*2.8. Antioxidant Activity*

#### 2.8.1. DPPH Radical Scavenging Activity

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of the extracts was measured with a modified version of the method of Blois (1958) [26] using 2,2-diphenyl-1-picrylhydrazyl and 80% methanol to prepare a 0.1 mM DPPH solution. The absorbance of the DPPH solution was adjusted to 0.650 ± 0.020 at 517 nm using 80% methanol. Extract (0.05 mL) was added to 2.95 mL of the adjusted DPPH solution, and the mixture was allowed to stand in a dark room at 23 ◦C for 30 min. Subsequently, the absorbance at 517 nm was measured. As standard and blank, ascorbic acid and distilled water were used, respectively. DPPH was calculated using the following Equation (3):

$$\% \text{ inhibition} = \frac{A\_{\text{reference}} - A\_{\text{sample}}}{A\_{\text{reference}}} \times 100 \tag{3}$$

where

<sup>A</sup>*reference* = absorbance of the blank <sup>A</sup>*sample* = absorbance of the sample <sup>A</sup>*reference* = mixture of 0.1 mL of 80% MeOH and 2.9 mL of DPPH radical solution.

#### 2.8.2. ABTS Radical Scavenging Activity

The 2,2--azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity was determined according to the following procedure [27,28]. The solutions of 1.0 mM 2,2--azobis-(2-amidinopropane) dihydrochloride and 2.5 mM 2,2--azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) were mixed in 100 mL PBS. The mixture was stirred at 70◦C for 30 min and then cooled to room temperature. The solution was filtered using a 0.45 μm syringe filter and diluted with PBS to achieve an absorbance of 0.650 ± 0.020 at 734 nm. The radical solution was stored at 37 ◦C. Extract (20 μL) and radical solution (980 μL) were mixed and reacted at 37 ◦C for 10 min, and the absorbance was measured at 734 nm. As standard and blank, ascorbic acid and distilled water were used, respectively.

#### *2.9. Total Phenolic Content*

Total phenolic content (TPC) was determined using the following procedure with some modifications [29,30]. To 2.6 mL of deionized water, 200 μL of Folin and Ciocalteu's phenol reagen<sup>t</sup> and 200 μL of extract were added and mixed. After 6 min, 2 mL of 7% Na2CO3 was added and mixed. The mixture was allowed to react at 25 ◦C for 90 min, and the absorbance was measured at 750 nm. Gallic acid was used as a standard, and deionized water was used as a blank.

#### *2.10. Total Flavonoid Content*

Total flavonoid content (TFC) was determined using the following method [31]. Distilled water (3.2 mL) and sample (0.5 mL) were mixed, and 0.15 mL of 5% NaNO2 was added. After 5 min, 0.15 mL of 10% AlCl3 was added. After 1 min, 1 mL of 1 M NaOH was added, and the absorbance was measured at 510 nm. Catechin was used as a standard, and distilled water was used as a blank.
