A-type proanthocyanidins from Spenceria ramalana alleviate inflammation through the dual inhibition of iNOS and COX-2 expression and catalytic activity

This study investigates the potent anti-inflammatory potential and the underlying dual-inhibition mechanism of two A-type proanthocyanidins, SR14 (dimer) and SR16 (trimer), isolated from the traditional medicinal plant Spenceria ramalana. Initial in vitro investigations revealed that SR14 and SR16 dose-dependently suppressed the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Mechanistically, these compounds downregulated the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) via the blockade of the upstream NF-κB signaling pathway. Crucially, cell-free enzymatic assays revealed a dual-action pharmacological profile, wherein these compounds directly inhibited the catalytic activities of existing iNOS and COX-2 enzymes with low-micromolar IC50 values, with SR14 exhibiting superior potency. To elucidate the structural basis of this direct inhibition, a rigorously validated in silico pipeline was employed. Molecular docking confirmed tight binding within the active pockets, which was further substantiated by extended 200-ns molecular dynamics (MD) simulations against controls. Principal Component Analysis (PCA) and MM/PBSA calculations thermodynamically validated the exceptional dynamic stability of the SR14-enzyme complexes, highlighting the energetic dominance of critical catalytic residues like ARG193 in iNOS. Prompted by these robust in vitro and in silico findings, in vivo evaluations were conducted using a transgenic zebrafish model at a safe, pre-validated dose (10 µM). Both compounds significantly attenuated LPS-induced neutrophil migration without causing morphological toxicity, exhibiting efficacies comparable to the corticosteroid dexamethasone. Collectively, these multidisciplinary findings establish SR14 and SR16 as highly promising natural scaffolds with a unique dual-inhibition mechanism for the targeted management of inflammatory disorders.