超声改性裂褶多糖结构表征和免疫活性关系研究

Schizophyllan has great Immunological activity. But, its application on the medical industry has been limited for the large molecular weight and poor solubility. Ultrasonic modification can reduce the molecular weight and increase the solubility of polysaccharides, which is considered to improve the structure properties of polysaccharides. Whereas, the active conformation and biological activity of polysaccharides have been reduced or lost after excessive ultrasonic modification treatment. Therefore, schizophyllan with which structural properties has best solubility and immunological activity, it is not clear at present. .In this study, schizophyllan prepared by solid fermentation with bran and corn cob is used as material and treated by ultrasonic technology to reduce the molecular weight. Varied schizophyllan fractions are collected after separation and purification processing. The solubility, immunological activity and structural properties of these schizophyllan fractions are measured and analyzed, and the structural properties of schizophyllan fraction with highest immunological activity are focused on, including molecular weight, primary and advanced structural characterization. On these bases, the correlation between structural properties and immunological activity of schizophyllan is investigated and evaluated finally. This study aims to provide the theoretical support to the research of immunological activity mechanism of schizophyllan and promote its application on medicine industry.

裂褶多糖具有良好免疫活性，但分子量大，溶解性差限制其医药应用。超声波改性处理能改善多糖结构，达到降低分子量、增加溶解性功效,但过度改性会导致多糖活性构象丧失，生理活性下降。具有何种结构特征的裂褶多糖溶解性和免疫活性能达到最佳，目前尚不明确。.本项目以麸皮和玉米芯固体发酵制备裂褶多糖为原料，采用超声波技术处理和分离纯化制备不同级分裂褶多糖；在此基础上开展溶解性、免疫调节活性和结构特性研究，重点表征高免疫活性裂褶多糖的结构特性；从分子量、一级结构和高级结构等方面探讨影响多糖免疫活性的结构特性，解析裂褶多糖结构与免疫活性之间的构效关系。本研究旨在为裂褶多糖免疫活性机理研究提供理论数据支撑，推动裂褶多糖医药产业应用。

裂褶多糖（SPG）具有良好免疫活性，但分子量大，溶解性差限制其医药应用。超声波改性处理能改善多糖结构，达到降低分子量、增加溶解性功效，但过度改性会导致多糖活性构象丧失，生理活性下降。具有何种结构特征SPG溶解性和免疫活性能达到最佳，目前尚不明确。.本项目以麸皮和玉米芯固体发酵制备SPG，采用超声波技术降解制备得到4个不同分子量SPG样品，在此基础上开展溶解性、免疫调节活性和结构特性研究，解析裂褶多糖结构与免疫活性之间构效关系。旨在为SPG免疫活性机理研究提供理论数据支撑，推动SPG医药产业应用。具体研究进展如下：.随超声时间和强度增强SPG分子量逐渐降低，降解动力学符合降解二级动力学模型。最终降解超声参数：时间10 min，超声强度796 W/cm2。结合乙醇分级沉淀和透析，最终得到4个SPG样品，分子量分别集中在107 Da、106 Da、105 Da和104 Da左右，样品纯度分别均为90%左右。.流变特性分析结果表明，SPG为假塑性流体，浓度10 mg/ml时表现为固体特性（储能模量G′>损耗模量G′′）。USPG40表现假塑性流体行为，USPG80表现出牛顿流体行为。.细胞和动物体内试验结果表明，不同分子量SPG样品均具有较好免疫及抗肿瘤活性。不同分子量SPG样品（25-600 µg/ml）在体外对促进脾淋巴细胞增殖、巨噬细胞RAW264.7的NO释放和抑制乳腺癌细胞T-47D活性具有良好功效；含多糖血清均可促进正常大鼠的脾淋巴细胞增殖和免疫因子IL-2和TNF-α释放；显著增强S180荷瘤小鼠的迟发型变态反应（DTH）、促进LPS诱导的B淋巴细胞增殖、提高NK细胞活性和肿瘤抑制率。整体看来，USPG60和USPG80综合表现最佳。.通过单糖分析、红外光谱、甲基化联合GC-MS、NMR和激光光散射等技术，解析了不同分子量SPG结构。研究结果表明，超声处理降低了多糖分子量，但并未破坏SPG样品一级结构。SPG是一种高支化的β-1,3-葡聚糖类多糖，在O-6 位置出现支链，为β-1–葡萄糖或β-1–葡萄糖、β-1,6-葡萄糖支链。.在此基础上初步探讨SPG生物活性和结构的关系。不同分子量的SPG样品存在显著免疫调节及抗肿瘤活性差异，但一级结构并没有差异，推测这种活性差异不仅仅跟多糖的一级结构相关，推测跟不同分子量SPG样品的流变特性和溶液性质相关。