Screening for natural inhibitors of 5-lipoxygenase from Zi-shen pill extract by affinity ultrafiltration coupled with ultra performance liquid chromatography-mass spectrometry

Abstract Ethnopharmacological relevance Zi-shen pill (ZSP), a traditional Chinese medicine, is widely used for the treatment of benign prostatic hyperplasia (BPH) and has remarkable curative effect. Aim of the study To screen the potential 5-Lipoxygenase(5-LOX) inhibitors from ZSP extract. Materials and methods A new approach based on affinity ultrafiltration-ultra performance liquid chromatography-mass spectrometry(UPLC-MS) was established and validated. Zileuton and glipizide were chosen as positive and negative control drug, respectively. For better screening result, the concentration of 5-LOX enzyme, incubation temperature and time, pH and ion strength were optimized. In addition, 5-LOX inhibitory assay in vitro and molecular docking technique were used for further verification. Results 20 compounds were characterized in the ultrafiltrate by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) and 16 ligands showed binding ability to 5-LOX. Among them, six ligands were deduced as high-potential 5-LOX inhibitors with their high specific binding values (>2.0). The inhibitory activities of anemarrhenasaponin I, timosaponin AI, nyasol and demethyleneberberine were confirmed by the 5-LOX inhibitory assay for validating the reliability of affinity ultrafiltration approach and the computer-simulated molecular docking technique further clarified the possible mechanism of action between the active compounds and the 5-LOX active sites.