Portion 4 (1.9 g) was subjected to silica gel CC and eluted with gradient petroleum ether-acetone (100:1C5:1) to generate five subfractions (Fr. cells in vitro and in vivo via activation of the mitochondria-mediated apoptotic signaling pathway.20 During the course of our search for bioactive natural products from Lindl., five phenanthrene derivatives were isolated and structurally characterized as nudol(1),21 confusarin (2),22 3,7-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (3),23 3,7-dihydroxy-2,4-dimethoxyphenanthrene (4),24 and 3,7-dihydroxy-2,4,8-trimethoxyphenanthrene (5)25 (Number 1) (Numbers S4-S13). Among them, nudol(1) was initially isolated from your orchids Linda, Gibson, and Lindl,21 and significantly inhibited the growth of HeLa cells (IC50=20.18 M).26 A preliminary screening of the anti-proliferative activity of 1C5 against osteosarcoma cells MG63 and U2OS revealed that nudol(1) exerted remarkable growth inhibitory effect (Number 2). Therefore, the aim of the present work was to perform an antitumor evaluation of nudol(1) toward the osteosarcoma cells and to investigate the essential factors for the cell growth inhibition including cell cycle, apoptosis, and migration. Open in a separate window Number 1 Chemical constructions of compounds 1C5. Open in a separate window Number 2 Effects of tested compounds on cell viability. Notes: (A, B) U2OS and MG63 cells were treated with 20 M compounds for 24 hrs, and cell viability VD2-D3 was VD2-D3 determined by MTT assay; DOX (20 nM) was used like a positive control. (C, D) Osteosarcoma cell viability by treatment with numerous concentrations of nudol(1) for 24, 48, and 72 hrs. (E) MDA-MB-231, MCF-7, and A549 cell viability following treatment with the various concentrations of nudol(1) for 48 hrs. Data were indicated as mean SD. * were purchased in April 2015 from Huoshan, Anhui Province, and were authenticated by Dr. Jinchuan Zhou from School of Pharmacy, Linyi University or college. A voucher specimen (No. DN20150420) has been preserved at VD2-D3 School of Biological Technology SKP1A and Technology, University or college of Jinan. Extraction and isolation The air-dried and powdered stems (2.5 kg) of were extracted with 95% EtOH (310 L, each for 5 days) at space temperature. After concentration under reduced pressure, the crude residue (180.3 g) was suspended in H2O (1.0 L) and partitioned with EtOAc (0.5 L3). The EtOAc extract (95.2 g) was subjected to silica gel CC and eluted with gradient petroleum ether-acetone (200:1C1:1) to afford six fractions (Fr. 1C6). Fr. 3 (1.2 g) was first chromatographed on a Sephadex LH-20 column (MeOH-CHCl3, 1:1) and then separated by an RP-18 silica gel column (MeOH-H2O, 5:5 to 9:1) to yield four subfractions (Fr. 3.1?3.4). Fr. 3.2 (30.2 mg) was purified by HPLC (MeOH-H2O, 60:40, 2.0 mL/min) to yield 1 (2.4 VD2-D3 mg, tR =20.4 mins, purity 96.2%) and 2 (2.6 mg, tR =23.7 mins). Portion 4 (1.9 g) was subjected to silica gel CC and eluted with gradient petroleum ether-acetone (100:1C5:1) to generate five subfractions (Fr. 4.1C4.5). Fr. 4.3 (260 mg) was further separated on a Sephadex LH-20 column (MeOH-CHCl3, 1:1), followed by purification on HPLC (MeOH-H2O, 70:30, 2.0 mL/min) to afford 3 (8.1 mg, tR =33.2 mins), 4 (6.9 mg, tR =34.6 mins), and 5 (13.4 mg, tR =45.9 mins). Cell tradition Cell lines MCF-7, MDA-MB-231, A549, U2OS, and MG63 were acquired from your Cell Standard bank of Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in high-glucose Dulbeccos Modified Eagles Medium (Thermo, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 IU/mL penicillin, and 100 g/mL streptomycin (both from Thermo Fisher Scientific Inc) inside a humidified atmosphere comprising 5% CO2 at 37C. The phenanthrene derivatives (1C5) were in the beginning dissolved DMSO (dimethylsulfoxide) to make a 20 mM stock remedy and diluted with tradition media to the final test concentrations, which contained no more than 0.05% DMSO. Doxorubicin hydrochloride (DOX, Sigma Chemical Co, purity 98%) was dissolved in distilled water and used like a positive control. Cell viability assay The cells were cultured in 96-well plates, and each well was seeded with 1104 cells. The viability of cells was measured from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 10 L of MTT (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 hrs at 37C. In this step, 100 L of DMSO was added to dissolve the formazan crystals. The optical denseness.