An improved 2D-HPLC-UF-ESI-TOF/MS approach for enrichment and comprehensive characterization of minor neuraminidase inhibitors from Flos Lonicerae Japonicae

[Display omitted] •A novel 2D-HPLC-UF-ESI-TOF/MS method was developed.•It was applied to the enrichment and characterization of minor neuraminidase inhibitors(NAIs) from Flos Lonicerae Japonicae (Jinyinhua).•Forty-four compounds were found with neuraminidase inhibition activity. Sixteen potent minor NAIs were confirmed by in vitro assays. Flos Lonicerae Japonicae(Jinyinhua) possesses clearing heat and detoxification activity, and has been used as a traditional Chinese medicine to treat influenza for many years. Due to the complex chemical composition and diverse content of Jinyinhua, especially the many trace ingredients, the effective components are unknown. In this study, an improved two-dimensional high performance liquid chromatography-ultrafiltration combined with electrospray ionization-time-of-flight/mass spectroscopy (ESI-TOF/MS) approach was designed and used for the enrichment, screening and characterization of minor neuraminidase inhibitors in Jinyinhua. In the first dimension, semi-prep-HPLC was employed for the preliminary separation of different polarity components and enrichment of low content components from Jinyinhua extract. In the second dimension, hydrophilic interaction liquid chromatography and reverse phase-HPLC were used to separate the different polar fractions, respectively. The fractions then underwent ultrafiltration and ESI-TOF/MS for the comprehensive screening and characterization of potential neuraminidase inhibitors. As a result, a total of 44 compounds were found to have neuraminidase inhibitory activity, and 22 of these compounds were preliminarily identified by accurate molecular weight and UV absorption data compared with standards and references. The activity of 16 of these compounds was verified by the neuraminidase inhibition assay. This study provides support for the rapid screening of minor neuraminidase inhibitors from complex natural medicines.