Lc 20ar pump

Analysis was carried out using an HPLC system (Shimadzu, Japan) equipped with an LC-20AR pump, SPD-20A detector, SIL-10AP auto-sampler, and an FRC-10A fraction calculator. Each of three different amounts (300, 540, and 1080 mg) of EAEM were diluted with 5 mL of ethanol and injected onto a C18 column (250 × 21.2 mm, 7 μm, PrepHT 300SB; Agilent, Santa Clara, CA, USA), respectively, and separated using a binary mobile phase composed of solvents A (water) and B (acetonitrile) at a flow rate of 10.0 mL/min. During this, a solvent gradient of 3% B for 0–5 min, 3%–29% B for 5–50 min, and 29%–56% B for 50–65 min was used. This was followed by a 15-min equilibrium period under initial conditions prior to sample injection. Chromatograms were monitored at 310 nm using the UV detector. Fractions were collected every 2 min and up to 33 fractions were collected in total. After fractionation, three sets of 33 fractions were merged into a single set of 33 fractions.

HPLC separation was performed on SHIMADZU LC-20AR pump and a SHIMADZU SPD-20A detector (Tokyo, Japan), using the COSMOSIL C₁₈ preparative column (250 × 20 mm) and YMC-pack Prep-ODS column (250 × 20 mm). HR-ESI-MS spectra (Agilent 6200 series Q-TOF spectrometer, United States) were in the m/z mode. Column chromatography: silica gel (SiO₂, 200–300 meshes), Sephadex LH-20 (Qingdao, China), and reversed-phase ODS (Kyoto, Japan). The 1D- and 2D-NMR were texted on a Bruker ARX-600 spectrometer (Bremen, Germany) in DMSO-d₆ (Sigma-Aldrich Company).