Three new C-27-carboxylated-lupane-triterpenoid derivatives from Potentilla discolor Bunge and their in vitro antitumor activities

Three new lupane-triterpenoids (1–3) along with six known compounds (4–9) were isolated from the ethanolic extract of whole plant of Potentilla discolor Bunge. The structures of Compounds 1–3 were established by extensive 1D and 2D NMR together with other spectrum analysis, indicating that their C-27 positions were highly oxygenated, which were rarely found in nature. Their in vitro anti-proliferative activities against HepG-2, MCF-7 and T-84 cell lines were evaluated by Cell Counting Kit-8 (CCK-8) assay, and the results showed different activities for three cell lines with IC50 values ranging from 17.84 to 40.64 μM. In addition, the results from Hoechst 33258 and AO/EB staining as well as annexinV-FITC assays exhibited Compound 1 caused a markedly increased HepG-2 cellular apoptosis in a dose-dependent manner. The further mechanisms of Compound 1-induced cellular apoptosis were confirmed that 1 induced the production of ROS and the alteration of pro- and anti-apoptotic proteins, which led to the dysfunction of mitochondria and activation of caspase-9 and caspase-3 and finally caused cellular apoptosis. These results would be useful in search for new potential antitumor agents and for developing semisynthetic lupane-triterpenoid derivatives with high antitumor activity.

本研究从翻白草（Potentilla discolor Bunge）全草的乙醇提取物中分离得到3个新的羽扇烷型三萜类化合物（lupane-triterpenoids）（1~3）及6个已知化合物（4~9）。通过详尽的一维核磁共振波谱（1D NMR）、二维核磁共振波谱（2D NMR）及其他谱学分析手段，确定了化合物1~3的结构，结果显示其C-27位点为高度氧化修饰位点，这类结构在自然界中较为罕见。采用细胞计数试剂盒-8（Cell Counting Kit-8, CCK-8）实验，评估了目标化合物对HepG-2、MCF-7及T-84细胞系的抗增殖活性，结果显示其对三种细胞系均表现出不同程度的抑制活性，半数抑制浓度（IC50）范围为17.84~40.64 μM。此外，通过Hoechst 33258染色、AO/EB染色及annexinV-FITC实验的结果表明，化合物1可呈剂量依赖性显著诱导HepG-2细胞凋亡。进一步的机制研究证实，化合物1通过诱导活性氧（Reactive Oxygen Species, ROS）生成、调控促凋亡蛋白（pro-apoptotic proteins）与抗凋亡蛋白（anti-apoptotic proteins）的表达失衡，引发线粒体功能障碍，激活半胱氨酸天冬氨酸蛋白酶-9（caspase-9）与半胱氨酸天冬氨酸蛋白酶-3（caspase-3）通路，最终诱导细胞凋亡。上述研究结果可为新型潜在抗肿瘤药物的研发，以及开发具有高抗肿瘤活性的半合成羽扇烷型三萜类衍生物（lupane-triterpenoid derivatives）提供重要理论依据。