Perccottus Glenii Research The Polysaccharide Extraction And Purification Of Anticoagulant

Perccottus glenii is a kind of economical freshwater fish,and it is often called as “LaoTou Fish”.P.glenii tastes delicious withrich variety of protein,trace elements and vita mins.Currently,the study of P.glenii confines to the breeding technology,living habits,drugresistance and other aspects.There is no report about the extraction and biological activity of P.glenii.This experiment makes first study on the extraction,purification and anticoagulant activity of polysaccharide in P.glenii and provides scientific basis for the comprehensive and high value utilization of P.glenii.The significance of the study is to compare and evaluate the study on the polysaccharide extraction and anticoagulant activity ofP.glenii.It makes beneficial evaluation on the study of the active substances of P.glenii.The specific research contents and results are as follows:1.Polysaccharide is extracted from heads of P.glenii by alkaline hydrolysis.After degreasing,centrif?galization,alkali solution,concentration,alcohol precipitation and freeze-drying,the coarse polysaccharide of P.gleniiis gotten at a rate of 5.23%.2.The method of animal experiments is used to identify whether the polysaccharide has the activity of extending the time of blood coagulation.It is gotten by the capillary method that: The blood coagulation time of the blank group is 64±3.14 s.The blood coagulation time of the high dose group is 125±5.56 s.It can be gotten by the tail test method that: The bleeding time of the blank group is 5.8±0.4 s,and the bleeding time of the high dose group is 12.21±0.8 s3.The method of vitro experiments is used to identify whether the polysaccharide has the activity of extending the coagulation time of the plasma: The blood coagulation indexes are deter mined by PUN-2048 type blood coagulation instrument.By analyzing the results,it can be found that the coarse polysaccharide of P.glenii can extend the activity of plasma clotting time.aPTT time is 62 s±4.1 at 50mg/mL.4.The P.glenii coarse polysaccharide and protein clearance test take four comparative ways of methods to screen,namely the sevage method,the TCA method and the combination method of enzymatic and sevage method respectively.Among them,the combination way of trypsin and sevage method has the best ability to clear protein with the protein clearance rate of 80.12% and the polysaccharide loss rate of 30.12%.5.The way of DEAE-52 cellulose column chromatography is used to separate the coarse polysaccharide(PGP)from P.glenii.Three main components of polysaccharide are gotten from the separation.The yield rates of PGP-1,PGP-2 and PGP-3 are 432.1 mg/g,291.6 mg/g and 172.2 mg/g respectively.Under the 4mg/mL concentration,PGP-1 and PGP-2 polysaccharide components have good anticoagulant effect.The testing time of aPTT is 64±2.3s and 57±2.6s respectively.Among them,PGP-3 has no obvious anticoagulant effect.6.The G-100 glucan gel column chromatography method is used to make purification on PGP-1 polysaccharide components.Three kinds of purified polysaccharide components are gotten respectively,including P-1,P-2 and P-3.The total sugar contents are 65.1%,63.53% and 66.43% respectively.The total protein contents are 1.64%,1.76% and 2.2% respectively.Under the 4mg/mL concentration,P-2 and P-3 polysaccharide components have good anticoagulant effect.The testing time of aPTT is 72±5.7s and 65±4.1s respectively.Among them,P-1 polysaccharide component has no obvious anticoagulant effect.7.The ultraviolet spectrum and Fourier are used to transform infrared spectroscopy.The P.gleniipolysaccharide components(P-1,P-2 and P-3)have absorption peak of polysaccharide material characteristic.And it is found that P-1 component is the polysaccharide which is combined with protein.8.The method of high performance liquid is used to analyze the monosaccharide composition of P-1 polysaccharides component in P.glenii.After comparing the samples with monosaccharide standards,it is concluded that the P-1 polysaccharide component in P.glenii consists of glucose,rhamnose and glucuronic acid,arabinose,galactose.According to the internal standard method,it can be calculated that the molarratio of the monosaccharide is: 0.64:24.6:17.15:21.49:35.12.