Structure And Bioactivity Analysis Of Polysaccharide From Acaudina Leucoprocta

Acaudina leucoprocta which belongs to Echinodermata,Holothuroidea,Molpiadida,Caudinidae,is a kind of sea cucumber and widely distributed in the coastal of Zhejiang province.In this paper,polysaccharides were extracted from the body of Acaudina leucoprocta.The structures of the polysacgharides were elucidated on the basis of chemical and modern spectroscopic analysis.And bioactivities of the polysaccahrides were investigated.The main results were as follows:The polysaccharide was extracted from the body of Acaudina leucoprocta by1%KOH at 60℃for 3 hours and enzymatic hydrolysis with 4%papain at 56℃for3 hours.The yield of the polysaccharides was 4%.Two polysaccharide fractions WS-1 and WS-2 were purified and obtained by Q Sepharose Fast Flow ion-exchange and Sephacryl S-200 gel-permeation chromatography.The physicochemical analysis showed that the total sugar contents of the WS-1 and WS-2 were 59%and 54%,protein were 8%and 4%,sulfate ester were 15%and 30%,repectively.The monosaccharide composition showed that WS-1 mainly consisted of fucose;WS-2was mainly composed of fucose,uronic acid and glucosamine.It indicated that WS-1 was a sulfated fucan and WS-2 was a fucosylated glycosaminoglycan.The average molecular weight of WS-1 and WS-2 by gel permeation chromatography was estimated to be about 593 kDa and 359 kDa.The structures of the two polysaccharide fractions were investigated by infrared spectroscopic,methylation,unsaturated disaccharide,nuclear magnetic resonance spectroscopic and mass spectroscopic.The results showed WS-1 was a sulfated fucan that was mainly composed of→3)-α-L-Fucp-（1→,and the sulfate groups were substiuted at O-2 or O-4 positions of the fucose.In detail,WS-1 consisted of Fuc2S4S（24%）,Fuc2S（24%）,Fuc0S（40%）and Fuc4S（12%）.On average,there were about seven sulfate groups on every eight fucose.WS-2 was a glycosaminoglycan,of which the backbone contained→3）-β-D-GlcpNAc6S-（1→4）-β-D-GlcpUA-（1→and→3）-β-D-GlcpNAc4S6S-（1→4）-β-D-GlcpUA-（1→,the ratio of these two kinds of disaccharide units were 1.5:1.And the branches were composed of fuco-oligosaccharides from dp1 to dp6 with the→3）-α-L-Fucp-(1→linkages,which linked to O-3 of GlcA residues in the main chain.The fucose side chains consisted of Fuc4S,Fuc3S4S,Fuc0S,Fuc2S4S and Fuc3S4S at the terminal.The anticoagulant activities of WS-1 and WS-2 were studied by APTT,TT and PT.WS-1 displayed intrinsic anticoagulant activity,while WS-2 displayed intrinsic and extrinsic anticoagulant activity.And WS-2 had high binding affinity to ATⅢ,and then inhibited the activities of FⅡa and FⅩa to achieve anticoagulant and antithrombotic effect through.RAW264.7 was adopted as macrophage to investigat the immunostimulatory effect of WS-1.The results showed WS-1 could enhance immunostimulatory activities of RAW264.7 cells and significantly promote the phagocytic activity and ROS production.Further experiments showed that WS-1 could increase the secretion levels of iNOs,TNF-α,IL-6,IL-1βand IL-12.Besides,It was found that toll-like receptor 4（TLR4）was involved in WS-1-mediated activation of macrophages.The result of antivirus showed WS-2 possessed high potent anti-HSV1 activity(IC₅₀=14.79±1.10μg/mL),and it had big safety index（SI=135.23）.Research results of this paper have great significance to the Acaudina leucoprocta polysaccharide research and development.It provided reference on the high-value utilization of Acaudina leucoprocta.