Liver Microsomes Extraction and Sample Treatment

Fresh mice liver samples were rinsed with pre-cooled sucrose solution at 4°C. Two times the volume of liver weight of sucrose solution was added to the liver samples. The solution was manually homogenized on ice, and centrifuged at 16,000 × g, at 4°C for 20 min. The collected supernatants were placed in ultra-high speed centrifuge tubes, and centrifuged at 100,000 × g, at 4°C for 60 min. Supernatants were discarded and the collected precipitate was washed with potassium pyrophosphate solution and centrifuged again at 100,000 × g, at 4°C for 60 min. The supernatant was discarded, and the microsomes were quantified by resuspension in Tris-HCl buffer.

Hepatic microsomes incubation and sample treatment: Mixed mouse liver microsomes and probe substrates (CHL and NIF), KET and 4-ME were added to the positive control group; methanol was added to the blank group, and OEB solutions (OEB-L 2.5 μg/ml, OEB-M 5.0 μg/ml, OEB-H 10.0 μg/ml) were added to the drug administration group. The system was pre-incubated at 37°C in a metal bath; the reaction was initiated by adding NADPH and incubating for 20 min at 37°C in a water bath; and terminated by adding cold ethyl glacial acetate. After adding 10 μl internal standard LOR solution (2 μg/ml) to the system, it was vortexed for 2 min, rested for 10 min and centrifuged at 3,500 rpm for 10 min. The supernatant was transferred and the organic solvent was evaporated. Before injection, 200 μl of 80% methanol water was added, vortex shaken for 1 min, and centrifuged at 16,000 rpm for 5 min to produce the sample.