Extraction,Purification And Activities Of Polysaccharides From Apostichopus Japonicus Sperm

Sea cucumber is a significant marine food in coastal area of china which has various functional ingredients and healthcare function.In recent years,sea cucumber polysaccharide,has antineoplastic activity and enhance immuno,become a research hotspot of sea cucumber gradually.In sea cucumber process,the sea cucumber sperm,a by-product of sea cucumber,was often be discarded to generate waste and contaminate the environment.Therefore,extraction and activities of polysaccharide,based on Apostichopus japonicus sperm,were studied in this research.Firstly,single factor experiments and response surface experiment were choosen to optimize enzyme hydrolysis by using two protease,and then purified components?SCSPA₁,SCSPA₂,SCSPB₁,SCSPB₂?were obtained by separating and purifing the sea cucumber sperm crude polysaccharide?SCSCP?.Finally,preliminary study were carried on the polysaccharide components,which include physical and chemical properties,antitumor activity and antioxidant activity,expecting provide theoretical basis and technical support for the production and application of sea cucumber sperm polysaccharide.The main studies and results are mentioned as follows:1.Extraction of polysaccharidesTwo widely used proteases were used to extract polysaccharides from Apostichopus japonicus sperm.After single factor experiments,factors such as enzyme dosage,extraction temperature and extraction time were taken into account to perform response surface experiment optimization according to polysaccharide yield.The results showed that the optimum extraction conditions of SCSCP were as follows: papain,enzyme dosage 3%,extraction temperature 49 ?,extraction time 6 h.Under these conditions the yield of SCSCP was 10.64‰.2.Purification and physical and chemical properties analysis of polysaccharidesTwo main fractions SCSPA₁ and SCSPA₂ of polysaccharides from SCSCP were further purified on DEAE Sepharose Fast Flow,and SCSPB₁ and SCSPB₂ were further purified onSephadex G-200 column chromatography respectively,determining the physical and chemical properties.The results were as follows: Firstly,the polysaccharides contents of SCSCP?SCSPA₁ ?SCSPA₂?SCSPB₁ and SCSPB₂ were 31.07%,40.03%,45.27%,44.62%,50.73%;The protein contents were 21.18%,19.43%,7.52%,10.36%,6.61%;The uronic acid contents were 3.81%,3.14%,1.73%,3.50%,2.97%;The sulfate ions contents were 12.39%,10.07%,17.71%,12.66 %,21.33%.Then,Ultraviolet and infrared spectrum scanning results showed that the five polysaccharides had typical characteristic absorption peaks of polysaccharides.Finally,SCSPB₁ and SCSPB₂ both contain mannose,glucosamine,lactose,galactose and fucose,SCSPB₂ contains galactosamine hydrochloride particularly.3.Antitumor activities of polysaccharidesThe inhibition of five Apostichopus japonicus sperm polysaccharides on Hela and HepG2 were test by using MTT assay,the results indicated that SCSCP and its purified components inhibited the survival rates of Hela cells and HepG2 cells,the order of cell-inhibiting capacity was SCSPB₂>SCSPA₂>SCSPB₁>SCSPA₁>SCSCP.At the concentration of 10 mg/mL,inhibition rates of SCSPB₂ on Hela and HepG2 at 72 h were 85.63 and88.46%,implying that Apostichopus japonicus sperm polysaccharides had a remarkable in vitro antitumor activity.4.Antioxidant activities of polysaccharidesThe antioxidant activity results of five Apostichopus japonicus sperm polysaccharides components showed the ability of scavenging DPPH·,OH· and O_2-·,but the ability of scavenging O_2-·was weaker than DPPH· and OH·.In the five components,SCSPB₂ has the strongest antioxidant activity,at the concentration of 4 mg/mL,inhibition rates of SCSPB₂ on DPPH·,OH· and O_2-· were 66.64%,52.12 U/m L,32.41 U/L.