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

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.