Study On The Anti-inflammatory And Metabolic Action Mechanism Of Sec-o-glucosylhamaudol In Vitro

LPS-stimulated RAW 264.7 murine macrophage cells were used to evaluate the anti-inflammatory activity of sec-O-glucosylhamaudol.The results showed that secO-glucosylhamaudol showed no significant cytotoxicity when the concentration reaches 100 μM and sec-O-glucosylhamaudol treatment markedly decreased the emergence of these inflammatory cytokines in a dose-dependent manner.Sec-Oglucosylhamaudol inhibited the expression of TNF-α and IL-6 via blocking the transcription of these genes.Sec-O-glucosylhamaudol via suppressing the MAPK phosphorylation and NF-κB activation in RAW 264.7 murine macrophages induced by LPS to play an anti-inflammatory role.Potato derived 5-lipoxygenase and human recombinant 5-lipoxygenase are important targets,which are closely related with human inflammatory disease.The mechanism of sec-O-glucosylhamaudol on the two different sources of 5-lipoxygenase were investigated using multi-spectroscopy methods combined with molecular docking studies.The results of fluorescence spectra showed that the two 5-lipoxygenase were quenched statically by sec-O-glucosylhamaudol,accompanied by non-radiative energy transfer.At the same temperature,the binding ability of sec-O-glucosylhamaudol to human recombinant 5-lipoxygenase was stronger.Synchronous fluorescence,threedimensional fluorescence,ultraviolet-visible spectroscopy,circular dichroism spectroscopy and infrared spectroscopy showed that the secondary structure of two 5-lipoxygenases was changed in the process of interaction between sec-Oglucosylhamaudol and the two 5-lipoxygenases.The results of molecular docking and fluorescence spectra showed that the main binding forces of sec-O-glucosylhamaudol to 5-lipoxygenase were electrostatic force and hydrogen bond.Cytochrome CYP3A4,CYP2D6 enzymes and human serum albumin were selected as targets to evaluate the metabolic mechanism of sec-O-glucosylhamaudol in vitro.The interaction mechanism of sec-O-glucosylhamaudol with CYP3A4,CYP2D6 and human serum albumin was studied by multi-spectroscopy methods and molecular docking techniques.The results suggested that CYP3A4,CYP2D6 and human serum albumin were quenched statically by sec-O-glucosylhamaudol.At the same temperature,sec-O-glucosylhamaudol had the highest binding capacity to CYP2D6,followed by CYP3A4,human serum albumin.Synchronous fluorescence,threedimensional fluorescence,ultraviolet-visible spectroscopy,circular dichroism spectroscopy,and infrared spectroscopy results indicated that sec-O-glucosylhamaudol changed their secondary structure in the process of interaction with CYP3A4,CYP2D6,and human serum albumin.Molecular docking results indicated that sec-Oglucosylhamaudol successfully entered into the active sites of CYP3A4 and CYP2D6,bound to the sub-domain IIA of human serum albumin.The interaction between secO-glucosylhamaudol with amino acid residues in the active site of CYP3A4,CYP2D6 and human serum albumin mainly through electrostatic attraction and hydrogen bonding.