Stable Isotope-Assisted Computational Mass Spectrometry Reveals Root-Specific Homopipecolic Acid-Containing Flavonoids in Glycyrrhiza Species

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Abstract Licorice (Glycyrrhiza) is a medicinal plant widely used in approximately 70% of traditional Japanese Kampo formulations and is known to produce a wide array of specialized metabolites with diverse pharmacological properties. Although hundreds of metabolites have been reported, the overall chemical diversity of Glycyrrhiza remains poorly characterized. Here, using mass spectrometry data obtained from fully 13C-labeled leaves and roots of Glycyrrhiza uralensis and Glycyrrhiza glabra, we determined the carbon number, followed by the molecular formula and substructure prediction in combination with MS/MS similarity-based molecular networking. After excluding redundant ions, including isotopic peaks, adducts, and in-source fragments, we extracted 3060 unique metabolite features with assigned carbon numbers. Among these, substructure information was assigned to 1015 features (33%) across the four plant tissues, revealing tissue-specific metabolome profiles. Furthermore, we discovered five previously unreported homopipecolic acid-conjugated flavonoids in the roots of G. uralensis and G. glabra and Glycine max, another member of the Fabaceae family. Two of these compounds were structurally characterized using nuclear magnetic resonance spectroscopy. We further proposed a biosynthetic route involving a spontaneous reaction between 1-piperideine and malonyl glycoside substrates and confirmed the formation of the conjugated product using authentic standards.