[Exploration of pharmacodynamic material basis and mechanism of Jingfang Mixture against influenza A (H1N1) based on UPLC-Q-Exactive-Orbitrap-MS and network pharmacology].

Abstract

This study systematically analyzed the components of Jingfang Mixture that entered the blood and target organ(lung) in both normal and influenza A(H1N1) virus-infected mice by ultra-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry(UPLC-Q-Exactive-Orbitrap-MS). Network pharmacology and molecular docking were employed to explore the pharmacodynamic material basis and potential mechanisms against influenza A(H1N1). Twenty-four male BALB/c mice were randomly allocated into normal control, normal administration, model control, and model administration groups. An influenza infection model was established by intranasal inoculation with the A/H1N1/PR8 virus strain. After gavage of Jingfang Mixture, serum and lung tissue samples were collected. The chemical components and the components entering the blood and lung were identified and analyzed by UPLC-Q-Exactive-Orbitrap-MS. SwissTargetPrediction was used to predict the targets of the absorbed components, and the influenza A(H1N1)-related targets were obtained from the GeneCard, OMIM, and TTD databases. The common targets were used to construct a protein-protein interaction(PPI) network, which was followed by Gene Ontology(GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. A "component-target-pathway" network was established from the result, and the key interactions were validated by molecular docking. A total of 271 compounds were identified from the Jingfang Mixture, including 108 flavonoids, 55 coumarins, 46 terpenoids, 24 organic acids, 16 phthalides, 13 chromones, and 9 others. In normal mice, 21 prototype components in the blood and 6 prototype components in the lung were detected, while in infected mice, the numbers increased to 37 and 29, respectively. This suggested that influenza virus infection may disrupt the alveolar epithelial and endothelial barriers, facilitating the entry of more active components into systemic circulation and the target organ, thereby enhancing anti-influenza efficacy. Network pharmacology screening identified 282 potential targets. Molecular docking results indicated that active ingredients such as nodakenetin, tangeretin, glycyrrhetinic acid, cimifugin, and glycyrrhizic acid may act on core targets including AKT1, TNF, IL-6, IL-1β, and STAT3, modulating signaling pathways such as PI3K/AKT, apoptosis, influenza A, CLRs, and TNF, thereby exerting synergistic anti-influenza effects. In conclusion, this study preliminarily reveals the pharmacodynamic material basis and the potential "multi-component, multi-target, and multi-pathway" mechanism of Jingfang Mixture against influenza A(H1N1), providing a scientific basis for the clinical application and quality control of the mixture.