Anti-Inflammatory Effects and Metabolomic Analysis of Ilex Rotunda Extracted by Supercritical Fluid Extraction

<i>Ilex rotunda</i> is a famous medicinal plant with many ethnopharmacological uses. It is traditionally employed for treating inflammation and cardiovascular diseases. In this study, we established green technology to extract the leaves and twigs of <i>I</i>. <i>rotunda</i>. The obtained extracts and their fractions were evaluated for their anti-inflammatory potential. In cytokine assays, the extract, <i>n</i>-hexane (H)<i>,</i> methylene chloride (MC), and EtOAc (E) fractions of the twigs of <i>I</i>. <i>rotunda</i> significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α production in RAW264.7 macrophages. Furthermore, the extract, H, and MC fractions of the leaves of <i>I</i>. <i>rotunda</i> modulated cytokine expression by downregulating LPS-induced NO, IL-6, and TNF-α production in RAW264.7 macrophages. Western blotting analysis revealed that the extracts and fractions of the leaves and twigs of <i>I</i>. <i>rotunda</i> inhibited inflammatory cytokines by inactivating nuclear factor kappa B (NFκB) action by reducing the phosphorylation of transcript factor (p65) and nuclear factor-kappa B inhibitor alpha (IκBα) degradation, or by inactivating mitogen-activated protein kinase (MAPK) through the p38 or ERK signaling pathways via the active ingredients of the leaves and twigs of <i>I</i>. <i>rotunda</i>. Ultra-high-resolution liquid chromatography-Orbitrap mass analysis (UHPLC-ESI-Orbitrap-MS/MS)-based molecular networking, in cooperation with social open platform-guided isolation and dereplication, led to the identification of metabolites in this plant. Our findings indicate that the leaves and twigs of <i>I</i>. <i>rotunda</i> could be promising candidates for developing therapeutic strategies to treat anti-inflammatory diseases.