Isolation And Purification Of Acetylcholiesterase Inhibitors From Fermented Bean

Fermented soybean products have many health care functions, such as lowering blood pressure, blood sugar and blood fat, dissolving (the) clots, anti-oxidation, anti-cancer, anti-Alzheimer, anti-bacterialand so on. Aiming at exploring the possibility for fermented soybean products to resist Alzheimers, This thesis Established a method for screening acetylcholinesterase(AchE) inhibitors from fermented soybean products. Optimized Extraction condition and discussed their separating and purifying approaches.Main results were as following:1 Establishment of the screening method Based on the spectrophotometry mothed(to detection of cholinesterase inhibitors in vitro). We Found that the concentration of AchE was directly proportional to OD value, That was, OD value may represent the enzymatic activity. Through the TLC- bioautography experiment showed that the introducing of Ellman's reagent as reaction reagent has a preferable coloration effect. We Choose spectrophotometry to detect the AchE activity from the different regions of douchi and fermented bean curd extracts. Results showed that the Douchi from Yongchuan performed the highest AchE inhibition rate, it was 52.36% when at the concentration of 1.0 mg/mL.2 Optimization of Extraction Condition We Use ethanol refluxing extraction method to extract AchE inhibitors from douchi. In order to optimize extraction condition, single factor experiment and orthogonal experiment were adopted And the Ellman's method was applied to determine AchE inhibitory ability in vitro; Results showed that the optimal condition for extraction is: extract time is 90 min, ratio of liquid/solid is 1:12, temperature is 75℃, ethanol concentration is 80% , the verification experiment was done by process optimization. The AchE inhibitions rate was 53.40% when Douchi extraction was at he concentration of 1.0 mg/mL.3 Isolation and purification We Choose Douchi from yongchuan chongqing as materials for separating, in order to separate the most active monomers. Macroporous adsorption resin enrichment, silica-gel column chromatography, Sephadex LH-20 chromatography and recrystallization method had been used. The crude extract was extracted by petroleum ether, methylene chloride , ethyl acetate extract and n-butyl alcohol respectively, and the active site was mainly rich in the petroleum ether, methylene chloride and ethyl acetate. After purifying of the petroleum ether components, Two highly active components attained were white crystal (1) and white powder (2) respectively, the AchE inhibitions rate were 95.27% and 100% respectively when at the concentration of 1.0 mg/mL. After purifying of the methylene chloride component. A highly active component which was light yellow powder had been collected. The AchE inhibition rate was 100% when the component at the concentration of 1.0 mg/mL. The activity has been greatly improved after purification.The paper comprehensively adopted the spectrophotometry, TLC-bioautography and modern chromatographic separation technology to isolate and purify AchE inhibitors from fermented soybean. The experiment results proved that the method could produce advisable results.