2.6.3. Hydroxyl radical scavenging assay

The hydroxyl radical scavenging activity was analyzed using a chemiluminescence method [22, 23]. A carbonate buffer solution (pH 8.5) with a concentration of 50 mmol/L and a luminol solution of 1 mmol/L was mixed at a ratio of 17:1 (working solution) away from light. For the assay, 10 μL of different concentrations of the spent substrate extract of C. militaris, 10 μL of a 6% H₂O₂ solution, 10 μL of a 1 mmol/L CuCl solution, 10 μL of a 1 mmol/L o-phenanthroline solution, and 150 μL of the working solution were added to a 96-well plate. The reaction time was set for 2 s. Chemiluminescence was measured and recorded continuously for 15 s to obtain the highest value, A1, with deionized water as blank control. The peak control value was measured and recorded as A0. Three replicate wells were used in each group, and the average value was calculated. The formula for hydroxyl radical clearance was: