Headache is a common episodic or chronic neurologic disorder. Treatment options and diagnosis are restricted by an incomplete understanding of disease pathology and the lack of diagnostic markers. Wu-Zhu-Yu decoction (WZYD), a traditional Chinese medicine (TCM) formula containing four TCM herbs, is commonly used in the treatment of headache in China. To deeply understand more about headache and investigate the pain-relief mechanism of WZYD, a comprehensive metabolomics study combined with multivariate data processing strategy was carried out. An LC-high resolution mass spectrometry-based metabolomics approach was applied to characterize metabolic biomarker candidates. Multiple pattern recognition including principal component analysis-discriminant analysis, partial least squares-discriminant analysis and hierarchical cluster analysis were used to determine groups and confirm important variables. A total of 17 potential biomarkers were characterized and related metabolic pathways were identified. The study demonstrated that the established metabolomics strategy is a powerful approach for investigating the mechanism of headache attack and WZYD. In addition, the approach may highlight biomarkers and metabolic pathways and can capture subtle metabolite changes from headache, which may lead to an improved mechanism understanding of central nervous system diseases and TCM treatment.

Uncaria rhynchophylla (Gouteng in Chinese, GT) is the main medicine in many traditional recipes in China. It is commonly used to alleviate central nervous system (CNS) disorders, although its mechanism in Alzheimer’s disease is still unknown. This study was designed to predict and validate the underlying mechanism in AD treatment, thus illustrating the biological mechanisms of GT in treating AD. In this study, a PPI network was constructed, KEGG analysis and GO analysis were performed, and an “active ingredient-target-pathway” network for the treatment of Alzheimer’s disease was constructed. The active ingredients of GT were screened out, and the key targets were performed by molecular docking. UHPLC-Q-Exactive Orbitrap MS was used to screen the main active ingredients and was compared with the network pharmacology results, which verified that GT did contain the above ingredients. A total of targets were found to be significantly bound up with tau, Aβ, or Aβ and tau through the network pharmacology study. Three SH-SY5Y cell models induced by okadaic acid (OA), Na₂S₂O₄, and H₂O₂ were established for in vitro validation. We first found that GT can reverse the increase in the hyperphosphorylation of tau induced by OA to some extent, protecting against ROS damage. Moreover, the results also indicated that GT has significant neuroprotective effects. This study provides a basis for studying the potential mechanisms of GT in the treatment of AD.

Aβ_25-35-induced PC12 cells were used as the in vitro injury model to evaluate the effects on PC12 cells after intervention with the “ginseng–polygala” drug pair. The results showed that the drug pair could significantly increase cell activity and reduce the level of reactive oxygen species and the concentration of inflammatory factors to improve the Alzheimer’s disease treatment process. Furthermore, to rapidly identify and classify complicated bioactive components of the drug pair, a liquid chromatography with quadrupole time-of-flight mass spectrometry method combined with a molecular network strategy was established. With this strategy, 40 constituents were preliminarily identified and a database of the compounds was successfully established. Among them, 12 compounds of different categories were accurately identified by comparison with reference substances. The content of the aforementioned active components was simultaneously determined by HPLC to control the quality of compatible medicinal materials, and the verification results of the analytical method met the content determination requirements. The results revealed that after compatibility, the content change of the components is not the simple addition of quantity but the comprehensive effect of the two medicines. In conclusion, this study could provide a generally applicable strategy for pharmacological activity, structural identification, and content determination in traditional Chinese medicine and its compatibility.

Hepatocellular carcinoma (HCC) is a lethal malignancy requiring novel therapeutic interventions. While Polyporus umbellatus exhibits anti-tumor properties, its specific bioactive pharmacophores and molecular mechanisms remain elusive. This study integrated network pharmacology, computational simulation, and experimental validation to decipher the anti-HCC efficacy of Polyporus umbellatus. Screening identified 11 bioactive sterols, with intersection analysis revealing 63 core targets. Clinical data stratified Checkpoint Kinase 1 (CHEK1) as a critical high-risk oncogene associated with poor prognosis. Molecular dynamics simulations (100 ns) demonstrated that polyporusterone E, a key constituent, forms a thermodynamically stable complex with CHEK1 via high-affinity hydrogen bonding. In vitro assays in HepG2 and HuH-7 cells confirmed that CHEK1 overexpression drives proliferation and metastasis, while its silencing reverses these phenotypes. Crucially, treatment with Polyporus umbellatus extract and purified polyporusterone E significantly compromised HCC cell viability and downregulated CHEK1 expression at transcriptional and translational levels. These findings suggest that polyporusterone E may downregulate CHEK1 expression and modulate CHEK1-associated signaling in HCC cells, providing preliminary evidence for the molecular basis of Polyporus umbellatus and highlighting its potential as a complementary therapeutic strategy for HCC management.