几种金银花果胶多糖的免疫调节活性及构效关系研究

Lonicera japonica possesses wide pharmacological effects, such as antivirus, anti-inflammation and immunomodulation, etc., with polysaccharide as one of its main active ingredients. Pectic polysaccharide structures of different plants are diversified, their bioactivity and application depend on change of their structures. Therefore is has important significance to illuminate structure-function relationship of pectic polysaccharide bioactivity of Lonicera japonica for deeply developing and utilizing Lonicera japonica. The project combines the locally superior resources Lonicera japonica of Suiyang to systematically perform trial of separation, purification, structure analysis and immunomodulatory activity to Lonicera japonica of three varieties, and discusses the similarities and differences and structure-function relationship of structure and activity. The research contents are as follows: ①extraction of total polysaccharides of Lonicera japonica for three places of origin, optimization of steps of separation and purification, establishment of stable fractionation system, as well as separation and purification of three kinds of total polysaccharides; ②degradation of fractionated polysaccharide products of Lonicera japonica, acquisition of pectic structural domains, and analysis of their fine structures, as well as comparison of differences and similarities of their structures; ③use of technological analysis methods such as immunology, analytical cytology, molecular biology, laboratory animal science, etc., comparison of immunomodulatory activity of pectic polysaccharides for three varieties of Lonicera japonica, and analysis of the structure-function relationship. The project results are expected to be in favor of research on structure-function relationship of immunomodulatory activity for pectic polysaccharides of Lonicera japonica, which will further enrich theory of glycobiology, providing basis for deep development and application of Lonicera japonica.

金银花具有抗病毒、抗炎及免疫调节等作用，多糖是其主要的活性成分之一。不同植物的果胶多糖结构呈多样性，生物活性和应用取决于其结构的变化，阐明金银花果胶多糖生物活性的构效关系对深度开发利用金银花具有重要意义。本项目结合本地优势资源绥阳金银花，对3个品种的金银花多糖进行系统地分离纯化、结构解析和免疫调节活性试验，探讨结构和活性的异同及构效关系。研究内容包括①提取3个产地的金银花总糖，优化分离纯化步骤，建立稳定的分级体系，将3种总糖进行分离纯化；②降解分级后的金银花多糖产物，获得各种类型的果胶结构域并解析其精细结构，比较其结构的异同；③采用免疫学、分析细胞学、分子生物学、实验动物学等技术观察、比较3个品种金银花果胶多糖的免疫调节活性，并分析其构效关系。本项目预期结果可填补金银花果胶多糖免疫调节活性构效关系研究，进一步丰富糖生物学理论，为金银花深度开发应用提供依据。

果胶多糖是金银花的主要活性成分之一，本课题组首先采用热水煮提和乙醇醇沉法制备了3个产地的金银花多糖（LJP），然后采用DEAE-纤维素、DEAE-Sepharose Fast Flow和Sepharose CL-6B对3个产地的金银花多糖进行了分离纯化，并通过HPLC、HPGPC、FT-IR、NMR法等对金银花果胶多糖子级分的结构特征进行了解析。结果表明，金银花多糖中的中性糖部分主要是淀粉样葡聚糖以及阿拉伯半乳聚糖，而酸性部分主要为果胶多糖，且结构较为复杂，平均分子量范围约为10 kDa至500 kDa。相同产地的果胶多糖子级份中LJP-A-2 ~ LJP-A-4的结构特征相似，主要由GalA和一些Glc、Gal、Ara等残基构成。其中LJP-A-3的精细结构为主链由(1→4)-连接的α-D-GalA组成，并含有微量的[→)4-α-D-GalpA-(1→2)-α-L-Rhap-(1→]n结构单元，且在Rhap的C4上连接有侧链，侧链由(1→4)-连接的半乳聚糖，(1→5)-连接和(1→3,5)-连接的阿拉伯聚糖或阿拉伯半乳聚糖构成，表明LJP-A-3主要是一种含有HG型结构域及少量带侧链的RG-I型结构域的果胶多糖。免疫调节活性实验结果表明，LJP的子级分，特别是LJP-A酸性糖组分，具有增强免疫的活性，能够显著改善环磷酰胺诱导小鼠的淋巴器官胸腺和脾脏的萎缩，显著增强巨噬细胞的吞噬功能，显著增加血清中细胞因子和免疫球蛋白水平的分泌，以及显著增强NK细胞的杀伤活性。此外，LJP能显著抑制鼻粘膜中的AR症状和嗜酸性粒细胞数量。LJP治疗AR小鼠后，血清中IgE、TNF-α、IL-1β和IL-17浓度显著降低。LJP治疗后IL-4、IL-5、IL-6、IL-17、IL-23和STAT3的mRNA表达量升高，ROR-γt和SOCS3降低等。本项目的研究结果将对金银花果胶多糖的免疫调节作用及构效关系研究提供新的实验证据，为金银花的深度开发和利用提供一定的参考价值。