Network pharmacology and UPLC/MS/MS metabolic profiling unveil the anti-inflammatory potential of Trifolium alexandrinum.

Trifolium alexandrinum, commonly known as berseem clover, has long been used in traditional medicine for its diverse therapeutic properties. In this study, we explore the anti-inflammatory potential of T. alexandrinum through an integrated approach combining network pharmacology and LC-MS/MS metabolic profiling. The ethanolic extract of T. alexandrinum was fractionated and analyzed, revealing a rich profile of phytoconstituents, including flavonoids, isoflavonoids, triterpenoid glycosides, and purine nucleosides. Network pharmacology analysis identified key bioactive compounds, such as tryptophan and adenosine, which exhibited strong interactions with inflammation-related genes, including TNF-α, IL-6, IL-1β, and INF-γ, as demonstrated from the "compound-target-pathway" constructed network. The arachidonic acid metabolism pathway, which plays a pivotal role in inflammation, was the top-listed pathway in the network. For the sake of confirmation, tryptophan and adenosine were isolated from the butanol fraction, and their structures were elucidated using (1)H-NMR, (13)C-DEPTQ, and HRESI-MS. In vitro studies using LPS-stimulated WI38 human fibroblast cells demonstrated that the butanol fraction of the extract significantly reduced the expression of pro-inflammatory cytokines, with adenosine and tryptophan showing particularly potent anti-inflammatory effects comparable to the synthetic drug piroxicam. These findings suggest that T. alexandrinum and its constituents, particularly polar compounds in the butanol fraction, hold promise as natural anti-inflammatory agents. This study not only elucidates the molecular mechanisms underlying the anti-inflammatory properties of T. alexandrinum but also highlights its potential as a functional food ingredient with both nutritional and therapeutic benefits.