Argon filled centrifuge tube

Total phenolics were extracted according to Kajdzanoska et al. (2011) , with slight modifications. Freeze-dried sample (0.5 g) was mixed with 10 mL of methanol solution containing acetic acid (H₂O:MeOH:HAc, 19:80:1, v/v/v) in a 15-mL argon-filled centrifuge tube (SARSTEDT, Nümbrecht, Germany) and stirred for 2 h at 8 °C in dim light on a MS2-Minishaker (IKA®, Königswinter, Germany). This mixture was centrifuged for 4 min at 13,000 rpm and 5 °C on a Sorvall RC-5B Plus centrifuge (Buch and Holm, Herlev, Denmark) and the resulting supernatant was filtered through a 0.45-µm RR Q-Max® nylon filter (Frisenette, Knebel, Denmark) into a brown 2-mL vial (VWR® INTERNATIONAL, Radnor, Pennsylvania, USA) prior to analysis. Quantification was performed using a microplate Folin-Ciocalteu method (Magalhaes, Santos, Segundo, Reis, & Lima, 2010 (link)). In brief, 50 µL of gallic acid or filtered sample was mixed with 50 µL Folin-Ciocalteu reagent (diluted 1:5 with H₂O, v/v) prior to by adding 100 µL 0.7 M sodium hydroxide. After 3 min, the absorbance was measured at 760 nm using a Synergy 2 multi-code microplate reader from BioTek (Vermont, USA). Water was used as a blank control. The total phenolic content was quantified as mg gallic acid equivalents per 100 g fresh weight (mg GAE/100 g FW).

Total monomeric anthocyanin was extracted according to Jensen et al. (2007) . Freeze-dried sample (0.5 g) was mixed with 20 mL ethanolic solution containing hydrochloric acid (H₂O:EtOH:HCl, 40.9:50:0.1, v/v/v) in a 50-mL argon-filled centrifuge tube (SARSTEDT) and stirred for 15 min at 8 °C in dim light. The mixture was centrifuged for 15 min at 13,000 rpm and 4 °C. The supernatant was filtered through a 0.22-µm Q-Max® acetate filter (Frisenette) directly into a 2-mL brown vial prior to analysis. Anthocyanins were quantified as the difference between sample absorption at 510 nm in pH 1.0 buffer (0.025 M KCl) (aq) and sample absorption at 700 nm in pH 4.5 buffer (0.4 M sodium acetate) (aq), as described by Lee et al. (2005) (link). In brief, two 75-µL aliquots of the filtered sample were mixed with either 125 µL pH 1 buffer or 125 µL pH 4.5 buffer. After 15 min, the absorbance was measured using a microplate reader. The total anthocyanin content was quantified as cyanidin-3-glucoside equivalents per 100 g FW (mg/100 g FW).