Drx 400

Flash column chromatographic separations were accomplished on Biotage Isolera Four system (Biotage, Uppsala, Sweden). NMR was recorded on DRX-400 and DRX-500 NMR spectrometers (Bruker, Rheinstetten, Germany). ESI-, APCI- and HR ESI-MS were recorded on Agilent 1290 LC-MS or Agilent G6530 TOF MS spectrometer (Agilent Technologies Inc., California, USA). Western blot results were visualized on Mini-HD9-Auto Biomolecular imager (Leader Oriental Technology. LTD, Beijing, China). Dichlone and flavonoids were purchased from Fisher Scientific (Fair Lawn, NJ, USA) and Ark Pharm, Inc. (Arlington Heights, IL, USA). Cell lines were gotten from the American Type Culture Collection (Manassas, VA). Caspase-3 (3G2) mouse mAb, caspase-3 antibody, cleaved caspase-3 (Asp175) (5A1E) rabbit mAb, PARP (46D11) rabbit mAb and horseradish peroxidase (HRP) were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA).

Where necessary, reactions were carried out under an inert argon atmosphere using standard Schlenk techniques. The solvents were dried by standard methods and freshly distilled before use. Et₄NF⋅2 H₂O, Ph₄PCl, AgCl, and KF were commercially available.
The NMR spectra were, unless otherwise stated, recorded at ambient temperature on Bruker DPX 300, DRX 400, DRX 500, AVANCE III HD‐400, AVANCE III HD‐600 and AVANCE III HD‐700 spectrometers. The chemical shifts δ are given in ppm and referenced to tetramethylstannane (¹¹⁹Sn), and trichlorofluoromethane (¹⁹F) and tetramethylsilane (¹H, ¹³C). Electrospray mass spectroscopy (ESI–MS) was recorded on a Thermoquest–Finnigan instrument using CH₃CN as the mobile phase. The samples were introduced as solution in CH₃CN through a syringe pump operating at 0.5 μL min⁻¹. The capillary voltage was 4.5 kV, whereas the cone skimmer voltage was varied between 50 and 250 kV. Identification of the expected ions was assisted by comparison of experimental and calculated isotope distribution patterns. The m/z values reported correspond to those of the most intense peak in the corresponding isotope pattern. Elemental analyses were performed on a LECO‐CHNS‐932 analyzer. Melting points were determined using a Büchi Melting Point M‐560. IR spectra were recorded on a PerkinElmer FTIR spectrometer.

The aerial parts of E. adenophorum were dried and crushed in a knife mill. Subsequently, 8 kg of E. adenophorum plant material was soaked in 95% ethanol (600 ml of 95% ethanol per 100 g plant material) for 30 min at room temperature. The material was then extracted twice with boiling ethanol under reflux, this step was 1 h per cycle. Then, the extracts were combined and concentrated by evaporation using a rotary evaporation apparatus. Finally, the ethanol extract was obtained. Then, we extracted ethanol extract with petroleum ether and obtained the petroleum ether extract of E. adenophorum (Nong et al. 2014a (link)).The petroleum ether extract was then chromatographed on a silica gel column using a gradient of ratios of petroleum ether to acetone (50:1 → 5:1) as the eluents to obtain the compound. We used ¹H NMR and ¹³C NMR (Wang et al. 2007 ) to identify the structures of the compound obtained from the E. adenophorum petroleum ether extract. The compound was subsequently identified as 9-oxo-10,11-dehydro-ageraphorone by nuclear magnetic resonance (NMR). NMR spectra were measured with a Bruker DRX-400 instrument with tetramethylsilane as the internal standard (Nong et al. 2014b (link)).