甘肃中药材内生真菌中α-葡萄糖苷酶抑制剂的筛选与发现

As the reduction of plant resources, endophytic fungi is increasingly remarkable for it’s a lot of advantages, for example, endophytic fungi is not fully developed, easy to discover new compounds, easy to expand cultivation, and subject to seasonal restrictions, et al. In this project, Astragalus, Arrowroot, Rhubarb and other traditional antidiabetic Chinese herbal medicine are choosed for the study. A rapid screening model is established to find endophytic fungi producing α-glucosidase inhibitor. The endophytic fungi are separated and screened using the established model, and then the fungi are identified. The activitive endophytic fungi are choosed to be fermented. The α-glucosidase inhibitors in the fermentation products are separated using column chromatography, high performance liquid chromatography, high-speed countercurrent chromatography et al, and the activities are tracked using a modified microplate-based screening method. Its hypoglycemic activity is evaluated using cell and animal models. Its structure of the activitive compounds were identified with NMR, IR, UV, and mass spectrometry. A compound library is established using all the α-glucosidase inhibitors. The relationship is analyzed between the compound structure and its hypoglycemic activity. It can provide the basis for the subsequent development of diabetes therapy. Through the implementation of the project, novel structure compounds with good hypoglycemic effect are expected to be found. It can provide a techniques and methods reference for the endophytic fungi studies of other medicinal plants in the future.

随着植物资源的减少，植物内生真菌以其未被充分开发、易于发现新化合物、易于扩大培养、不受季节限制等优势日渐引人瞩目。项目选择甘肃产黄芪、葛根、大黄等传统降糖中药为研究对象，建立产α-葡萄糖苷酶抑制剂内生真菌的快速筛选模型，运用建立的快速筛选模型分离筛选内生真菌，并鉴定菌种；发酵活性内生真菌，采用柱层析、高效液相色谱、高速逆流色谱等分离发酵产物中的α-葡萄糖苷酶抑制剂，改进微孔板法跟踪监测活性；并运用细胞和动物降糖模型评价其体内外降糖活性；结合核磁、红外、紫外、质谱法分析鉴定其结构，建立α-葡萄糖苷酶抑制剂化合物库。分析活性化合物结构与活性的构-效关系，为后续糖尿病治疗药物开发提供依据。通过项目的实施，期望发现结构新颖、具有良好降糖作用的先导化合物，为今后其它药用植物内生真菌的开发提供技术和方法上的参考，为甘肃中药材经济发展提供支持。

基于现场实地采集样本和文献论著调研法，明确了潜在的具有糖尿病治疗作用的中草药资源，并从甘草、党参、黄芪、地木耳等组织部位分离、纯化和鉴定了内生细菌800余株，内生真菌500余株。通过初级筛选获得了α-糖苷酶抑制活性较好的菌株资源76余株。利用色谱纯化、红外光谱和核磁等技术等手段完成了甘草内生真菌小鼻枝霉属（Rhinocladiella. sp G-207）的乙酸乙酯部位和Trichoderma sp. G-504的正丁醇部位化合物的纯化和结构解析，明确了较好活性化合物结构6个。采用分子对接法评价了其中2个化合物抑制α-糖苷酶潜在的活性位点，为后续糖尿病治疗药物开发提供了依据。同时，对地木耳水溶性纯化多糖进行了结构表征和抗氧化、α-糖苷酶抑制等活性评价；基于动物创伤模型，评价了地木耳多糖复合微囊对小鼠背部创面修复的生物学机制，为地木耳多糖的医学生物材料应用提供了数据支撑。