"To calculate the DPPH IC50 value, you should start with an initial concentration of 1mg/ml and dilute it to 25, 50, 100, 150, and so on."

Dear Ankit , yes but concentrations change in the reaction medium , for example: concentration 1000 ug/ml become 200 ug/ml if volume of extract in reaction is 40 ul and other dpph with methanol in reaction is 160 so the total volume will be 200 ul .. so the initial concentration will change .

For IC50, a reaction mixture containing different concentrations of given plant extract (25, 50, 75 & 100 µg/ml) was added to a DPPH reaction mixture. The reaction mixture was shaken tremendously and kept in the dark for 30 minutes. Finally, take the absorbance of the reaction mixture at 517 nm.