Centipede Anticoagulant Peptide Extraction And Separation And Analysis

Objective:To study the process condition of bionic enzymatic hydrolysis of Centipede,explore the effective parts of anticoagulation of Centipede Hydrolysates. Further separation and analysis of its component structure to provide a reasonable basis for the pharmaceutical preparationsMethod:(1) Extraction Process:Taken activated partial thromboplastin time (APTT) and fibrinogen plate assay as the index, Comparison of bionic enzymatic hydrolysis, water extraction and alcohol precipitation, alcohol extraction and alcohol precipitation and water boiling, selected the extraction method of centipede; taken activated partial thromboplastin time (APTT) and fibrinogen plate assay as the index too, further Comparison of pepsin Pepsin hydrolysis method, trypsin hydrolysis method, Bionic enzymatic hydrolysis method and Combined enzymatic hydrolysis methods, Select the best hydrolysis method. Single factor explored the process condition of Centipede's extraction with hydrolysis degree as the index,to determine the best conditions of bionic enzymatic hydrolysis. (2) anticoagulant peptide separation and analysis:Taken activated partial thromboplastin time (APTT) as the index, separate the anti-coagulate active components from the pepsin enzymolysis liquid of Centipede by SephadexG-50, SephadexG-25 gel filtration chromatography and C18 reversed phase chromatography, and supplemented by high performance liquid chromatography, MAILDIA-TOF-MASS mass spectrometry, gel electrophoresis and thin layer chromatography methods for analysis.Results:(1) Bionic enzymatic hydrolysis is the best extraction method for Centipede, The best extracting technique of them as follows:Centipede was firstly hydrolyzed for 4hours in an artificial gastric liquid at 37℃which contained 4.0% pepsin (pepsin to substrate). Then the residue was hydrolyzed for 4 hours in artificial intestine liquid at the same temperature and contained 5.0% trypsin (trypsin to substrate) (2) A component F3-3 can separated by SephadexG-50 and SephadexG-25 gel filtration chromatography, showed strong anticoagulant activity. It can further purified by C18 reversed phase chromatography and get four components, the second Fraction F3-3-2 has a strong anticoagulant effect, but it is not a single component; (3) In addition,the third component F3-3-3 is a relatively pure material, but the anticoagulant effect is not obvious.Conclusion:The bionic enzymatic method is suitable for extracting the effective parts of anticoagulation and thrombolysis.Used gel and C18 reversed phase column can effectively separate the anti-coagulate active components from the pepsin enzymolysis liquid;MAILDIA-TOF-MASS MS show, the anticoagulant of centipede is a small molecule peptide,and they are not a single substance,but some Peptide in the context of a certain molecular weight.