Identification Of α-glucosidase Inhibitors From Pueraria Lobata By Ligand Fishing Based On Magnetic Mesoporous Silicon Combined With Knock-out/Knock-in Technology

Objective:Magnetic mesoporous silicon was synthesized and used as the carrier ofα-glucosidase.Subsequently,α-glucosidase modified magnetic mesoporous silicon was used to screen its inhibitors from Pueraria lobata.At the same time,the knock-out/knock-in technique was used to verify the interaction of several active ingredients from a holistic perspective.It is expected to discover compounds that can inhibitα-glucosidase activity and clarify their role in P.lobata,so as to provide more stable and reliable lead compounds for clinical treatment of diabetes.Methods:1.Fe₃O₄was used as the core material,tetraethyl orthosilicate was first used for stability modification.Subsequently,the template agent Hexadecyltrimethylammonium chloride was added at the same time as the second stability modification with tetraethyl silicate.The magnetic mesoporous silicon can be obtained from the modified material after removing the template agent with acidified ethanol.Then,the-NH₂modified magnetic mesoporous silicon was synthesized using（3-Aminopropyl）triethoxysilane as material.Finally,α-glucosidase was modified to a magnetic mesoporous silicon surface modified with-NH₂using glutaraldehyde as a cross-linking agent.Fourier infrared spectroscopy and transmission electron microscopy were used to characterize the synthesized materials.2.Using Allure red to study the surface-NH₂content of magnetic mesoporous silicon with different sizes,non-mesoporous magnetic nanomaterials and magnetic mesoporous silicon without-NH₂modification.The effect of the amount of reactants on the amount of-NH₂on the surface of the material and the activity of the immobilized enzyme compared with the free enzyme at different temperatures and p H were also investigated.3.Potentialα-glucosidase inhibitors were screened from P.lobata by ligand fishing technique,and the structure of ligands was identified by UPLC-Q-TOF-MS/MS.4.Knock-out/knock-in technology was used to analyze and verify the effect of the important components in P.lobata which played a role in inhibitingα-glucosidase activity,and their inhibition kinetics was studied.The activity ofα-glucosidase inhibitors was verified by molecular docking method.Results:1.The infrared spectra ofα-glucosidase modified Fe₃O₄@n Si O₂@m Si O₂showed characteristic peaks at 1557 cm^-1and 1650 cm^-1,which were due to the stretching vibration of C=O and the bending vibration of N-H,indicating thatα-glucosidase has been successfully modified on the Fe₃O₄@n Si O₂@m Si O₂surface.The surface morphology of Fe₃O₄,Fe₃O₄@n Si O₂and Fe₃O₄@n Si O₂@m Si O₂was analyzed by transmission electron microscopy,and it was found that Fe₃O₄@n Si O₂@m Si O₂had dense pores on the surface,indicating that the magnetic mesoporous silicon was successfully synthesized.2.Through the study of the adsorption performance of materials,it was determined that the surface of magnetic mesoporous silicon with small size could carry the largest number of-NH₂,and which indicating that the surface could be fixed the largest amount of enzymes.At the same time,when the amount of reactant added to the material was 0.2 m L,0.4 m L and 6 m L for two stability modifications and one functional modification,respectively,the amount of-NH₂that could be loaded on the surface of the material was the largest,and the immobilized enzyme was more stable than the free enzyme at different temperatures and p H.3.Using ligand fishing technique based on magnetic mesoporous silicon combined with UPLC-Q-TOF-MS/MS,ten possibleα-glucosidase inhibitors were screened from P.lobata,which were 3’-hydroxypuerarin,puerarin,3’-methoxy puerarin,puerarin-7-xyloside,puerarin 6"-O-xyloside,daidzin,genistin,ononin,daidzein and glycitein.4.Puerarin,daidzein and daidzein were used for knock-out/knock-in analysis.Daidzein and puerarin showedα-glucosidase inhibitory activities with IC₅₀of 0.088±0.003 mg/m L and 0.414±0.005 mg/m L,respectively.Among them,puerarin,which accounted for more than 40%of the total content,showed synergistic effects with other components and was the main contributor to theα-glucosidase inhibitory activity of P.lobata.In addition,compounds selected by ligand fishing were verified by molecular docking,and the results showed that all of the 10 compounds could bind toα-glucosidase.Conclusion:In this research,magnetic mesoporous silicon with large surface area and excellent biological affinity was successfully synthesized and used as a carrier for immobilizedα-glucosidase.Meanwhile,a ligand fishing method based on magnetic mesoporous silicon was established to rapidly screenα-glucosidase inhibitors from P.lobat.Finally,10 ligands were successfully screened out and the structures were identified by UPLC-Q-TOF-MS/MS.In addition,to determine the potential synergistic or antagonistic effect of multiple bioactive compounds,the activity of the ligands was verified using a knock-out/knock-in method.Puerarin,daidzin,and daidzein enhanced the inhibitory effect of other components.Among them,puerarin was identified as the key constituent responsible for the inhibitory effect of P.lobata onα-glucosidase activity.The inhibitory kinetics and mechanism were also studied.Taken together,ligand fishing based on magnetic mesoporous silicon combined with a knock-out/knock-in strategy was an efficient approach for identifyingα-glucosidase inhibitors and evaluating their multiple-component interactions in P.lobata.