Studies On Separation,Purification And Bioactivities Of Polypeptide From Hirudinaria Mannillensis

In this study,we focused on the isolation from Hirudinaria manillensis.And the anticoagulant activity peptides were obtained.First,the basic components of Hirudinaria manillensis powder including moisture,ash,protein,and fat were measured in the study.The result indicated that the protein content(68.1±0.10%)was higher than other contents.Hirudinaria manillensis was extracted by the pH precipitation,acetone extraction,water leaching,alkaline protease,and ammonium sulfate precipitation by alkaline protease.The anticoagulant activity of the alkaline protease extraction was the highest,which was 35.47±0.55 U/g.To optimize the extraction process of the alkaline protease,we investigated the effects of enzyme content(U/g),time(min),ratio of material to liquid,and the pH.The optimum conditions determined by the orthogonal experiment were:enzyme content was 2000 U/g,the time of enzymolysis was 250 min,the ratio of material to liquid was 1:15,and the pH was 9.Under these conditions,the anticoagulant activity was 72.3 U/g and the degree of hydrolysis was 31.6%.In addition,the study investigated the antioxidant activity of the anticoagulant substance by the determination of the hydroxyl radical scavenging,the determination of the reducing ability,and the determination of the superoxide free radical scavenging.The result indicated that the anticoagulant active peptides released from Hirudinaria manillensis had a certain degree of the scavenging ability to hydroxyl radical,a certain reducing power,and an obvious scavenging ability to superoxide anion.In order to further separate and purify the extracted substances,the anion exchange chromatography DEAE Sephadex A-50 and the gel filtration chromatography Sephadex G-50 were used.Three components of dry products were obtained by the method of the DEAE Sephadex A-50,and the result demonstrated that the main peak HA was active and the activity was 202.5±2.15 U/g.HA2 obtained by the further purification using the gel filtration chromatography Sephadex G-50,which had a higher activity with 358.96±1.56 U/g.The eluting peak was collected and freeze-dried.The results showed that the extracted liquid had the maximum absorption peak at 295 nm.The IR spectra of amide A,amide B,amide Ⅰ,and amide Ⅱ in the range of 500-4000 cm-1 was obtained by Fourier transform infrared spectroscopy.The specific activity of the purified peptide was 461.33 U/mL when the anticoagulant activity was 358.96 U/mL,and the concentration of the polypeptide was 0.7781 mg/mL.The stability result indicated that the anticoagulant active substances were asily dissolved under the neutral or weak acid conditions,and the higher temperature led to the inactivation of anticoagulant active substances.So the active substances should be stored in a low-temperature environment.