Abstract: Objective　To provide theoretical references for developing the medicinal value of Clinacanthus nutans (Burm.f.) Lindau by separating and purifying its flavonoid glycosides and studying its antitumor effects on epithelial ovarian cancer. Methods　The research time was from October 2019 to March 2022.The flavonoid glycosides were isolated and purified by macroporous adsorption resins and semi-preparative high performance liquid chromatography, and their structures were identified by nuclear magnetic resonance spectrum. The cell counting kit-8 (CCK 8) method was used to determine the effect of the extract on the activities of the SKOV3, OVCAR3, and A2780 epithelial ovarian cancer cells, and flow cytometry was used to detect the apoptosis. The effects of the extract on the expressions of Beclin-1 and Bcl-2 mRNA in the epithelial ovarian cancer cells were detected by fluorescence quantitative polymerase chain reaction (PCR). Its effect on the expressions of Beclin-1 and Bcl-2 proteins in the epithelial ovarian cancer cells was detected by Western blot. The statistical softwares SPSS 22.0 and GraphPad Prism were used to analyze the experimental results. The one-way ANOVA was used to determine the cell 50% inhibiting concentration (IC₅₀) value. t test was applied. Results　Three flavonoid glycosides were isolated and purified from Clinacanthus nutans (Burm.f.) Lindau, and they were identified as saponarin, schaftoside, and vitexin. The results of the CCK8 experiment showed that vitexin significantly inhibited the proliferation of the epithelial ovarian cancer cells (P < 0.05). The IC₅₀ values (semi-inhibitory concentrations) of the SKOV3, OVCAR3, and A2780 epithelial ovarian cancer cells were 33.777 μg/μl, 34.114 μg/μl, and 31.968 μg/μl. The results of fluorescence quantitative PCR showed that compared with those in the blank group, the relative expression of Beclin-1 mRNA increased and the expression of Bcl-2 mRNA decreased in the SKOV3, A2780, and OVCAR3 epithelial ovarian cancer cell groups after vitexin treatment, with statistical differences (all P<0.01). The results of apoptosis test showed that the inhibitory effect of vitexin on the growth of the SKOV3 epithelial ovarian cancer cells significantly increased with the vitexin concentration (P<0.01). Western bolt showed that after vitexin treatment, the expression of Beclin-1 protein increased significantly (P<0.01), and the expression of Bcl-2 protein decreased significantly (P<0.01). Conclusions　Vitexin shows significant anticancer activity by inhibiting the proliferation and inducing the apoptosis of epithelial ovarian cancer cells. The anticancer activity may be related to regulating the interaction between Bcl-2 and Beclin-1, controlling the autophagy level of tumor cells, and promoting autophagy-dependent cell death.

Key words: Clinacanthus nutans(Burm.f.)Lindau；Epithelial ovarian cancer；Bcl-2,  , Beclin-1；Apoptosis；Autophagy

摘要： 目的　对忧遁草中的黄酮苷成分进行分离与纯化，研究其对上皮性卵巢癌的抗肿瘤作用，为开发其药用价值提供理论依据。方法　研究时间：2019年10月至2022年3月。通过大孔吸附树脂结合半制备型高效液相色谱对忧遁草中的黄酮苷进行分离纯化，核磁共振波谱鉴定化合物的结构；采用CCK-8（cell counting kit-8）法测定忧遁草黄酮苷提取物对上皮性卵巢癌细胞SKOV3、OVCAR3、A2780细胞活性的影响，流式细胞学技术检测细胞凋亡情况；采用荧光定量聚合酶链式反应（polymerase chain reaction，PCR）技术检测忧遁草黄酮苷提取物对上皮性卵巢癌细胞中Beclin-1、Bcl-2 mRNA表达的影响；采用蛋白印迹（Western blot）技术检测其对上皮性卵巢癌细胞中Beclin-1、Bcl-2蛋白表达的影响。使用统计软件SPSS 22.0、GraphPad Prism对实验结果进行统计学分析，单因素方差分析进行细胞半抑制浓度（IC₅₀）值的测定，采用t检验。结果　自忧遁草中分离纯化得到3种黄酮苷化合物，分别为肥皂草苷、夏佛塔苷和牡荆苷；CCK-8实验结果显示，牡荆苷对上皮性卵巢癌细胞具有明显增殖抑制作用（P<0.05），对上皮性卵巢癌细胞SKOV3、OVCAR3、A2780的半抑制浓度IC₅₀值分别为33.777 μg/μl、34.114 μg/μl、31.968 μg/μl。荧光定量PCR检测结果表明，和空白组相比，经牡荆苷处理后，上皮性卵巢癌细胞SKOV3、A2780、OVCAR 3组Beclin-1 mRNA相对表达量增加，Bcl-2 mRNA相对表达量降低，差异均有统计学意义（均P<0.01）。以上皮性卵巢癌细胞SKOV3为研究对象，细胞凋亡检测结果显示，随着牡荆苷浓度的增加，其对上皮性卵巢癌细胞SKOV3的生长抑制作用越显著（P<0.01）；Western bolt结果显示，经牡荆苷处理后，上皮性卵巢癌细胞SKOV3中Beclin-1蛋白表达显著升高（P<0.01），Bcl-2蛋白表达明显减少（P<0.01）。结论　牡荆苷可以抑制上皮性卵巢癌细胞的增殖，并诱导其凋亡，具有明显的抗肿瘤作用，可能是通过调节Bcl-2与Beclin-1的交互作用发挥抗癌活性，控制肿瘤细胞自噬水平，促进自噬基因依赖性细胞死亡。

关键词: 忧遁草, 上皮性卵巢癌, Bcl-2, Beclin-1, 细胞凋亡, 自噬