DPPH free radical scavenging assay

The free radical scavenging activity of the test sample was estimated based on the previously described method (Blois 1958) with some modifications. Briefly, 1 mL of DPPH solution (0.1 mM of DPPH in methanol) was mixed with 3 mL of various concentrations of the test sample (0–128 µg/mL). The mixture was incubated at room temperature for 30 min, and the absorbance was measured at 517 nm in a UV-Visible spectrophotometer (Shimadzu UV2600i, Kyoto, Japan). Reaction mixtures without the test sample and with BHA were used as negative and positive controls, respectively. The experiments were conducted in three biological replicates. IC₅₀ was calculated by analysing the linear regression of the obtained data.