Betanin Extraction and Purification Protocol

The HPLC apparatus consisted of an LC-20A Prominence (Shimadzu^®, Kyoto, JPN) equipped with a quaternary pump and a DAD model SPD-M20A (Shimadzu^®). A 15 µm Phenomenex C18 column 250 × 21.2 mm I.D. (Torrance, CA, USA) connected to a fraction collector FRC-10A (Shimadzu^®, Kyoto, Japan). Elution was performed with a mixture of solvents according to Silva al. [6 (link)] with small modifications: solvent A was 1% formic acid, and solvent B, 80% methanol at a linear gradient (0–25 min, 11–55% of solvent B). The sample injection volume was 400 µL at 3.5 mL/min flow rate, and separations were monitored at 536 nm. Red-violet-colored fractions containing betanin were concentrated by rotary evaporation, and pure and concentrated betanin was suspended in deionized water, freeze-dried, and stored at −80 °C for further analysis (Figure S1).