Study On The Interactions Between The Main Components Of Forsythia Suspensa And Acetylcholinesterase

Alzheimer's disease?AD?is a neurodegenerative disorder characterized by learning,cognitive and memory dysfunction.Acetylcholinesterase?AChE?inhibitors are the main means of treating the disease.Currently,the AChE inhibitors used in clinical use have some disadvantages such as low bioavailability,varying degrees of toxicity and side effects.Therefore,further searching for new,efficient and less toxic AChE inhibitors is one of the research focuses.Natural Chinese herbs are rich in variety and they are less toxic and less costly in the treatment of diseases,and therefore more and more attention is paid to them by pharmacologists.Forsythia suspensa?Thunb.?Vahl is a kind of Natural Chinese herbal medicine,it has been studied from animal experiments that forsythiaside can improve learning and memory in AD animal model,but it is not clear whether Forsythia suspensa and its main components can affect the neuroprotective effect of cholinergic system.In view of the problems mentioned above,Forsythia suspensa was chose to study in this thesis.First of all,from the perspective of chemistry,we will use the method of Ellman to study the inhibitory effects of Forsythia suspensa and its main components on the enzyme activity of AChE.Secondly,the interaction mechanism between forsythiaside or rutin and AChE was studied by molecular docking techniques and various spectral techniques,and the interaction between the two compounds was compared.Finally,from the biological point of view,the neuroprotective effect of forsythiaside on A?-induced apoptosis was further studied with PC12 cells as a model.The main research contents and results are as follows:1.Study on the inhibitory effects of Forsythia suspensa and its main components on AChE.The effect of Forsythia suspensa and its main components on the activity of AChE was studied by the method of Ellman,and the inhibition ability was compared by IC₅₀0 value.Results showed that the inhibition effectof Q2 was stronger than the inhibition of Q3 and the inhibition of Q4 on AChE.However,Q1,Q5 and Q6 had no inhibitory effect on AChE.According to the analysis of liquid phase diagram,the peak area of forsythiaside was 68.9%.As a result,the effect of Q2 on enzyme inhibition was better.It was suggested that forsythiaside may be the effective component to inhibit AChE.Knockout experiment confirmed that forsythiaside played a major role to inhibit AChE in Q2.Kinetic experiment results showed that the inhibition of forsythiaside on AChE was mixed-type inhibition,and the inhibitory constant was 47.68?M.2.Study on the interactions of forsythiaside or rutin with AChE.Quenching mechanism,binding constant,binding site and binding distance were studied by spectral method and molecular docking technique.The experimental results showed that the interaction of forsythiaside or rutin with AChE induced in the complete quenching of AChE fluorescence.The quenching mechanism was mainly the static quenching of the formation of complex;both combined with AChE at 1:1;Van der Waals force,hydrogen bond and hydrophobic force were the main interaction forces between them with AChE;molecular docking technique further verified the interaction force;the binding distance between both of them was less than 7 nm.The two compounds also showed some different properties:at the test temperature,the binding constant K of rutin is smaller and the interaction between rutin with AChE was weaker than that of forsythiaside.Forsythiaside showed strong interaction and quenching,and its binding constant K was slightly larger.According to the comparison of Total_Score displayed by molecular docking software,it was concluded that the interaction ability of forsythiaside with AChE was stronger than that of rutin.These results showed that forsythiaside has strong fluorescence quenching effect on AChE and has high affinity.3.Protective effects of forsythiaside on amyloid beta-induced apoptosis by downregulating AChE.MTT,ELISA kit,Hoechst33258 staining,flow cytometry and Western blot were used.The results showed that the decrease of cell survival rate and the increase of AChE activity and expression and cell apoptosis induced by A?_25-355-35 were improved by forsythiaside.It was proved that protective effects of forsythiaside on amyloid beta-induced apoptosis in PC12 cells by downregulating AChE.This discovery makes forsythiaside a new potential drug for the treatment of AD.