Juglone analogues have been isolated from numerous medicinal plants as active constituents, which exhibited many biological activities such as anti-viral, anti-bacterial, anti-inflammatory, and anti-cancer activities [16, 17]

Juglone analogues have been isolated from numerous medicinal plants as active constituents, which exhibited many biological activities such as anti-viral, anti-bacterial, anti-inflammatory, and anti-cancer activities [16, 17]. in holding malignancy stemness of HCC CSCs, which are essential for hepatoma initiation, relapse, metastasis FLJ13165 and drug resistance. Therefore, STAT3 has N-desMethyl EnzalutaMide been validated as a novel anti-cancer drug target and the strategies targeting HCC CSCs may bring new hopes to HCC therapy. This study aimed to isolate and identify small-molecule STAT3 signaling inhibitors targeting CSCs from the ethyl acetate (EtOAc) extract of the roots of and to evaluate their in vitro anti-cancer activities. Methods The chemical components of the EtOAc extract and the subfractions of were isolated by using various column chromatographies on silical gel, Sephadex LH-20, and preparative HPLC. Their chemical structures were then decided N-desMethyl EnzalutaMide on the basis of spectroscopic data including NMR, MS and IR analysis and their physicochemical properties. The inhibitory effects of the isolated compounds against STAT3 signaling were screened by a STAT3-dependent luciferase reporter gene assay. The tyrosine phosphorylation of STAT3 was examined by Western Blot analysis. In vitro anti-cancer effects of the STAT3 pathway inhibitor were further evaluated on cell growth of human HCC cells by a MTT assay, on self-renewal capacity of HCC CSCs by the tumorsphere formation assay, and on cell cycle and apoptosis by flow cytometry analysis, respectively. Results The EtOAc extract of the roots of was investigated and a novel juglone analogue 2-ethoxystypandrone (1) along with seven known compounds (2C8) was isolated. Among the eight isolated compounds 1C8, 2-ethoxystypandrone was a novel and potent STAT3 signaling inhibitor (IC50?=?7.75??0.18?M), and inhibited the IL-6-induced and constitutive activation of phosphorylation of STAT3 in HCC cells. Moreover, 2-ethoxystypandrone inhibited cell survival of HCC cells (IC50?=?3.69??0.51?M ~?20.36??2.90?M), blocked the tumorspheres formation (IC50?=?2.70??0.28?M), and induced apoptosis of HCC CSCs in a dose-dependent manner. Conclusion A novel juglone analogue 2-ethoxystypandrone was identified from the EtOAc extract of the roots of and was demonstrated to be a potent small-molecule STAT3 signaling inhibitor, which strongly blocked STAT3 activation, inhibited proliferation, and induced cell apoptosis of HCC cells and HCC CSCs. 2-Ethoxystypandrone as a STAT3 signaling inhibitor might be a promising lead compound for further development into an anti-CSCs drug. Electronic supplementary material The online version of this article (10.1186/s12906-019-2440-9) contains supplementary material, which is available to authorized users. Sieb. et Zucc. as STAT3 signaling inhibitors [14] and found that 2-methoxystypandrone inhibited both STAT3 and NF-B pathways dramatically by inhibiting Janus kinase 2 (JAK2) and IB kinase (IKK) [15]. Juglone analogues have been isolated from numerous N-desMethyl EnzalutaMide medicinal plants as active constituents, which exhibited many biological activities such as anti-viral, anti-bacterial, anti-inflammatory, and anti-cancer activities [16, 17]. Because of an interest in juglone analogues with STAT3 pathway inhibitory activities, the EtOAc extract of the roots of was re-examined and a novel juglone analogue 2-ethoxystypandrone (1) along with seven known compounds (2C8) were isolated. These isolated compounds were screened for their inhibitory effects on a STAT3 luciferase reporter gene in HepG2 cells. 2-Ethoxystypandrone (1) strongly blocked STAT3 activation (IC50?=?7.75??0.18?M) and inhibited the IL-6-induced as well as constitutive activation/phosphorylation of STAT3 in HCC cells. Moreover, 2-ethoxystypandrone (1) inhibited cell growth of HCC cells (IC50?=?3.69??0.51?M ~?20.36??2.90?M), blocked the tumorspheres formation (IC50?=?2.70??0.28?M), and induced apoptosis of HCC CSCs in a dose-dependent manner. Methods General details The 1H (400 and 500 MHz) and 13C NMR (100 and 125 MHz) spectra were decided on Avance 400 and Avance 500 Bruker spectrometers (Brucker, Germany). The chemical shifts were expressed in ppm as values relative to tetramethylsilane (TMS) as an internal standard. Mass spectra were recorded on DSQ ESI-mass spectrometer (Thermo, USA) and LC-MS-IT-TOF-mass spectrometer (Shimadzu, Japan). Analytical thin layer chromatography (TLC) was performed on silica gel 60 and visualized using Camag TLC visualizer by UV at 254 and 366 nm. Column chromatography was carried out on silica gel (Qindao Marine Chemical, China). Analytical HPLC was performed on a Agilent 1200 HPLC system (Agilent, USA) equipped with C18 column (250??4.5?mm i.d. stainless steel, 10 m; Waters, USA); Preparative HPLC was performed on a Elite.