Kinetex

High performance liquid chromatography (HPLC) analysis was performed on a Hewlett Packard/Agilent series 1100 (Agilent, Santa Clara, CA, USA) equipped with an analytical C18 reverse phase column (Kinetex, 75 × 4.6 mm, 2.6 μm, Phenomenex, Torrance, CA, USA). Nuclear magnetic resonance (NMR) studies were performed on a Varian 500 system (400 MHz). The sizes and polydispersity of micelles were determined on a Delsa Nano C particle analyzer (Beckman Coulter, CA, USA). Fluorescence microscopy was conducted on a FSX 100 (Olympus, PA, USA). 8453 UV–Vis spectrophotometer (Agilent, CA, USA) was used to acquire UV–Vis spectra and Cary Eclipse fluorescence spectrophotometer was used to measure fluorescence. For transmission electron microscopy (TEM) image, a 2.0 μL aliquot of the micelle solution was deposited on a copper grid coated with a carbon film. The sample was dried at room temperature and then imaged on a Tecnai G2 Spirit BioTWIN transmission electron microscope, operating at 100 kV (note that this drying procedure employed in TEM analysis may cause considerable changes in the size and shape of the micelles since they only exist in solution).

UHPLC–DAD analysis was performed according to a published protocol 29. The utilized system was an Agilent 1290 series HPLC instrument, equipped with a quaternary pump, autosampler, column oven, and a photodiode array detector (Agilent, Waldbronn, Germany). A Phenomenex Kinetex 1.7 µm XB‐C18 column (50 × 2.10 mm) guarded with an in‐line filter was used as stationary phase, and water (A) and acetonitrile (B), each fortified with 0.1% formic acid, as mobile phase solvents. The applied gradient was as follows: 95% A at 0 min, 85% A at 0.5 min, 75% A at 5 min, 65% A at 6 min, and 2% A at 6.1 min and held at this composition for 0.9 min (total runtime: 7 min); then the column was equilibrated for 5 min under the initial conditions. Flow rate, temperature, and injection volume were adjusted to 0.45 mL/min, 45°C, and 1 µL, respectively. Detection wavelengths were set to 234 and 350 nm.

ESVR was analyzed in an ultrafast liquid chromatograph (UFLC, Shimadzu) coupled to a diode array detector (DAD, Shimadzu) and electrospray ionization time-of-flight mass spectrometer (ESI-QTOF-micrOTOF QII, Bruker Daltonics; operating in the positive and negative ionization modes, 120-1200 Da). A C-18 column was used (Kinetex, 2.6 μm, 150 × 2.2 mm, Phenomenex), protected by a guard precolumn of the same material. The mobile phase was water (solvent A) and acetonitrile (solvent B), both with 0.1% formic acid, in a gradient of 0-2 min 3% B, 2-25 min 3-25% B, and 25-40 min 25-80% B, followed by the washing and reconditioning of the columns (8 min). The flow rate was 0.3 mL/min, and 1 μL of the extract (1 mg/mL) was injected. The other micrOTOF QII parameters were as follows: temperature, 200°C; N₂ gas flow rate, 9 L/min; nebulizer, 4.0 bar; capillary voltage, 3500 V (negative), +4500 V (positive); and internal calibration with sodium trifluoroacetate (TFA-Na) injected at the end of the chromatographic analysis. The catechin and piceatannol authentic standards were purchased from Sigma-Aldrich with ≥95% purity. The metabolites present in ESVR were identified based on the interpretation of mass and UV absorption spectra and based on comparison with the literature. When available, the compounds were confirmed by comparison with authentic standards.

Detection and quantification of amino acids and dipeptides was done by LC–MS/MS, using a Vanquish HPLC system coupled to a TSQ Altis mass spectrometer (both Thermo Fisher Scientific), employing the Selected Reaction Monitoring (SRM) mode and positive polarity. In brief, dried samples were resolved in 0.1% formic acid in water and 1 µL was injected onto a Kinetex (Phenomenex) C18 column (100 Å, 150 × 2.1 mm), employing a 9-min-long linear gradient from 100% A (1% acetonitrile, 0.1% formic acid in water) to 90% B (0.1% formic acid in acetonitrile) at a flow rate of 100 µL/min. Retention times, SRM transitions, and optimal collisional energies were determined by authentic standards and degenerate dipeptide libraries. All data interpretation was performed using Xcalibur (Thermo Fisher Scientific).