Excretion Experiments Using MDCKII-UGT1A1/MRP2 Cells

Before experiments, the MDCKII-MRP2/UGT1A1 cells were washed twice with pre-warmed (37°C) HBSS buffer (Hank’s balanced salt solution, PH=7.4). The cells adapted itself to the condition in 37°C HBSS buffer for 30 minutes. Then the cells were incubated with a HBSS buffer containing these flavones(3HF, 3′HF, 6HF, 7HF, 4′HF, 3,3′DHF, 3,7DHF, 3,5,7THF, 3,6,4′THF, 3,7,4′THF, 3,5,7,4′QHF, 3,7,3′4′QHF and Quercetin) (defined as the “loading solution”). The aglycone substrate concentrations in the range of 0.3125–20um were used unless method sensitivity or substrate solubility necessitated otherwise. In the loading solution, the proper concentration was diluted from 1000X concentrated stock solutions in organic solvent (DMSO/Ethanol=1:4), so that the final concentration of organic solvents were 0.1% (v/v) of the experimental media. The sampling times were selected to ensure that the amounts excreted vs. time plots stay in the linear range. At each time point, 200μl of incubating media from each well was collected and the same volume of loading solution was used to replenish each well. The collected incubating media were mixed with 50 μl of “Stop Solution” consisted of 94%acetonitrile and 6% acetic acid containing 100uM testosterone as the internal standard. Supernatants were ready for UPLC analysis after centrifugation (15 min at 15,000 rpm). All experiments were performed in triplicates.