| Coronary heart disease was regarded as the number one killer of threatening human health,traditional chinese medicine has unique advantages in treatment and therapeutics.As one of the representative medicines of Mongolian medicine,Choerospondias axillaris has been recorded in the treatment of coronary heart disease as early as the 8th century.However,due to its complex chemical components and diverse structures,there are few studies on the specific functional components of the specific pharmacological activities and corresponding targets.Therefore,how to carry out multi-target extraction of active components from Choerospondias axillaris,with the help of some advanced technical means and scientific methods,to clarify its pharmacodynamic material basis was necessary for the prevention and treatment of coronary heart disease.In this thesis,the new materials of magnetic nano-microspheres were synthesized,and the surface of the microspheres was individually functionalized and characterized according to the different targets.Then,with the help of the combination of magnetic ligands fishing and UPLC-Q-Exactive orbitrap-MS/MS,the alcohol extract of Choerospondias axillaris was used to isolate and identify the active components according to the relevant ion data and reports.Therefore,taking three enzymes related to coronary heart disease as examples,it can be fully demonstrated that the combination of the enzyme-immobilized magnetic nano-microsphere and magnetic ligand fishing can provide a prospective solution for the rapid screening of anti-coronary heart disease components in Choerospondias axillaris.The specific research contents are as follows:1.Magnetic ligand fishing using immobilized COX-2 for identification and screening of anti-coronary heart disease ligands from Choerospondias AxillarisIn this study,a cyclooxygenase-2（COX-2）functionalized magnetic nanosphere（MGO@Si O2-COX-2）was synthesized and used as an adsorbent in magnetic ligand fishing.And combined UPLC-Q-Exactive Orbitrap-MS/MS,the new method of rapid screening COX-2 ligands from Choerospondias axillaris was established.Firstly,the MGO@Si O2-COX-2 nanospheres and the intermediates were synthesized and characterized using Fourier transform infrared spectroscopy（FT-IR）,vibrating sample magnetometer（VSM）,scanning electron microscopy（SEM）,and transmission electron microscopy（TEM）.Under the positive and negative ion modes of UPLC-Q-Exactive Orbitrap-MS/MS,21 compounds consisting of 6 organic acids,8 flavonoids,and 7 others were separated and characterized from Choerospondias axillaris,which were reported to inhibit the production of COX-2 in vivo to varying degrees mainly through the p38MAPK/NF-κB pathway.Taking the known COX-2inhibitor celecoxib as the target,specific fishing was carried out using MGO,MGO@Si O2,MGO@Si O2-COX-2,and MGO@Si O2-inactive COX-2,respectively.And the results showed that the MGO@Si O2-COX-2 has high specificity and good reusability for screening COX-2ligands.Meanwhile,the storage stability and synthesis reproducibility of the MGO@Si O2-COX-2 were investigated,and the results showed that the relative standard deviation values of the stability and reproducibility were lower than 8%（n=5）and 10%（n=3）,respectively.Therefore,the MGO@Si O2-COX-2 nanomaterials synthesized in this study were successfully applied to the extraction and identification of COX-2 ligands in Choerospondias axillaris.And this method could provide a prospective solution for the extraction and identification of active pharmaceutical ingredients and the rapid screening of some enzyme inhibitors in the complex mixtures.2.Magnetic ligand fishing using immobilized PPAR-γfor identification and screening of anti-coronary heart disease ligands from Choerospondias AxillarisIn this study,we have been proposed a new method based on MGO@Si O2@PPAR-γligand fishing for facile and rapid screening PPAR-γligands from complex natural products,and then UPLC-Q-Exactive orbitrap-MS/MS analysis was employed for structural elucidation.Initially,the magnetic nano-microspheres with functionalized modification of PPAR-γhave been successfully synthesized and characterized by VSM,SEM,TEM.And with the help of rosiglitazone,the properties of the specificity,reusability,and reproducibility of the MGO@Si O2@PPAR-γwere investigated.Additionally,to improve the specific adsorption efficiency of the analytes,the incubation temperature,and the p H of the buffer solution in the magnetic ligand fishing were optimized.The final adsorption conditions were as follows:when the incubation temperature was 40°C and the p H of the buffer was 6,the adsorption efficiency of rosiglitazone was the highest.Finally,with the aid of UPLC-Q-Exactive Orbitrap-MS/MS in both positive and negative ion modes,16 active compounds composed of9 organic acids,5 flavonoids,and 2 phenols were isolated and identified from the ethanolic extracts of Choerospondias axillaris.Therefore,this method can be used to immobilize macromolecular targets and facilitate the screening of many specific biologically active compounds from complex natural products.3.Magnetic ligand fishing using immobilized ACE for identification and screening of anti-coronary heart disease ligands from Choerospondias AxillarisIn this study,the new method consisted of magnetic ligand fishing with the adsorbent of MGO@Si O2-ACE and HPLC-Q-TOF-MS/MS for component identification was proposed to single out the ACE ligands from Choerospondias axillaris.In the experiment,MGO@Si O2-ACE was synthesized and characterized by SEM,FT-IR,TEM,and VSM.And regarded as the target compound,lisinopril was used to investigate the specificity,reusability,stability,and reproducibility of MGO@Si O2-ACE,and optimize the important parameters of magnetic ligand fishing,such as incubation time,incubation temperature,and the p H of buffer solution.The result showed that the same amount of MGO@Si O2-ACE nanomaterials was incubated at 30°C for 30 min,after washing with the p H of PBS buffer solution at 5 and dissociating in the dissociation solution for 1 h,and the content of lisinopril in the supernatant was the highest.Under the above conditional,5 active compounds were successfully detected from the alcohol extract of Choerospondias axillaris,in both positive and negative ion modes of HPLC-Q-TOF-MS/MS.Therefore,the study can provide a successful precedent for realizing the designated extraction and rapid isolation of target-specific active ingredient groups in the complex mixtures. |
