Dissecting tissue-specific metabolite profiling and jasmonic acid-induced calotropin in Calotropis procera: Biotechnological production and analytical validation

Calotropis procera is a medicinal plant known to produce cardiac glycosides, a class of specialized bioactive metabolites with significant therapeutic potential, among which calotropin is one of the most pharmacologically relevant. However, the tissue-specific distribution of these compounds, their inducible biosynthesis, and reliable platforms for calotropin production remain poorly characterized. Here, we address these gaps by performing LC–MS/MS-based metabolite profiling across multiple tissues, and by evaluating jasmonic acid (JA) elicitation in both hydroponically grown seedlings and adventitious root cultures (ARC) to enhance calotropin accumulation. Metabolite profiling across tissues revealed a wide variety of metabolites, including cardiac glycosides, phenolics, amino acids, organic acids, lipids, and other compounds. Comparative analysis highlighted pronounced tissue-specific distribution: cardenolides were highly enriched in fruits and latex, moderately present in leaves, and relatively depleted in stems and flowers, while flavonoids and phenolic acids accumulated mainly in leaves, flowers, and fruits. JA elicitation significantly enhanced calotropin levels in both whole-plant and in vitro systems. Calotropin was subsequently isolated, its structure unambiguously confirmed by NMR and MS/MS, and a validated quantitative method was developed. Together, these data provide a detailed metabolic framework of the spatial distribution of metabolites and establish a foundation for future biosynthetic, ecological, and biotechnological studies of C. procera . • LC–MS 2 metabolomics mapped tissue-specific metabolites across Calotropis procera . • Cardiac glycosides were strongly enriched in fruits and latex. • Flavonoids and phenolic acids accumulated mainly in leaves and flowers. • JA enhanced calotropin levels in both seedlings and adventitious root cultures. • Calotropin was isolated, NMR-confirmed, and quantified using a validated method.