*3.6. Antioxidant and Cytotoxic Activities*

#### 3.6.1. Free Radical Scavenging Activity

The free radical scavenging activity of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was determined using the DPPH (1,1-diphenyl-1-picrylhydrazyl) method [38]. The samples were tested at different concentrations (0.0625–2 mg/mL). An aliquot (0.5 mL) of solution containing different amounts of sample was added to 3 mL of daily prepared methanol DPPH solution (0.1 mM). The optical density change at 517 nm was measured, 20 min after the initial mixing, with a model UV-1601 spectrophotometer (Shimadzu). Butylated hydroxytoluene (BHT) was used as reference.

The scavenging activity was measured as the decrease in the absorbance of the samples versus DPPH standard solution. Results were expressed as the radical scavenging activity percentage (%) of the DPPH, defined by the formula [(Ao − Ac)/Ao] × 100, where Ao is the absorbance of the control and Ac is the absorbance in the presence of the sample or standard.

The results, obtained from the average of three independent experiments, are reported as mean radical scavenging activity percentage (%) ± standard deviation (SD) and mean 50% inhibitory concentration (IC50) ± SD. The IC50 value is a parameter calculated as the concentration of extract needed to decrease the initial DPPH concentration by 50%. Thus, the lower IC50 value, the higher the antioxidant activity of the sample.

#### 3.6.2. Reducing Power Assay

The reducing power of *Em*-L, *Em*-A, *Es*-L, *Es*-A, and *Cv*-L extracts was evaluated by the spectrophotometric detection of Fe3+-Fe2+ transformation method [39]. The extracts were tested at different concentrations ranging from 0.0625 to 2 mg/mL. Solutions of different concentrations of extracts in 1 mL solvent were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide [K3Fe(CN)6], and the resulting mixture was incubated at 50 ◦C for 20 min. The solution was cooled rapidly, mixed with 2.5 mL of 10% trichloroacetic acid, and centrifuged at 3000 rpm for 10 min. After centrifugation, the supernatant (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% fresh ferric chloride (FeCl3). The absorbance of the solution was measured at a wavelength of 700 nm after 10 min. An increase in the absorbance of the reaction mixture indicates an increase in its reducing power. An equal volume (1 mL) of water mixed with a solution prepared as described above was used as a blank. Ascorbic acid and BHT were used as references. The results averaged from three independent experiments were expressed as mean absorbance values ± SD. The reducing power was also expressed as ascorbic acid equivalent (ASE/mL); when the reducing power is 1 ASE/mL, the reducing power of 1 mL extract is equivalent to 1 μmol ascorbic acid.
