From Plant to Target: Uncovering a Novel Anti-Inflammatory Compound in Pouzolzia pentandra via Phytochemical, Cellular, and Computational Studies

Phytochemical investigation of the ethyl acetate extract from the aerial parts of Pouzolzia pentandra led to the isolation and identification of fourteen compounds (1–14). These include known compounds such as β-sitosterol (1), bauerenol (2), oleanolic acid (3), 3β-friedelanol (4), kaempferol (5), quercetin (6), 2′,6′-dihydroxy-3′,4′-dimethoxychalcone (7), friedelan-3-one (8), dipterocarpol (9), 3β-hydroxyolean-12-en-28-one 3-p-coumarate (10), daucosterol (11), astilbin (12), 3-methoxy-4-hydroxybenzoic acid (13), and pouzolignan F (14). Among these, compound 14 displayed the most potent inhibitory activity on nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophages, with an IC50 value of 10.54 ± 0.4 µM. Mechanistic studies further revealed that compound 14 significantly suppressed the LPS-induced release of key pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Furthermore, it inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by preventing the nuclear translocation of its p65 subunit. Molecular docking studies were performed to evaluate the anti-inflammatory potential of compound 14 against cyclooxygenase-2 (COX-2) and phosphodiesterase-4 (PDE4). The compound exhibited binding affinities of −6.138 kcal/mol and −9.361 kcal/mol for COX-2 and PDE4, respectively. Subsequent molecular dynamics (MD) simulations confirmed the formation of a stable complex with the active site of PDE4. Collectively, these integrated in vitro and in silico findings demonstrate that pouzolignan F acts as a multi-target anti-inflammatory agent, likely through the inhibition of inflammatory mediators, cytokines, and the NF-κB pathway.