食品科学 ›› 2019, Vol. 40 ›› Issue (11): 139-145.doi: 10.7506/spkx1002-6630-20180610-122

• 营养卫生 • 上一篇    下一篇

泡叶藻聚糖低分子质量降解片段组成特征及其体外免疫诱导活性

何萍萍1,韦敬柳乙1,姜泽东1,2,3,4,*,朱艳冰1,2,3,4,倪 辉1,2,3,4,李清彪1,2,3,4   

  1. 1.集美大学食品与生物工程学院,福建 厦门 361021;2.福建省食品微生物与酶工程重点实验室,福建 厦门 361021;3.厦门南方海洋研究中心经济海藻资源化利用与深加工重点实验室,福建 厦门 361021;4.厦门市食品生物工程技术研究中心,福建 厦门 361021
  • 出版日期:2019-06-15 发布日期:2019-06-28
  • 基金资助:
    国家自然科学基金青年科学基金项目(31501441);福建省高校杰出青年科研人才培育计划项目(B16166)

Composition and in Vitro Immunostimulatory Activity of Low-Molecular-Mass Fractions of Chemically Degraded Ascophyllan from Ascophyllum nodosum

HE Pingping1, WEI Jingliuyi1, JIANG Zedong1,2,3,4,*, ZHU Yanbing1,2,3,4, NI Hui1,2,3,4, LI Qingbiao1,2,3,4   

  1. 1. College of Food and Biological Engineering, Jimei University, Xiamen 361021, China; 2. Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering, Xiamen 361021, China; 3. Key Laboratory of Systemic Utilization and In-depth Processing of Economic Seaweed, Xiamen Southern Ocean Technology Center of China, Xiamen 361021, China; 4. Research Center of Food Biotechnology of Xiamen City, Xiamen 361021, China
  • Online:2019-06-15 Published:2019-06-28

摘要: 目的:对从泡叶藻(Ascophyllum nodosum)中提取的泡叶藻聚糖进行化学法降解,将得到的低分子质量降解产物进行分离纯化,分析其基本组成特征和体外免疫诱导活性,为泡叶藻聚糖或其他海藻多糖的生物活性和基本结构特征研究提供参考。方法:通过酸水解法和双氧水氧化降解法制备泡叶藻聚糖低分子质量降解片段,利用超滤法和Sephadex G-50凝胶柱层析分离纯化得到4 个泡叶藻聚糖低分子质量片段:HCl-F1、HCl-F2、H2O2-F1、H2O2-F2;采用对氨基苯甲酸乙酯柱前衍生样品后进行高效液相色谱及化学方法分析其单糖组成和化学组成;采用高效分子排阻色谱法测定其重均分子质量;通过小鼠巨噬细胞RAW264.7模型分析HCl-F1、HCl-F2、H2O2-F1、H2O2-F2的体外免疫诱导活性。结果:泡叶藻聚糖低分子质量片段的组成特征表明HCl-F1、HCl-F2、H2O2-F1、H2O2-F2均为杂多糖,且单糖组成存在较大差异,其重均分子质量分别为4.80、4.20、5.30、2.30 kDa。免疫活性分析细胞模型结果表明,HCl-F1、HCl-F2、H2O2-F2在所测定质量浓度范围(0~200 μg/mL)内能明显诱导RAW264.7细胞活化,释放NO和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),而H2O2-F1诱导RAW264.7细胞释放NO和TNF-α的活性明显低于HCl-F1、HCl-F2和H2O2-F2。结论:泡叶藻聚糖低分子质量降解片段(HCl-F1、HCl-F2和H2O2-F2)具有明显的免疫诱导活性,其中HCl-F1和H2O2-F2的免疫诱导活性明显高于泡叶藻聚糖。结合其化学组成分析的结果,提示泡叶藻聚糖低分子质量降解片段的免疫诱导活性是由单糖组成和硫酸根质量分数共同决定的。

关键词: 泡叶藻聚糖, 低分子质量降解片段, 分离纯化, 化学组成, 体外免疫诱导活性

Abstract: Objective: To separate and purify low-molecular-mass fractions from the chemical degradation products of the sulfated polysaccharide ascophyllan extracted from the brown seaweed Ascophyllum nodosum and to analyze the monosaccharide composition and in vitro immunostimulatory activity of each of these fractions for the purpose of providing useful information for the studies on the biological activities and structural characteristics of ascophyllan and other seaweed polysaccharides. Methods: The degraded products of ascophyllan, prepared separately by treatment with hydrochloric acid or hydrogen peroxide, were fractionated sequentially by ultrafiltration and Sephadex G-50 gel filtration chromatography into four low-molecular-mass fractions (HCl-F1, HCl-F2, H2O2-F1 and H2O2-F2). The monosaccharide composition of each fraction was determined by high-performance liquid chromatography (HPLC) with pre-column ethyl-p-aminobenzoate (ABEE) derivatization and some of their chemical groups were analyzed. Their weight-average molecular masses were evaluated by high-performance size exclusion chromatography (HPSEC), and their in vitro immunostimulatory activities were investigated by using mouse RAW264.7 macrophages as a model. Results: All these compounds were heteropolysaccharides with significant differences in their monosaccharide composition and sulfation level. The weight-average molecular masses of HCl-F1, HCl-F2, H2O2-F1 and H2O2-F2 were estimated to be 4.80, 4.20, 5.30 and 2.30 kDa, respectively. The in vitro immunomodulatory activity revealed that HCl-F1, HCl-F2, and H2O2-F2 significantly induced the activation of RAW264.7 macrophages to produce immune factors such as nitric oxide (NO) and tumor necrosis factor-a (TNF-a) in the test concentration range (0–200 mg/mL), whereas the ability of H2O2-F1 to induce the production of NO and TNF-a in RAW264.7 cells was significantly lower than that of any other polysaccharide fraction. Conclusion: All the low-molecular-mass polysaccharide fractions (HCl-F1, HCl-F2 and H2O2-F2) derived from ascophyllan possessed potent immunostimulatory activity, with HCl-F1 and H2O2-F2 being much stronger than ascophyllan. Taken together, the immunostimulatory activity is likely determined by a combination of monosaccharide composition and sulfate content.

Key words: ascophyllan, low-molecular-mass fractions, separation and purification, chemical composition, in vitro immunostimulatory activity

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