Elucidating the active compounds and anti-inflammatory mechanisms of Wubi Shanyao pill through random forest-based spectrum-effect relationship analysis and UPLC-MS/MS-based network pharmacology.

Abstract

This study integrated the spectrum-effect relationship with UPLC-MS/MS-based network pharmacology to identify the active compounds and clarify the anti-inflammatory mechanism of Wubi Shanyao Pill (WSP). The chemical fingerprint of WSP was established using liquid chromatography-tandem mass spectrometry (LC-MS), and its anti-inflammatory properties were evaluated in lipopolysaccharide (LPS) and interferon-gamma (IFN-γ)-induced bone marrow macrophages (BMDM) by assessing four inflammatory factors. A random forest (RF) algorithm associated phytochemical components with anti-inflammatory activity, screening the top 10 potential active compounds. Network pharmacology was further utilized to predict the core targets and pathways, followed by molecular docking and in vitro validation. WSP significantly inhibited inflammatory factor production in BMDMs (P < 0.0001). Spectrum-effect relationship analysis screened 16 potential bioactive compounds, with echinacoside, acteoside, forsythoside I, schisantherin A, and schizandrin B ranked as the top five. Meanwhile, 106 compounds were identified from WSP, primarily lignans, triterpenoids, and phenylethanol glycosides. Network pharmacology predicted key anti-inflammatory targets based on these components including AKT1, PIK3R1 and PIK3CA, with the PI3K-Akt pathway identified as the key. Molecular docking confirmed stable binding of the top five compounds to core targets (binding energies < -5.0 kcal/mol). In vitro experiments confirmed that the predominant compound, echinacoside, markedly suppressed the phosphorylation of PI3K and AKT proteins (P < 0.05), thereby attenuating systemic inflammatory responses. This study reveals the anti-inflammatory mechanisms of WSP, demonstrating that its primary active ingredient, echinacoside, exerts anti-inflammatory effects by modulating the PI3K-Akt signaling pathway. Overall, the results suggest that WSP possesses promise as a therapeutic option for inflammatory disorders.