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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50~250μg/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。
The structure and structure of Acanthopanax black fungus acidic polysaccharide (EAP) were purified by ion exchange chromatography and gel chromatography. The structure of the polysaccharides was analyzed by high performance liquid chromatography (HPLC) and Fourier transform infrared spectroscopy (FT-IR) (MTT) assay, Griess method and enzyme-linked immunosorbent assay to determine the release of nitric oxide (NO), IL-6, IL-10 and TNF-α and other cytokines and evaluate the effect of EAP on RAW264.7 Immunomodulatory effect. The results showed that EAP was a β-type heteropoly acid with a molecular weight of 925.9 kDa and consisted mainly of mannose, glucuronic acid, xylose, glucose and galactose in molar ratio 64.8:14.7:14.2:3.2:2.0. EAP at 50 ~ 250μg / mL concentration of RAW264.7 mouse macrophages did not produce significant toxicity, and can significantly increase the activity of macrophages (p <0.01); EAP at a concentration of 50μg / m L , NO release from macrophages and secretion of cytokine IL-10 were significantly increased (p <0.01), which were 17.2μmol / L and 171.5 pg / ml, respectively. When the concentration was 200μg / ml, Increased TNF-α and IL-6 secretion (p <0.01). Conclusion: Acanthopanax senticosus polysaccharides have the potential to enhance the immunocompetence.