions. The scavenging activity was measured as the decrease in absorbance of the DPPH, and it is expressed as percent inhibition of DPPH radicals calculated according to Equation (3). Additionally, antioxidant concentration corresponding to 50% inhibition of the DPPH radical (EC50 DPPH), which was calculated from the graph of scavenging percentage versus concentration of the antioxidant (mg/mL) tested using linear regression.

Ferric reducing ability was evaluated based on FRAP assays [40]. The FRAP reagent was prepared from acetate buffer (pH 3.6), 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) solution in 40 mM HCl, and 20 mM iron (III) chloride solution in proportions of 10:1:1 (v/v/v). EGCG or the nanoparticles (20 μL) were added to 280 μL of the FRAP reagent. A Trolox standard solution was prepared and assayed under the same conditions. The absorbance of the reaction mixture was recorded at 638 nm after 30 min using a microplate reader (Veloskan™ LUX, Thermo Fisher Scientific, Waltham, MA, USA). The results are expressed as micromoles of Trolox equivalents per g of dry weight (μmol TE/g).