Withanolides from Physalis minima as potential anti-inflammatory agents: From isolation to in vitro and in vivo validation.

Ten withanolides (1-10) were isolated from Physalis minima L., a medicinal and edible plant. Among them, five (1-5) are previously undescribed. Their chemical structures were established through comprehensive spectroscopic and spectrometric analyses. In vitro bioassays revealed that compounds 2-5 and 8 significantly inhibited nitric oxide production in lipopolysaccharide (LPS)-stimulated murine RAW 264.7 cells, exhibiting anti-inflammatory effects with IC50 values ranging from 4.29 to 25.54 μM. Compound 2, which was identified as phyminiolide B and was the most abundant and potent one, potently reduced inflammatory cytokines release and downregulated COX-2 and iNOS expression. Mechanistically, it inhibited the activation of the NF-κB and MAPK pathways by blocking the phosphorylation of IKKα/β, IκBα, ERK, JNK, and p38. Furthermore, molecular docking and molecular dynamics simulation revealed that phyminiolide B exhibited strong binding affinity for the TLR4/MD2 complex, with a calculated binding energy of -9.0 kcal/mol. Additionally, in vivo experiments confirmed that phyminiolide B markedly alleviated LPS-induced acute lung injury in animal models, underscoring its therapeutic potential for inflammatory diseases. The findings provided valuable insights into the structural diversity and anti-inflammatory mechanisms of withanolides from P. minima.