Isolera one

For the isolation of cardenolides, 15 g of freeze-dried, milled G. physocarpus flowers and 25 g of freeze-dried, milled G. fruticosus flowers were extracted in 450 or 750 ml methanol, respectively, for 24 h under occasional stirring and then filtered through Whatman Grade 1 filter paper. Extracts were dried on a rotary evaporator until complete removal of the solvent.
A cardenolide and a flavonoid fraction were prepared from the extracts of both Gomphocarpus species using flash chromatography on a Biotage Isolera One (Biotage, Sweden) system with a SNAP Ultra C18 cartridge. Extracts were re-dissolved in 5 ml of 80% methanol, and loaded on top of the column. The solvent gradient run comprised two column volumes of 10% methanol in water followed by a linear gradient from 10% methanol in water to 100% methanol over nine column volumes and a final two column volumes of 100% methanol at a constant flow rate of 50 ml min⁻¹. UV absorbance spectra of the eluate were monitored at 220 nm (peak absorbance of Gomphocarpus cardenolides) and 350 nm (flavonoid peak absorbance), and the eluate was fractionated by distinct UV absorbance peaks. Fractions were analysed via HPLC-MS (see conditions described for analysis on a Velos-Pro mass spectrometer, in section ‘Compound identification and quantitative comparison’ above), and fractions containing cardenolides or flavonoids combined separately.

The crude extract was pre-fractionated on a reversed phase C₁₈ flash column (10 g, 15 ml) using an Isolera One automated flash system (BIOTAGE, Uppsala, Sweden). The gradient was 10% stepwise (15 column volumes) from 30–100% methanol (MeOH) buffered with 20 mM formic acid with a flow of 15 ml/min. Nine fractions were collected manually every 10% step. MeOH was of HPLC grade and water was purified and deionized using a Millipore system through a 0.22 μM membrane filter (Milli-Q water).
4-Hydroxybenzoic acid (fraction 1E) was purified from the Isolera fraction (fraction 1) on a Waters semi-preparative HPLC, with a Waters 600 Controller (Milford, MA, USA) coupled to a Waters 996 Photodiode Array Detector. Separation was achieved on a Luna II C18, 5 μm, 250 × 10 mm column (Phenomenex, Torrance, CA, USA) with a flow of 5 ml min⁻¹ using a linear gradient 5% MeCN in Milli-Q water with 50 ppm TFA going to 35% MeCN in 24 min, from 35–45% MeCN in 2 min, 45–100% MeCN in 2 min, kept for 5 min at 100% MeCN and down to the starting conditions in 2 min. MeCN was of HPLC grade.

One- and two-dimensional nuclear magnetic resonance (NMR) spectra were obtained using an Avance 400 instrument (Bruker Co., Ltd., Bremen, Germany), with a solvent signal of CDCl₃ as the internal reference. Gas chromatography-mass spectrometry (GC-MS) analyses were performed using an Agilent 7890A-5975C MSD instrument (Agilent Technologies, Santa Clara, CA, USA). Desorption electron ionization (DEI-MS) was measured using JEOL JMS-K9 (JEOL Co. Ltd., Tokyo, Japan). Headspace-gas chromatography (HS-GC) analyses were performed using an Agilent 6890 GC instrument (Agilent Technologies), which was equipped with a pulsed flame photometric detector (PFPD, model 5380, Agilent Technologies). Liquid chromatography-mass spectrometry (LC-MS) analyses were performed using an ultra-performance liquid chromatography-mass spectrometry system (LCMS-8050, Shimadzu, Kyoto, Japan), which was equipped with Lab Solutions Version 5.75. Flash column chromatography was performed on an Isolera One instrument (Biotage, Uppsala, Sweden).

The melting point was taken using an Electrothermal 9300 (Electrothermal Engineering LTD). UV spectra were obtained from a Hewlett-Packard HP8453 diode array spectrometer. NMR experiments were carried out with a Bruker AVANCE III 700 spectrometer (Ettlingen, Germany). Chemical shifts (δ) are reported in parts per million (ppm), referencing the solvent used. ESIMS data were obtained on a Waters Acquity Ultra Performance LC-MS system LCA 048 (Milford, MA, USA). LRFABMS and HRFABMS spectra were obtained on a JMS-700 M Station Mass Spectrometer (JEOL Ltd., Tokyo, Japan). Column chromatography and MPLC were performed on Sephadex LH-20 (25–100 μm, Sigma-Aldrich, St. Louis, MO, USA) and Biotage Isolera One equipped with Biotage^® SNAP ULTRA C18 Cartridges (Uppsala, Sweden), respectively. Thin-layer chromatography was conducted on TLC Silica gel 60 F₂₅₄ and TLC Silica gel 60 RP-18 F_254S (Merck, Darmstadt, Germany). Fluorescence microscopy was carried out with an Olympus 1X70 microscope (Japan). Image J (NIH, Bethesda, MD, USA) and Focus Lite (Focus Co., Seoul, Republic of Korea) were used for image analysis.

NMR spectra were recorded on a JEOL JNM‐LA400 instrument at 400 MHz for ¹H NMR and at 100 MHz for ¹³C NMR. Mass spectra (MS) were measured with a JEOL JMS‐T100LC AccuToF (ESI). Preparative HPLC was performed on an Inertsil ODS‐3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU‐2080, JASCO) and a detector (MD‐2015 or FP‐2025, JASCO). LC‐MS analyses were performed on a Waters Acquity UPLC (H class)/QDa quadrupole MS analyzer or Acquity UPLC (H class)/Xevo TQD quadrupole MS/MS analyzer equipped with an Acquity UPLC BEH C18 column (Waters). Column chromatography using silica gel was performed on a MPLC system (Yamazen Smart Flash EPCLC AI‐5805 (Tokyo, Japan)). Reversed‐phase MPLC purification was performed on an Isolera One (Biotage) equipped with a SNAP Ultra C18 30 g (Biotage).

For the purification of streptothricin aqueous extracts produced by squeezing freeze/thawed agar/cell slurry, generated by growth of E222 on Medium I plates [82 ], through muslin had washed Amberlite™ XAD16N resin beads (Sigma) added (20 g l⁻¹) to adsorb bioactive compounds. The compounds were released by washing the resin with a volume of methanol equal to the volume of the original aqueous extract and the methanol was removed by rotary evaporation (Büchi) to obtain a water-based concentrate. The concentrate, after filtering through Whatman filter paper and the addition of methanol to 10%, was passed through a pre-packed “reverse phase” C18 25 g SNAP “flash” chromatography column (Biotage) and the bound compounds eluted in a 90–0% water—methanol gradient using a Biotage “Isolera One” chromatography machine. Fractions containing bioactive compounds were identified by spotting 30 µL aliquots onto filter paper discs, see above (filter paper assay). Active fractions producing a halo of cells with the expected characteristic phenotypes were pooled and the methanol removed by a GeneVac Series II system equipped with a GeneVac VC3000TA condenser unit. Material from active fractions was analysed/further purified by HPLC.