Methanol

Cell-free supernatants obtained from cell culture experiments were mixed in a ratio of 1:2 (v/v) with ice-cold methanol (Fisher Chemical, Schwerte, Germany) containing 10 µL of deuterium-labelled standards as internal reference. Deuterated standards include 200 nM d₈-5S-hydroxyeicosatetraenoic acid (HETE), d₄-LTB₄, d₅-lipoxin A₄, d₅-resolvin D2, d₄-PGE₂ and 10 µM d₈-arachidonic acid. Before proceeding with purification steps, proteins were precipitated at -20 °C overnight. After centrifugation (1200 × g, 10 min, 4 °C), the supernatant was collected and acidified by addition of 7 mL Milli-Q water (pH 3.5) for solid phase extraction. C18 solid phase cartridges (Waters, Eschborn, Germany) were washed with 6 mL methanol and equilibrated with 2 mL Milli-Q water before sample addition. Subsequently, columns were washed with 6 mL of Milli-Q water to return to neutral pH ~ 7.0. Following another washing step with 6 mL of n-hexane (Fisher Chemical), eicosanoids, docosanoids and PUFAs were eluted with 6 mL of methyl formate (Acros Organics, Schwerte, Germany). Samples were evaporated until dryness with a moderate stream of nitrogen (TurboVap LV, Biotage; Uppsala, Sweden) and resuspended in 100 µL of a mixture of methanol and Milli-Q water (50:50, v/v).

A total of 100 berries of Grignolino, previously weighted and stripped of seeds, were homogenized (Di Stefano et al., 1998 ). The suspension was then centrifuged (4000g for 15 min) and the supernatant was transferred to a 300-ml volumetric flask, washed, and brought up to volume with tartaric acid buffer (pH = 3.0, 0.04 M). Three replicates of all samples were analyzed. The isolation of grape heterosides was performed as previously reported for a grape and wine matrix (Mateo et al., 1997 (link); Asproudi et al., 2016 (link); Asproudi et al., 2018 (link)) after appropriate modifications for better extraction of target compounds from Grignolino grape extracts. Briefly, 250 ml of extract was passed through a 5-g C18 End Capped cartridge (Biotage AB, Uppsala, Sweden) previously activated with methanol and distilled water in sequence. After washing (water and dichloromethane), the glycosides were recovered with 25 ml of methanol (Sigma Aldrich Co., St. Louis, MO, USA). C18 sorbents were indicated to be more suitable for selective extraction of less polar precursors such as terpenic and norisoprenoid precursors. Furthermore, the same standardized procedure was applied for all samples to obtain reliable comparison results between vineyards and vintages (Jesús Ibarz et al., 2006 (link); Liu et al., 2017 (link)).