Study On Mechanisms Of Drug Metabolism Of Okanin

The effect of okanin on the activities of CYP3A4 and CYP2D6 enzymes was studied by the combined method of ultra performance liquid chromatography and triple quadrupole mass spectrometry for the first time.The results suggested that okanin has an effective inhibitory effect on the two enzymes,with IC₅₀ of 3.661μM and 6.330μM,respectively.Okanin showed a mixed inhibitory effect on CYP3A4,with K_i and K_isvalues of 10.649 and 34.927μM,respectively,while the inhibitory type for CYP2D6was non-competitive inhibition with K_i value of 19.729μM.The Multi-spectral methods and molecular docking technology were used to study the molecular mechanism of inhibition effect of CYP3A4 and CYP2D6 by okanin for the first time.The results indicated that the quenching mechanism of the two enzymes by okanin was static quenching accompanied by non-radiative energy transfer.At the same temperature,the binding ability of okanin to CYP3A4 was stronger than that of CYP2D6.Synchronous fluorescence spectroscopy,three-dimensional fluorescence spectroscopy,ultraviolet-visible spectroscopy and circular dichroic spectroscopy confirmed that the protein conformation and microenvironment of the two enzymes were changed after CYP3A4 and CYP2D6 combined with okanin.The molecular docking results showed that okanin successfully docked into the active site and hydrophobic cavity of CYP3A4 enzyme,surrounded by heme molecule（HEM508）and residues such as PHE213 and PHE215;similarly,okanin entered into the active site and hydrophobic cavity of CYP2D6 and was embraced by the heme molecule（HEM508）,PHE120,PHE483,LEU213,LEU484,VAL370,VAL347,VAL308 residues.Fluorescence spectroscopy and molecular docking experiments proved that hydrophobic force and hydrogen bond were the main forces for the binding of okanin to the two enzymes.The Multi-spectral methods and molecular docking technology were used to study the interaction mechanism between okanin and human serum albumin for the first time.The results indicated that the quenching mechanism of the human serum albumin by okanin was static quenching,accompanied by non-radiative energy transfer.The result of ultraviolet-visible spectroscopy showed that okanin interacted with HAS and formed a complex.Fluorescence spectroscopy and molecular docking experiments indicated that the forces between okanin and human serum albumin were mainly hydrophobic forces and hydrogen bonds.The results of site competition and molecular docking showed that okanin binds to the SiteⅠof human serum albumin.The results of synchronous fluorescence,three-dimensional fluorescence,circular dichroism spectroscopy and infrared spectroscopy indicated that microenvironment and secondary structure of human serum albumin were changed by okanin.The ultra performance liquid chromatography coupled with a triple-quadrupole tandem mass spectrometry method was established through the specificity,linearity,precision,accuracy,recovery rate,matrix effect and stability experimental investigation.Bavachalcone was used as an internal standard（IS）.This method was used to analyze and determine the changes in the main pharmacokinetic parameters of okanin in rat plasma after oral administration of 1 mg/m L okanin monomer.Drug metabolism mechanism of okanin in vivo was discussed in depth.The precise molecular weight of okanin in rat plasma was determined by high-resolution mass spectrometry,and the fragmentation law of okanin in mass spectrometry was inferred.