云南产红花蜂花粉多糖的分离分析与抗肿瘤活性研究

Yunnan province riches in biological resources but the economy, science and technology development are relatively lag. Effective use of the new medicine resources is one of the main tasks of regional development. Bee pollen of Carthamus tinctorius is the special agricultural products in western area of Yunnan. It is a kind of precious new resource of new medicines. Our previous study of anti-tumor activity of the total polysaccharide showed that it can significantly inhibit the S180 tumor growth and markedly increase serum TNF-α in tumor-bearing mice. We also purified and identified a polysaccharide from bee pollen of Carthamus tinctorius PBPCII which has anti-tumor activity both in vivo and in vitro. On the base of previous study, this program plans study the following aspects: ① To isolate and purify polysaccharides from bee pollen of Carthamus tinctorius by the way of fractional extraction. In order to confirm which polysaccharide components have anti-tumor activity, each purified homogeneous polysaccharide is selected using anti-tumor methods both in vivo and in vitro. ②The possible mechanisms of anti-tumor of the polysaccharides are also researched on the cellular and molecular levels. ③Taking chemical, spectrum analysis and physical analysis methods, the primary structure and configuration of the active polysaccharides will be studied. Then the structure-effect relationship of the active polysaccharides is preliminarily explored. The research results will illuminate the anti-tumor mechanisms, structure-effect relationships of the polysaccharides and will lay the foundation of further study on more effective, low toxic antitumor drugs.

云南生物资源丰富但经济、科技发展相对滞后，有效地利用生物药物资源是地区发展的主要任务之一。红花蜂花粉是云南滇西地区特产，是一种宝贵的新药物资源。 我们前期研究发现，红花蜂花粉总多糖能抑制小鼠S180 肉瘤的生长，升高血清TNF-α含量，并已纯化出一个具有体内、体外抗肿瘤活性的红花蜂花粉多糖PBPCII。在已有研究的基础之上，本课题拟开展三方面的研究。①从红花蜂花粉中分级分离、纯化多糖的不同均一级分，用体内、体外抗肿瘤实验方法，对不同多糖级分进行抗肿瘤活性筛选，以明确具有抗肿瘤作用的活性多糖级分。②在细胞水平、分子水平上研究活性多糖级分的抗肿瘤作用机制。③采用化学分析、波普分析及物理方法对抗肿瘤级分进行一级结构与空间构象解析，探索其抗肿瘤作用与结构之间的构效关系。 研究结果将阐明红花蜂花粉多糖的抗肿瘤作用机制与构效关系，为进一步研发高效、低毒的抗肿瘤药物奠定重要基础。

红花蜂花粉是云南滇西地区特产，是一种宝贵的新药物资源。本项目从云南产红花蜂花粉中分离、纯化出4种蜂花粉多糖PBPCIa、PBPCⅡ、PBPCⅢ、PBPCⅣ，用响应面实验方法，探索出了其最佳脱蛋白工艺。采用凝胶柱层析、HPLC、GC、红外光谱、GC-MS、高碘酸氧化、Smith降解、部分酸水解、1D、2D核磁共振谱、原子力显微镜扫描、圆二色谱及X射线扫描等方法，测定了4种多糖的分子量、糖醛酸含量，分析了PBPCIa、PBPCⅡ、PBPCⅢ、PBPCⅣ 的单糖组成、糖苷键连接方式、糖残基的连接顺序及主链及支链糖链的结构，解析出了4种多糖的一级结构，并观察了其空间结构，初步探索了其构效关系。采用MTS法观察了4种多糖的体外抗肿瘤活性，表明其具有明显的体外抗肿瘤活性，其作用机制为提高Bax, p53的表达，抑制bcl-2 , c-myc表达而促进肿瘤细胞凋亡。对H22与S180小鼠移植性肿瘤模型的体内抗肿瘤实验结果发现，4种多糖中，PBPCIa、PBPCⅡ具有明显的抗肿瘤作用，且呈剂量依赖关系。用ELISA、RT-qPCR、Western blot实验表明，其抗肿瘤分子机制为抑制PI3K/Akt信号通路、上调Bax、P53、Cleaved casepase-3、蛋白表达、下调Bcl-2表达，并促进胸腺、脾脏的免疫功能、血清免疫相关细胞因子的分泌等多种途径而抑制肿瘤细胞的分裂增殖。本研究分析了红花蜂花粉多糖的结构、体内外抗肿瘤活性及其在基因与蛋白水平的作用机制，为其科学开发利用提供了理论基础，也为寻找高效低毒的抗肿瘤多糖药物的研究提供基础数据和新途径。