This study aims to analyze the differences in chemical constituents before and after the ginger-processing of Anemarrhenae Rhizoma(AR), screen for key differential components, and elucidate their anti-inflammatory mechanisms. Furthermore, it seeks to clarify the principles of processing and the scientific rationale behind the synergistic enhancement achieved through ginger processing. This research provides scientific support for the preservation of traditional processing techniques and the rational clinical application of ginger-processed Anemarrhenae Rhizoma (GAR). The Ultra Performance Liquid Chromatography-Quadrupole-Time of Flight-Tandem Mass Spectrometry (UPLC-Q-TOF-MS/MS) technology was employed for qualitative analysis of the chemical constituents present in AR and GAR in both positive and negative ion modes. By comparing the variations in constituent levels before and after ginger-processing, as well as identifying newly emerged characteristic peaks, we prioritized novel and upregulated differential components with well-documented pharmacological activities as target compounds. The key differential components were subsequently validated quantitatively using High Performance Liquid Chromatography (HPLC) technology. Furthermore, molecular docking technology was employed to predict the binding affinity of these key differential components with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and cyclooxygenase-2 (COX-2) proteins. An inflammatory model was established utilizing lipopolysaccharide (LPS)-induced RAW264.7 macrophages, and the CCK-8 (Cell Counting Kit-8) assay was performed to determine the optimal concentrations of the key differential components and LPS. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell supernatants were measured using ELISA, while the expression levels of IκBα (Inhibitor of nuclear factor kappa B alpha), NF-κB, phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells (p-NF-κB), nuclear NF-κB, and COX-2 proteins were assessed through Western blot (WB) analysis. UPLC-Q-TOF-MS/MS analysis identified 40 compounds in both AR and GAR under both positive and negative ion modes, predominantly comprising saponins. Key differential components (mangiferin, timosaponin BⅡ, 6-gingerol) were screened based on changes in the chemical profile before and after ginger-processing, as well as pharmacodynamic literature. HPLC quantitative analysis confirmed that the components timosaponin BⅡ and mangiferin were increased following ginger-processing, while 6-gingerol was identified as a unique component in GAR, a novel compound generated during ginger-processing. Molecular docking results indicated a strong binding affinity of these components with NF-κB and COX-2 proteins. CCK-8 assays demonstrated that at an LPS concentration of 1 µg/mL, mangiferin at 100 μmol/L, timosaponin BⅡ at 2 μmol/L, and 6-gingerol at 20 μmol/L, the cell viability exceeded 90%, with no significant cytotoxicity observed. ELISA results indicated that compared to the model group, the treatment groups treated with timosaponin BⅡ, mangiferin, and 6-gingerol significantly inhibited the release of TNF-α and IL-6 (P < 0.05). Furthermore, Western blot analysis confirmed that these compounds markedly upregulated the protein expression of IκBα while significantly downregulating the expression levels of NF-κB, p-NF-κB, nuclear NF-κB, and COX-2 (P < 0.05). This study demonstrates that GAR introduces 6-gingerol, a key anti-inflammatory component found in ginger, while also increasing the levels of mangiferin and timosaponin BⅡ. These differential components may enhance the anti-inflammatory efficacy of GAR by inhibiting the activation of the NF-κB signaling pathway and downregulating COX-2 protein expression, which in turn reduces the release of pro-inflammatory mediators such as IL-6 and TNF-α. By following the sequence of "component variation-enhanced efficacy-mechanism clarification," this study systematically elucidates the material basis, action mechanisms, and processing rationale that underlie the enhanced anti-inflammatory effects of GAR. Furthermore, it provides scientific support for the preservation of traditional processing techniques of GAR and its clinical applications.
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