Computational metabolomics reveals flavonoid architectures underlying species-specific chemical identity in medicinal Plectranthus

Introduction : Plant metabolomes encode biochemical information underlying species diversity, ecological adaptation, and pharmacological potential. Within the genus Plectranthus , diterpenoids are widely regarded as both the primary bioactive constituents and species-specific chemical signatures; however, the extent to which other bioactive metabolite classes contribute to species-specific chemical signatures remains poorly understood. Objective : To characterize and compare the leaf metabolomes of Plectranthus hadiensis and Plectranthus ambiguus , two medicinally important species used in South African ethnopharmacology. Methods: Methanolic leaf extracts of P. hadiensis and P. ambiguus were analysed usinguntargeted LC–MS/MS platform. The spectral data were mined and annotated using feature-based molecular networking followed by comparative metabolomics and GNPS library matching, MS2Query; and MS2LDA 2.0-enabled structure and substructure-level annotations. Results : The findings revealed extensive metabolic diversity beyond diterpenoids. While the prenol lipid chemical class, consisting of diterpenoids, was confirmed as a major discriminatory chemical class, flavonoids emerged as key drivers of species-level chemical differentiation. Distinct structural patterns were observed, with glycosylated flavones predominating in P. hadiensis , whereas methylated flavones and glycosylated flavonols were enriched in P. ambiguus . These compositional differences reflect underlying variation in flavonoid biosynthetic modifications, suggesting divergent metabolic regulation between species. Conclusions : Computational metabolomics revealed that flavonoid structural diversity constitutes a previously underappreciated layer of chemical specialization in Plectranthus plants and contributes substantially to species-specific chemical identity beyond the emphasized diterpenoids. By moving beyond targeted phytochemical screening, this work establishes a scalable framework for resolving species-specific metabolic architectures in medicinal plants. These findings have implications for chemotaxonomy, natural product discovery, and the rational exploration of plant-derived bioactives, contributing to a deeper understanding of the molecular basis of ethnomedicinal efficacy.