| Summary: | Four new phenolic components, eurylophenolosides A (<b>1</b>) and B (<b>2</b>), eurylolignanosides A (<b>3</b>) and B (<b>4</b>), along with twelve known compounds were isolated from the roots of <i>Eurycoma longifolia</i> Jack. The structure of these components was elucidated by using various spectral techniques and chemical reactions. Among the known isolates, syringaldehyde (<b>12</b>), 3-chloro-4-hydroxybenzoic acid (<b>13</b>), 3-chloro-4-hydroxyl benzoic acid-4-<i>O</i>-β-<span style="font-variant: small-caps;">d</span>-glucopyranoside (<b>14</b>), and isotachioside (<b>15</b>) were isolated from the <i>Eurycoma</i> genus for the first time. Further, the NMR data of <b>14</b> was reported here firstly. Meanwhile, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 40 μM. As results, piscidinol A (<b>6</b>), 24-<i>epi</i>-piscidinol A (<b>7</b>), bourjotinolone A (<b>10</b>), and scopoletin (<b>16</b>) were found to play important role in suppressing NO levels without cytotoxicity. Furthermore, the Western blot method was used to investigate the mechanism of compounds <b>6</b>, <b>7</b>, <b>10</b>, and <b>16</b> by analysing the level of inflammation related proteins, such as inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in LPS-stimulated RAW264.7 cells. Consequently, compounds <b>6</b>, <b>7</b>, <b>10</b>, and <b>16</b> were found to significantly inhibit LPS-induced protein expression of IL-6, NF-κB and iNOS in NF-κB signaling pathway. Moreover, it was found that the protein expression inhibitory effects of <b>6</b>, <b>7</b>, and <b>16</b> exhibited in a dose-dependent manner. The mechanism may be related to the inhibition of the iNOS expressions through suppressing the IL-6-induced NF-κB pathway.
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