2.7.7.2. ABTS Radical Scavenging Activity

The mixture of ABTS (7 mM) and potassium persulfate (2.45 mM) with a volume ratio of 1:1 generates a green-blue reagent (ABTS+) after 12–16 h incubation in the dark, and has maximum absorption at 734 nm. When this cationic radical is exposed to the hydrogen donating compound, the green-blue is decolorized and the color intensity is measured at 734 nm. To measure ABTS radical scavenging activity, 40 μL of the prepared hydrolysate concentration was mixed with 4 mL of diluted ABTS solution, vortexed vigorously for 30 s, and incubated in the dark for 6 min. The absorbance was measured at 734 nm. The ABTS radical scavenging activity was calculated by the following equation [20]:

$$\text{ABTS radical saving activity (\%)} = \frac{A\_{\text{control}} - A\_{\text{sample}}}{A\_{\text{control}}} \times 100\tag{7}$$

#### 2.7.7.3. Ferric Reducing/Antioxidant Power (FRAP) Assay

The capability of hydrolysate to reduce Fe+3 ions present in the complex to a Fe+2 form with 2,4,6-tri (2-pyridyl)-s-triazine (TPTZ) was determined by the ferric ion reducing capacity (FRAP) assay as described previously. The FRAP reagent was freshly prepared by mixing TPTZ (10 mM) dissolved in 40 mM HCL, iron (III) chloride hexahydrate (20 mM) dissolved in water, and acetate buffer (0.3 mM) at pH 3.6 at a ratio of 1:1:10 (*v/v/v*) and warming it to 37 ◦C. Then, 900 μL of the working solution was mixed with 100 μL of different concentrations of hydrolysate and the corresponding nano-formulated system, followed by incubation of the mixture at 37 ◦C for 30 min, and the resulting blue color absorbance at 595 nm was recorded. Different concentrations of FeSO4, in the range 0–1 mM, were used as the calibration curve [21].
