仿刺参肠高抗凝血活性多糖筛选及其结构解析

doi:10.7506/spkx1002-6630-20240329-217

食品科学 ›› 2025, Vol. 46 ›› Issue (6): 47-53.doi: 10.7506/spkx1002-6630-20240329-217

仿刺参肠高抗凝血活性多糖筛选及其结构解析

毕英豪，都红芳，贾爱荣，王凌英，张绵松，崔婷婷，徐振鲁，刘雪

（1.齐鲁工业大学（山东省科学院），山东省科学院生物研究所，山东济南 250103；2.威海人生药业集团股份有限公司，山东威海 264200；3.山东省海洋功能食品技术创新中心，山东威海 264305；4.威海人生药业有限公司，山东威海 264200）

出版日期:2025-03-25 发布日期:2025-03-10
基金资助:
山东省自然科学基金面上项目（ZR2023MC065）；山东省科技型中小企业创新能力提升工程项目（2023TSGC0681）；齐鲁工业大学（山东省科学院）-威海市产学研协同创新基金项目（2022CXY-04；2022KC04）；山东省自然科学基金青年基金项目（ZR2022QC085）；校（院）人才科研项目（2023RCKY228）；山东省重点研发计划（重大科技创新工程）项目（2021TZXD008）

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

BI Yinghao, DU Hongfang, JIA Airong, WANG Lingying, ZHANG Miansong, CUI Tingting, XU Zhenlu, LIU Xue

(1. Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250103, China; 2. Weihai Rensheng Pharmaceutical Group Co. Ltd., Weihai 264200, China; 3. Shandong Provincial Marine Functional Food Technology Innovation Center, Weihai 264305, China; 4. Weihai Rensheng Pharmaceutical Co. Ltd., Weihai 264200, China)

Online:2025-03-25 Published:2025-03-10

摘要/Abstract

摘要： 以仿刺参肠为原料，提取得到粗多糖，经Q-Sepharose Fast Flow阴离子交换色谱柱分离得到3 种多糖（SCG1～SCG3）。采用PMP柱前衍生高效液相色谱法、高效凝胶渗透色谱法对3 种多糖的单糖组成、分子质量进行分析；采用活化部分凝血活酶时间（activated partial thromboplastin time，APTT）、凝血酶时间（thrombin time，TT）、凝血酶原时间（prothrombin time，PT）、纤维蛋白原（fibrinogen，FIB）含量评价多糖的抗凝血活性，筛选高抗凝血活性多糖；通过理化性质分析、红外光谱分析、核磁共振波谱分析对高抗凝血活性多糖进行结构分析。结果显示，仿刺参粗多糖的提取得率为2.75%，按照多糖SCG1～SCG3的顺序，岩藻糖的比例逐渐升高，SCG3仅含有岩藻糖；分子质量逐渐升高，其中SCG3分子质量为171.5 kDa。3 种多糖对APTT延长作用逐渐增强，SCG3的延长作用高于阳性对照低肝素，20 µg/mL时可达500 s以上；SCG3对PT延长作用最强，200 µg/mL时可达500 s以上，SCG1、SCG2作用不明显；SCG2、SCG3对TT均有显著延长作用，且SCG3作用最明显，50 µg/mL时可达500 s以上，SCG1几乎没有延长作用；SCG3质量浓度超过50 µg/mL时可显著降低FIB的含量。因此，SCG3是一种多机制高抗凝血活性多糖。结构分析结果显示，SCG3的总糖质量分数为50.36%，蛋白质量分数为0.67%，硫酸基质量分数为28.31%，表明SCG3是一种硫酸化岩藻多糖，即岩藻聚糖硫酸酯。红外光谱、核磁共振波谱进一步验证SCG3是一种高纯度岩藻聚糖硫酸酯，主要由糖基→3)-α-L-Fucp-(1→、→3)-α-L-Fucp(2SO4)-(1→及→3)-α-L-Fucp(2,4SO4)-(1→组成。

关键词: 仿刺参肠；抗凝血活性；岩藻聚糖硫酸酯；结构解析

Abstract: 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 →.

Key words: intestine of Apostichopus japonicus; anticoagulant activity; fucoidan; structural analysis

中图分类号:

TS201.2

毕英豪，都红芳，贾爱荣，王凌英，张绵松，崔婷婷，徐振鲁，刘雪. 仿刺参肠高抗凝血活性多糖筛选及其结构解析[J]. 食品科学, 2025, 46(6): 47-53 https://doi.org/10.7506/spkx1002-6630-20240329-217

BI Yinghao, DU Hongfang, JIA Airong, WANG Lingying, ZHANG Miansong, CUI Tingting, XU Zhenlu, LIU Xue. Screening and Structural Analysis of a Polysaccharide with High Anticoagulant Activity from the Intestine of Apostichopus japonicus[J]. FOOD SCIENCE, 2025, 46(6): 47-53 https://doi.org/10.7506/spkx1002-6630-20240329-217

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仿刺参肠高抗凝血活性多糖筛选及其结构解析

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

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