Integrated MALDI-MSI and UHPLC-OE-MS for Spatial Visualization and Biosynthetic Pathway Elucidation of Bioactive...

Lilium lancifolium Thunb. is an important economic crop widely cultivated and traded across Asia and has significant pharmacological activity. Despite decades of research on their chemical composition, the spatial distribution patterns of characteristic secondary metabolites within the bulbs remain poorly understood. In this study, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) technology to characterize and spatially visualize multiple metabolites within the bulb for the first time. Additionally, ultra-high-performance liquid chromatography-Orbitrap Exploris mass spectrometry (UHPLC-OE-MS) was used to obtain comprehensive metabolite information from the bulbs. Using spatial metabolomics, we successfully identified nine steroidal saponins, three phenolic acid glycerides, and six other metabolites. Subsequently, we analyzed the spatial distribution of steroidal saponins and phenolic acid glycerides, which are key bioactive components. The analysis revealed that most of the steroidal saponins and phenolic acid glycerides, such as deacylbrownioside and regaloside A, exhibited a similar distribution pattern, mainly being enriched in the outer regions (A2, B2) and basal regions (B1, B2) on an individual scale. Further metabolomic and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that 11 substances detected in the bulbs, including diosgenin, phenylalanine, and acetyl-CoA, were jointly associated with 39 metabolic pathways, including "phenylpropanoid biosynthesis" and "terpenoid backbone biosynthesis". Based on the above findings, we propose biosynthetic pathways and accumulation patterns of steroidal saponins and phenolic acid glycerides in bulbs. This study provides a basis for precise resource utilization of L. lancifolium bulbs and a methodology to elucidate the biosynthesis of plant metabolites.