2.1.3. Antioxidant Activity

The antioxidant activity was also measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavening method, described by Brand-Williams et al. [21] and Sánchez-Moreno et al. [22]. Each extract was diluted with water or ethanol in a 1000 ppm solution. A DPPH radical solution was prepared with 0.0063 g DPPH in 250 mL ethanol. Then, 3.9 mL of DPPH radical solution was added to 100 μL of sample and a standard solution of 500 μM Trolox. After incubation in the dark at room temperature for 30 min, the absorbance was measured at 515 nm. The % chelating activity was calculated using the following formula: [(Abs DPPH − Abs Sample)/Abs DPPH] × 100 [23].

Radical cation scavenging capacity against ABTS+ radical (2,2-Azinobis (3-ethylbenzothiazolin) -6-sulphonic acid) was measured as by Re et al. [24]. Each extract was diluted with water or ethanol in a 1000 ppm solution. ABTS radical cations were prepared by reacting 7 mM ABTS (2,2-Azinobis (3-ethylbenzothiazolin) -6-sulphonic acid) with 2.45 mM potassium persulphate (1:1, *v*/*v*) pH = 7.4. This solution was diluted with distilled water to an absorbance of 0.7000 at 734 nm. Then, 1 mL of ABTS solution was added to 100 μL of sample and standard solution of 500 μM Trolox. After incubation for 2 min, the absorbance was measured at 734 nm. The % chelating activity was calculated using this formula: [(Abs ABTS − Abs Sample)/Abs ABTS] × 100.

The hydrophilic antoxidant capacity was measured using the ORAC (Oxygen Radical Absorbance Capacity) method described by Prior et al. [25]. The reaction was carried out in a phosphate buffer (0.075 M, pH 7.0). For this, 20 μL of sample, at different concentrations, and Trolox standard solutions (6.25, 12.5, 25, 50 μM), were pipetted into the wells of a 96-well black microplate. When the microplate was ready, 200 μL of 0.04 μM fluorescein were dispensed into each well. Samples were incubated for 15 min at 37 ◦C in the dark and the reaction was started by adding 20 μL of 40 mM AAPH (2,2-Azobis(2-amidinopropane)dihydrochloride) to each well. The flourescence decay was measured every 2 min at 37 ◦C using the microplate reader Biotek Synergy HT at 485 nm excitation and 538 nm emission until zero fluorescence was detected. All samples were prepared in triplicate and at a minimum of three different concentrations. The antioxidant activity of the sample was expressed as μM of Trolox Equivalents (TE) per g of extract, with the following formula: *(C* × *DF)/a;* where *C* is obtained from the area under the fluorescence decay curve of fluorescein in the presence of sample (AUC net = AUC sample − AUC blank); *DF* is the dilution factor; and *a* is the weight of the sample. A Biotek Synergy HT fluorescent microplate reader (Biotek Instruments, Winooski, VT, USA) was used with an excitation wavelength of 485 nm and an emission wavelength of 528 nm; and a UV2 spectrophotometer (Pye Unicam Ltd., Cambridge, UK) at different wavelengths depending on the method to be performed.

The total antioxidant activity was also determined using the method described by Benzie and Strain [26] with some modifications. Each extract was diluted with water or ethanol in a 1000 ppm solution. Then, the FRAP reagen<sup>t</sup> was prepared with 20 mL 300 mmol/L acetate buffer pH = 3.6, 2 mL 20 mmol/L FeCl3·6H2O and 2 mL 10 mmol/L TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mmol/L HCl. Then, 1 mL of the FRAP reagen<sup>t</sup> was added to 100 μL of sample and a standard solution of 500 μM Trolox. After incubation for 4 min, the absorbance was measured at 593 nm. The antioxidant power was expressed as μM Trolox Equivalents (TE) per g extract.
