*2.7. Antioxidant Activity*

#### 2.7.1. Preparation of Sample

The solvent in the test samples (SDEO, FV, GBV and GBAF) were removed under a nitrogen stream. The resulting residues were dissolved in *n*-pentane:diethyl ether (1:1). BHA, BHT and ascorbic acid all diluted to a concentration of 1000 μg per mL in methanol were used as positive controls for the antioxidant activity assays.

#### 2.7.2. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) Free Radical-Scavenging Activity

DPPH radical scavenging activity was determined according to the method described by Thaipong et al. [39] with some modifications. For calculation of e ffective concentration EC50 value, a stock solution of DPPH was freshly prepared by dissolving 240 mg DPPH in methanol (1000 mL) and the working solution was prepared by diluting stock solution with methanol to obtain an absorbance of 1.1 ± 0.02 units at 517 nm using an ultraviolet–visible (UV–vis) spectrophotometer (Shimadzu UV-1601, Osaka, Japan). 100 μL of the samples (SDEO, FV and TBAF) and chemicals were allowed to react with 0.1M Tris-HCl bu ffer (900 μL) and 500 μM DPPH solution (1000 μL) for 20 min at RT in the dark. Then absorbance was taken at 517 nm using UV–vis spectrophotometer. The EC50 (μg/mL) were calculated from the regression curves using six di fferent concentrations (10–100 μg/mL) of samples and chemicals. The results were expressed as EC50 value (μg/mL). As a blank, the test was repeated using bu ffer instead of samples, and the DPPH radical-scavenging activity of the extracts was calculated against a blank as follows:

#### DPPH radical-scavenging activity (%) = (1 − A0/A1) × 100

where A0 and A1 are absorbance values of the test sample and control, respectively. 2.7.3. ABTS (2,2-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid)) Free Radical-Scavenging Activity

ABTS free radical scavenging activity was determined by the methods of Thaipong et al. [39] with some modifications. Briefly, a mixture of ABTS (7.4 mM) solution and potassium persulfate (2.6 mM) solution in 1:1 ratio was kept at room temperature for 12 h under dark condition to form ABTS cation. The solution was diluted by adding methanol to obtain an absorbance of 1.1 ± 0.02 at 734 nm. All the required solutions were freshly prepared for each assay. 100 μL of the samples and chemicals were added to 1400 μL of the diluted ABTS solution and the mixture was incubated at room temperature for 2 h in a dark. After the reaction, its absorbance was measured at wavelength of 734 nm. The results were expressed as RC50 value (μg/mL), and also ABTS radical scavenging activity (%) was calculated with the following equation:

> ABTS radical scavenging activity (%) = (1 − A0/A1) × 100

where A0 and A1 are absorbance values of the test sample and control, respectively.

2.7.4. Ferric-Reducing Antioxidant Power (FRAP)

Ferric-reducing power was determined using FRAP assay [40] with some modification. The FRAP reagen<sup>t</sup> was prepared by mixing 10 volume of 300 mM acetate buffer (pH 3.6) with 1 volume of 10 mM TPTZ solution in 40 mM HCl and 1 volume of 20 mM ferric chloride solution. Sample extract (75 μL) was added to 1425 μL of FRAP reagent. The reaction mixture was then incubated at RT for 30 min in a dark. The reducing power was expressed as absorbance at 593 nm and RC50 values (μg/mL) of FRAP were calculated from the regression lines using six different concentrations (10–100 μg/mL) in triplicate.
