Chemical Profiling and Anti-Inflammatory Mechanisms of Heracleum millefolium Diels Revealed by Molecular Networking, Network Pharmacology, and Cellular Validation

Abstract

Background/Objectives: Heracleum millefolium Diels (HMD) is a traditional Tibetan medicinal herb that has been reported to possess analgesic, anti-edematous, and anti-inflammatory activities. However, its chemical composition and anti-inflammatory mechanisms remain unclear. Methods: In this study, ultra-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS), combined with database searching and “seed”-based molecular networking, was used to systematically characterize the chemical constituents of HMD. Furthermore, an integrated strategy combining network pharmacology, molecular docking, and cellular validation was applied to explore potential anti-inflammatory mechanisms associated with HMD. Results: A total of 472 compounds were identified or tentatively identified from HMD. Network pharmacology analysis predicted five key targets, namely TNF, IL-6, IL-1β, GAPDH, and AKT1, together with four potential bioactive constituents, including velutin, artemitin, kaempferol, and naringenin. Pathway enrichment analysis indicated that the potential anti-inflammatory effects of HMD were mainly associated with lipid and atherosclerosis and the AGE-RAGE signaling pathway. Molecular docking suggested potential favorable interactions between the selected constituents and key targets. Moreover, cellular experiments demonstrated that the four compounds significantly inhibited inflammatory responses in LPS-stimulated RAW264.7 macrophages. Conclusions: This study systematically characterized the chemical profile of HMD and preliminarily explored its potential anti-inflammatory mechanisms, providing a scientific basis for further investigation of its bioactive constituents and pharmacological properties.