Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopusscaber, on lung adenocarcinoma (A549) cells

OBJECTIVE： Deoxyelephantopin, a sesquiterpene lactone from Elephantopus scaber, showed inhibition of the growth of various tumor cells in vitro. In the present study, we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma （A549） cells. METHODS： The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay and 50% inhibitory concentration （IC50） value was determined. The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay. Cellular morphology of deoxyelephantopin-treated cells was observed using phase- contrast microscopy. The induction of apoptosis was evaluated using acddine orange and ethidium bromide staining, Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling （TUNEL） assay, DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry. Activation of caspases was detected using fluorogenic substrate specific to caspases 2, 3, 8 and 9 and flow cytometric analysis. The total cellular DNA content and expression of cleaved poly （ADP-ribose） polymerase was also analyzed. RESULTS： Deoxyelephantopin exhibited cytotoxicity to A549 cells （IC50 = 12.287 μg/mL）, however, there was no toxicity towards normal human lymphocytes. Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner. Acridine orange, ethidium bromide and Hoechst 33342 staining showed cell shrinkage, chromosomal condensation and nuclear fragmentation, indicating induction of apoptosis. Deoxyelephantopin increased apoptosis of A549 cells, as evidenced by more TUNEL-positive cells. DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population. Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through both extrinsic and intrinsic pathways. CONCLUSION： These results suggest that deoxyelephantopin has great potential as a new chemotherapeutic agent to be developed further for the treatment of lung cancer.

Deoxyelephantopin induces ROS-mediated autophagy and apoptosis in human colorectal cancer in vitro and in vivo

Objective:To systematically map the stepwise events leading to deoxyelephantopin-induced cell death of HCT116 human colorectal cancer cells and evaluate the effectiveness of deoxyelephantopin in vivo.Methods:HCT116 cells were treated with deoxyelephantopin at various concentrations and time points.Autophagy was confirmed by the detection of autophagosomes and autophagosomal proteins by electron microscopy and Western blotting assays,respectively,and then validated by siRNA knockdown.In addition,apoptosis was confirmed by the detection of apoptosis-related proteins.The intracellular reactive oxygen species(ROS)level was measured using flow cytometry.The growth inhibitory effect of deoxyelephantopin was further evaluated in vivo using a mouse xenograft model.Results:Deoxyelephantopin firstly elevated ROS production,which then triggered autophagic flux with the accumulation of autophagosomal proteins including LC3 A/B,ATG5,and ATG7,followed by the induction of apoptosis via the intrinsic and extrinsic pathways.Pre-treatment with N-acetyl-L-cysteine,a ROS inhibitor,reversed both apoptosis and autophagy.The knockdown of LC3 prevented apoptosis induction which confirmed that deoxyelephantopin induced autophagy-dependent apoptosis in HCT116 cells.Accumulation of ROS also activated apoptosis via the mitogen-activated protein kinases signaling pathway.Furthermore,deoxyelephantopin also inhibited the PI3 K/AKT/mTOR pathway,which then released the inhibition of autophagy.In vivo study further showed that deoxyelephantopin significantly suppressed the growth of HCT116 subcutaneous xenograft in nude mice.Conclusions:Our findings revealed that deoxyelephantopin elevates oxidative stress and induces ROS-dependent autophagy followed by apoptosis in HCT116 cells via the concerted modulation of multiple signaling pathways.These findings further support the development of deoxyelephantopin as a therapeutic agent for colorectal cancer.

抗肿瘤的植物成分——C19-甲基柔毛地胆素（Molephantinin）和脱氧地胆素（Deoxyelephantopin）

作者曾从菊科地胆草属植物分离出具有细胞毒作用的柔毛地胆素（molephantin）和凡托母灵 Phanto-molin。这两个化合物都属于大环内酯（germacranolide）化合物,具有倍半萜的结构。最近,作者又分别分离出抗肿瘤的成分 C19-甲基柔毛地胆素（molepbantinin）和脱氧地胆素。C19-甲基柔毛地胆素是从菊科柔毛地胆草（Elephantopus mollis H.S.K.）分离出。这个化合物在2.5mg/kg 剂量下对大鼠 W-256有抑制作用,T/C 为397%。该化合物熔点为223～225℃,分子式为 C20H24O6,经红外,核磁共振及质谱分析证明,其结构如图所示。

去氧地胆草素对人大细胞肺癌H460细胞株的抑制作用研究

为进一步研究去氧地胆草素(Deoxyelephantopin,DET)对人大细胞肺癌H460细胞株的抑制作用,本研究通过网络药理学分析,确定去氧地胆草素影响人大细胞肺癌H460细胞株的潜在信号通路。再通过细胞增殖和凋亡实验,研究不同浓度去氧地胆草素对人大细胞肺癌H460细胞株增殖和凋亡的影响。网络药理学分析表明,去氧地胆草素可能通过影响凋亡相关蛋白诱发细胞凋亡从而抑制细胞增殖。MTT和细胞凋亡实验结果显示,去氧地胆草素能有效抑制H460细胞的增殖,能促进H460细胞的凋亡,且均呈剂量依赖效应。

