Lu Min-Ren, Huang Huey-Lan, Chiou Wen-Fei, Huang Ray-Ling
Department of Life Science, National Taitung University, Taitung, Taiwan, ROC.
Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan, ROC.
Pharmacogn Mag. 2017 Oct-Dec;13(52):702-706. doi: 10.4103/0973-1296.218113. Epub 2017 Nov 13.
Traditional Chinese herb , belonging to the Compositae family, has long been applied for the treatment of liver diseases. In recent years, many reports also indicated that it possesses hepato-protective, anti-inflammatory, and anti-cancer activities.
In this study, we evaluated whether is an effective therapy for hepatocellular carcinoma (HCC).
Dry leaves of were first extracted in ethyl acetate, then further fractionated by different ratio of -hexane-ethyl acetate (8:2→0:1) or methanol as fractions 1-6 (Td-F1 to Td-F6), respectively. We first showed that the ethyl acetate extracts of leaves (Td-L-EA) exhibits growth inhibition on human hepatoma HepG2 cells. To further check the extracts-induced apoptosis, microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, flow cytometry, and Western blot analysis were performed.
After isolating the effective fractions from Td-L-EA, we found strong cytotoxic effects of fraction-2 (Td-F2). By further analyzing the mechanisms of cytotoxic activities using microscopic observation, fragmented chromosomal DNA electrophoresis, apoptotic DNA-detection ELISA assay, and flow cytometry, we found that induction of apoptosis such as DNA fragmentation increased the apoptosis rate and the apoptosis sub-G1 populations in Td-F2-treated HepG2 cells. In addition, we also confirmed Td-F2-induced degradation of caspase-8, caspase-9, caspase-3, and caspase-3 substrate PARP. Besides, Td-F2 also increased the Bcl-2 proapoptotic family protein Bax expression.
In short, our results clearly showed the induction of apoptosis by ethyl acetate extracts of leaves in human hepatoma HepG2 cells, suggesting its potential application as an antitumor agent.
leaves were first extracted in ethyl acetate, then further fractionated by different ratio of -hexane/ethyl acetate (8:2→0:1) or methanol.These extracts exhibit growth inhibition on human hepatoma (HCC) HepG2 cells.-Hexane/ethyl acetate (6:4) extract (Td-F2) induces apoptosis of HCC. , ; HCC, Hepatocellular carcinoma DMEM, Dulbecco's modified Eagle's medium; DMSO, dimethyl sulfoxide; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; OD, optical density; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel electrophoresis; PARP, Poly (ADP-ribose) polymerase; PBS, phosphate buffered saline; PI, propidium iodide.
传统中药，属于菊科，长期以来一直用于治疗肝脏疾病。近年来，许多报告还表明它具有肝脏保护、抗炎和抗癌活性。
在本研究中，我们评估了[中药名称未给出]是否是治疗肝细胞癌（HCC）的有效疗法。
[中药名称未给出]的干燥叶片首先用乙酸乙酯提取，然后分别用不同比例的正己烷 - 乙酸乙酯（8:2→0:1）或甲醇进一步分级分离，得到馏分1 - 6（Td - F1至Td - F6）。我们首先表明[中药名称未给出]叶片的乙酸乙酯提取物（Td - L - EA）对人肝癌HepG2细胞具有生长抑制作用。为了进一步检查提取物诱导的细胞凋亡，进行了显微镜观察、染色体DNA片段电泳、凋亡DNA检测ELISA测定、流式细胞术和蛋白质印迹分析。
从Td - L - EA中分离出有效馏分后，我们发现馏分2（Td - F2）具有很强的细胞毒性作用。通过使用显微镜观察、染色体DNA片段电泳、凋亡DNA检测ELISA测定和流式细胞术进一步分析细胞毒性活性的机制，我们发现Td - F2处理的HepG2细胞中诱导的凋亡如DNA片段化增加了凋亡率和凋亡亚G1期细胞群。此外，我们还证实了Td - F2诱导的半胱天冬酶 - 8、半胱天冬酶 - 9、半胱天冬酶 - 3和半胱天冬酶 - 3底物PARP的降解。此外，Td - F2还增加了Bcl - 2促凋亡家族蛋白Bax的表达。
简而言之，我们的结果清楚地表明[中药名称未给出]叶片的乙酸乙酯提取物在人肝癌HepG2细胞中诱导细胞凋亡，表明其作为抗肿瘤剂的潜在应用。
[中药名称未给出]叶片首先用乙酸乙酯提取，然后分别用不同比例的正己烷/乙酸乙酯（8:2→0:1）或甲醇进一步分级分离。这些提取物对人肝癌（HCC）HepG2细胞具有生长抑制作用。正己烷/乙酸乙酯（6:4）提取物（Td - F2）诱导肝癌细胞凋亡。[中药名称未给出]；HCC，肝细胞癌；DMEM，杜尔贝科改良伊格尔培养基；DMSO，二甲基亚砜；HRP，辣根过氧化物酶；MTT，3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐；OD，光密度；SDS - PAGE，十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳；PARP，聚（ADP - 核糖）聚合酶；PBS，磷酸盐缓冲盐水；PI，碘化丙啶。
