Characterization of major metabolites of polymethoxylated flavonoids in Pericarpium Citri Reticulatae using liver microsomes immobilized on magnetic nanoparticles coupled with UPLC/MS–MS

The peels of citrus fruits (Pericarpium Citri Reticulatae, PCR) have long been used in traditional Chinese medicines (TCMs). Polymethoxylated flavonoids (PMFs) were found to be the main components present in PCR extracts, but their metabolism remains unclear which restrain the utilization of this TCM. In the present work, rat liver microsomes were immobilized on magnetic nanoparticles (LMMNPs) for in vitro metabolic study on the whole PMFs of PCR. LMMNPs were characterized by transmission electron microscope and Fourier-transform infrared spectrum. The relative enzyme binding capacity of LMMNPs was estimated to be about 428 μg/mg from thermogravimetric analysis. Incubation of LMMNPs with PMFs produced demethylated metabolites of PMFs, six of which were identified by ultrahigh pressure liquid chromatography–mass spectrometry (UPLC–MS/MS). The 3′-hydroxylated tangeretin (T3) was detected from the metabolites of tangeretin for the first time, which suggested that 4′-demethylated and 3′-hydroxylated derivative of tangeretin (3′-hydroxy-5,6,7,8,4′-pentamethoxyflavone, T4) was not only derived from 4′-demethylated tangeretin (T2) as previously reported, but also from T3. This is the first investigation of the metabolism of the whole PMFs, which may shed light on the mechanism of action of PCR.