Structural Characterization And Biological Activity Analyses Of Polysaccharides From Two Sea Cucumbers

Sea cucumbers Acaudina molpadioidea and Holothuria nobilis belong to Holothuroidea:Molpadioidea:Caudinidae and Holothuroidea:Aspidochirotida:Holothuriidae,respectively.They are widely distributed in China.Market prices and edible value of the two type sea cucumbers are far below that of sea cucumber Stichopus japonicus.As a result,they have not yet been fully explored.The polysaccharides are the main active ingredient in the body wall of sea cucumbers,divided into fucosylated chondroitin sulfate(FucCS)and fucan in general.FucCS is very important hetero-polysaccharide from the body walls of sea cucumbers.Besides that,FucCS from different sea cucumbers has different structural characterizations and activities.The studies on the primary structure,the anti-coagulation and anti-tumor activities of the two FucCS from Acaudina molpadioidea and Holothuria nobilis have finished in our laboratory.To further perfect the researches on the structural characterizations and activities of the two FucCS,the advanced structure and the antioxidant activity of the two FucCS were explored.In final,we selected to continuously study the FucCS from the sea cucumber Holothuria nobilis after the comparison of the structural characterizations and activities of the two FucCS.The optimization degradation conditions of the FucCS and the primary structure of the oligosaccharide were performed.The main results are followed:1.Advanced structures of FucCS.Advanced structural characterization of the two FucCS ACP(from A.molpadioides)and HOP(from H.nobilis)including solution behavior and superficial morphology was conducted by specific rotation analysis,circular dichroism spectroscopy(CD),atomic force microscopy(AFM),intrinsic viscosity analysis,scanning electron microscope(SEM),respectively.The specific rotations of ACP and HOP are-46° and-22°,respectively.The intrinsic viscosity[η]of ACP and HOP was 145.52 ml/g and 7.30 ml/g,respectively.The conformation of ACP and HOP were be influenced by thermal treatment and the change of pH values.The change of pH values had more significant influence on ACP and HOP than thermal treatment.Especially,the alkali treatment would cause the mutation of the conformation.And there were no highly-ordered double or triple helix structure in ACP and HOP.SEM and AFM images revealed that ACP had a rough surface with lots of particles which stacked together and the diameter of the aggregates was approximately 60 nm.HOP exhibited a smooth and homogeneous surface.The degree of HOP aggregation was low and the height of HOP molecules was about 2 nm.2.Antioxidant assays of FucCS in vitro.The antioxidant activities were evaluated via different methods,including DPPH scavenging assay,hydroxyl,and superoxide radicals scavenging assay.Results showed ACP and HOP had good antioxidant activity,but the activity was lower than a synthetic antioxidant-VC.The concentration of ACP and HOP didn’t show the significant linear relation with the scavenging activity.At a low concentration,the antioxidant activity of ACP and HOP increased with concentration.However,the scavenging activity increased slowly when reached a certain value.At 0.5 mg/ml,VC,ACP and HOP achieved the most scavenging activity.3.Preparation and preliminary structural identification of HOP oligosaccharideIn this study,HOP was degraded by H2O2.And the conditions of HOP degradation were optimized using fractional factorial design(FFD),central composite design(CCD)and response surface methodology(RSM).We investigated the effects of several processing variables on the oxidative degradation of HOP,such as metal ion,R(H2O2-HOP),H2O2 flow rate,reaction pH,reaction time,reaction temperature and HOP concentration.Our data showed that the optimum conditions with Fe2+ were found as follows:R(H2O2-HOP)of 0.53;reaction pH of 6.91;H2O2 flow rate of 0.40 mL/min;reaction time of 2 h;reaction temperature of 30°C and HOP concentration of 4 mg/mL.According to the optimum conditions of HOP,the fraction with a molecular weight of 7.9 kDa(D-HOP)was collected.Monosaccharide composition was determined by HPLC using pre-column derivation by PMP and TFA hydrolysis method.Sulfate content was carried out using the BaCl2-gel turbidimetric method.The absolute configurations of the oligosaccharide were assessed by GC analysis using(-)-2-butanol.The results showed that oligosaccharide consisted of glucuronic acid(GlcUA),N-acetyl-galactosamine(GalNAc),fucose(Fuc).And the ratio of GlcUA to GalNAc was approximately 1:0.80.The monosaccharide composition and the ratio of GlcUA to GalNAc were same as that of HOP.The skeleton structure of oligosaccharide was-→4GlcA1→GalNAcβ1→.The absolute configurations of the monosaccharides in oligosaccharide were determined to be D for GlcUA,D for GalNAc and L for Fuc.Compared with 1 molar Fuc,the sulfate content in oligosaccharide was 3.27.In addition,the molecular weight of oligosaccharide was 7.9 kDa by high performance gel permeation chromatography(HPGPC).NMR spectra provided more detailed information about the preliminary structural information of oligosaccharide.In the 1H NMR spectra,the chemical shifts at 5.0-5.7 ppm were the anomeric protons which were consistent with the presence of sulfated fucose residues.We found that sulfated patterns of oligosaccharide were Fuc2,4S,Fuc3S,Fuc4S and FucOS.However,Fuc4S didn’t appear in the 1H NMR spectra of HOP.13C NMR spectra presented the information about the backbone of oligosaccharide.The result of 13C NMR spectra verified that oligosaccharide consisted of glucuronic acid(GlcUA),N-acetyl-galactosamine(GalNAc),fucose(Fuc).The carbon skeleton of oligosaccharide was in accordance with that of HOP.