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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 />Non-targeted metabolite profiling<br />
The samples extracted with methanol were reconstituted in a mixture of methanol/deionized water/formic acid [75:24.85:0.15 (v/v/v)] and filtered through a 0.2-μm filter. Chromatographic separation was achieved on an Acquity UPLC System (Waters, Milford, USA) using a CSH C18 2.1 × 150 mm, 1.7 µm column maintained at 40 °C. The samples were injected (10 μl) and elution of compounds was performed at a flow rate of 0.3 ml min−1, the gradient comprised mobile phase A: deionized water containing 0.1% formic acid; and mobile phase B: acetonitrile containing 0.15% formic acid. The gradient program was isocratic for the first 30 s, then a linear gradient increase from 30 to 80% of solvent B at 6 min, 100% of B at 24.9 min, and hold for 1 min for column washing and 4 min at 30% of solvent B for column re-equilibration. The mass spectrometer comprised an orthogonal QTOF Synapt G1 (Waters, UK) operated under the following conditions: positive electrospray ionization mode, capillary voltage at 3.0 kV, cone voltage 45 V, extractor voltage 4.0 V, source and desolvation temperature were 150 and 350 °C, respectively. Cone and desolvation gas flow was nitrogen at a flow rate of 20 l h−1 and 700 l min−1. Leucine-enkephalin (M + H)⊕ = 556.2771 was infused at a flow rate of 5 μl min−1 at concentration of 2 ng ml−1 during acquisition as internal mass calibrant. MS data were acquired on continuum mode and processed with MassLynx (version 4.1, Waters, USA) and Progenesis QI for small molecules (Nonlinear Dynamics version 2.3, Waters, USA) software, using Progenesis MetaScope Database, with Biomolecules, LipidsMAPS, and LipidBlast as searching parameters, accepting precursor tolerance of 10 ppm for putative identifications. ce of 10 ppm for putative identifications.
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