Dionex ultimate 3000 instrument

Spent hops extracts type I: Four solvents with different polarities were used, i.e. methylene chloride, ethyl acetate, acetone and methanol. Spent hops (250 g) were shaken with 1 L of each solvent for 24 h on a rotary shaker. Extracts were filtered with Whatman filter paper 1. Plant material was washed with a portion of 400 mL of solvent and collected filtrates were evaporated under vacuum at the temperature below 50 °C. The residues obtained were stored at −20 °C for further use.
Spent hops extracts type II: spent hops extracts type I were chromatographed on a Sephadex LH–20 column using methanol as eluent. Fractions contained a majority of xanthohumol (1) were removed and the rest fractions were collected, evaporated under vacuum at the temperature below 50 °C. The residues obtained were stored at −20 °C for further use.
The all spent hops extracts were analysed for xanthohumol (1) content by HPLC on a Dionex Ultimate 3000 instrument (Thermo Fisher Scientific, Waltham, MA, United States) with a diode array detector (detection at 360 nm wavelength) using the analytical HPLC column Agilent ZORBAX Eclipse XDB 5 µm (4.6 × 250 mm). Elution was carried out with a gradient of 40–100% solvent (1% formic acid in MeCN) in solvent A (aqueous 1% formic acid) in 15 min at the flow rate of 0.8 mL/min after initial 2 min at 40% solvent B.