4.6. Antioxidant Activity Study

ABTS radical scavenging activity of AE/ChS, GCBE/ChS, AE, and GCBE was assessed by measuring the disappearance of the bluish-green color of the aqueous ABTS radical cation solution [52]. First of all, ABTS was dissolved in water to make a 7 mM solution. ABTS radical cation (ABTS^•+) was produced by reacting ABTS stock solution with 2.45 mM potassium persulfate in equal quantities (V/V) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. As ABTS and potassium persulfate react stoichiometrically at a ratio of 1 to 0.5, this will result in incomplete oxidation of the ABTS. For the study of natural extracts and their complexes with ChS, the ABTS^•+ solution was diluted with phosphate buffer solution pH 4.1, to adjust a light absorbance at 734 nm to 1 ± 0.02 a.u. A total of 0.00183 g of AE/ChS or 0.00125 g of GCBE/ChS dried powder was mixed with 50 mL of diluted ABTS^•+ solution and stirred at 300 rpm at room temperature. At certain periods of time, the absorbance of ABTS^•+/phosphate buffer solution at 734 nm was measured with a visible light spectrophotometer T60 Vis (PG Instruments Ltd., Wibtoft, UK). The ABTS^•+/phosphate buffer solution free radical scavenging activity (RSA, in%) was calculated using the following equation [52]:

where A_initial is the absorbance of the initial ABTS^•+/phosphate buffer solution, and A_t is the absorbance of ABTS^•+/phosphate buffer solution containing the sample at the time t.

In the same way, experiments with AE and GCBE extracts were performed.