Separation, Purification, Preliminary Structural Characterization And Biological Activity Analyses Of Polys Accharides From Two Sea Cucumbers

Sea cucumbers Acaudina molpadioidea and Holothuria nobilis belong to Holothuroidea:Molpadioidea:Caudinidae and Holothuroidea:Aspidochirotida: Holothuriidae, respectively. They are very rich in resources. A. molpadioidea and H. nobilis widespreadly exist from the coast of Shandong province to Hainan Island. However, market prices of these two type sea cucumbers are far below the price of sea cucumber Stichopus japonicus. And they have not yet been fully explored. The main active ingredients of sea cucumbers are polysaccharides. Fucosylated chondroitin sulfate (FucCS) is an important and active heteropolysaccharide from the body walls of sea cucumbers. However, polysaccharides isolated from various sea cucumbers have different structural characterizations and activities of FucCS. So far, the research of academe on the FucCS from these two sea cucumbers is very little and is absence of comparison and analysis. And the studies of the FucCS oligosaccharides from these two sea cucumbers are also not performed. In order to explore the relationship between the biological activity and structure of the FucCS in sea cucumber A. molpadioidea and H. nobilis, studies on the nutrition components of the body walls, separation, structure identification and anti-coagulation and anti-tumor activities of the FucCS were performed. The main results are as follows:1. Nutritional contents of the body walls.The proximate composition, amino acid, fatty acid, polysaccharide compositions as well as mineral and trace element contents of the body wall of sea cucumbers A. molpadioides and H. nobilis were analyzed and compared to assess their nutritional quality. Results showed that these two sea cucumbers share similar characteristics: high protein contents and low lipid content. The protein content of the dry weight was 80.22% for A. molpadioidea and 70.81% for H. nobilis, respectively; the crude fat content of the dry weight was 0.50% for A. molpadioidea and 1.33% for H. nobilis, respectively. However, the fatty acid profiles of these two sea cucumbers were in a great difference. A. molpadioides contained more unsaturated fatty acids and less saturated fatty acids than H. nobilis. The amino acid distributions of these two sea cucumbers were both balanced and more than half were flavor amino acids (FAA), which gives the sea cucumber a delicious taste. At the same time, the two sea cucumbers both contained high contents of Fe (16.25-21.55 mg/kg) and Zn (12.45-18.95 mg/kg) elements.2. Separation and purification of FucCS.After preliminary extraction and separation, the mixtures of polysaccharides were obtained from sea cucumber A. molpadioidea and H. nobilis. The yield ratios of crude polysaccharides from sea cucumber A. molpadioidea and H. nobilis were 7.60% and 8.29% by dry weight, respectively. Pure FucCS ACP (from A. molpadioides) and HOP (from H. nobilis) were obtained, respectively, after separation and purification by the anion exchange chromatography DEAE Sepharose Fast Flow and gel filtration chromatography Sephadex G-100. The yield ratios of ACP and HOP from crude polysaccharides were 32.56% and 35.22%, respectively.The purity and homogeneity of the obtained polysaccharides were further confirmed by CAME using chondroitin sulfate A as a control. Results showed that each polysaccharide exhibited a single band. The molecular weights of ACP and HOP were 94.707 and 136.238 kDa, respectively, which were obtained by high performance gel permeation chromatography (HPGPC).3. Preliminary structural identification.Monosaccharide composition was assessed by HPLC using pre-column derivation by PMP and chemical methods. Sulfate content was determined using the BaCl2-gel turbidimetric method. Results showed that ACP and HOP consisted of glucuronic acid (GlcUA), N-acetyl-galactosamine (GalNAc), fucose (Fuc) and sulfate groups. And the ratio of GlcUA to GalNAc was approximately 1:0.80. The total amount of the sulfate group