虎杖多糖的化学组成及免疫增强活性研究

罗娅; 尹昭俊; 伍根瑞; 李继兴; 李翠红; 尹文洁; 王重娟

doi:10.13748/j.cnki.issn1007-7693.20233582

虎杖多糖的化学组成及免疫增强活性研究

Study on Chemical Composition and Immunoenhancement Activity of Polysaccharide from Polygonum Cuspidatum Sieb. et Zucc

摘要

摘要:
目的初步研究虎杖多糖(Polygonum cuspidatum Sieb. et Zucc polysaccharide)的化学组成及其对小鼠巨噬细胞和免疫抑制小鼠的调节作用，为虎杖多糖的开发利用提供依据。
方法通过苯酚-硫酸法、硫酸-四硼酸钠法、考马斯亮蓝染色法测定虎杖多糖中中性糖、糖醛酸和蛋白质含量；HPLC分析其单糖的组成。采用MTT实验，研究虎杖多糖对Raw264.7细胞活力的影响；采用中性红染色和流式细胞术，研究虎杖多糖对Raw264.7细胞吞噬功能的影响；通过ELISA法探索研究虎杖多糖对Raw264.7细胞免疫因子分泌水平的影响。利用环磷酰胺建立免疫抑制小鼠模型，观察虎杖多糖对胸腺指数和小肠派氏结数量的影响，以初步评估虎杖多糖对环磷酰胺产生的免疫抑制的对抗作用。
结果虎杖多糖由葡萄糖(Glc)、甘露糖(Man)、鼠李糖(Rha)、半乳糖醛酸(GalA)、半乳糖(Gal)、阿拉伯糖(Ara) 6种单糖组成，主要为Glc，占比为76.44%；虎杖多糖中中性糖、糖醛酸、蛋白质含量分别为52.31%、13.47%、6.90%。25~800 μg·mL⁻¹虎杖多糖能够增强巨噬细胞活力；200、400 μg·mL⁻¹虎杖多糖可以促进巨噬细胞的中性红吞噬能力；100~400 μg·mL⁻¹虎杖多糖可以促进巨噬细胞吞噬FITC-dextran荧光微球；此外，虎杖多糖还能刺激巨噬细胞分泌TNF-α和IL-6；动物实验进一步证明，虎杖多糖使免疫抑制小鼠的胸腺指数升高，使其肠道内派氏结数量增多。
结论虎杖多糖的主要单糖组成为Glc，其可以增强巨噬细胞的免疫功能，对抗环磷酰胺产生的免疫抑制。

Abstract:
OBJECTIVE To study the chemical composition and the modulating effect of Polygonum cuspidatum Sieb. et Zucc polysaccharide(PCPS) on macrophages and immunosuppressed mice, to provide a basis for the development and utilization of PCPS.
METHODS The content of neutral sugar, uronic acid and protein were determined by phenol-sulfuric acid method, sulfuric acid-sodium tetraborate method and coomassie brilliant blue staining method, respectively. Monosaccharide composition of PCPS was analyzed by HPLC. The effects of PCPS on the viability of Raw264.7 cells were evaluated through MTT experiment. Neutral red staining and flow cytometry were used to study the effects of PCPS on the phagocytic function of Raw264.7 cells. The effects of PCPS on the secretion level of immune factors in Raw264.7 cells were determined by ELISA. Furthermore, cyclophosphamide(CTX) was used to establish an immunosuppressive mouse model, and the effects of PCPS on the thymus index and the number of small intestinal Peyer's patches were observed to evaluate the resistance of PCPS to CTX induced immunosuppression.
RESULTS PCPS was composed of six monosaccharides, including glucose(Glc), mannose(Man), rhamnose(Rha), galacturonic acid(GalA), galactose(Gal) and arabinose(Ara). Among them, Glc was the main monosaccharide, accounting for 76.44%. The contents of neutral sugar, uronic acid and protein in PCPS were 52.31%, 13.47% and 6.90%, respectively. PCPS within a concentration range of 25−800 μg·mL⁻¹ could enhance the vitality of macrophage; PCPS at 200 and 400 μg·mL⁻¹ could promote the neutral red phagocytic ability of macrophages, and PCPS at 100−400 μg·mL⁻¹ could promote the phagocytosis of FITC-dextran fluorescent microspheres. In addition, PCPS could also stimulate the secretion of TNF-α and IL-6 by macrophages. Animal experiments further proved that PCPS increased the thymus index and small intestinal Peyer's patches of immunosuppressed mice.
CONCLUSION The main monosaccharide composition of PCPS is Glc, which can enhance the immune function of macrophages and resist the immunosuppression induced by CTX.

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