The transcytosis pathway of IPMs across Caco-2 cell monolayers was studied as reported previously with a little modification [8]. In short, the Caco-2 cell monolayers were pre-incubated with 0.5 mL of HBSS containing four kinds of endocytosis inhibitors such as genistein (100 μM), chlorpromazine hydrochloride (CMZ, 10 μg/mL), amiloride (100 μM), and methyl-β-cyclodextrin (MβCD, 10 mM) at 37 °C for 30 min. Afterwards, the HBSS in the AP side was replaced with prewarmed IPMs solution (1.0 mg/mL) in HBSS containing the corresponding inhibitors, and then incubated for another 4 h at 37 °C. The medium collected from BL side was treated with 1% acetic acid solution (10 μL) and β-glucurondase (45 μL) followed by incubation for 1 h at 37 °C, lyophilized and redissolved in methanol (175 μL). The amount of transported icaritin was determined by HPLC as described earlier.

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