B 490

All reagents were purchased from Sigma-Aldrich and used without further purification. All solvents were anhydrous or analytical grade (Sigma-Aldrich) and used without further purification. The reaction progress was monitored by GC–MS (using the procedure given in Section 4.4). Solvents were removed under reduced pressure using a Büchi Rotavapor R-200 and Büchi B-490 heating bath set to 40 °C. Mixtures were further dried under high vacuum using an Alcatel Pascal 2005SD vacuum pump. NMR spectra were recorded on a Bruker AVANCE-400 instrument (¹H NMR: 400 MHz, ¹³C NMR: 101 MHz) or a Bruker AVANCE-600 instrument equipped with a cryoprobe (¹H NMR: 600 MHz, ¹³C NMR: 150 MHz) using CDCl₃ and C₆D₆. The ¹H NMR chemical shifts were referenced to the residual protonated solvent peaks at δH 7.26 for chloroform-d and 7.15 for benzene-d₆. ¹³C NMR chemical shifts were referenced to the central solvent peaks of bulk solvent at δC 77.16 for chloroform-d and 127.68 for benzene-d₆. J values are given in Hz.

In order to extract and purify PAHs, the same method proposed earlier by Perugini et al., in 2007 was followed [32 (link)]. This determination was carried out in composite pools of tissues dissected from 30 fish (five samples, each constituted by tissues of six specimens). After being thawed, the assay sample (2 g dw) was inserted into a l00mL round-bottomed flask with a 10 mL of 1 M KOH in an ethanolic solution. The mixture was placed in a reflex system in a temperature of 80°C for 3 hours. The liquid phases were transferred to a separation funnel and were extracted with a 10 mL of cyclohexane. They were then shaked rigorously for 30 minutes. The cyclohexane KOH phase was drained and discarded. Next, the liquid phases were rinsed with 10 mL cyclohexane once more. The samples were allowed to pass through the anhydrous sodium sulphate column. After that, the organic phase was concentrated in a rotary evaporator (Model Buchi B-490) to a volume of 5 mL under a reduced pressure. The samples passed through a column filled with florisil and concentrated in rotary evaporator (30°C, 19–21 kPa) to volume of 1 mL. Finally, the extracts were evaporated with a gentle stream of nitrogen at room temperature and were then reconstituted in 1 mL of acetonitrile.

The plant leaves after the collection were dried under shade and crushed to a fine powder. About 1.2 kg powder was macerated in 60% methanol (Sigma Aldrich, USA) for 15 days. After 15 days the plant extract was further filtered via Whatman filter paper. After filtration, the extract was concentrated using a rotary evaporator (B-490, Buchi, Switzerland) at 45 °C and obtained 122 g greenish A. parviflora hydromethanolic (APHM) leaves extract [18 (link), 19 (link)]. The extract was stored at 4 °C for further studies.

Removal of ethanol from the MOS preparations was critical to avoid any interference of residual ethanol with the sensory properties of the final MOS products. Ethanol removal was achieved by solvent removal followed by water washing three times using a rotary evaporator (Büchi Rotovapor R-205, Büchi Labortechnik AG) equipped with a 55° water bath (Buchi B-490) and vacuum pump (Chemglass Scientific Apparatus/10 Torr). Concentrated MOS preparations were stored at -23°C until drying by lyophilization in a VirTis CONSOL 4.5 freeze dryer.

In order to obtain an extract including total polyphenols, the extraction was performed using the acid MeOH, according to the method published by Genskowsky et al. [22 (link)], with slight modifications. The initial sample consisted of 50 g lyophilized powdered wild maqui fruit (seed and pulp) from a packaged and commercialized product (Isla Natura de Chile^®, Chiloé, Chile). In total, 250 mL of MeOH/H⁺ (0.1% HCl) at pH 1 were added to the sample, and then homogenized with an ultrasound device (Hielscher Ultrasound Technology UP400S, Teltow, Germany). The extract was centrifuged at 4000 rpm for 10 min at 4 °C, and the supernatant was collected. This procedure was repeated 5 times. The supernatants were mixed in a round-bottomed flask and evaporated until dryness using a rotary evaporator (Büchi B-490, Hampton, VA, USA). The dried extract was dissolved in distilled water and centrifuged at 4000 rpm for 4 min. Finally, it was passed through 2 filtering processes (100–150 MM and 40–100 MM) and then lyophilized in a Telstar Cryodos Freeze Dryer (Tokyo, Japan). The final extract was stored at −20 °C. All experiments were carried out under darkness and controlled temperature.

All chemicals were of analytical reagent grade and used as received. Methanol was purchased from Panreac (Barcelona, Spain) and acetic acid from Sigma-Aldrich (Steinheim, Germany). Double-deionized water with conductivity lower than 18.2 MΩ was obtained with a Milli-Q system (Millipore, Bedford, MA, USA). The evaporation to dryness of the extracts was performed using a rotary vacuum evaporator model R-200 coupled to a heating bath model B-490, both from Büchi Labortechnik (Flawil, Switzerland.)