Uv vis 1600 microprocessor double beam spectrophotometer

For in vitro DPPH radical scavenging activity assay, 12 mg of a stable free radical, 1,1-diphenyl-2-picryhydrazyl (DPPH), was accurately weighed using a Shimadzu analytical balance and dissolved in 100 mL of analytical grade methanol to give 0.3 mM solutions. One milliliter of the methanolic solution of 0.3 mM DPPH was added to 2.5 mL of each of the extract concentrations (1000, 100, 10, 1, 0.1 and 0.01 μg/mL). The mixtures were shaken and incubated for 15 minutes in the dark, at room temperature. After incubation, absorbance (A) was measured at λ₅₁₇ nm with a Shimadzu UV-VIS (1600) microprocessor double beam spectrophotometer. Percentage of the radical scavenging activity (% RSA) was calculated using the formula described by Brand Williams et al⁴⁸ :
DPPH Solution (2.5 mL) plus methanol (1 mL) was used as a negative control while methanol (2.5 mL) plus sample solution (1 mL) was used as a blank. In addition, L-ascorbic acid at concentrations equivalent to that of the test samples (100, 10, 1, 0.1, and 0.01 μg/mL) was used as positive control.⁴⁸