Establishment Of Monoamine Oxidase-A-immobilized Magnetic Beads Based Ligand Fishing Method And Its Application In Natural Products

In recent years,the incidence of depression has been increasing,monoamine oxidase-A(MAO-A)is considered an important therapeutic target in depression.Screening of MAO-A inhibitors is expected to develop new drugs for the treatment of depression.The commonly used anti-depression drugs toloxatone and moclobemide are both anti-depression drugs obtained through the traditional screening methods of active ingredients.The traditional screening methods have the disadvantages of labor-intensive,expensive,time-consuming and inefficient,therefore,it is necessary to develop a simple,reliable and high-throughput screening method for MAO-A inhibitors under the guidance of new screening strategy.The immobilization of enzyme appears multitudinous advantages,such as high stability,improved efficiency in recovery,convenience in handling and reuse of enzymes,therefore,the cost can be drastically reduced.Ligand fishing based on enzyme-immobilized magnetic beads(MBs)is an affinity-based technology in which specific binding of ligands to target enzymes allows rapid separation from unbound compounds of complex mixtures,ligand fishing has been proved to be an effective method and increasingly applied to screen inhibitors from natural products.In the first chapter,the related researches on depression and MAO-A were reviewed.The screening approaches of active compounds from natural products and immobilized enzyme technology were introduced.In addition,the immobilized enzyme technology and the research innovations of this study were put forward.In the second chapter,the immobilization of amine-terminated MBs and MAO-A was achieved by covalent crosslinking with glutaraldehyde as crosslinking agent.A series of systematic studies were carried out on the key factors affecting the enzyme activity in the immobilization process,and the physicochemical characterization showed that the prepared MAO-A reactor had better enzyme activity.Furthermore,a activity testing method of MAO-A was established by the prepared MAO-A reactor combined with HPLC-UV chromatograrphy system,the results show that screening method has the potential for further application.In the third chapter,based on the immobilized MAO-A reactor and the testing method of immobilized enzyme activity,a screening method of MAO-A inhibitor was established which was suitable for mixed reference substance,and then applied for the screening of 12 natural products.The results not only presented good reusability,validity and stability,but also confirmed the desired anti-MAO-A activity of some alkaloids and flavonoids.In the fourth chapter,the MAO-A-immobilized MBs combined with HPLC-Q-TOF-MS/MS method was established and used for screening MAO-A inhibitors from natural products.The ligand fishing method was established and verified using a mixture to testify the efficiency of the method.The the ethyl acetate fraction extract of Corydalis Rhizome and Coptis Chinensis were selected for further ligand fishing,seven active compounds were screened by MAO-A-immobilized MBs from the extract of Corydalis Rhizome,six active compounds were screened by MAO-A-immobilized MBs from the extract of Coptis Chinensis.These results proved that the ligand fishing method could specifically capture MAO-A inhibitors from natural products.In the fifth chapter,all the research contents were summarized,the future research work was prospected.