苦豆子活性多糖的空间结构表征及分子构象和功能性机制研究

The studies on structural characterization and functional property of bioactive polysaccharides and relationships between them are hot points in food and life science at present. However, there is a lack of systematic study of polysaccharides from Sophora alopecuroides L., a traditional Chinese herb, mainly distributed in northwest China. The spatial structure, melecular configuration and functional mechanism of bioactive polysacchrides from Sophora alopecuroides L. are investigated and determined in this project. The physicochemical properties of polysaccharide fractions from Sophora alopecuroides L. are analyzed and antitumor bioactivities of fractions both in vitro and in vivo are carried out using medical and biological methods based on preparation of polysaccharides using extracting alkaloids waste as material. The primary structure of bioactive fractions are determined using partial acid hydrolysis and methylation analysis, combined with HPAEC-PAD, GC-MS and 1D & 2D NMR spectroscopy techniques. The monosaccharide compositions, linkages of glycosidic bonds and side chains attached to the polysaccharide backbone are obtained. Advanced structural characterizations of bioactive polysaccharides are described by reasoning the characteristic parameters of polysaccharide chains, such as symmetry and screw pitch. The morphology, gel network and subcellular structure of fraction molecules are investigated using the atomic force microscope (AFM). The spatial configurations of polysaccharide molecules in solution are investigated in the molecular level by dynamic and static light scattering techniques. The weight average molecular weight (Mw), radius of gyration (Rg), the second virial coefficient (A2) and hydrodynamic radius (Rh) of the molecules are derived, and molecular conformations are obtained. The effects of degree of polymerization, substitution pattern and distribution of side chains and configuration of polysaccharide molecules on antitumor bioactivities are studied by molecular modification techniques. The functional mechanisms and relationships between structure and function of bioactive polysaccharides are elucidated, and meanwhile the important elements and bioactive sites of polysaccharide molecules are determined.

活性多糖的结构表征与功能性质以及二者之间构效关系机制的研究是目前生命科学研究的热点，而苦豆子多糖的研究缺乏系统性。本项目在苦豆子多糖前期制备的基础上，分析苦豆子多糖组分的物化性质，应用医学和生物学方法研究多糖组分体外和体内抗肿瘤活性，确定活性多糖组分。通过现代仪器分析结合部分酸水解和甲基化分析等化学方法表征活性多糖的一级结构，确定糖苷键连接方式以及多糖主链上的取代等。采用仪器分析和计算机模拟推理获得多糖分子链的特征参数如对称性、螺距等，观察多糖分子的形貌、凝胶网络和糖的亚细胞结构，揭示活性多糖的高级结构。通过光散射技术确定多糖分子尺寸和流体动力学半径等参数，从分子水平上研究多糖分子在溶液中的空间构象。通过分子修饰技术研究多糖链的聚合度、取代方式、支链分布及分子构象等对多糖抗肿瘤活性的影响，诠释多糖的功能机制和结构与功能之间的构效关系，明确多糖的活性部位和影响活性的关键因素。

目前国内外苦豆子研究热点主要集中在生物碱上，而对苦豆子活性多糖缺乏系统研究。本课题对生物活性成分苦豆子多糖提取分离纯化、空间结构、分子构象和构效关系研究。响应面分析获得苦豆子多糖超声波提取优化工艺参数，对其分离纯化和乙醇分级获中性多糖组分F15、F25、F35和F60，F25得率最高，为63.92%，分析研究各组分的物化性质。功能实验显示，F25对H22肿瘤细胞体外增殖抑制作用高于其它组分，抑制率可达到47.75%；F25体内肿瘤抑制率达到50.46%，且体内外活性均具有剂量依赖性。结构研究发现F25是一种半乳甘露聚糖，由β-D-Manp以(1→4)糖苷键连接构成骨架，并以大量T-α-D-Galp（86.82%）和/或少量α-(1→4)-D-Galp（13.18%）作为侧链随机分布在β-D-Manp O-6位上（M/G = 1.48-1.84）。通过3D数学建模获得F25分子的三维空间结构模型，发现F25分子空间构象具有较强刚性。经过分子模拟计算获得F25多糖分子链构象，推得多糖分子链具有一定的柔顺性，具有与溶液浓度紧密相关的缠绕结构。多糖分子在水、尿素和NaNO3稀溶液中均有聚集，但在0.1 M NaOH溶液中能消除分子聚集，测得重均分子量（Mw 302.54 KDa）、旋转半径（Rg 25.64 nm）和流体动力学半径（Rh 20.32 nm），系数ρ为1.26，表明多糖分子在稀NaOH溶液中是多分支紧凑的无规线团构型。F25的MHS方程指数α为0.66，表明F25为紧凑的无规线团的分子链，ρ和α反映的分子构型基本一致。多糖高级结构发现，一定浓度多糖在溶液中交联形成凝胶网络状，浓度降低分子逐渐分散，但1.0 μg/mL仍存在分子缠绕。在低浓度NaOH溶液中有大量分子分散，测得糖链长2.0-3.4 μm、高1.0-8.1 nm和宽19.9-27.9 nm。F25的结晶度为25.7%，分子中存在大量无定形结构，表观结晶尺寸为7.6 nm，表明F25分子结构排列具有较高规则性。经研究，F25抗肿瘤活性主要通过半乳甘露聚糖的半乳糖支链表达，并通过甘露聚糖的主链进行调节；不同的酸水解和酶解程度会使多糖分子链形成不同的空间构象，影响其抗肿瘤活性。系统研究苦豆子活性多糖的空间结构、分子构象和构效机制，对于改善人类膳食营养，推动中草药传统资源的开发利用有着极其重要的科学意义。