In order to solve the bottleneck of reference standards shortage for comprehensive quality control of traditional Chinese medicines (TCMs), a series of strategies, including one single reference standard to determine multi-compounds (SSDMC), quantitative analysis by standardized reference extract (QASRE), and quantitative nuclear magnetic resonance spectroscopy (qNMR) were proposed, and Mahoniae Caulis was selected as an example to develop and validate these methods for simultaneous determination of four alkaloids, columbamine, jatrorrhizine, palmatine, and berberine. Comprehensive comparisons among these methods and with the conventional external standard method (ESM) were carried out. The relative expanded uncertainty of measurement was firstly used to compare their credibility. The results showed that all these three new developed methods can accurately accomplish the quantification by using only one purified reference standard, but each of them has its own advantages and disadvantages as well as the specific application scope, which were also discussed in detail in this paper.
Objective Mahoniae Caulis (MC) has long been documented in Chinese medical texts for the treatment of "Ru Yong" (acute mastitis). Previous research has affirmed its effectiveness in addressing breast inflammatory conditions. However, the underlying mechanisms by which MC treats acute mastitis remain unclear. This study employed a multidimensional approach, combining systems biology and ethnopharmacology, to explore the topical anti-mastitis mechanism of MC. Methods The composition and batch-to-batch consistency of MC were analyzed using ultra-high-performance liquid chromatography-quadrupole-exactive mass spectrometry (UHPLC-QE-MS). Potential therapeutic targets were identified through integrated summary-data-based Mendelian randomization (SMR) and network pharmacology. A lipopolysaccharide (LPS)-induced murine mastitis model was established to evaluate therapeutic efficacy via histological examination using HE staining. Myeloperoxidase (MPO) activity was measured through immunohistochemistry-ELISA hybridization. Pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) were quantified by RT-qPCR and ELISA, while protein levels of COX-2, iNOS, and key components of the cGAS-STING-NF-κB pathway were analyzed using Western blotting. Molecular docking assessed interactions between MC’s key compounds and critical pathway targets. Results UHPLC-QE-MS identified 25 characteristic components in the MC, demonstrating batch-to-batch consistency. Multi-omics integration revealed NF-κB signaling as a central therapeutic target. Topical application of MC significantly reduced LPS-induced mammary erythema, neutrophil infiltration, and vascular congestion. Notable decreases in MPO activity and pro-inflammatory cytokine levels (IL-1β, IL-6, TNF-α) were observed, alongside downregulation of COX-2 and iNOS protein expression. Molecular docking indicated favorable binding affinities between MC’s key components and proteins of the cGAS-STING-NF-κB axis, with pathway inhibition further confirmed by Western blot analysis. Conclusion This study provides the first evidence that MC may mitigate mammary inflammation by inhibiting the cGAS-STING-NF-κB signaling pathway, offering a scientific basis for its clinical application in the management of acute mastitis.
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