20a series hplc system

Silica gel 60A and gel filtration resin (Sephadex LH-20) were obtained from Millipore-Sigma (Burlington, MA, USA). Reversed-phase high-performance liquid chromatography (HPLC)-UV analysis of antioxidants was carried out on a Shimadzu 20A series HPLC system (Kyoto, Japan) (Core-facility center, Jeju, Korea). LPS from E. coli was obtained from InvivoGen (San Diego, CA, USA). Cell proliferation was done using the EZ-cytox Cell Viability Assay Kit (Wellbio, Seoul, Korea). We used 13-KODE purified from S. herbacea L. extracts. All other chemicals were obtained from Millipore-Sigma (Burlington, MA, USA).

A Shimadzu 20A series HPLC system (Shimadzu, Tokyo, Japan) coupled with an automatic degasser (DGU-20A 3R), quaternary pump (LC-20AD), auto-sampler (SIL-20 HT) and diode array detector (SPD-M20A) was used for the HPLC-DAD analyses. The separations were performed on the Agilent Zorbax Eclipse XDB-C18 (3.5 µm, 4.6 × 150 mm, Santa Clara, CA, USA) column. The conditions for the HPLC were as follows: gradient of solvents: water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) were used as the mobile phases. The following gradient procedure was adopted: 0–45 min, 15% of B; 45–50 min, 70–95% B; 50–60 min 15% (B). The total time of analysis was 60 min with a stable flow rate at 1 mL/min. The injection volume for extracts was 10 μL. The concentration of xanthohumol present in the samples was determined from the response factors at 370 nm using a five-point calibration curve obtained with xanthohumol standard (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) (5–250 μg/mL, y = a × 5.19E + 12, R² = 0.9995). Dry extracts and α-AEF were weighed and dissolved in methanol resulting in concentrations of 10 mg/mL. Separations were performed for all extracts and α-AEF samples in triplicate.

A Shimadzu 20A series HPLC system (Shimadzu, Tokyo, Japan) coupled with an automatic degasser (DGU-20A 3R), quaternary pump (LC-20AD), auto-sampler (SIL-20 HT) and diode array detector (SPD-M20A) was used for the HPLC-DAD analyses. The separations were performed on a Phenomenex Gemini NX-C18 110A (250 mm × 4.6 mm, 5 μm) column with the mobile phase composed of water (A) and methanol (B). The following gradient was applied: 0 min—50% B, 5 min—60% B, 20 min—80% B, 25 min—100% B, and 26–35 min—50% B. The flow rate was 1 mL/min, the column temperature was 25 °C, the injection volume was 10 μL, and the DAD spectra were recorded at 254 and 300 nm.