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

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 ¹H-NMR, ¹³C-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.