*2.6. Antioxidant Activity*

2.6.1. Evaluating the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Assay

The free radical scavenging capacity of *G. littoralis* samples was measured using a DPPH radical scavenging assay, following the method Chung et al. [35] described previously. In triplicate, an aliquot of 200 μL of different sample extracts was mixed with 4.5 mL DPPH (0.004% in methanol). The reaction mixture was homogenized manually and incubated at room temperature (25 ◦C) for 40 min. The mixture was shaken and kept in dark conditions for 45 min. Then, the absorbance value was taken using a spectrophotometer (Jasco V530 UV-VIS spectrophotometer, Tokyo, Japan) at 517 nm. A blank was prepared by replacing DPPH with 80% methanol in the reaction medium. BHT was used as the positive control.

The free radical scavenging ability of the sample was measured from the following equation:

$$\text{DPPH} \text{ scavenging activity (\%)} = (\text{Abs}\_{\text{control}} - \text{Abs}\_{\text{sample}}) / \text{Abs}\_{\text{control}} \times 100\%$$

where Abscontrol represent the absorbance of the mixture + methanol, and Abssample represent the absorbance of the mixture + plant extract. The antioxidant activity was expressed as the capacity to scavenge or reduce the DPPH radical by 50%; that is, the amount of antioxidant compounds required to scavenge or reduce the initial concentration of DPPH by 50%.

#### 2.6.2. Evaluation of the 2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) Assay

The antioxidant activity was determined using the ABTS radical method described elsewhere [35]. Briefly, the ABTS solution used in the experiments was formed by mixing 7.4 mM ABTS and 2.6 mM potassium persulphate (1:1, *v/v*). The reaction solution was incubated for 12 h at room temperature. The solution was then diluted with 80% methanol until a solution with an absorbance of 0.70 ± 0.01 was achieved. Then, 1 mL diluted ABTS was mixed with 100 mL of the sample. Then, the absorbance was taken using a spectrophotometer (Jasco V530 UV-VIS spectrophotometer, Tokyo, Japan) at 734 nm. Trolox was used as a positive control. The standard curve was prepared from various concentration of trolox (500 μM, 600 μM, 700 μM, 800 μM, 900 μM, and 1000 μM).

The ABTS radical scavenging ability of the samples was measured from the following equation:

$$\text{ABTS savinging activity} = \text{(Abs}\_{\text{control}} - \text{Abs}\_{\text{sample}}) / \text{Abs}\_{\text{control}} \times 100$$

where Abscontrol represent the absorbance of the ABTS solution + methanol, and Abssample represent the absorbance of the ABTS solution + test sample.
