生附片多糖的理化性质及其抗炎与免疫调节作用研究

Study on physicochemical properties and anti-inflammatory and immunomodulatory effects of Shengfupian polysaccharides

以生附片为研究对象,采用回流提取粗多糖,利用Sevag除蛋白,通过DEAE-52纤维素柱和Sephadex G-100葡聚糖凝胶柱对其中的中性多糖片段分离纯化,利用紫外光谱、红外光谱、离子色谱、凝胶渗透色谱等仪器对生附片多糖结构表征;以LPS刺激RAW264.7细胞建立炎症损伤模型,以M1型细胞表面抗体CD86表达、巨噬细胞功能、上清液NO、IL-6含量为指标,考察生附片多糖的抗炎活性;建立H22荷瘤化疗小鼠免疫抑制模型,以抑瘤率、免疫器官指数与功能、血清细胞因子水平为指标,评估生附片多糖的免疫调节活性。结果表明,生附片多糖是由阿拉伯糖、半乳糖、葡萄糖、果糖以物质的量比0.004∶0.018∶0.913∶0.065组成,相对分子质量为80251的多糖,扫描电镜下呈现光滑的块状形态;在25~200μg·mL^(-1),生附片多糖对RAW264.7细胞未表现出明显的细胞毒性并可抑制细胞向M1型极化,降低细胞上清液中NO与IL-6含量,在25μg·mL-1下能抑制细胞吞噬功能,100~200μg·mL^(-1)增强RAW264.7细胞吞噬功能;200 mg·kg^(-1)生附片多糖能减轻环磷酰胺所致的脾脏损伤,使小鼠血清中IL-1β、IL-6水平增加,TNF-α水平降低。综上所述,生附片多糖具有一定的抗炎作用与较弱的免疫调节作用,可能是附子散寒止痛的物质基础之一。

In this study,the crude polysaccharide was extracted from Shengfupian,and purified by Sevag deproteinization.Then,a purified neutral polysaccharide fragment was obtained by the DEAE-52 cellulose chromatography column and Sephadex G-100 column.The structure of polysaccharides was characterized by ultraviolet spectrum,infrared spectrum,ion chromatography and gel permeation chromatography.To investigate the anti-inflammatory activity of Shengfupian polysaccharides,we established an inflammatory model by stimulating RAW264.7 cells with LPS.The expression of the CD86 antibody,the function of macrophages,and the content of NO and IL-6 in the supernatant were examined.An immunosuppressive model of H22 tumor-bearing mice was established,and the immunomodulatory activity of Shengfupian polysaccharides was evaluated by tumor inhibition rate,immune organ index and function,and serum cytokine levels.Research indicated that Shengfupian polysaccharide was composed of arabinose,galactose,glucose and fructose with a molar ratio of 0.004:0.018:0.913:0.065 and the molecular weight was 80251 Da.It displayed a smooth and lumpy surface under the scanning electron microscope.In the concentration range of 25-200μg·mL^(-1),the Shengfupian polysaccharides exhibited little or no cytotoxicity on RAW264.7 cells and could inhibit the polarization of cells to the M1-type and reduce the NO and IL-6 content in the cell supernatant.It could inhibit the phagocytosis of cells at the concentration of 25μg·mL^(-1),while enhancing the phagocytosis of RAW264.7 cells within the concentration range of 100-200μg·mL^(-1).200 mg·kg^(-1)Shengfupian polysaccharide could alleviate the spleen injury caused by cyclophosphamide.And it also could increase the levels of IL-1βand IL-6,and decrease the levels of TNF-αin the serum of mice.In conclusion,Shengfupian polysaccharide has a certain anti-inflammatory effect and weak immunomodulatory effect,which may be one of the material basis of Fuzi for dispelling cold and relieving pain.