Study On The Extraction,Isolation,purification,structure And Bioactivities Of Polysaccharides From Notarchus Leachii Freeri Eggs

Notarchus leachii freeri eggs is a marine animal belonging to anaspidea opisthobranch gastropod mollusk belonging to the family of Aplysiidae.Notarchus leachii freeri eggs are known as sea powder and sea noodles which have used as the traditional nutritious food source and medicine.Sea hare and its eggs contain various bioactive substances including peptides,proteins,secondary metabolites and the structures and biological activities of them have been widely reported.However,the research on polysaccharides mainly focused on the structure and activity of sea hare muscle polysaccharides,but the current papers on sea hare egg polysaccharides are very scarce.In this work,the extraction,purification,structure of polysaccharides from Notarchus leachii freeri eggs were investigated.Furthermore,the immunomodulatory,antioxidant and antibacterial activities of Notarchus leachii freeri eggs polysaccharides were evaluated.The main results are as follows:(1)Single factors experiment and response surface analysis were used and the optimal extraction conditions for NLCEP were Na Cl solution concentration of 2.96 %,ratio of raw material to liquid of 1 : 40 g/m L,extraction time of 2 h and extraction temperature of 69 °C,and the actual extraction rate was 4.20 %.The factors affecting the yield of polysaccharides were in the following order: extraction time > ratio of liquid to material >extraction temperature > concentration of Na Cl solution.(2)The crude polysaccharides of the Notarchus leachii freeri eggs(NLCEP)was obtained after deproteinizing with Sevage method and alcohol precipitation.Two fraction,NLCEPs-1 and NLCEPs-2 were obtained after purification by a DEAE-Cellulose 52 column chromatography and a Sephadex G-100 column chromatography.Structural analysis indicated that NLCEPs-1and NLCEPs-2 were homogeneous polysaccharides.NLCEPs-1 had an average molecular weight of 31.4 k Da and was consisted of rhamnose,glucose,galactose,xylose and arabinose with the ratio of 11.13 : 63.77 : 5.44 : 6.59 : 13.08.NLCEPs-2 had an average molecular weight of 4176.8 k Da and was consisted of rhamnose,glucuronic acid,glucose,galactose and xylose at the ratio of 1.24 : 8.35 : 10.01 : 65.74 : 14.66.By FI-IR and NMR analysis,the glycoside linkage types of NLCEPs-1 consisted of ?)4-?-D-Glcp(1?,?6)-?-D-Glcp(1?,?1)-?-D-Galp and ?-D-Galp-(1?;the glycoside linkage types of NLCEPs-2 consisted of ?)4-?-D-Galp A6Me(1?,?-D-Glc-(1? and ?6)-?-Glcp-(1?.(3)Murine macrophage RAW 264.7 cells were used as the cell model to measure the immunomodulatory activities of polysaccharides from Notarchus leachii freeri eggs in vitro.The results showed that NLCEP,NLCEPs-1 and NLCEPs-2 could significantly promote the pinocytic capacity of RAW264.7 cells,the productions of NO and cytokines(IL-6 and TNF-?).NLCEPs-1 could also increase the expression of i NOS,IL-6 and TNF-?corresponding m RNA.The immunomodulatory capacity of NLCEPs-1 was stronger than other two polysaccharides.(4)The antioxidant activities of polysaccharides from Notarchus leachii freeri eggs were measured by free radical scavenging ability and reducing power.The results showed that both NLCEP,NLCEPs-1and NLCEPs-2 could scavenge DPPH radical,hydroxyl radical and superoxide anion radical,and all of them had certain reducing power.The antioxidant capacity of the three polysaccharides was evaluated comprehensively as follows: NLCEP,NLCEPs-1,NLCEPs-2.The bacteriostatic effect of polysaccharides from Notarchus leachii freeri eggs was determined by the size of bacteriostatic zone of polysaccharides on common bacteria.In terms of antibacterial activity,except for Bacillus subtilis,the three polysaccharides had a certain inhibitory effect on different bacteria.In general,the purified polysaccharide NLCEPs-2 from Notarchus leachii freeri eggs had the best bacteriostatic effect on Gram-positive bacteria,and the crude polysaccharide NLCEP from sea hare eggs had the best antibacterial effect on Gram-negative bacteria.The bacteriostatic effect of purified polysaccharide NLCEPs-1 from Notarchus leachii freeri eggs was relatively weak.