Lowering cholesterol bioaccessibility and serum concentrations by saponins: in vitro and in vivo studies

I. Methods. S1. Extraction of the non-hydrolyzed TG and reaction products by chloroform. The used procedure is taken from ref. (26). Briefly, after stopping the lipolysis reaction with Orlistat granules, the reaction mixture was allowed to cool down to room temperature and its pH was decreased to pH = 2 by adding HCl (to decrease the solubility of the reaction products in the aqueous phase). Next, 6 mL chloroform was added and the sample was sonicated for 15 min. After every 5 min of sonication, the sample was agitated by vigorous hand shaking. The obtained complex dispersion was centrifuged for 30 min at 3620 g (4500 rpm), which led to separation of clear aqueous and organic phases, indicating that the oily drops were entirely extracted by the chloroform phase. The recovery of cholesterol, FA, MG, DG and TG by this extraction procedure was ≥ 90 %. The same extraction procedure was applied to the aqueous phases, separated by filtration or centrifugation. The obtained chloroform phase was further analyzed by GC.