氧化石墨烯与氮掺杂氧化石墨烯量子点负载去氧地胆草内酯抑制肿瘤细胞的研究

以膀胱肿瘤细胞UMUC-3为模型,研究了氧化石墨烯(GO)和氮掺杂氧化石墨烯量子点(NGQD)负载抗癌药物去氧地胆草内酯(DEO)体外抑制肿瘤细胞的效果。分别用Hummers法制备GO,球磨法制备NGQD,并通过超声负载的方法将DEO与GO、NGQD进行接枝。采用傅里叶变换红外光谱仪分析DEO与GO、NGQD的接枝机理,通过扫描电子显微镜分析其微观结构和元素分布。使用MTT比色法测试肿瘤细胞在GO-DEO、NGQD-DEO作用下的增殖情况,使用免疫荧光技术进一步分析NGQD-DEO抑制率更高的原因。结果表明,GO与NGQD均可通过共价键接枝的方式负载DEO,同时对比得到,GO-DEO、NGQD-DEO负载组均可通过破坏细胞骨架的方式提高对肿瘤细胞的抑制率,其中NGQD负载15μmol/L DEO且时间为48 h和72 h时对肿瘤细胞骨架的破坏效果最佳。

NOESY技术在地胆草倍半萜内酯化合物结构鉴定中的应用(I)

目的 明确从地胆草中分离得到的两对牛儿内酯型的立体异构体即地胆草种内酯(scabertopin)、异地胆草种内酯 (isoscabertopin)、异去氧地胆草内酯 (isodeoxyelephantopin)和去氧地胆草内酯 (deoxyelephantopin)的C 2相对构型 ;方法 NOESY技术 ,并结合这 4个化合物的1 HNMR ,1 3CNMR ,HMQC谱 ;结果 两对立体异构体的NOESY谱有明显差异 ;结论通过分析NOESY谱 ,找寻H 1相关信息 ,若发现H 1与H 8,H 9β和 1 4 CH3有NOE相关 ,可以判断化合物的C 2为α构型 ,若H 1与H 5 ,H 7,H 3α及H 9α之间有NOE相关 ,则化合物的C

地胆草中去氧地胆草内酯的定性和定量方法研究

目的建立地胆草中有效成分去氧地胆草内酯的定性和定量分析方法。方法采用薄层色谱法进行定性鉴别,薄层板为硅胶G板,展开剂为石油醚(60~90℃)-乙酸乙酯(2∶3),展开后喷以10%香草醛硫酸溶液,105℃加热至斑点清晰;采用反相高效液相色谱法测定含量,色谱柱为Kromasil C18色谱柱(250 mm×4.6 mm,5μm),流动相为乙腈-水(35∶65),流速:1.0mL·min-1,检测波长208 nm,柱温:30℃。结果供试品色谱中,在与对照药材色谱和对照品色谱相应的位置上,显相同颜色的斑点;去氧地胆草内酯在0.005 915~0.059 15 g·L-1范围内线性关系良好(r=1.000),平均回收率(n=6)为98.75%,样品含量范围为0.072%~0.15%。结论所建立的方法简单灵敏,准确可靠,重复性好,可用于地胆草的质量控制。

HPLC法同时测定神农茶颗粒中的夏佛塔苷、异夏佛塔苷、去氧地胆草内酯和4,5-二咖啡酰奎宁酸

目的：建立同时测定神农茶颗粒中夏佛塔苷、异夏佛塔苷、去氧地胆草内酯、4,5-二咖啡酰奎宁酸4个成分的高效液相色谱测定方法。方法：采用Hypersil C18色谱柱（200 mm ×4.6 mm,5μm）；以乙腈-0.025 mol·L-1磷酸溶液为流动相进行梯度洗脱,体积流量：1.3 ml·min-1,检测波长：270 nm（夏佛塔苷和异夏佛塔苷）、208 nm（去氧地胆草内酯）、327 nm（4,5-二咖啡酰奎宁酸）,柱温为室温。结果：夏佛塔苷、异夏佛塔苷、去氧地胆草内酯和4,5-二咖啡酰奎宁酸分别在5.850~117.000,4.650~93.000,4.160~83.200,5.470~109.400μg · ml-1范围内线性良好, r 值均大于0.999,平均加样回收率分别为97.70%、96.87%、97.53%、99.29%,RSD分别为1.40%、1.13%、1.69%、1.01%（n=6）。结论：该方法简便,结果准确,可用于神农茶颗粒的含量测定。