Study On Manufacture Of Anticoagulant Peptide From Hirudo Nipponica Whitman By Controled-Enzymatic Hydrolysis

Medicinal leech has been used as a remedy for blood stasis and traumatic. At the present time,it is a main kind of medicine used in Traditional Chinese Medicine for promoting blood circulation by removing blood stasis,and often, it is used after being processed.In modern medicine,medicinal leech is used in the treatment of the thrombus.Because of its off-flavor problem and complicated components,the use of medicinal leech is confined.In this work,we focused our attention on the leech Hirudo nipponica Whitman.Under the theory of chinese medicine,medicine biological techniques,including controlled-enzymatic hydrolysis,ultrafiltration,ion exchange chromatography,are introduced to purify low molecule weight anti-coagulate active peptides from crude drug samples.The anti-cerebral ischemia effect of these highly purified peptides was studied by using MCAO. The main research contents are as follows:1.Controlled-enzymatic hydrolysis techniqueTaking Anticoagulant activity as the key indicator,we analyze the influence of enzymatic methods based on the types of protease,enzyme dosage, enzyme solution pH,and temperature and enzyme substrate concentration. Optimum experiment results are:the protease is trypsin,the enzyme dosage is 5000NFU·g^-1,enzyme substrate concentration is 10 percent,enzyme solution pH is 8.5,temperature is 50 centigrade and enzymolysis time is 8 hours respectively.2.Separation and Purification of anticoagulant peptide(HAP) from Hirudo nipponica WhitmanThe conditions of organic solvent concentrations,ultrafiltration membrane molecular weight and chromatograph conditions were compared by taking Anticoagulant activity as the key indicator,the Optimized HAP separation and purification conditions was identified:firstly retrieve enzymolysis liquid Supernatant to which added double volume Acetone,then filtrated at 0.22μm and ultra-filtrated at 5kD;The anticoagulant peptides were separated from supernatant by Q Sepharose^TM Fast Flow strong anion exchange chromatography which equilibrated by diethanolamine-hydrochloric acid(pH8.9),desalted by G10.The purified peptide takes up 53.3 percent of products' weight.The major structures of peptide was determined by ultraviolet-visible(UV-Vis),infrared spectra(IR) and Mass Spectrometry (MS).3.Anticoagulation of HAPResearch results indicate that both the HAP group of 16mg·kg^-1 and 8 mg·kg^-1,comparing with the model group,can significantly improve the neurological symptoms and reduce the infarct size.Markedly,group 16mg·kg^-1 and 8mg·kg^-1 have an increasing effect on the levels of SOD,GSH and GSH-PX;in contrary,the level of MDA,LDH and NO decreased.In the model of respiration time of decapitated mice after burden swimming,HAP group 20 mg·kg^-1,10 mg·kg^-1 and 5 mg·kg^-1 can significantly increase the respiration time of decapitated mice after burden swimming.In vitro test,we observed that the HAP has a role in thrombolysis.In artery vein bypass thrombosis,HAP group 16mg·kg^-1,8mg·kg^-1 and 4mg·kg^-1 can reduce the wet weight of thrombus in vein and artery loops to varying degrees.We observed that the HAP can extend the healthy human plasma APTT and PT duration in vitro,and have an inhibit effect on ADP-induced platelet aggregation in healthy people.In conclusion,the major achievements of this research were as follows:HAP which take up 53.5 percent of product content conforms to the rule of "herbs and natural medicine registration of technical requirements for Category 5' from The Drugs Register To Manage The Way. Multi-model were taken into the study of HAP pharmacodynamic test, proving it has unique therapeutic effects on ischemic stroke and laying a good foundation for the further development of HAP.