2.10.6. MAO-B Activity Assay

PD-induced model cells were treated with serial dilutions of the SC derivatives (65.2–8000 µg/mL) and control samples (no treatment) and incubated for an additional 24 h (Scheme 1). Cells were then lysed by the frost-defrost method followed by differential centrifugation to isolate samples containing the MAO-B enzyme from cellular debris. The collected extracts were subsequently transferred to a new container for storage at −80 °C until further use.

The functional MAO-B activity in 25 µL samples of the cell lysates (around 0.4 mg/mL of total protein) was determined using the Fluoro:MAO^TM kit from Cell Technology, following the manufacturer’s recommendations, with minor modifications. The substrate for the enzymatic reaction was Tyramine. The MAO-B inhibitor pargyline was used as positive control, while the reaction sample buffer was the negative one. The product formed was determined from a hydrogen peroxide (H₂O₂) calibration curve. One unit of MAO-B catalyzes the formation of 1 µmole of (H₂O₂) per minute under assay conditions (Unit/mL = µmol/min/mL). MAO-B activity is presented relative to the negative control, which corresponds to 0% inhibition (i.e., 100% MAO-B activity).