Here is the procedure i am following for the DPPH assay. I am not sure on how do I prepare the 500 microM DPPH in ethanol like how many mg of DPPH should be dissolve to get this molarity. I am confused as well with the sequence, it says to take an aliquot of 100 microL added to reaction medium so that I can get to the desired concentrations. I was thinking that I should first reach the desired concentrations before adding it to the reaction medium (with DPPH soln).

Antioxidant activity was evaluated by the DPPH assay as described by Blois [38].

Firstly, a stock solution (2 mg extract/mL ethanol) was prepared. From this solution several

dilutions were carried out. An aliquot (100 microL) of each dilution was added to the reaction

medium to reach concentrations between 3.33 microg/mL and 66.67 mcg/mL Subsequently,

1 mL of acetate buffer at pH 6.0, 1.4 mL of ethanol and 500 microL of DPPH (500 microM in ethanol) were added. The solutions were stirred for 30 s and afterwards left for 30 min in the dark.

Absorbance at 517 nm was measured against a blank using a UV-visible spectrophotometer

(Cintra 101). A control solution containing 500 microL of DPPH (500 microM in ethanol), 1 mL of

acetate buffer at pH 6.0 and 1.5 mL ethanol was used. The extract antioxidant activity was

evaluated from the EC50 value.