Amberlite xad 16n

Xylaria sp. and D. endophytica were subjected to an alternative extracellular extraction procedure to obtain a more purified extract of each axenic culture grown on PDB. Hence, the mycelium was separated by filtration from the fermentation broth, after which the exhausted medium was extracted with polymeric resin Amberlite® XAD16N (Sigma-Aldrich, Darmstadt, Germany), previously washed with distilled water (1% w/v), methanol (1% w/v), and distilled water (0.5% w/v) again for 30min with each solution, respectively. This method was adapted from Caicedo et al. (2011) (link) and Narmani et al. (2018) (link). A ratio of 0.06g resin/ml of the exhausted media was applied and continuously mixed in an Erlenmeyer flask (100–500ml, depending on the supernatant volumes) using agitation plates (120rpm) at room temperature for 24h. For the extraction, 25ml of methanol (MeOH) and EtOAc per gram resin were added and continuously mixed for 4h. Then, the resin was filtered, and the organic solution was reduced by vacuum-evaporation (40°C, 40mbar) to 5ml, and then vacuum-dried to obtain the extracts finally in Caicedo et al. (2011) (link). These last followed the same antibacterial activity assessment procedure, which was performed for the primary screening.

Once the optimal extraction conditions were defined, the polyphenols were concentrated using an open chromatographic column (25 mm × 1000 mm) packed with Amberlite XAD-16N (Sigma-Aldrich, St. Louis, MO, USA). The separation and purification of the bioactive compounds were carried out using the methodology of De la Rosa Hernández [58 ]. Firstly, an elution with water was carried out to remove water-soluble compounds, and then an elution with 70% ethanol was performed to recover a fraction rich in bioactive compounds. The aqueous fraction was discarded and the ethanolic fraction was recovered and concentrated in a rotavapor. The purified ethanolic fraction obtained was called the purified polyphenol fraction, and it was stored at 4 °C until use. It is worth mentioning that only the extract obtained under the expected extraction conditions (previous point) was concentrated and purified, and only a single fraction of purified polyphenols was obtained; this was characterized and its biological activity was measured.