Cellular Antioxidant Activity (CAA)

Cellular antioxidant activity (CAA) was evaluated in VERO cell line culture (European Collection of Cell Cultures, ECACC 84113001) using 2´,7´-dichlorodihydrofluorescein diacetate (DCFH₂-DA) as a fluorescent probe.⁶² The cells were plated in sterile, white, polystyrene, flat-bottom 96-well microplates (Nunc, Denmark) at a concentration of 50,000 cells per well and incubated for 24 h at 37 °C and 5% CO₂ in RPMI 1640 culture medium. The cells were washed with 150 mL of phosphate buffered saline (PBS) pH 7.4 and incubated for 1 h with 100 µL of RPMI 1640 containing 20 µM. The aqueous extract and AgNPs-Sm were added in a final concentration of 21 mg mL⁻¹. After 2 h of incubation, the medium was discarded, and the cells were gently washed twice with 100 µL of PBS. Then, they were incubated with 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) (Sigma‐Aldrich 97%, St Louis, MO, USA) at a final concentration of 600 µM in PBS. Fluorescence was measured immediately after addition in a spectrofluorometer (Bio‐Tek Instruments, Winooski, VT, USA) in 96-well plates at 37 °C using an excitation of 485 nm and an emission of 538 nm. The evaluation was carried out every min for 1 hr, and the CAA values were calculated with Equation (1):

where F is the fluorescence intensity in the presence of free radicals without the extract, and F_AH is the fluorescence intensity in the presence of free radicals, extract, and AgNPs-Sm. All measurements were done in the same period.