Development of a method to screen and isolate lipoxidase inhibitors from Radix Saposhnikoviae via ultrafiltration liquid chromatography combined with metablism in vitro

Abstract

Introduction: Radix Saposhnikoviae is an excellent source of chromones, the consumption of which has been shown to positively affect human health, such as anti-inflammatory and antioxidant activities in vivo, and it also has been used to treat headache, inflammation, and cancer as a herbal medicine or functional soup.

Objective: Isolation and screening of lipoxidase (LOX) inhibitors from Radix Saposhnikoviae. As a result, the metabolism of the active ingredients in cytochrome P450 (CYP450) in vitro was identified.

Methods: Screen and identify the LOX inhibitors from Radix Saposhnikoviae by ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS). Then take the isolation and purification of the inhibitors by high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (HPLC). Furthermore, the active ingredients were used to study the metabolism in vitro in CYP450.

Results: Subsequently, the specific binding ligands, prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminol (with purities of 95.28, 92.33%, and 97.89, respectively) were separated by HSCCC. Semi-preparative HPLC was applied to separate the four major compounds in Radix Saposhnikoviae and identify them as prim-O-glucosylcimifugin, 5-O-methylvisamminol, sec-O-glucosylhamaudol, and hamaudol, which were obtained with relatively high purity. Furthermore, the three chromone possible metabolism pathways were obtained.

Conclusion: The results demonstrated that UF-LC-MS combined with HSCCC and semi-preparative HPLC is not only a powerful tool for screening and isolating LOX inhibitors in complex samples, but also a useful platform for discovering bioactive compounds for anti-inflammatory activity. This study provides information regarding the active ingredients of chromones in Radix Saposhnikoviae and a framework for anti-inflammatory activity treatment.

Keywords: Radix Saposhnikoviae; high-speed counter-current chromatography; lipoxidase inhibitors; metabolism; semi-preparative high-performance liquid chromatography; ultrafiltration.