刺五加黑木耳酸性多糖化学组成及免疫活性研究
Structural properties and immunostimulatory activity of an acidic polysaccharide from Auricularia auricular-judae cultivated on Eleutherococcus senticosus
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摘要: 利用离子交换色谱与凝胶色谱纯化获得刺五加黑木耳酸性多糖(EAP),使用高效液相色谱(HPLC)、傅里叶红外光谱(FT-IR)等方法分析其组成结构;通过噻唑蓝(MTT)实验、Griess法及酶联免疫吸附法测定一氧化氮(NO)释放量和IL-6、IL-10、TNF-α等细胞因子含量,评价EAP对小鼠巨噬细胞RAW264.7的免疫调节作用。结果表明:EAP是一种分子量为925.9 k Da的β型酸性杂多糖,主要由甘露糖、葡萄糖醛酸、木糖、葡萄糖及半乳糖以摩尔比例64.8∶14.7∶14.2∶3.2∶2.0组成。EAP在50250μg/m L浓度下对小鼠巨噬细胞RAW264.7未产生明显毒性,且可极显著提高巨噬细胞的活性(p<0.01);在EAP处理浓度为50μg/m L时,巨噬细胞的NO释放量和细胞因子IL-10分泌量极显著提高(p<0.01),分别为17.2μmol/L和171.5 pg/m L;当浓度200μg/m L时,EAP可极显著提高TNF-α和IL-6的分泌量(p<0.01)。结论:刺五加黑木耳酸性多糖具有增强免疫活性的潜力。Abstract: An acidic polysaccharide named EAP from Auricularia auricular-judae cultivated on Eleutherococcus senticosus was purified by iron chromatography and gel chromatography.The chemical composition and structure of EAP were analyzed by high performance liquid chromatography ( HPLC) and fourier transform infrared spectoscopy ( FT-IR) .The immunomodulatory effects of EAP on RAW264.7 macrophage were investigated by determining the production of NO and secretion of cytokines ( IL-6, IL-10, and TNF-α) . The result showed that EAP was an acidic polysaccharide with a molecular weight of 925.9 k Da and a βconfiguration.It was composed of mannose, glucose, glucuronic acid, xylose and galactose in the mole ratio of 64.8∶ 14.7∶ 14.2∶ 3.2∶ 2.0.At 50~250 μg/m L, EAP not only was cytotoxic to RAW264.7 cells, but also could significantly promoted the cell viability ( p <0.01) . The secretion of NO and IL-10 induced by EAP at 50 μg/m L was significantly improved ( p < 0.01) , reached17.2 μmol/L and 171.5 pg/m L, respectively.Moreover, EPA at 200 μg/m L significantly promoted the secretion of TNF-α and IL-6 ( p < 0.01) .It was concluded that the acidic polysaccharide from Auricularia auricular-judae cultivated on Eleutherococcus senticosus had good potential of immune enhancement.