Screening and Structural Analysis of a Polysaccharide with High Anticoagulant Activity from the Intestine of Apostichopus japonicus

In this study, three polysaccharides, namely SCG1, SCG2 and SCG3, were separated from the intestine of the sea cucumber Apostichopus japonicus by Q-Sepharose Fast Flow anion-exchange chromatography. The monosaccharide compositions and molecular masses of the polysaccharides were analyzed by pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by high performance liquid chromatography (HPLC) and high-performance gel permeation chromatography (HPGPC). Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and fibrinogen (FIB) levels were assessed to evaluate the anticoagulant activity of the polysaccharides and to select those with high anticoagulant activity. Physicochemical and structural characterization of the polysaccharide with high anticoagulant activity was conducted by Fourier transform infrared (FTIR) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the yield of crude polysaccharides from A. japonicus was 2.75%. The proportion of fucose in the three polysaccharides followed an increasing order of SCG1 < SCG12 < SCG3, and SCG3 consisted solely of fucose. Their molecular masses increased in the order of SCG1 < SCG12 < SCG3, and the molecular mass of SCG3 was 171.5 kDa. The increasing order of the effects in prolonging APTT was SCG1 < SCG12 < SCG3, and SCG3 was more effective than low-molecular-mass heparin, prolonging APTT to more than 500 s at 20 µg/mL. Additionally, SCG3 was the most effective in prolonging PT, prolonging it to more than 500 s at 200 µg/mL, while SCG1 and SCG2 had insignificant effects. Both SCG2 and SCG3 significantly prolonged TT, and SCG3 was more effective, prolonging TT to more than 500 s at 50 µg/mL, while SCG1 hardly prolonged TT. SCG3 significantly reduced FIB levels at a concentration above 50 µg/mL. Based on the results, SCG3 exerted anticoagulant activity through multiple mechanisms. Structural analysis showed that SCG3 contained 50.36% total sugar, 0.67% protein, and 28.31% sulfate group and was identified as a fucoidan. FTIR and NMR spectroscopy corroborated that SCG3 was a high-purity fucoidan, predominantly consisting of glycosyl→3)-α-L-Fucp-(1→,→ 3)-α-L-Fucp (2SO₄)-(1→ and → 3)-α-L-Fucp (2,4SO₄)-(1 →.