4.5.1. Antioxidant Activity Assay by DPPH Method

The antioxidant effect of new compounds was determined according to the well-known DPPH assay based on the fact that the antioxidant samples react with stable 2,2-diphenyl-1- (2,4,6-trinitrophenyl)hydrazin-1-yl (2,2-diphenyl-1-picrylhydrazyl, DPPH), an effective free radical trap with an absorption band at 517 nm, which converts to its reduced form, i.e., 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine (2,2-diphenyl-1-picryl hydrazine), with a color change from purple to yellow accompanied by a decrease in absorption at 517 nm [84].

The antioxidant potential of the tested compounds was investigated according to the method previously described by Blois [87–89], with some modifications, and compared with the free radical scavenging activity of AA, BHA, and BHT standards.

Briefly, 2 mL of a solution of DPPH in ethanol at a concentration of 400 μM was added to 2 mL of each solution of the tested compound in DMSO at a concentration of 500 μM. After maintaining the samples in the dark at room temperature for 30 min, the absorbance of each sample at the wavelength of 517 nm was measured on a Specord 40 UV/VIS

spectrophotometer (Analytik Jena AG, Jena, Germany). Then, the radical scavenging activity (*RSA*) in percent (%) was calculated using the following formula:

$$RSA\ (\%) = (1 - \frac{A\_{\text{sample}} - A\_{\text{blank sample}}}{A\_{\text{DPP control}}}) \times 100$$

where *A*sample is the absorbance of the tested compound solution with the DPPH solution, *A*blank sample is the absorbance of the tested compound solution (without the DPPH solution), and *A*DPPH control is the absorbance of the DPPH solution (without the tested compound solution) [90]. Each analysis was performed on three replicates and the results were averaged.

## 4.5.2. Antioxidant Activity Assay by ABTS Method

The ABTS discoloration test is based on the ability of the antioxidant agents to scavenge the long-life ABTS radical cation, ABTS•<sup>+</sup> [91]. The stable green-blue radical cationic chromophore, ABTS•+, which has an absorption maximum at the wavelength of 734 nm, by reaction with most antioxidants turns into its colorless neutral form, i.e., 2,2 -azino-bis(3 ethylbenzothiazoline-6-sulfonic acid) (ABTS) [85].

The antioxidant capacity of the tested compounds was assessed as described earlier by Re et al. [91], with some modifications. The ABTS•<sup>+</sup> is prepared by oxidation of ABTS with radical-initiator potassium persulfate. Initially, the stock solution of ABTS was obtained by dissolving it in water at a concentration of 7 μM. The ABTS radical cation (ABTS•+) was then generated by reacting the 7 μM ABTS stock solution with a 2.45 μM K2S2O8 solution (1:1, *V*/*V*) and kept in the dark at room temperature for 12–16 h. Before analysis, this solution was diluted with ethanol and equilibrated at 30 ◦C to have absorbance at 734 nm, *A*<sup>734</sup> of 0.7000 ± 0.02. Subsequently, 2 mL of ABTS•<sup>+</sup> solution was added to 2 mL of 500 μM tested compound solution in DMSO. After 6 min, the absorbance of each sample was read at 734 nm with a UV/VIS spectrophotometer (Specord 40, Analytik Jena AG, Jena, Germany) and converted into the percentage radical scavenging activity, %*RSA*, using the formula:

$$RSA\ (\%) = (1 - \frac{A\_{\text{sample}} - A\_{\text{blank sample}}}{A\_{\text{AETS}^+} \text{ control}}) \times 100$$

where *A*sample is the absorbance of the tested compound solution with the ABTS•<sup>+</sup> solution, *A*blank sample is the absorbance of the tested compound solution (without the ABTS•<sup>+</sup> solution), and *A*ABTS•<sup>+</sup> control is the absorbance of the ABTS•<sup>+</sup> solution (without the tested compound solution) [89,90]. All determinations were undertaken in triplicate and the results were averaged. The same antioxidant agents (AA, BHA, and BHT) were used as positive controls.
