Spatial metabolome of Polygala tenuifolia and Polygala sibirica roots by matrix-assisted laser desorption/ionization mass spectrometry imaging

The therapeutic efficacy of medicinal plants is intrinsically linked to the spatial distribution of their bioactive constituents, yet this critical aspect has been largely uncharacterized due to technical limitations in metabolite localization. Here, we employ high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at 50 μm resolution to investigate the spatial metabolomics of P. tenuifolia and P. sibirica roots, two pharmacologically important species with documented neuroprotective properties. Our analysis revealed distinct species-specific chemotypes: P. tenuifolia showed predominant accumulation of oleanane-type triterpenoid saponins, including tenuifolin, senegenin and their biosynthetic precursor polygalic acid, with specific localization in the secondary phloem and periderm. In contrast, P. sibirica displayed a broad distribution of flavonoid glycosides (e.g., hyperoside and quercitrin) and xanthones (e.g., polygalaxanthone XI), along with enrichment of sucrose esters such as 3′,6-disinapoylsucrose. Multivariate statistical analysis confirmed a clear metabolic divergence between the species (OPLS-DA: R²Y = 0.999, Q² = 0.981), identifying ten key biomarkers with VIP > 1.0. Spatial co-localization and segmentation analyses further indicated greater complexity and finer regulation of metabolite distribution in P. tenuifolia compared with the relatively simplified organization in P. sibirica , suggesting species-specific metabolic regulation strategies. Functional validation in BV-2 microglial cells demonstrated that both extracts (60 μg/mL) significantly suppressed LPS-induced NO overproduction and reduced microglial activation, supporting their anti-neuroinflammatory potential. This integrated strategy not only clarifies tissue-specific localization of bioactive metabolites but also establishes a scientific basis for quality assessment, species authentication, and optimized utilization of these medicinal resources. Our findings underscore spatial metabolomics as a transformative approach for linking phytochemical profiling with pharmacological validation in medicinal plant research. • Spatial metabolomics reveals species-specific accumulation of saponins and flavonoids in Polygala roots. • P. tenuifolia shows finer metabolic compartmentalization than P. sibirica, indicating divergent regulation. • Ten biomarkers clearly discriminate the two species' metabolomes. • Root extracts suppress neuroinflammation by inhibiting NO production and microglial activation.