Screening And Identification Of Lipase Inhibitors In Scutellaria Baicalensis By Combining Magnetic Metal Organic Frameworks With UPLC-Q-TOF-MS/MS Technology

Objective:Potential pancreatic lipase inhibitors were rapidly screened from traditional Chinese medicine Scutellaria baicalensis by using the immobilized enzyme of magnetic metal organic framework materials,which was expected to obtain safe and effective ingredients and to provide lead compounds for the treatment of obesity.The method combining immobilized enzyme with UPLC-Q-TOF-MS/MS technology could provide a new platform for rapid screening and identification of effective active ingredients from natural medicines.Methods:1.Carboxyl-modified Fe₃O₄magnetic nanoparticles were synthesized by a solvothermal method,and then was encapsulated in the metal-organic framework（UiO-66-NH₂）toformcore-shellcomposite （Fe₃O₄-COOH@UiO-66-NH₂）by solvothermal method at high temperature and pressure by combining Fe₃O₄with 2-aminoterephthalic acid and Zr Cl₄in a specific ratio.Using glutaraldehyde as a cross-linking agent,pancreatic lipase was immobilized on the surface of Fe₃O₄-COOH@UiO-66-NH₂nanoparticles toformtheimmobilizedpancreaticlipasenamely Fe₃O₄-COOH@UiO-66-NH₂@PPL.The synthesized materials were characterized by Fourier transform infrared spectroscopy,X-ray diffraction and Transmission electron microscope.2.By using Coomassie brilliant blue method,the amount of immobilized enzyme loaded under different conditions was determined with Bovine serum albumin as the standard protein.The p-nitrophenol method was used to measure the activity of immobilized enzyme synthesized under different conditions, respectively.In this way,the optimal synthesis conditions of the immobilized enzyme were determined.3.Potential pancreatic lipase inhibitors were fished out from Scutellaria baicalensis by means of ligand fishing with immobilized enzymes,and the screened active ingredients were identified by UPLC-Q-TOF-MS/MS technology.4.The enzyme inhibition activity of the screened possible pancreatic lipase inhibitors was verified by p-nitrophenol method and molecular docking experiments.Results:1.The infrared spectrum of the synthesized Fe₃O₄@UiO-66-NH₂@PPL showed that the characteristic peak at 1651 cm^-1of UiO-66-NH₂was shifted to 1620 cm^-1indicating that the lipase had successfully immobilized on the surface of Fe₃O₄-COOH@UiO-66-NH₂nanoparticles.The X-ray diffraction pattern of the synthesized Fe₃O₄-COOH@UiO-66-NH₂revealed that it contained both the characteristic peaks of Fe₃O₄and UiO-66-NH₂,representing that the core-shell magnetic nano-metal-organic framework composites were successfully synthesized.In addition,the core-shell image of Fe₃O₄-COOH@UiO-66-NH₂, was clearly displayed in the transmission electron microscope,which showed that MOFs had been successfully wrapped on the surface of Fe₃O₄.2.The enzyme loading and enzyme activity of the immobilized enzyme were determined under the conditions of different carrier and enzyme mass ratio, cross-linking time,cross-linking agent concentration,etc.The results showed that the immobilized enzyme had the highest enzymatic activity when the mass ratio of carrier to enzyme was 2:1,the cross-linking time was 2 h and the cross-linking agent concentration was 150 m M.3.Using immobilized enzyme combined with UPLC-Q-TOF-MS/MS technology, eight possible pancreatic lipase inhibitors were screened and identified from Scutellaria baicalensis,which were 2ˊ,3,5,6ˊ,7-pentahydroxyflavanone, scutellarein,baicalin,oroxyloside,wogonoside,skullcapflavoneⅡ,wogonin and oroxylin A.4.From the compounds screened by ligand fishing,baicalin,wogonin and oroxylin A were selected to verify the inhibitory activity of pancreatic lipase in vitro.The results showed that comparing with the positive drug orlistat （1.9±0.6μM）,these three compounds all exhibited inhibitory activity with IC₅₀ values of 242.2±5.6μM,168.5±8.4μM and 82.7±3.6μM,respectively.In addition,the molecular docking study was carried out on the eight compounds screened by ligand fishing,and the results showed that these eight compounds could bind to pancreatic lipase.Conclusion:In this research,eight pancreatic lipase inhibitors were screened and identified from Scutellaria baicalensis by the method of ligand fishing with magnetic metal organic framework materials immobilized enzyme.This work was expected to provide an effective basis for the follow-up research of traditional Chinese medicine on obesity treatment.Compared with the traditional separation methods,the screening method established in this work could simply,efficiently and directly screen and identify potential active small-molecules from traditional Chinese medicine.Additionally,this method combined the advantages of magnetic nanoparticles and metal organic frameworks,which improved the protein load and stability of immobilized enzyme.Taken together,it provided a new avenue for discovering enzyme inhibitors from natural sources,which had certain application values.