2.8.1. DPPH• Antioxidant Assay

The determination of free radical scavenging activity was determined according to the DPPH^• method previously described [14]. Firstly, a 24 µM ethanol solution of DPPH^• was prepared. Then, 450 µL of DPPH^• reagent previously prepared was mixed with 50 µL of extract (1.25–25 mg/mL) and they were incubated for 10 min at room temperature. After incubation, the absorbance was measured at 523 nm using a Microplate Spectrophotometer (PowerWave HT, BioTek Instruments, Inc., Winooski, VT, USA).

The scavenging effect of DPPH^• was measured using the formula

where A_control is the absorbance of the control (DPPH^• solution), and A_sample is the absorbance of the test sample (DPPH^• solution plus 50 µL of extract). The median inhibitory concentration (IC₅₀) was determined using linear regression. The scavenging activity was expressed as the IC₅₀ that represents the V. sphaerocephala extract concentration (mg/mL) needed to reduce by 50% the initial DPPH^• absorbance.