4.6. P-gp ATPase Activity Assay

P-gp ATPase activity was measured using P-gp-Glo™ assay systems kit according to the manufacturer’s instructions, the method detail of which was also described in our previous study [29]. The P-gp-Glo™ assay system kit mainly contains recombinant human P-gp membranes, ATP detection substrate, ATP detection buffer., P-gp-Glo™ assay buffer, Mg-ATP, verapamil (a positive control substrate of P-gp), and sodium orthovanadate (Na₃VO₄, a P-gp ATPase inhibitor). This study was performed to observe the effect of OPD on P-gp ATPase activity and a total of 6 groups (n = 3 for each group) were included as follows: the untreated group, the Na₃VO₄ group, the verapamil 100 µmol/L group, and OPD at concentrations of 3.5, 17.5 and 70 μmol/L groups. Those treatments were added into the solution containing 25 mmol/L Mg-ATP and 1.25 mg/mL recombinant human P-gp membranes, respectively and incubated at 37 °C for 40 min. After 40 min, ATP detection reagent was added, respectively. The unconsumed ATP detection reagent reacted with luciferase at room temperature for 20 min and the chemiluminescence intensity was detected. The average relative light units (RLU) was calculated as follows:

ΔRLU_basal, the decrease in luminescence of the untreated group (RLU_NT) compared to the Na₃VO₄ group (RLU_Na3VO4), represents the basal P-gp ATPase activity. ΔRLU_TC, the decrease in luminescence of the test compound-treated group (RLU_TC) compared to the Na₃VO₄ group (RLU_Na3VO4), indicates the P-gp ATPase activity in the presence of the test compound. The decrease in luminescence reflect ATP consumption by P-gp and is proportional to P-gp activity.