2.7.1. Superoxide-Radical Scavenging Activity Assay

The superoxide-radical scavenging ability of the chitosan nanoparticles was assessed following Tan’s methods [16]. Firstly, the tested samples were dissolved in deionized water to ensure the initial concentration of 10 mg/mL. Different volumes of initial sample solution (30, 60, 120, 240, and 480 μL) were then transferred to a centrifuge tube and deionized water was added to ensure a volume of 1.5 mL. Afterwrad, the 3 mL of total reaction mixture, involving 1.5 mL of tested samples, 0.5 mL of nitro blue tetrazolium (NBT, 72 μM), 0.5 mL of phenazine methosulfate (PMS, 30 μM), and 0.5 mL of nicotinamide adenine dinucleotide reduced (NADH, 338 μM) in Tris-HCl buffer (16 mM, pH 8.0), was incubated at 25 °C for 5 min. The absorbance was measured at 560 nm, and three replicates for every tested sample, control, and blank were recorded. The superoxide-radical scavenging effect was calculated using the following equation:

where A_{sample 560 nm} is the absorbance of the samples, A_{control 560 nm} is the absorbance of the control (NADH was substituted with distilled water), and A_{blank 560 nm} is the absorbance of the blank (samples were substituted with distilled water).