in HOP was higher than that of ACP. Compared with 1 molar Fuc, the sulfate content in ACP and HOP was 2.64 and 4.30, respectively. The absolute configurations of the monosaccharides in ACP and HOP were determined to be D for GlcUA, D for GalNAc and L for Fuc by the gas analysis with capillary column SE-30 using (-)-2-butanol.Much structure information can be abtained by fourier transform infrared spectroscopy (FT-IR), especially in the fingerprint region. In the fourier transform infrared spectroscopy,1,253 cm-1 was assigned to S=O antisymmetric stretching vibration; the absorptions at 820/852/853 cm-1 can be attributed to the symmetric stretching vibration of C-O-S; 584 cm’1 was assigned to the S-O stretching vibration. All of these assignments can indicate the existence of sulfate groups. However, HOP only showed absorptions at 852 cm"1, suggesting the absence of some types of sulfation patterns. The characteristic absorptions at 820/852/853 cm-1 also indicated that a-configurations existing in ACP and HOP.Methylation analysis on desulfated ACP and HOP was used to to determine the linkage pattern of the sugar residues. PMAAs of ACP and HOP were produced after reduction, desulfuration and derivatization. After GC-MS analysis, results showed that ACP and HOP were mainly composed of 1,4-Linked-GalNAc,1,3,4-Linked-GlcUA, 1-Linked-Fuc. And the proportions of GalNAc, GlcUA and Fuc were consistent with the results of monosaccharide composition analysis. ACP and HOP also contained a small amount of 1-Linked-Gal,1,4-Linked-GlcUA.NMR spectra provided more detailed information about the structural characteristics of ACP and HOP. In the 1H NMR spectra, the chemical shifts of the anomeric protons signals at 5.0-5.8 ppm were consistent with the presence of sulfated fucose residues. We found apparent differences in the anomeric proton region (5.0-5.8 ppm), revealing various sulfated patterns of FucCS. By comparison and the integral operations, the sulfated patterns in ACP were Fuc2,4S, Fuc3,4S, Fuc4S and FucOS and the ratio was 0.16:0.16:0.08:0.27; the sulfated patterns in HOP were Fuc2,4S, Fuc3S and FucOS and the ratio was 0.08:0.24:0.36. The assignments were also confirmed by two dimensional’H-’H correlation spectroscopy (COSY).13C NMR spectra presented the information about the backbone of ACP and HOP. Moreover, the backbone carbon signals observed in the spectra of ACP and HOP were very similar to each other. Both ACP and HOP consisted of β-D-glucuronic acid, P-D-N-acetyl-galactosamine and a-L-fucose, which was consistent with the results of monosaccharide composition analysis. And the (3 anomeric configuration of both GlcUA and GalNAc can be deduced from the H-1/H-2 coupling constants 6.5 and 7.4 Hz, respectively. The assignments were confirmed by the two-dimensional NMR spectra HSQC and HMBC of oligosaccharides o-ACP and o-HOP.4. Anticoagulant and anti-tumor assays of FucCS in vitro.The anticoagulant activities of ACP and HOP in vitro were determined by the action on human plasma. The human plasma was platelet-poor and prepared from the fresh blood of a healthy man. The measurement indicatiors are activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FBG). Results showed that ACP and HOP can significantly prolong the clotting time and the anticoagulant activities were increased in a concentration dependent manner during the 0-60 g/mL range. The anticoagulant activity of ACP and HOP was even higher than that of heparin, and HOP exhibited a stronger anticoagulant activity compared with ACP. The in vitro inhibitory effects of ACP and HOP on human cervical carcinoma Hela cells, human hepatocellular carcinoma Hep G2 cells, human gastric carinoma SGC-7901 cells and human leukemia cells HL60 were investigated using the colorimetric MTT assay. ACP and HOP both presented medium-efficiency inhibition on Hela cells, SGC-7901 cells and HL60 cells, while ACP and HOP both presented weak-efficiency inhibition on Hep G2 cells. Results indicated that the inhibition effect of HOP is better than ACP on human gastric carinoma SGC-7901 cells in vitro.