稀有人参皂苷Rk1的定向生物转化及抗肿瘤机制研究

Ginsenosides are derived from Panax ginseng, Panax quinquefolius and Panax notoginseng, and so on. Ginsenoside Rk1, dihydric ginsenoside, is a derivative obtained of deglycosylation by physicochemical or biological transformation, and it has significantly anti-tumor activity. However, it is very difficult to obtain Ginsenoside Rk1 using conventional preparation technology, and little is known about its anti-tumor mechanism. In this project, ginsenoside Rk1 was prepared by biotransformation of ginsenoside Rb1, in the presence of Lactobacillus. And compared with the rare ginsenosides that anti-tumor activity has been identified, such as 20 (R)-ginsenoside Rg3, and clinical first-line anti-tumor drugs, we investigated the effect of ginsenoside Rk1 on the inhibitory effect of various tumor cells, cell apoptosis, cell cycle arrest and cell differentiation. And we studied its function role in promoting the immune organs and cells, determined its inhibitory effects on tumor cell invasion and metastasis, simultaneously, and obtained dose-dependent relationship of its anti-tumor effect against xenograft model in mice. The difference between the anti-tumor activity, the target of action and the mechanism of action of ginsenoside Rk1 and isomeric ginsenoside Rg5 was compared, and the relationship between the double bond position and the anti-tumor activity and the mechanism of action was analyzed. These results provide an important reference for the study of anti-tumor activity of rare ginsenosides and drug development.

人参皂苷来源于人参、西洋参和三七等药材，人参皂苷Rk1是二醇型人参皂苷通过理化或生物转化等方法去糖基化得到的衍生物，抗肿瘤活性良好，常规技术获取人参皂苷Rk1非常困难，其抗肿瘤机制研究也很少。本项目以乳杆菌定向生物转化人参皂苷Rb1制备人参皂苷Rk1，并与20（R）-人参皂苷Rg3等抗肿瘤活性已确认的稀有人参皂苷及临床一线抗肿瘤药物为对照，考察对多种体外肿瘤细胞的抑制和诱导凋亡、对细胞周期的阻滞和对细胞分化的影响；研究人参皂苷Rk1对多种肿瘤模型荷瘤动物肿瘤增长的抑制，考察其对给药动物免疫器官及细胞有无功能促进，并确定其对肿瘤细胞侵袭和转移有无抑制，同时获得其对模型动物抗肿瘤作用的量效关系；比较人参皂苷Rk1与同分异构体人参皂苷Rg5的抗肿瘤活性、作用靶点和作用机制的差异，分析二者分子结构中双键位置与抗肿瘤活性及作用机制的关系。为系列稀有人参皂苷抗肿瘤活性研究和药物开发提供重要参考。

项目研究团队筛选出一株野生型乳杆菌Lac-F140307并对其进行驯化，其在发酵过程中以体外无抗肿瘤活性的人参皂苷Rb1为底物，定向转化为稀有人参皂苷Rk1，通过优化发酵工艺，可显著提高转化效率。体外细胞学实验和动物学药效实验结果显示人参皂苷Rk1和人参皂苷Rg5等几种稀有人参皂苷对多个肿瘤细胞具有显著的抑制和诱导凋亡作用，对肿瘤模型动物的肿瘤体积增大有很强的抑制作用，且安全性良好，此外，通过药理学实验证明人参皂苷Rk1和Rg5 通过线粒体介导的细胞凋亡和自噬细胞死亡显着抑制肿瘤增殖。 LC3B-GFP/Lysotracker 和 mRFP-EGFP-LC3B 用于显示人参皂苷Rk1和 Rg5 诱导自噬体-溶酶体融合。蛋白质印迹分析进一步表明，人参皂苷Rk1和Rg5 降低了乳腺癌中 PI3K、Akt、mTOR 和 Bad 的磷酸化水平，并抑制了 PI3K/Akt 信号通路。此外，PI3K/Akt 抑制剂 LY294002 可以显着增强人参皂苷Rk1和Rg5 诱导的细胞凋亡和自噬。揭示了人参皂苷Rk1和Rg5作用靶点和作用途径，为系统研究稀有人参皂苷的抗肿瘤作用机制提供重要参考，也为新的抗肿瘤药物开发提供基础研究数据，具有重要的理论意义和实践意义。