地骨皮中氮杂糖类成分的抗糖尿病作用、机理和构效关系研究

Previous studies has revealed that the water-soluble fraction from Cortex Lycii and its alkaloid fraction have a significant inhibitory activity on α-glucosidase and an anti-diabetes effect, and these fractions contain a series of iminosugars. This project focuses on the studies on systematic isolation, identification, anti-diabetes activities of iminosugar constituents in the water-soluble fractions from Cortex Lycii (L. Chinese and L. barbarum) and root bark from the other Lycii plants as well as the discovery of new bioactive iminosugar compounds. We use various ion exchange chromatography and other chromatography techniques to isolate and purify the water-soluble constituents, and use various spectroscopic methods such as MS, NMR, and X-ray diffraction to determine the chemical structures for all the isolated compounds. We also measure the inhibitory activities on various glycosidase for all the isolated compounds, and investigate their structure-activity relationship (SAR) with the computer-aiding analysis of interaction between ligand-enzyme. We do bioassay on the iminosugars’ inhibiting some glycosidases from cultured human Caco-2 cell lines and from rat intestinal brush border membrane, and the promoting effect of isulun secretion on INS-1 cell line for all the compounds，and evaluate the in vivo anti-hyperglycaemia effect of the main isolated compounds on mice, in order to find out the active constituents against hyperglycaemia, and to reveal the efficacious substances and their action mechanism of Cortex Lycii on anti-diabetes, to establish the foundation for quality evaluation of crude materials of Cortex Lycii, and for the development of innovate products of Chinese patent drug to cure diabetes.

本项目在前期研究表明地骨皮水部位及其中生物碱部位具有明显的α-葡萄糖苷酶抑制活性和抗糖尿病作用、且具有多种氮糖类成分的基础上，旨在对该活性部位及同属其它种植物根皮中氮杂糖类成分进行深入的分离鉴定和抗糖尿病相关生物活性研究，并发现活性新生物碱成分。采用离子交换层析等多种色谱技术分离纯化水溶性生物碱成分，采用多种核磁和质谱等光谱手段及单晶衍射技术鉴定化合物结构。对各单体化合物进行多种糖苷酶抑制活性等的评价，并进行计算机辅助的酶－配体（氮杂糖生物碱成分）构效关系研究。对分离化合物进行人源Caco-2细胞和小鼠小肠绒膜等来源多种糖苷酶抑制作用和对INS-1细胞的胰岛素分泌促进分析，并主要化合物进行小鼠在体降血糖作用实验研究，发现降血糖作用相关的活性化合物，阐明地骨皮抗糖尿病的物质基础和作用机制，为该种类药材品质评价和抗糖尿病创新中药产品研发奠定研究基础。

地骨皮为茄科植物枸杞(Lycium chinense Mill)或宁夏枸杞（Lycium barbarum L)的干燥根皮，其功能主治为内热消渴等。而消渴症是多饮、多食、多尿，身体消瘦，或尿浊、尿有甜味的病症，与西医的糖尿病相对应。现有研究并未揭示其抗糖尿病的作用物质基础和作用机制。本项目研究首先聚焦地骨皮水提物中的成分研究，并重点关注氮糖类化合物及糖苷酶抑制活性，研究发现其含有4个托品烷形氮糖类化合物，但抑制糖苷酶活性弱。因此，及时调整研究内容，从分离研究成分上，不限制成分类型，从研究材料上，扩展到地骨皮资源植物枸杞和宁夏枸杞植物全株和同属云南枸杞，从活性测试上，增加与抗糖尿病相关的抗炎活性（NO释放抑制）等。我们对地骨皮水提取物、及枸杞、宁夏枸杞和云南枸杞醇提取物进行了系统的活性成分分离和结构鉴定，共分离鉴定化合物100余个，其中新化合物9个，从结构上看，属于新结构类型木质素杂合咖啡酰胺类似物4个，咖啡酰胺类二聚体1个，倍半萜类化合物4个。其他已知化合物结构类型为氮糖类、倍半萜、咖啡酰胺类及其二聚体或与木脂素杂合体、香豆素、环肽、木脂素及其苷、酚酸、黄酮苷、甾体，其中以咖啡酰胺类最结构多样，且多个化合物具有NO释放抑制活性。本研究极大地丰富了地骨皮及其资源植物的化学成分信息，为地骨皮抗糖尿病物质基础、真伪鉴别和质量控制研究奠定了基础。本研究能发表至少10篇以上学术论文，SCI论文和中文论文各5篇，现已发表论文共8 篇，其中SCI 论文4 篇、中文核心期刊论文4 篇，另有英文SCI、中文论文各一篇在正投稿审稿中；通过本项目研究，已培养硕士研究生5名毕业、产生了硕士学位论文5篇。