DPPH Assay
For the DPPH radical scavenging assay, 20 μL of extract diluted appropriately in DMSO was mixed
with 180 μL of DPPH in methanol (40 μg/mL) in wells of a 96-well plate. The plate was kept in the
dark for 15 min, after which the absorbance of the solution was measured at 540 nm in a Multiskan
Ascent plate-reader (Thermo Electron Corporation, Basingstoke, UK). Appropriate blanks (DMSO)
and standards (quercetin solutions in DMSO) were run simultaneously. Extracts were first tested at a
single concentration of 4 mg/mL, and those showing good evidence of antioxidant activity were tested
over a range of concentrations to establish the EC50 (the concentration reducing DPPH absorbance by
50%). This method follows closely that used by previous workers [4–7].