Guidelines for evaluating the antioxidant activity of lignin via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

The most widespread procedure to measure the antioxidant activity of lignin is via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. So far, different experimental procedures (i.e., different solvent, time, etc.) have been used to implement the DPPH methodology without estimating the effect of such modifications on the experimental procedure. To overcome this issue, the impact of the solvent, the time, and the type of substrate on the evaluation of the antioxidant activity (AoA) of lignin via the DPPH assay was investigated in this work. We found that multiple different parameters affect the evaluation of the AoA of lignin: i) the stability of the DPPH radical and the lignin solubility in a given solvent; ii) the importance of reaching steady state (the effect of time); iii) the background noise associated with lignin absorbance at λ = 515 nm (used to monitor the DPPH radical scavenging); iv) lignin structure; v) providing a normalized radical scavenging index (nRSI); vi) comparing nRSI vs. inhibition percentage (IP) values. Overall, our investigation allowed us to provide guidelines on how to perform the DPPH assay for a more reliable evaluation of lignin AoA.