酸枣仁三萜皂苷的分离及对树突状细胞功能调控机理的研究

Dendritic cells (DCs) play an important role in the immunologic process. DCs as target cells help to filter the active ingredients of Traditional Chinese Medicine and promote the research and development of efficient, safe and novel immunoadjuvant. Based on the early phytochemical and pharmacological study, eleven triterpenoid saponins were obtained from Semen Ziziphi Spinoae and the immunoenhancemental effects were discovered. By the help of UPLC-Q-TOD-MSn analysis and pharmacological active screening, several unknown active saponins were detected from the extract of the seeds. The objective of this project based on the former study is to discovered novel and immunological effective saponins, and study the immunological adjuvant activities and their mechanism on DCs using the pharmacological, immunological and molecular biological methods such as MTT, ELISA, flow cytometer, RT-PCR, gene chips, two-dimensional electrophoresis, as well as Western Bolt. Then lead compounds with good activity will be selected by the exoteric experiment. This project not only provides new idea to discover active immunoenhancemental components but alos has significant sense and practical value for the discoverment of novel immunoadjuvant to the provention and control of animal deseases.

树突状细胞在机体免疫过程中发挥重要作用，以其为靶细胞筛选中药活性成分有助于研发高效、安全的新型免疫佐剂。申请人已经从酸枣仁中分离到十一种三萜皂苷类化合物并发现部分皂苷具有较好的增强免疫活性，UPLC-Q-TOF-MSn分析和药理活性筛选提示酸枣仁中仍存在未知的活性三萜皂苷。本项目拟在前期工作的基础上从酸枣仁中定向寻找和分离新的活性三萜皂苷，并以树突状细胞为靶细胞，综合运用MTT、ELISA、流式细胞仪、RT-PCR、基因芯片、双向电泳、Western Blot等药理学、免疫学和分子生物学的技术探索未知活性三萜皂苷的体外免疫促进作用和机理，并通过体内实验验证筛选出活性较强的先导化合物。本研究不仅为寻找增强免疫作用的活性先导化合物提供思路，而且对研制新型免疫增强剂、提高动物疫病的防控效果有重要意义和实用价值。

树突状细胞是一类重要的动物免疫细胞。本项目对酸枣仁、酸枣肉的化学成分进行了较为系统的分离，从中分离得到一系列化合物。通过波谱学等方法对其中大部分化合物进行了结构分析，鉴定了其中60个化合物，发现新化合物2个。本课题成功建立了小鼠骨髓源和鸡骨髓源原代树突状细胞的分离培养方法，并以此为细胞模型，分别筛选了酸枣仁总皂苷、酸枣仁皂苷A、酸枣仁皂苷B和淫羊藿多糖、硫酸化淫羊藿多糖的体外免疫活性。同时，运用正交试验的方法，研究并优化了大枣多糖的超声辅助提取方法。研究发现，酸枣仁总皂苷、酸枣仁皂苷A和酸枣仁皂苷B不具有体外调节小鼠树突状细胞免疫活性的作用，淫羊藿多糖和硫酸化淫羊藿能够体外调节鸡树突状细胞的免疫活性。同时，超声时间为20min，超声功率为120W，提取温度为85℃，提取时间为1.5h，料液比为1：15为大枣粗多糖提取的最佳工艺条件。