基于功能化离子液体靶向富集的甘草中异戊烯基黄酮的制备分离及其诱导自噬构效关系研究

Recent studies of our group and other researchers have suggested that prenylated flavonoids in licorice possess significant anti-tumor activity, since they could induce autophagy of tumor cells. Thus, they are important antineoplastic natural products with good medical prospect. However, the content of prenylated flavonoids in licorice is relatively low, and thus it is difficult to prepare them. Ionic liquids are new environmentally-sound solvent, and their structures can be flexibly designed as required. In this project, we will employ imidazolium-based functionalized ionic liquids for fast targeted preconcentration of prenylated flavonoids from licorice, which aims to discover natural autophagy inducer with small molecular weight and investigate their structure-activity relationship. Firstly, according to chemical structures and properties of prenylated flavonoids, a series of functionalized ionic liquids will be designed and synthetized to extract prenylated flavonoids from licorice. Based on the obtained extracting solution, a functionalized ionic liquid/saline solution aqueous biphasic system will be directly constructed for the further extraction and preconcentration. We will screen out the optimal functionalized ionic liquid with the highest specificity, and the total prenylated flavonoids with relatively high purity will be obtained. Next, preparative HPLC will be used to prepare a series of pure compounds, and their structures will be fully identified. Finally, the activities of pure prenylated flavonoids in inducing autophagy will be respectively evaluated, and the structure-activity relationship will be systematically summarized. We will also evaluate in vivo anti-tumor activity of high bioactive compounds. This project will provide a new idea for fast targeted preconcentration of active ingredients from herb medicines, and also provide lead compounds for discovery of new anti-tumor agents with the activity of inducing autophagy.

近年，申请人及其他课题组均发现甘草的异戊烯基黄酮类成分具有显著的抗肿瘤活性，其机制与诱导肿瘤细胞自噬有关，是一类具有药用前景的抗肿瘤天然产物。然而，该类成分在甘草中的含量较低，制备困难。离子液体是一种新型绿色溶剂，结构具有可设计性。本项目将利用咪唑类功能化离子液体高效靶向富集甘草的异戊烯基黄酮类成分，发现高活性的天然小分子自噬诱导剂并探讨构效关系。首先，根据异戊烯基黄酮的极性和结构特点，设计合成一系列功能化离子液体用于甘草提取，并直接构建离子液体/盐溶液双水相体系进一步萃取，筛选出特异性最高的离子液体，靶向富集高纯度的异戊烯基黄酮部位。其次，采用制备HPLC分离纯化单体化合物并鉴定结构。最后，评价化合物的诱导自噬活性，系统阐明构效关系，发现高活性分子并开展体内抗肿瘤药效研究。本项目将为中药活性成分的高效靶向富集研究提供新的思路，也将为诱导自噬新型抗肿瘤药物的发现提供先导化合物。

甘草中的异戊烯基黄酮类成分可以通过诱导自噬而发挥抗肿瘤作用，但其含量低、制备困难。本项目根据异戊烯基黄酮的疏水性特点，设计合成了一系列烷基功能化的疏水性咪唑类离子液体。以5个代表性异戊烯基黄酮（isoangustone A、甘草香豆素、甘草双氢异黄酮、甘草西定和光甘草定）的提取效率作为评价指标，通过离子液体的阳离子和阴离子筛选，建立了甘草中异戊烯基黄酮类成分的靶向提取方法，并通过响应曲面法等方法优化了提取条件（提取时间、固液比、提取温度等）。该方法以1-辛基-3-甲基咪唑四氟硼酸盐（[C8MIM]BF4）为提取溶剂，采用超声提取法，可以高选择性地提取出甘草中的异戊烯基黄酮类成分，提取液中几乎不含有其他类型化学成分，这是传统的有机溶剂难以实现的。同时，进一步研究了离子液体靶向提取的机理，扫描电子显微镜观察表明[C8MIM]BF4-超声辅助提取可以大量破环植物细胞，更重要的是，分子动力学模拟研究表明[C8MIM]BF4与异戊烯基黄酮具有较强的相互作用力，包括范德华力、静电相互作用力等，显著高于传统溶剂甲醇。另外，本项目尝试了固相萃取和分子排阻开口柱色谱用于异戊烯基黄酮类化合物的回收，其平均回收率分别为78.92%和57.07%，并且，离子液体表现出良好的回收再利用能力。将此方法用于提取甘草药材的异戊烯基黄酮类成分，结合半制备HPLC等传统植物化学研究方法，共分离得到45个异戊烯基黄酮单体化合物（1−45），包括3个新化合物。以SW480人结肠癌细胞为研究对象，首次发现一系列具有诱导自噬活性的异戊烯基黄酮类化合物（15、24、26、28、29、30、38、43等），构效关系分析表明，异戊烯基可以显著增加该类化合物诱导自噬的活性。最后，选取两个代表性化合物6,8-diprenygenistein (15)和lupalbigenin (30)，确认它们可以通过诱导SW480细胞自噬而产生细胞毒作用。同时，以接种移植瘤的裸鼠为研究对象，发现它们具有较强的体内抗肿瘤药效。本项目结果为中药活性成分的靶向富集研究提供了新思路，也为诱导自噬新型抗肿瘤药物的发现提供了先导化合物。