目的 合成羽扇豆醇-3-吡啶季铵盐衍生物，并进行其体内外抗乳腺癌MCF-7活性及作用机制研究。方法 通过酯化、酰化和成盐反应得到目标化合物。采用紫外分光光度法在305 nm下测定羽扇豆醇和羽扇豆醇-3-吡啶季铵盐的水溶性；以羽扇豆醇-3-吡啶季铵盐0、1.25、2.5、5、10、20、40 μmol/L作用乳腺癌MCF-7细胞24、48、72 h，使用MTT法对其细胞增殖抑制能力进行测定；采用羽扇豆醇-3-吡啶季铵盐（7、14、28 μmol/L）作用MCF-7细胞48 h后，采用流式细胞术（AnnexinV-FITC/PI法）检测细胞凋亡，JC-1染色法检测细胞线粒体膜电位，DCFH-DA染色法检测细胞活性氧（ROS）水平；同时在激光共聚焦显微镜下观察细胞凋亡、线粒体膜电位变化以及ROS水平；Western blotting法检测MCF-7细胞中线粒体凋亡途径相关蛋白表达变化。通过构建4T1小鼠荷瘤模型，设置对照组和羽扇豆醇-3-吡啶季铵盐（20、40 mg/kg）组，与对照组进行比较，观察羽扇豆醇-3-吡啶季铵盐在体内抑制乳腺癌的活性。结果 合成羽扇豆醇-3-吡啶季铵盐，其结构经¹H-NMR、¹³C-NMR和HR-ESI-MS确证。紫外分光光度法结果显示羽扇豆醇-3-吡啶季铵盐相对羽扇豆醇水溶性显著提高；MTT提示羽扇豆醇-3-吡啶季铵盐对乳腺癌MCF-7细胞有显著的抑制能力（P＜0.01、0.001），且抑制率呈浓度、时间相关趋势；流式细胞术以及激光共聚焦显微镜观测结果表明羽扇豆醇-3-吡啶季铵盐能够有效诱导细胞凋亡，线粒体膜电位显著下降，活性氧水平明显升高；羽扇豆醇-3-吡啶季铵盐能够显著上调B细胞淋巴瘤2（Bcl-2）相关X蛋白（Bax）、细胞色素C（Cyt C）、Bax、cleaved Caspase-9、cleaved Caspase-7蛋白表达，下调Bcl-2蛋白表达（P＜0.01、0.001）；羽扇豆醇-3-吡啶季铵盐在体内抑制肿瘤生长效果显著，并未引起明显毒性。结论 成盐后的羽扇豆醇-3-吡啶季铵盐水溶性明显增加，并显著提高了体内外抗乳腺癌活性，本研究拓展了羽扇豆醇抗肿瘤研究的新思路。

Objective To study the effect and mechanism of a quaternary ammonium salt derivative of lupeol pyridine on breast cancer in vitro and in vivo. Methods The target compound was obtained by esterification, acylation and salt forming reactions. The water solubility of lupeol and lupeol-3-pyridine quaternary ammonium salt were determined by ultraviolet spectrophotometry at 305 nm. Breast cancer MCF-7 cells were treated with lupeol and lupeol-3-pyridine quaternary ammonium salt (0, 1.25, 2.5, 5, 10, 20, 40 μmol/L) for 24, 48, 72 h, and the cell proliferation inhibition ability was measured by MTT assay. MCF-7 cells were treated with 7, 14, and 28 μmol/L lupeol and lupeol-3-pyridine quaternary ammonium salt for 48 h, and the apoptosis was detected by flow cytometry (AnnexinV-FITC/PI method). The mitochondrial membrane potential was detected by JC-1 staining, and the reactive oxygen species (ROS) level was detected by DCFH-DA staining. At the same time, apoptosis, mitochondrial membrane potential changes and ROS levels were observed under confocal laser microscopy. Western blotting was used to detect the expression of proteins related to mitochondrial apoptosis pathway in MCF-7 cells. The tumor bearing model of 4T1 mice was established, and the control group and lupinol-3-pyridine quaternary ammonium salt (20 and 40 mg/kg) groups were set up to compare with the control group, and the anti-breast cancer activity of lupinol-3-pyridine quaternary ammonium salt in vivo was observed. Results Lupeol-3-pyridine quaternary ammonium salt derivative was synthesized and the structure was confirmed by ¹H-NMR, ¹³C-NMR and HR-ESI-MS. UV spectrophotometry showed that the water solubility of lupeol-3-pyridine quaternary ammonium salt was significantly higher than that of lupeol. MTT assay showed that lupeol-3-pyridine quaternary ammonium salt had a significant inhibitory effect on breast cancer MCF-7 cells (P < 0.01, 0.001), and the inhibition rate was concentration-time dependent. Flow cytometry and laser confocal microscopy showed that lupeol-3-pyridine quaternary ammonium salt could effectively induce cell apoptosis, significantly decrease mitochondrial membrane potential, and significantly increase the level of reactive oxygen species. Lupeol-3-pyridine quaternary ammonium salt could significantly up-regulate the expression of Bax, Cyt C, Bax, cleaved Caspase-9, and cleaved Caspase-7 proteins, and down-regulate the expression of Bcl-2 protein (P < 0.01, 0.001). Lupeol-3-pyridine quaternary ammonium salt significantly inhibited tumor growth in vivo without causing obvious toxicity. Conclusion The water solubility of Lupeol-3-pyridine quaternary ammonium salt significantly increased, and improved the anti-breast cancer activity in vivo and in vitro. The study has expanded the new idea of anticancer research of lupeol.

R284.3;R285.5

黑龙江省教育厅基本科研业务费基础研究项目(2020-KYYWF-0024)