Characterization and Isolation of Ginger Phenols from <i>Zingiber officinale</i> Rhizomes through in Silico-Based Reverse Annotation and Molecular Networking

Ginger is a rich source of ginger phenols with an anti-inflammatory property. Antineuroinflammatory pathways represent a promising therapeutic strategy for neurodegenerative diseases. Ginger was selected as our research object to discover bioactive constituents responsible for the antineuroinflammatory effect. An in silico-based reverse annotation strategy, in conjunction with molecular networking, was employed for the large-scale annotation of ginger phenols from ginger. A total of 174 ginger phenols and 235 ginger phenol dimers were annotated. Five new ginger phenols (<b>1-5</b>) and 18 known ginger phenols (<b>6-23</b>) were isolated following this proposed strategy, among which compound <b>1</b> was a new natural product and compounds <b>2-3</b> were novel ginger phenol dimers. Their structures were elucidated using extensive spectroscopic data and semisynthesis methods. Compounds <b>2</b>, <b>8</b>, <b>12</b>, <b>18</b>, and <b>19</b> exhibited a capacity to inhibit nitric oxide (NO) production and demonstrated antineuroinflammatory activity in lipopolysaccharide (LPS)-induced BV2 cells at the concentrations of 10, 20, and 40 μM. Enzyme-linked immunosorbent assay (ELISA) showed that compounds <b>2</b>, <b>18</b>, and <b>19</b> could significantly reduce the expression levels of cyclooxygenase-2 (COX-2) and interleukin-1β (IL-1β). In addition, compounds <b>2</b> and <b>18</b> displayed a certain inhibitory effect on the expressions of interleukin-10 (IL-10) and interleukin-6 (IL-6). The Western blot analysis revealed that compounds <b>18</b> and <b>19</b> effectively attenuated the expression of IL-1β, while only compound <b>2</b> inhibited the expression of phosphorylated nuclear factor kappa B (p-NF-κB) in BV2 cells. This study demonstrates the great potential and prospect of the proposed strategy in the utilization of ginger resources. Additionally, it reveals that ginger contains diverse ginger phenols with antineuroinflammation activity.