Study On The Inhibitory Effect Of Several Natural Elastase Inhibitors

Elastase overexpression can trigger a variety of pathological changes,and the study of elastase inhibitors is a direction for the development of drugs to treat a variety of inflammatory conditions,lung diseases,psoriasis and cancer.The study of natural elastase inhibitors has received extensive attention.In this thesis,the inhibition activities of natural components were screened and evaluated by establishing an in vitro target enzyme activity screening model,analyzed by spectroscopic,and also studied the inhibitory effects of five flavonoids and two organic acids on elastase using molecular docking simulation techniques to clarify their conformational relationships,and the results of the study are as follows.The inhibitory effects of five flavonoids and two organic acids on elastase were studied by establishing an in vitro screening model of target enzyme activity,spectroscopic analysis and molecular docking simulation.The results are as follows:The in vitro enzyme target activity screening model was established,and the inhibitory IC₅₀ of quercetin,hyperoside,rutin,luteolin,luteoloside on elastase were53.2,31.01,27.12,18.22 and 61.32μM,respectively.The inhibitory capacity was luteolin,rutin,hyperoside,quercetin and luteoloside in order from the largest to the smallest.The five flavonoid monomers showed good inhibition of elastase.The results of spectral study showed that five flavonoids had fluorescence quenching effect on elastase,and the quenching was static.The monomer and elastase can form a 1:1complex.The main binding forces of luteolin,rutin and hyperoside are hydrogen bond and van der Waals force,while the main electrostatic interaction of quercetin and luteoloside is.According to F?rster’s theory,non-radiative energy transfer occurs between each flavonoid monomer and elastase;UV,synchrotron fluorescence and Fourier transform infrared spectroscopy results show that each monomer can alter the microenvironment of the luminescent group around elastase.Analysis of CD spectra shows that the action of the five monomers results in a decrease in theα-helix andβ-fold content of elastase,an increase in the random coil content,a loosening of the enzyme structure and a change in the secondary structure of the enzyme.Molecular docking simulations showed that the benzene ring,heterocyclic ring,conjugated system and polyhydroxyl structure of the five active monomeric flavonoid molecular backbones bound well to the amino acid residues of elastase,all with hydrogen bonding,van der Waals forces,electrostatic interactions,π-cationic interactions and hydrophobic interactions.Among them,the main force between quercetin and luteoloside was electrostatic interaction,and that between luteolin,rutin and hyperoside was hydrogen bond and Van der Waals force.Combined with molecular docking results and activity screening:the presence of hydroxyl group in the C ring C-3 of flavonoids was unfavorable to the inhibition of elastase.If the C ring C-3 changed from hydroxyl group to glycoside,it was favorable,and the double glycoside was more favorable than the single glycoside,and the 7-O-glycosylation of the A ring was unfavorable to the inhibition effect.The in vitro screening model of enzyme target activity was established,and the inhibitory effect of inhibitors was studied by enzyme reaction kinetics.The inhibition rates of two organic acid compounds chlorogenic acid and caffeic acid on elastase were determined to be 23.32%and 13.34%at 1 m M,K_i（chlorogenic acid）=1.69 m M,K_i（caffeic acid）=2.59 m M.Chlorogenic acid had stronger inhibition ability than caffeic acid,and the two organic acids had better inhibition ability to elastase.The spectral results showed that chlorogenic acid and caffeic acid had a static fluorescence quenching effect on elastase.The monomer and elastase can form 1:1 complex.According to the calculation,the main binding force of chlorogenic acid and caffeic acid is electrostatic interaction.According to F?rster’s theory,both monomers and elastase undergo non-radiative energy transfer;the results of UV spectra,synchrotron fluorescence spectra and Fourier transform infrared spectra show that each monomer can cause changes in the microenvironment of the luminescent groups around elastase.CD spectroscopy analysis showed that the action of the two monomers decreased theβ-fold content of elastase,increased theα-helix,increased the random coil content,the enzyme structure became loose,and the secondary structure of the enzyme was changed.Molecular docking simulation showed that hydrophobic interaction,electrostatic interaction,hydrogen bond and van der Waals force existed between the two organic acids and elastase.There is alsoπ-cation interaction between chlorogenic acid and ARG217A.Combined with molecular docking results and activity screening:chlorogenic acid,a derivative of caffeic acid,has stronger inhibitory ability,that is,the substitution of carboxylic hydrogen of caffeic acid by quinic acid is favorable for inhibiting elastase.Five natural flavonoids and two organic acids belong to the derivatives of flavonoids and caffeic acid.The skeleton structure of flavonoids and caffeic acid is the basic structural unit of potential elastase inhibitors.According to the results,the presence of glycosidic bond of flavonoid monomer C-3 is favorable for the inhibition of elastase,the presence of hydroxyl group is unfavorable,and the glycosidization of C-7is unfavorable for the inhibition of elastase.Organic acid monomers can introduce effective functional groups on the basis of caffeic acid and improve their inhibitory activity.This study can provide flavonoids,organic acids elastase inhibitor screening,modification,alteration,design,build,to provide the reference.