Study On The Extraction And Separation Of Acetylcholinesterase Inhibitors From Fermentation Soybean

At present, the trend of aging population in the world is becoming increasingly serious,the incidence of Alzheimer’s disease (AD) is also increasing year by year, how to preventand treat Alzheimer’s disease is causing more and more attention. In recent years, somestudies have shown that the active ingredient in some special foods and medicinal plantshelps reduce the risk of Alzheimer’s disease, therefore, the development of a functionalfood for the prevention and treatment of AD has important significance. There is a strongacetylcholinesterase (AChE) inhibition of the active ingredient found in homemade-fermented soybean, by comparing the different materials, different fermentation times,filter out the most suitable raw material and fermentation time for fermentation; usingultrasonicassisted extraction to optimize the extraction process of acetylcholinesteraseinhibitors in fermented soybeans; to determine the effective polar fractions by fractionalextraction of the ethanol extracts; macroporous resins for the initial separation andenrichment of the extract components, to provide the theoretical foundation andexperimental basis for the development of medicinal and edible fermented soy products toprevent and reduce the risk of onset of AD. The main contents and results are as follows:1. Compared fermentation samples of different raw materials (soybeans, defattedsoybean meal, black beans, peeled black beans) and soaking and fermenting at differenttimes (0h,24h,48h,72h,96h,120h), by measuring acetylcholinesterase IC50found that thesoybean raw was hardly with inhibition of acetylcholinesterase, but after fermenting theinhibition was significantly enhanced, among all the samples, the peeled black beanssample which fermented96h had the best inhibitory effect and its IC50could reach to0.045mg/mL.2. Through single factor experiment and three factors and three levels’ response surfaceexperiment, studied the effects of ethanol concentrations, solid-liquid ratios, ultrasonicpowers, ultrasonic times and ultrasonic temperatures on the IC50of the acetylcholinesteraseinhibitors extract; built the regression mathematical model of the ethanol concentrations,solid-liquid ratios and ultrasonic times to IC50:IC50=0.041－0.016*A－0.093*B－0.0009250*C＋0.035*A*B＋0.0003500*A*C＋0.006800*B*C＋0.011*A2＋0.093*B2－0.009630*C2 Applying response surface analysis, studied the correspondence between the variousfactors and response value to determine the optimum conditions of the extraction processas follows: ethanol concentration70%, solid-liquid ratio1︰60, ultrasonic time45min,ultrasonic power140W, ultrasonic temperature55°C.3. Four different polarity organic solvents of petroleum ether, methylene chloride, ethylacetate, water-saturated n-butanol were used to fractional extraction of Ethanol extract. Theresults showed that the first three of the extracted components had the inhibitory activity ofacetylcholinesterase, and the water saturated n-butanol’ inhibitory activity was not detected.Presumably, the fermented soybeans’ acetylcholinesterase inhibitory activity might becaused by the performance of several substances synergistically.4. By using filtering macroporous resins to preliminarily separate acetylcholinesteraseinhibitors in the ethyl acetate fraction, adsorption property and desorption property,determined the ADS-17macroporous resin for the separation of acetylcholinesteraseinhibitors resin, the static adsorption rate and resolution liquid inhibition rate were96.65%and64.24%. The suitable analytical solution of ADS-17macroporous resin was80%ethanol solution; After static saturated adsorption, using80%ethanol solution to parse theresin, elution rate was1.0mL/min, the AChE inhibition rate of eluent was62.64%.