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

Abstract

Phytochemical investigation of the ethyl acetate extract from the aerial parts of <i>Pouzolzia pentandra</i> led to the isolation and identification of fourteen compounds (<b>1</b>-<b>14</b>). These include known compounds such as <i>β</i>-sitosterol (<b>1</b>), bauerenol (<b>2</b>), oleanolic acid (<b>3</b>), 3<i>β</i>-friedelanol (<b>4</b>), kaempferol (<b>5</b>), quercetin (<b>6</b>), 2',6'-dihydroxy-3',4'-dimethoxychalcone (<b>7</b>), friedelan-3-one (<b>8</b>), dipterocarpol (<b>9</b>), 3<i>β</i>-hydroxyolean-12-en-28-one 3-<i>p</i>-coumarate (<b>10</b>), daucosterol (<b>11</b>), astilbin (<b>12</b>), 3-methoxy-4-hydroxybenzoic acid (<b>13</b>), and pouzolignan F (<b>14</b>). Among these, compound <b>14</b> 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 <b>14</b> significantly suppressed the LPS-induced release of key pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-<i>α</i>) and interleukin-6 (IL-6). Furthermore, it inhibited the activation of the nuclear factor-kappa B (NF-<i>κ</i>B) signaling pathway by preventing the nuclear translocation of its p65 subunit. Molecular docking studies were performed to evaluate the anti-inflammatory potential of compound <b>14</b> 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-<i>κ</i>B pathway.