Yufeng Ningxin Tablet (YNT) is a traditional Chinese medicine formula, that has been used clinically to treat migraine for many years. It is composed of one herb Pueraria lobata var. lobata (Willd.) Ohwi (Relevant Chinese name: Gegen). Previously, it has been recorded by traditional Chinese doctor that Gegen could be used as medicine to treat migraine. However, the underlying mechanism of action remains to be investigated. It was to explore the effect and mechanism of YNT on migraine based on network pharmacology and experimental verification. First, with the network pharmacology, the effective chemical components and target genes of YNT were filtrated, the YNT-compound-migraine-targets network was constructed. The protein-protein interaction network (PPI) and literature reports were combined to identify potential targets of YNT in the treatment of migraine. Then, the representative compounds of YNT were characterized by LC-MS/MS and the major effect components were identified. Finally, the prediction results of network pharmacology were verified by animal and cell experiments. 7 bioactive components of YNT could act on 97 migraine potential targets. The 5 bioactive components could be characterized comprehensively of YNT. The key therapeutic targets and pathways were collected including 5-HT, CGRP, inflammation and nociceptive factors, and NF-κB signaling pathway. Animal experiments showed that YNT could increase the expression level of 5-HT and reduce the expression of CGRP, NF-κB, c-fos and IL-1β. YNT could inhibit LPS-induced neuroinflammation by NF-κB in BV2 cells in vitro. Western blotting analysis results showed YNT inhibited the NF-κB and phospho-NF-κB levels. It is the first time to verify the consistency between the metabolic components of YNT by LC-MS/MS and the active components predicted by network pharmacology. Meanwhile, the potential mechanism of YNT in the treatment of migraine was studied by combining network pharmacology and in vitro and in vivo experiments.

Magnolol, an essential bioactive natural lignans isolated from Magnolia Officinalis, has various pharmacological activities, such as antibacterial, anti-inflammatory, antioxidation and anti-tumor. In this study, a practical strategy based on ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was established to reveal the metabolic fate of magnolol in rat plasma and urine. First of all, in the data-dependent scanning (DDS) acquisition mode, the ESI-MSⁿ data sets of biological samples and reference standard were obtained by using a high-quality online data analysis method. Subsequently, off-line data-mining techniques including multiple mass defect filters (MMDFs) and high-resolution extracted ion chromatograms (HREICs) were applied to screen major-to-trace metabolites of magnolol at the full-scan ESI-MS¹ level. Finally, a total of 177 metabolites (prototype compound included) were preliminarily observed and characterized according to accurate mass measurement, the characteristic fragmentation patterns, chromato-graphic behaviors, and corresponding Clog P values. The results showed that magnolol experienced a variety of biotransformation reactions in rats, including hydroxylation, methoxylation, dehydrogenation, carboxylation, sulfonation, glucuronide conjugation, glucose conjugation, N-acetylcysteine (NAC) conjugation, glutathione conjugation and their composite reactions. In conclusion, this study not only greatly expanded the understanding of the therapeutic material basis and pharmacological mechanism of magnolol, but also provided new ideas for its toxicity evaluation, safety monitoring and drug delivery forms design.