Research On High Performance Heparin Extraction Separation And Purification Technology

Heparin is an anticoagulant traditionally prepared from province small intestine and ox lung, besides antithrombotic it posseses some other bology functions like regulation blood fat, anti-inflammatory and ananaphylaxis. The purpose of our research was to difine the crisis technology parameters of heparin high proformance extraction and purification, to set up a technology condition of heparin high proformance extraction and purification. In this experiment, took the porcine small intestine mucous membrane as raw materials, by using single factor experiment, multi factor combination experiment and RSM analysis software to research the extraction, purification and resin absorption and separaqtion conditions of heparin, meanwhile, the ultraviolet spectra, infrared spectra, HPLC and PAGE electrophoresis were applied to detect the characters of heparin.1. Research on enzyme hydrolysis method extraction heparin. Single factor experiment were used to study the effect of enzyme hydrolysis time, enzyme hydrolysis temperature and the ratio of material to solution on the yield of heparin, and the quadratic rotary combinational design and SAS analysis software were used to optimize the extraction technology parameters. The optimized results were: extraction temperature 55℃, extraction time 2 hours, ratio of material to solution 1︰15, the heparin extraction yield was 206.83mg/kg. The yield of heparin increased 32% than traditional salt fraction, and the extraction time were shortened 2 hours.2. Research on ultrasonic assisted salt fraction method extraction heparin. The effections of ultrosonic power, ultrosonic time, ultrosonic temperature, salt concentration, ratio of material to solution on the yield of heparin were researched with single factor experiment, and the quadratic rotary combinational design and SAS analysis software were used to optimize the extraction technology parameters of heparin. The optimized results were:ultreosonic power 300W, ultreosonic time 40min, ultreosonic temperature 40℃, salt concentration 3%, ratio of material to solution 1︰15, the heparin extraction yield was 189.19mg/kg. If exacted heparin with two methods meanwhile, the yield of heparin was 230.81mg/kg, it increased 40% than traditional salt fraction, and shortened the extraction time to 2h. The method of ultrasonic extraction and enzymic hydrolysis extraction carrying out at the same time was determined the best extraction method.3. Research on technology conditions of ion-exchange resin absorption and separation of heparin. Using D208 strong alkalinous anion resin to absorb and separate heparin from crude heparin extract liquid, single factor experiment were carried out to study the effect of salt concentration, adsorption temperature and adsorption time on the yield of heparin, and orthogonal experiment and SAS analysis software were used to optimize technology parameters, the optimized results were: adsorption temperature 40℃,adsorption time 3h, salt concentration 2M, the heparin potency was 115U/mg, it increased 15% than traditional salt fraction.4. Research on heparin purification and structure verification. In this experiment, Sephadex G-75 gel column and Sepharose-QFF ion-exchange chromatography were applied to purify heparin steply. Firstly, the crude heparin were purified by Sephadex G-75 gel column chromatography, collected the eluting peaks and purified the main eluting peak with Sepharose-QFF ion-exchange chromatography, the purified heparin potency was 150U/mg. The purified heparin were analyzed by UV spectra, IR spectra, HPLC and PAGE electrophoresis. UV spectra results showed that no protein or nucleic acid substance exsited in purified heparin. IR analysis results showed that heparin saccharied chain contained hydroxy group, carboxy group and amide linkage, which means heparin structure were not changed. HPLC results showed that the main disaccharide of purified heparin were the same as standard heparin. The PAGE result showed that electrophoresis strip of Sephadex G-75 purified heparin were two main strips, however, the electrophoresis strip of Sepharose-QFF purified heparin was single strip, and the average heparin molecular was 15 KDa.