Discovery of novel anti-inflammatory compounds from Borojó via bioactivity-guided zebrafish phenotyping screening and mechanistic validation.

Five new compounds, (3R,4S)-4-(2-methoxy-2-oxoethyl)cyclopent-1-ene-1,3-dicarboxylic acid (1), (3R,4S)-4-methylcyclopent-2-ene-1,3-dicarboxylic acid (2), (3R,4S)-4-(carboxymethyl)cyclopent-1-ene-1,3-dicarboxylic acid (3), tans-3-oxo-3,3a,6,6a-tetrahydro-1H-cyclopenta[c]furan-4-carboxylic acid (4) and tans-3,3a,4,6a-tetrahydro-2H-cyclopenta[b]furan-5-carboxylic acid (5), along with 4 known secondary metabolites including dihydroxybenzoic acid (6), trans-sinapic acid (7), furan-2-carboxylicacid (8) and 5-hydroxylmethylfuraldehyde (9) were isolated from Borojó (Borojoa patinoi Cuatrecasas). Their structures were established by spectroscopic data (UV, IR, MS, NMR, CD). Anti-inflammatory evaluation in zebrafish models revealed that both the crude extract and all isolates suppressed neutrophil recruitment, with 1 being the most active. Mechanistic studies showed that 1 inhibited LPS-induced inflammation by modulating the STAT3 and the TNF-α/NF-κB p65/IκBα pathways, reducing inflammatory cell infiltration, cytokine expression (IL-6, TNF-α), and nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65. Molecular docking confirmed its binding to STAT3 and TNF-α. The discovery of novel anti-inflammatory compounds in Borojó, with the mechanisms validated, provides a molecular basis for its traditional use and supporting its development as a functional food or therapeutic agent.