青藏高原引种树莓多糖抗肿瘤免疫活性及其构效关系研究

Owing to the remarkable immunoregulatory activity, safety and nontoxic properties, polysaccharides show significant advantage in the development of antitumor drug or immunologic adjuvant. Influenced by the unique geographical environment and climatic condition, polysaccharide from raspberry cultivar in Qinghai-Tibet Plateau may exhibit distinct structure and bioactivities. In the previous study, we found that raspberry pulp polysaccharide exhibited significant antitumor immunostimulatory activity and chemotherapy enhancement. However, the structure of raspberry pulp polysaccharide was still unclear. The structure of natural product performs a key function in their bioactivities. Therefore, to determine the relationship between the structure and antitumor immunostimulatory activity of raspberry pulp polysaccharide is the basis of any other further research. With raspberry pulp crude polysaccharide that extracted in the previous study as material, this project will obtain homogeneous polysaccharide component through column chromatography. The primary structure of polysaccharide component was determined through a variety of method and technologies such as hydrolysis reaction, spectrum technology and so on. The polysaccharide component and their fragment were compared for the antitumor immunostimulatory activity in vitro and vivo using macrophage and murine tumor-bearing model to illuminate the relationship between the structure and the antitumor immunostimulatory activity, and to screen out polysaccharide component and fragment with high antitumor immunostimulatory. The successful implementation of this project will provide an important platform for the further researches and give some guidance for antitumor drug or chemotherapy adjuvant agent development.

植物多糖凭借其显著的免疫调节活性和安全无毒的特性在抗肿瘤免疫治疗方面具有突出优势。受独特地理环境和气候条件的影响，来源于青藏高原引种树莓果实的多糖可能在结构和生物活性方面也具有其独特性。前期研究发现，该多糖具有显著的体内抗肿瘤免疫活性并与化疗药物产生明显的协同作用，但结构尚不明确。结构决定着天然产物的活性。因此，探明树莓多糖的构效关系是进行深入研究的基础。本项目以前期提取的树莓粗多糖为材料，采用柱层析技术获得均一多糖组分。然后，综合利用多种水解反应、色谱、光谱、波谱等结构分析方法和技术着重研究树莓多糖的一级结构。通过细胞和荷瘤小鼠模型比较并分析不同多糖组分及片段的抗肿瘤免疫活性，揭示树莓多糖一级结构与其抗肿瘤免疫活性之间的内在联系，并筛选出活性突出的多糖组分或片段。本项目的顺利实施将为树莓多糖的深入研究奠定坚实的基础，为植物多糖抗肿瘤免疫治疗药物或免疫佐剂的开发提供重要参考。

天然来源的植物多糖凭借其显著的免疫调节活性及安全无毒的特性成为了功能性食品、免疫佐剂、药物等产品开发的优质原料。而多糖的结构是决定其生物活性的重要基础。前期研究发现来源于青藏高原引种栽培的树莓果肉粗多糖具有显著的体内抗肿瘤免疫活性。本项目旨在从树莓果肉粗多糖中获得具有显著免疫调节活性的植物多糖组分及片段，并探究它们一级结构与免疫调节活性之间的内在联系。项目利用柱层析法对树莓果肉粗多糖进行分离纯化，采用酸水解法得到多糖片段；结合分子量、单糖组成、甲基化分析及核磁共振波谱等数据表征树莓多糖组分和片段的一级结构；采用小鼠巨噬细胞RAW264.7检测树莓多糖组分与片段的体外免疫调节活性；通过人工神经网络模型分析树莓多糖组分及片段一级结构与其免疫调节活性的联系。最终共获得6个树莓多糖组分和3个树莓多糖片段，其中多糖组分RPP-1、RPP-2a和RPP-3a是具有全新一级结构的来源于树莓的多糖。活性研究结果显示，6个树莓多糖组分和3个树莓多糖片段都具有明显的体外免疫增强活性，它们均能明显增强RAW264.7细胞的增殖。但不同多糖组分或片段对细胞上清液中NO、TNF-α、IL-6和IL-1β含量的提高作用存在差异。多糖一级结构与体外免疫活性的构效关系分析结果显示，分子量越大（7000-100000 Da），阿拉伯糖、半乳糖、→3)-Arap-(1→、→4)-Galp-(1→和Araf-(1→单糖连接方式的占比越高的树莓多糖组分或片段具有越强的体外免疫增强作用。本项目的顺利实施明确了来源于青藏高原引种栽培的树莓果肉多糖的一级结构特点，为其抗肿瘤免疫机理的深入研究提供了重要依据，也为具有免疫调节功能的天然植物多糖的开发与应用提供了新的候选物。此外，基于机器学习方法的多糖一级结构与体外免疫活性的构效关系分析方法为此类问题的研究提供了有价值的参考。