Effects of fractions derived from HPHU, CTH or FP on cholesterol micellar solubility in vitro

The effects of HPHU fractions, CTH or FP on cholesterol micellar solubility were evaluated in vitro using a previously described method^11,37. The [4-¹⁴C]-labeled micellar solutions (1.0 mL) containing the following components were mixed by sonication (Ultrasonic Homogenizer, Model VP-5; Taitec, Tokyo, Japan): 0.74 kBq [4-¹⁴C]-cholesterol (2.1 Gbq/mmol; PerkinElmer Life Sciences, Yokohama, Japan), 0.1 mM cholesterol (Sigma Aldrich, St. Louis, MO, USA), 6.6 mM sodium taurocholate (Sigma Aldrich), 1 mM oleic acid (Sigma Aldrich), 0.6 mM phosphatidylcholine (Sigma Aldrich), 0.5 mM monoolein (Sigma Aldrich), 132 mM NaCl, and 15 mM sodium phosphate buffer (pH 7.4). After incubation at 37 °C for 24 h, the sample (5 g/L, respectively) was added to the micellar solution, solubilized by sonication, incubated at 37 °C for 1 h, and centrifuged at 100,000 × g for 60 min at 37 °C. The supernatant was collected for the determination of the [¹⁴C]-cholesterol content using a liquid scintillation counter.