2.3. Preparation of Liver Microsomes and Microsomal Incubation

The in vitro metabolism study of pterostilbene was developed in rat liver microsomes (purchased from NEWGAINBIO). The microsomes used in this paper are subcellular components of rat liver organelles prepared by differential centrifugation, which are part of the organelle endoplasmic reticulum and have intact phase I metabolic enzymes, phase II metabolic enzymes, and esterases. A reaction mixture was carried out in a 1 M phosphate buffer (pH = 7.4) containing rat liver microsomes (1 mg/mL) and MgCl₂ (3 mM). Pterostilbene was diluted with the above solvent to a concentration of 0.1 mg/mL. To a separate 6-well plate, 900 μL of above mixture was added to a drug well. Simultaneously, incubation without pterostilbene served as blank control and incubation absent from NADPH as negative control. Preincubation was performed at 37 °C for 5 min before adding 100 μL of NADPH (25 mg/mL) to start the reaction. The reactions were incubated for 5, 10, 15, 30, 45, 60, 120, and 240 min at 37 °C. After that, 100 μL of different system solutions was removed and terminated using 200 μL of cold acetonitrile. Finally, the acetonitrile solutions were collected and dried under nitrogen at room temperature.