*2.4. Cytotoxicity Assay*

The cytotoxicity of *R. acetosa* extract on Caco-2 cells and HEK293 cells was measured using an EZ-Cytox cell viability assay kit (Daeil Lab Service, Seoul, Korea). The cells were cultured in DMEM containing 10% FBS, 1% NEAA, 10-mM HEPES, 100 U/mL of penicillin and 100-μg/mL streptomycin without phenol red. The seeding density was 3 × 10<sup>4</sup> cells/well for Caco-2 cells and 2.5 × 10<sup>4</sup> cell/well for HEK293 cells, respectively. The Caco-2 cells were incubated for 7 days and the HEK 293 cells were incubated for 24 h after seeding. The medium was replaced with 50 μL of new medium containing *R. acetosa* extract at the concentrations of 1, 2, 5, 10, 20, 50 and 100 μg/mL achieved the 0.5% of DMSO content. After 15 min of incubation, 5 μL of EZ-Cytox reagen<sup>t</sup> (water-soluble tetrazolium) was added to the cells, and the cells were incubated for 3 h. Cell viability was calculated as a percentage of the absorbance at 450 nm compared to untreated cells.

#### *2.5. P-gp Inhibition Test of Anthraquinones and R. acetosa Extract*

The *P*-gp inhibition effect of anthraquinones from *R. acetosa* was evaluated via MDR assay kit using Caco-2 cells. It was reported that verapamil has concentration-dependent inhibition effects on absorptive and secretory transporters. Accordingly, 100-μM verapamil was used as a positive control [28]. Caco-2 cells were cultured in 96-well plates at a density of 5 × 10<sup>5</sup> cells/mL and incubated in humidified 5% CO2 at 37 ◦C for 24 h. They were treated with 6 test compounds (10 μM) [18] or *R. acetosa* extract in HBSS and incubated for 15 min. The concentration levels of *R. acetosa* extract were 5, 10, 25 and 50 μg/mL. The MDR dye-loading solution was added at a volume of 100 μL and incubated. Fluorescence intensity was detected with a microplate reader Synerge H1 (Biotek, Winooski, VT, USA) at a wavelength of 490 nm for the excitation and 525 nm for the emission.

#### *2.6. Fexofenadine Uptake Test Using OATP1A2*/*SLCO1A2 Transfected HEK293 Cells*

The seeding density of the OATP1A2 overexpressed HEK293 cells was 10<sup>5</sup> cells/well. Verapamil was used as a positive control with a concentration of 100 μM [28]. The cultured cells were washed twice with warmed HBSS with 5-mM MES after removing the medium, then 15-μM fexofenadine was treated with *R. acetosa* extract of 10, 20 and 50 μg/mL. After 15 min of incubation in 8% CO2 with low humidity at 37 ◦C, they were washed twice with cold HBSS. They were gently shaken after adding 120 μL of 50-ng/mL terfenadine in 80% acetonitrile. Terfenadine was used as an internal standard. After centrifugation at 10,000× *g* for 5 min, 50 μL of supernatant was mixed with 50 μL of 5-mM ammonium formate (pH 4). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the fexofenadine uptake amount [28,29].
