Combination of preparative HPLC and HSCCC methods to separate phosphodiesterase inhibitors from Eucommia ulmoides bark guided by ultrafiltration-based ligand screening.

Phosphodiesterase (PDE) inhibitors are widely used because of their various pharmacological properties, and natural products are considered the most productive source of PDE inhibitors. In this work, a new ultrafiltration-high-performance liquid chromatography (HPLC)-diode-array detection-mass spectrometry based ligand screening was developed for the first screening of PDE inhibitors from Eucommia ulmoides bark, and then the target bioactive compounds were prepared by combination of stepwise preparative HPLC and high-speed countercurrent chromatography (HSCCC) methods. Experiments were conducted to optimize the parameters in ultrafiltration, stepwise preparative HPLC, and HSCCC to allow rapid and effective screening and isolation of active compounds from complex mixtures. Seven lignans with purity over 97 % were isolated and identified by their UV, electrospray ionization mass spectrometry, and NMR data as (+)-pinoresinol-4,4'-di-O-β-D-glucopyranoside (1), (+)-pinoresinol-4-O-β-D-glucopyranosyl(1 → 6)-β-D-glucopyranoside (2), (+)-medioresinol-4,4'-di-O-β-D-glucopyranoside (3), (+)-syringaresinol-4,4'-di-O- β-D-glucopyranoside (4), (-)-olivil-4'-O-β-D-glucopyranoside (5), (-)-olivil-4-O-β-D- glucopyranoside (6), and (+)-pinoresinol-4-O-β-D-glucopyranoside (7). Compound 2 was first isolated from the genus Eucommia. Lignan diglucopyranosides (compounds 1-4) shower a greater inhibitory effect than lignan monoglucopyranosides (compounds 5-7). The method developed could be widely applied for high-throughput screening and preparative isolation of PDE inhibitors from natural products.