In Silico and In Vitro Insights into the Pharmacological Potential of Pouzolzia zeylanica

The present study involves the isolation, structural elucidation, and biological evaluation of eight compounds from <i>Pouzolzia zeylanica</i>. From the <i>n</i>-hexane and ethyl acetate extracts of the plant, eight compounds were successfully isolated and identified: oleanolic acid (<b>1</b>), ursolic acid (<b>2</b>), 2<i>α</i>-hydroxyursolic acid (<b>3</b>), 3<i>β</i>-O-acetyl-12-oleanen-28-oic acid (<b>4</b>), 5-hydroxy-6,7-dimethoxyflavanone (<b>5</b>), 4'-methoxytectochrysin (<b>6</b>), 3,4',5,7-tetrahydroxyflavanone-3-O-L-rhamnopyranoside (<b>7</b>), and 3,3',5,5',7-pentahydroxyflavanone-3-O-L-rhamnopyranoside (<b>8</b>). These compounds were evaluated for in vitro antioxidant activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) and lipid peroxidation inhibition (TBARS) assays, as well as anti-inflammatory activity via inhibition of nitric oxide (NO) production and the secretion of pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-<i>α</i>) and interleukin-6 (IL-6) in RAW 264.7 macrophages. It was observed that compound <b>3</b> exhibited the strongest antioxidant activity with IC₅₀ values of 18.52 ± 1.50 µM (DPPH) and 10.34 ± 0.93 µM (TBARS), whereas compounds <b>2</b>, <b>5</b>, and <b>6</b> showed moderate to weak effects. Meanwhile, compound <b>8</b> demonstrated the most potent anti-inflammatory effect with IC₅₀ values of 16.25 ± 0.95 µM (NO inhibition), 12.97 ± 0.88 µM (TNF-<i>α</i> inhibition), and 22.52 ± 1.98 µM (IL-6 inhibition). Furthermore, in silico approaches were employed, including density functional theory (DFT) calculations to predict the antioxidant mechanisms of compounds <b>1</b> and <b>3</b> and molecular docking to assess the cyclooxygenase-2 (COX-2) and phosphodiesterase-4B (PDE4B) inhibitory potentials of compounds <b>4</b>, <b>7</b>, and <b>8</b>. Computational results aligned well with experimental data, supporting the potential of these compounds as natural antioxidant and anti-inflammatory agents.