Purification Of Glycoprotein From Nostoc Flagelliforme And Its Physiological Activities

Nostoc flagelliforme is a kind of terrestrial nitrogen fixation Nostoc cyanobacteria(blue-green algae),mainly districted in Ningxia,Inner Mongolia and Gansu region(arid and semi-arid zone),has great ecnomic and medical value.In recent years,the researches on the distribution,physiology and artificial cultivation having been carrying out,mostly about exopolysaccharide.Glycoprotein is an efficient biological substance,proved that it has function of antioxidant,immune enhancement and tumor inhibition.So glycoprotein was extracted from Nostoc flagelliforme cells,and further exploring its properties and functional activity,which has important theoretical and practical significance.The response surface methodology was used to extract the glycoprotein from Nostoc flagelliforme cells,The crude glycoprotein was purified by DEAE-52 cellulose column chromatography and gel filtration chromatography,the properties was characterized and its antioxidant activity and immune activity were analyzed.The main results were:1,on the basis of single-factor test,the response surface methodology was used to investigate the effects of processing parameters,including extraction temperature,time and ratio of water to raw material,the extraction rate of glycoprotein was choosen as response value,and establish the regression model,the equation analog effect was good.The optimal conditions were as follows,extraction temperature is 95℃,time is4 h,the ratio of water to raw material is 1:15,under this condition,extraction yield is0.173%.2,The crude glycoprotein was purified by DEAE-52 cellulose column chromatography and gel filtration chromatography,the molecular weight is about40.0KDa.The Phenol-sulfuric acid and Bradford analysis show that NGP-1 sugar content of 76.25%,the protein content of 15.62%.The composition of Monosaccharide and amino acid were analyzed by Ion chromatograph and Amino Acid Auto Analyzer,NGP-1 was composed of Rha,Ara,Gal,Glc,Suc,Fru,Xyl,Man,GalA and Glc A.Seventeen amino acids were identified in NGP-1,the total is 13.42%.3,β-elimination reaction indicated it has O-glycopeptide bond,FT-IR showed it was Pyran type,and DSC indicated the thermal denaturation temperature was about140℃.4,The result showed that the NGP-1 has significant antioxidant activity,when the concentration of NGP-1 was 4mg/mL,its reducing power,phenylhydrazine radical and hydroxyl free radical scavenging abilities reached the maximum.When it was180μg/m L,the superoxide anion free radical reached the maximum.5,Three doses of NGP-1 was gavaged to mice,at lower and middle doses,NGP-1can significantly and markedly increase the index of immune organs including thymus and spleen.At 5mg/kg and 10mg/kg,NGP-1 can markedly increase the level of serum hemolysin,and has certain effect relationship with increasing concentration.The lower dose has no significance on spleen cells,there was a trend of increasing gradually from the middle dose.Only when the concentration of NGP-1 was 5mg/kg,it has markedly significance on the transformation of peritoneal macrophages,the lower and higher concentration has no significance.