Ligand Fishing With Cellular Membrane-coated Filter Papers And Preliminary Study On Extraction Of Sulforaphane

Purposes: 1.Establish the fishing method of cell membrane-coated filter papers(CMFPs),which is used for screening of potential active ingredients in natural products and provide a new method for drug research and development.2.Extract sulforaphane(SFN)from broccoli seeds and provide preliminary preparation for the preparation of SFN.Methods: Use rabbit red blood cells as raw materials and obtain cell membranes(CMs)by hypotonic swelling method.Immobilize the red blood cell membranes on filter papers according to the principle of physical adsorption.Select the preferably filter papers with the largest amount of the immobilized CMs by comparing the difference of immobilized amount of CMs between before and after immobilization.Dye the CMs with fluorescein isothiocyanate(FITC)and 1,1'-dioctadecyl-3,3,3',3'-tetramethy-lindocarbocyanine perchlorate(Di I)and observe fluorescence of dyed CMs,filter papers and dyed CMFPs under an inverted fluorescence microscope to confirm that the CMs have been immobilized onto the filter papers.Perform the selective evaluation of fishing method of CMFPs with verapamil and thiourea.Apply CMFPs to fish the potential active ingredients in Angelica dahuricae.Study the active ingredients by Network pharmacology and compare with the fishing method of CMs.Extract SFN for the preparation of SFN preparations.highest content of SFN is in the broccoli seeds according to literatures.SFN is extracted by hydrolysis and identified by high performance liquid chromatography.Purify the SFN crude by silica gel column chromatography.Analyze the purified SFN by mass spectrometry and determine the recovery by high performance liquid chromatography.Results: By comparing the difference of immobilized amount of CMs between before and after immobilization,We found that Grade 2 filter paper has the largest amount of cell membranes immobilized.The observation of the inverted fluorescence microscope showed that the cell membranes were immobilized onto the filter papers successfuly.The chromatograms of CMFPs incubated with verapamil and thiourea mixed liquids demonstrated that they can be specifically combined with verapamil in the mixed liquid and the fishing method of CMFPs is selective.In the chromatogram results of CMFPs and Angelica Dahurica extract incubation,three compounds were found and analyzed by mass spectrometry.They are the potential active ingredients that specifically bind to the cell membranes after analyzed by networked pharmacology and molecular docking evaluation.Repeat selective evaluation and fishing in Angelica dahuricae with fishing method of CMs,more impurity peaks were found in the chromatogram results and the operation is complicated,time-consuming and laborious.Extract the SFN crude by the hydrolytic method.The same peaks are present in the crude and the standard by comparing the HPLC chromatograms of the crude and the standard.We found that when the p H value is 5,the yield of SFN is the most by comparing different hydrolysis conditions.Its purity exceeded the standard after the purification by silica gel column chromatography and its recovery rate was 0.28%.The purified product was SFN confirmed by mass spectrometry.Conclusions: We optimized the type of filter paper with the largest amount of immobilized CMs,and succesully demonstrated that the CMs were immobilized on the filter papers.The selective evaluation of verapamil and thiourea demonstrated that CMFPs can bind specifically to positive drugs and can be used to screen active ingredients in natural products.the fishing method of CMFPs has advantages compared with the fishing method of CMs.Apply the fishing method of CMFPs to screen the active ingredients in the natural product Angelica dahurica.Three active ingredients were succesully screened out,which provided a new method for screening potential active ingredients in natural products.We used a hydrolysis method to crudely extract SFN from broccoli seeds.We extract the SFN and compare it with the standard.Purify the SFN by silica gel column chromatography and identify the product by mass spectrometry.Finally,determine the recovery of SFN.Provide preliminary preparation for the preparation of SFN.