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[摘要]
目的 从赤芝Ganoderma lucidum中提取、分离和纯化获得碱提多糖组分,在表征其基本理化性质和精细结构的基础上,对其体外免疫调节活性进行研究。方法 赤芝子实体干燥粉碎后,利用碱提醇沉法提取粗多糖,经Q-Sepharose Fast Flow离子交换色谱柱纯化得到灵芝多糖组分(LZJ-0.15)。采用PMP柱前衍生-高效液相色谱法(HPLC)及高效凝胶渗透色谱-十八角度激光光散射联用法(HPGPC-MALLS)测定LZJ-0.15的单糖组成及相对分子质量特征,并通过核磁共振氢谱(1H-NMR)、碳谱(13C-NMR)及二维谱(1H-1H COSY和1H-13C HSQC)对LZJ-0.15的精细结构进行表征。采用小鼠巨噬细胞RAW264.7吞噬中性红实验对灵芝多糖LZJ-0.15进行免疫活性评价。结果 LZJ-0.15的相对分子质量、分子半径及Mw/Mn分别为24 700、46.6 nm和1.019,其单糖组成分析显示以葡萄糖为主(92.3%),核磁谱图显示LZJ-0.15为→3) Glc (β1→及→6) Glc (β1→连接为主的葡聚糖。RAW264.7细胞吞噬中性红实验显示LZJ-0.15具有较好的免疫调节活性。结论 赤芝碱提灵芝多糖相较于水提多糖组分具有较优的免疫活性,有望开发成重要的免疫调节剂。
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[Abstract]
Objective To extract, separate, and purify polysaccharide from Ganoderma lucidum with alkali from the residue after water extraction, characterize the basic physicochemical properties and structural features in detail, and study the immunomodulatory activity in vitro.Methods The polysaccharide LZJ-015 was isolated and purified from the dry fruiting bodies of G. lucidum by alkaline extraction and ethanol precipitation following Q-Sepharose Fast Flow ion exchange chromatography column. Monosaccharide composition and molecular weight of LZJ-0.15 was analyzed by high performance liquid chromatography (HPLC) with PMP precolumn derivatization and high performance gel permeation chromatography-multiple angle laser (HPGPC-MALLS), respectively. The detailed structure of polysaccharide LZJ-0.15 was characterized by 1H-NMR, 13C-NMR, 1H-1H COSY, and 1H-13C HSQC spectrum. The immunomodulatory activity of G. lucidum polysaccharide was test by RAW264.7 cells phagocytose neutral red experiment.Results The molecular weight, molecular radius, and Mw/Mn of LZJ-0.15 were determined to be 24 700, 46.6 nm, and 1.019, respectively. The monosaccharide composition was confirmed to be mainly composed of glucose (92.3%). LZJ-0.15 was →3) Glc (β1→and→6) Glc (β1→linked glucan indicated by NMR spectrum. Moreover, G. lucidum polysaccharide exhibited good immunomodulatory activity in our study.Conclusion G. lucidum polysaccharide LZJ-0.15 from alkaline extraction showed better immune activity than the polysaccharide extracted from water, which would be potentially developed as an effective immunomodulatory agent.
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