Research On The Preparation And Structural Characterization Of Low Molecular Weight Heparin

Heparin,consisted of hyaluronic acid and glucosamine residues connected by 1,4 glycosidic bond connection is a kind of highly sulfa ted linear glycosaminoglycan which is widely existed in the tissue cells of liver, lung and intestine mucosa.Due to the large molecular weight, complex composition and the dispersion of the length of the sugar chain, heparin has low bio-availability, unpredictable pharmacokinetic charact-eristics,large individual differences in drug use and bleeding. In comparing to the normal heparin, low molecular weight heparin (LMWH) had several advantages such as simpler structure and lower molecular. These characteristics made it more resistance to FXa (clotting factor), low anti FⅡa (thrombin) effect and can reduce bleeding. The preparation of heparin with low molecular weight by acid degradation was studied in this paper. The purpose was to obtain heparin with high anticoagulant activity, the main research methods are as follows:The crude heparin was obtained from pig small intestinal mucosa powder by water extraction, alcohol precipitation and Sevag purification method. The yield was calculated to be 11.24%. The heparin content was measured to be 30.46% by the phenol sulfuric acid method. The content of soluble protein is 20.63%.The heparin was purified from the crude extract by macroporous resin D208. the heparin content increased to 70.83%. The content of glucuronic acid is 45.26%. The sulphate content is 32.54%. The anti-Xa activity is 188.36 IU/mg and anti-Ⅱa activity is 185.16 IU/mg. The specific rotation is +50°.The degradation condition was optimized with the purpose of obtaining heparin with the highest anticoagulant activity. The optimum reaction conditions according to the single factor orthogonal experiment were listed as follows: 0.5% NaNO2 concentration, pH value of 2.8, the degradation temperature of 25℃ and 4.5 h of reaction period. Afterward, the heparin derived from the optimum condition was purified by SephadexG-50. The content of glucuronic acid is 43.62% The sulphate content is 30.48%. The anti-Xa activity is 182.48 IU/mg and anti-Ⅱa activity is 121.23 IU/mg.HPGPC analysis was carried out to study its purity. Finally, its structural characteristics was studied by the optical rotation measurement, the specific rotation is +15°. Infrared spectrum and nuclear magnetic resonance(NMR) analysis. The molecular weight was measured to be 4600 Da by the mass spectrometry (MALDI - TOF MS) method.