Studies On Affinity Screening Acetylcholinesterase And α-Glucosidase Inhibitors From Traditional Chinese Medicines And Interactions With Enzymes

Enzymes are important catalysts in vivo,which are closely related to human metabolism.If the function of enzymes is abnormal in vivo,it will lead to the occurrence of diseases.In modern medicine,enzymes are not only important indicators for disease diagnosis,but also significant targets for drug discovery.Acetylcholinesterase(ACh E)and α-glucosidase(α-Glu)are important targets for Alzheimer’s disease and diabetes mellitus,respectively.Enzyme inhibitors can prevent and treat diseases by inhibiting the activity of corresponding enzymes.Currently,enzyme inhibitors,such as donepezil and rivastigmine(ACh E inhibitors),acarbose and voglibose(α-Glu inhibitors),have been clinically used for the treatment of related diseases.However,most of these inhibitors are synthetic drugs,which may cause obvious side effects if taken in large quantities for a long time,so their usage is limited to some extent.Traditional Chinese medicines(TCM)is a promising source for the discovery of new lead compounds due to the wide varieties,diverse chemical structures,significant activity and high safety,which is suitable for the screening and development of natural enzyme inhibitors.However,due to the complexity of the components,efficient and accurate screening of enzyme inhibitors from extracts of TCMs has become an urgent problem to be solved.In this study,based on the affinity interaction between active small molecules and target enzyme proteins,using glucosidase as the target enzyme,affinity screening methods combining affinity ultrafiltration,immobilized target enzyme screening,and high-performance liquid chromatography-mass spectrometry(UPLCQTOF-MS/MS)were established to rapidly screen and identify natural enzyme inhibitors from extracts of Terminalia chebula fruits and Cyclocarya paliurus leaves.In addition,inhibition interactions between potential inhibitors and enzymes were systematically elucidated by enzyme inhibition kinetics,combination of various spectral methods and molecular docking technique.The main contents are as follows:1.Affinity ultrafiltration screening methods combined with UPLC-QTOF-MS/MS were developed to screen and identify 17 potential acetylcholinesterase(ACh E)inhibitors from Terminalia chebula fruits,mainly gallotannins and ellagitannins,and 36 potential α-glucosidase(α-Glu)inhibitors from Cyclocarya paliurus leaves,mainly damarane-type triterpenoid saponins.Then,the enzyme inhibition assay in vitro was used to evaluate inhibitory activities of active compounds against enzyme,and molecular docking was performed to predict inhibition mechanisms between active compounds and enzyme,further verifying the reliability and practicability of the affinity ultrafiltration screening method.2.Affinity screening methods based on cellulose filter paper(CFP)-immobilized target enzymes were developed.Cellulose filter paper(CFP)as the carrier was modified with chitosan to be introduced to amino groups,and then ACh E was modified on the modified CFP through a Schiff base reaction with glutaraldehyde as a cross-linking agent to obtain CFP-immobilized ACh E.The CFP was modified by polydopamine/polyethyleneimine(PDA/PEI)co-deposition to form a uniform positive charge coating on the surface,and then α-Glu was immobilized on the modified CFP by electrostatic adsorption to obtain CFP-immobilized α-Glu.By virtue of the instantaneous separation characteristic of CFP,novel affinity screening methods based on CFP-immobilized target enzymes combined with UPLC-QTOF-MS/MS were developed to screen and identify 25 potential ACh E inhibitors from Terminalia chebula fruits,and 36 potential α-Glu inhibitors from Cyclocarya paliurus leaves.Then,the enzyme inhibition assays in vitro were used to estimate inhibitory activities of active compounds against enzymes,and molecular docking was carried out to predict inhibition mechanisms between active compounds and enzyme,further verifying the reliability and practicability of the CFP-immobilized target enzymes screening methods.3.Inhibition interactions of four ellagitannin compounds,punicalagin,chebulinic acid,geraniin and corilagin,from Terminalia chebula fruits aganist ACh E were investigated systematically by a combination of inhibition kinetics,multi-spectroscopic method and molecular docking.Results showed that punicalagin,chebulinic acid and geraniin exhibited strong reversible inhibitory effects on ACh E in an uncompetitive manner,while corilagin inhibited ACh E activity in a mixed type.Four ellagitannin compounds could significantly quenched the intrinsic fluorescence of ACh E though a static quenching along with non-radiative energy transfer,and showed strong affinity to ACh E.The hydrogen bonding and Van der Waals forces might make a great contribution to the formation of inhibitor-ACh E complexes.Moreover,four ellagitannin compounds could lead to alterations in the micro-environment and secondary structure of ACh E,and thus the conformational change of ACh E.4.Inhibition interaction of arjunolic acid from Cyclocarya paliurus leaves aganist α-Glu was studied systematically by a combination of inhibition kinetics,multi-spectroscopic method and molecular docking.Results showed that arjunolic acid had strong reversible inhibitory effects on α-Glu in an uncompetitive manner,and could significantly quenched the intrinsic fluorescence of α-glucosidase though a static quenching and non-radiative energy transfer.Arjunolic acid exhibited strong affinity to α-glucosidase,and the hydrophobic force and Van der Waals force might play an important role in the formation of inhibitor-enzyme complexes.Moreover,arjunolic acid could cause alterations in the micro-environment and secondary structure of α-Glu,and thus the conformational change of α-Glu.