| Blood coagulation is an important physiological defense process in human bodies, butpathological thrombosis is very harmful to human health. Currently, ordinary heparin, lowmolecular heparin and warfarin are the main anticlotting drugs for clinical use either at homeor abroad, which have side effects like thrombocytopenia, bleeding and time consuming.While peptide anticlotting drugs, such as hirudin, which are the key development direction ofanticlotting drugs, have prominent advantages like high time efficiency and few side effects.Hirudinaria manillensis, a natural traditional Chinese medicine which，is called soft gold andcan produce hirudin with the functions of anticoagulation, antiatheroscloresis, antitumor andcardiovascular protection. Using Hirudinaria manillensis as main raw material, the enzymatichydrolysis and relations between the activity and hydrophobicity of the products weresystematically studied in this dissertation. This study was not only of high theoretical value,but also provided a new method for the production of a safe, green and natural alternative toanticlotting drugs with high anticoagulant activities.In view of the great influences of environmental factors and human factors, severalsignificant conditions during the process of in vitro anticoagulant activity determination areoptimized:(1) sample concentrations should be diluted to10~15mg/mL for titration, and thenumber of titration should be kept below4, or the samples should be diluted twice;(2) the pHof reaction system should be adjusted to7.0;(3) thrombin should be stored and determined at0℃;(4) fibrinogen solution should be used within4hours right after it was ready.In the process of enzymatic hydrolysis, using anticoagulant activity, hydrolysis degreeand yield of soluble peptide as indexes, Alcalase AF2.4L, which has best hydrolytic activity,was selected as the enzyme. The best reaction conditions of Alcalase AF2.4L were: substrateconcentration12%, temperature55℃, time2h, pH8.5. Under these conditions, theanticoagulant activity of product can reach702ATU/g, hydrolysis degree21.34%, solublepeptide yield86.22%. The molecular weight distribution of product ranged from700.315to 4472.065Da.In order to explore the relationship between anticoagulant activity and hydrophobicity,macroporous adsorption resin chromatography was chosen to purify the product. According tothe static adsorption and desorption experiment, both the quantity and rate of adsorption ofDA201-C resin were the highest. Different alcohol concentrations were adopted as elutionagents successively. Hydrophobic value of each component increased with ethanolconcentration. The relationships between anticoagulant activities and hydrophobicities ofpeptides and content of hydrophobic amino acid were compared, results showed that theanticoagulant activity of component T3-75which was eluted by75%ethanol was the highest,but component T3-25,eluted by25%ethanol, was the lowest. Their anticoagulant activiteswere830ATU/g and324ATU/g, respectively. Anticoagulant activity increased withhydrophobicity and content of hydrophobic amino acid, and there are significant correlationbetween them.Hirudinaria manillensis is very expensive. In order to save cost, three cheap rawmaterials—earthworm, Whitmania pigra and small intestine from pig were chosen to prepareanticoagulant peptides by pepsin, trypsin and Alcalase AF2.4L with anticoagulant activity,hydrolysis degree and yield of soluble peptide determined. The results showed that:(1) Theanticoagulant peptide from earthworm by trypsin has the strongest activity554ATU/g;(2)The anticoagulant peptide from Whitmania pigra and small intestine by Alcalase AF2.4Lboth has the strongest activity,444and400ATU/g, respectively. Anticoagulant activity ofthese three kinds of peptides is lower than that of Hirudinaria manillensis but a little, whichstills proves that the method of enzymatic preparation by various cheap raw materials isfeasible. |
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