Isolation,Puirfication And Screening Of Analgesic Active Parts Of Porcine Brain Protein

Purpose: Using procine brain as raw material, optimize the process of brain proteinhydrolyzate, investigate their physiochemical and structural properties, screening out theanalgesic active parts and structural studies of the compounds that have been isolated.Method: Inspected each of the key factors on the enzymatic hydrolysis, optimize theprocess of brain protein hydrolyzate, to determine its optimal process; Enzymatic the porcinebrain, preparation of different molecular weight fragments. Study their physiochemical andstructural properties through the Lowry method, molecular weight determination and HPLCetc.Analgesic activity parts were screened by the mouse acetic acid writhing experiments andits structures were researched by the HPLC-MS methods.Result: To inspect the key factors of the enzymatic hydrolysis,determined the bestprocess of enzyme hydrolysis. Pharmacological experiment results show that the partsisolated from porcine brain through enzymatic method were no effect to the mouse hot-plateanalgesia,but significant analgesic effect to the mouse acetic acid writhing;hydrolysis supernatant analgesic effect is obvious,with dose-effect relationship;brain proteinpowder has the trend of decrease,with dose-effect relationship. Use macroporous resinseparated the part of enzymatic supernatant,collected water elution,50%ethanol elution partand95%ethanol elution part.Molecular weight were determinated of the three parts by HPLCshow that Water elution part around1500-300Da,50%ethanol elution part around3000-1500Da,95%ethanol elution part less than1000Da.Mouse writhing analgesicexperiments show that the trend of water elution part and50%ethanol elution part reducedobviously, and with dose-effect relationship. Protein content determination of the three partsby Lowry method show that the protein content of water elution part was40.5%,50%ethanolelution part was97.5%,95%ethanol elution part was10.6%. Sephardex LH-20was used inthe fine separation of the50%ethanol elution part--the highest protein content, a high purity sample was got(Sample A),it was determined as tryptophan by HPLC-MS.Conclusion: In this study,we inspected the factors of hydrolysis, to optimize thehydrolysis process, explored a safe, low cost, quality controlled hydrolysis process, greatlyimprove the efficiency of hydrolysis; The analgesic part was screened and the effective partswas determined by pharmacological experiments. Depth study of physical and chemicalproperties were carry out utilized HPLC,Lowry method,HPLC-MS etc.