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

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.

To characterize and compare the leaf metabolomes of Plectranthus hadiensis and Plectranthus ambiguus, two medicinally important species used in South African ethnopharmacology.

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.

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.

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.