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Plant metabolites extraction<br />
A … Plant metabolites extraction<br />
After cell saturation was achieved F. hygrometrica cultures were transferred to plastic containers, flash-frozen with liquid nitrogen and subsequently freeze-dried. Dried plant material was ground with a Mixer Mill MM 301 (Retsch®) at 30 Hz for 15 s. Plant powder (20 mg of every sample) was extracted with 1.5 ml of HPLC grade methanol for 25 min in ultrasound bath at room temperature. Extracts were centrifuged at 15,870g for five min at room temperature and the supernatants were filtered using a 0.2 µm nylon syringe filter. Filtrates were subjected to UPLC–MSn analysis.<br />
<br />Chromatography/mass spectrometry conditions<br />
Funaria hygrometrica extracts were analyzed using an ultra performance liquid chromatography electrospray mass spectrometry (UPLC–ESI–MSn) system Accela LCQ Fleet Ion trap (Thermo Finnigan). The compound mixture was separated on a Hypersil Gold C18 column (50 × 2.1 mm, 1.9 µm particle size). 10 µl were injected per sample. The mobile phase consisted of H2O with 0.1% (v/v) formic acid (solvent A) and methanol with 0.1% (v/v) formic acid (solvent B). The solvent gradient program was as follows: 30% B, 0–0.5 min; 30–80% B, 0.5–6 min; 80–100% B, 6–24.9 min; 100–30% B, 24.9–25 min; 30% B, 25–30 min. The flow rate was 400 µl min−1. Column oven temperature was maintained at 35 °C. Spectra were acquired in full scan mode 50–1000 m/z range, operating in positive mode; the scan time was 500 ms (3 micro-scans). The ESI source parameters were set as follows: capillary temperature 320 °C; capillary voltage 20 V; spray voltage 4.5 kV; tube lens 40 V; nitrogen sheath gas 25 arbitrary units (AU); auxiliary gas 15 AU. For the MSn analysis, collision energy (CID) was normalized to 35. llision energy (CID) was normalized to 35.
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