Reaction time and DPPH concentration influence antioxidant activity and kinetic parameters of bioactive molecules and plant extracts in the reaction with the DPPH radical

The antioxidant activity of lemon and pomegranate juice, infusion of Java green tea, rosemary oil and pure chemicals was studied by the DPPH method. The chemical composition of the samples was determined by liquid chromatography-mass spectrometry (LC-MS). The kinetics of the reaction is different among extracts. The reaction of DPPH with lemon juice is complete in 3 min; 87 and 98 min are necessary for green tea infusion and pomegranate juice, respectively; 283 min are needed for rosemary essential oil. In samples with an intermediate and slow kinetic behavior, the antioxidant activity measured after 2 h of reaction is significantly higher than that measured after 30 min. These differences are 18% for Java green tea infusion, 21% for rosemary essential oil, 23% for pomegranate juice. The results demonstrate that the percentage of remaining DPPH decreases with increasing initial DPPH concentrations (between 25 and 200 μM), while keeping constant antioxidant/DPPH ratio. Moreover the time necessary to reach the steady state is dependent, while keeping the same antioxidant/DPPH ratio, on the initial DPPH concentration, showing that longer time intervals are required when using lower DPPH concentrations (25 μM). These results confirm the necessity of standardizing the method to be able to compare results from different laboratories.