Revisiting DPPH (2,2-diphenyl-1-picrylhydrazyl) assay as a useful tool in antioxidant evaluation: a new IC100 concept to address its limitations

The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay has been widely used in antioxidant evaluation. However, it suffers from certain limitations that are addressed in this contribution. The limitations discussed in this work were the ratio of DPPH radicals to antioxidants and the presence of pigments in the reaction medium which interferes with absorbance readings. To do so, we used eight different concentrations of DPPH solution. The modified DPPH assay proposes a new concept, IC100, that is defined as the amount of DPPH radical required to oxidize all antioxidants present in the reaction medium. The modified DPPH assay does not suffer from an underestimation of antioxidant activity found in the original DPPH procedure due to the decrease in the ratio of DPPH radicals to antioxidants. Moreover, the modified method was not influenced by interference from coexisting pigments in the measurement of radical scavenging potential of extracts. To the best of our knowledge, this is the first attempt to effectively resolve the above-mentioned limitations of the DPPH assay.