Metabolomic Analysis of Heliopsis longipes Roots Across Phenological Stages

Heliopsis longipes, commonly known as chilcuague, is a Mexican medicinal plant recognized for its diverse biological activities, largely attributed to its alkamide content, particularly affinin. However, metabolic variations associated with plant development remain poorly understood. This study evaluated the influence of phenological stage on the phytochemical profile and vasodilatory activity of H. longipes roots. Dichloromethane root extracts from plants at different developmental stages were analyzed using metabolomics based on 1H NMR spectroscopy, complemented by GC–MS profiling. Major alkamides were isolated and structurally characterized as analytical standards. Notably, three alkamides, N-isobutylundeca-2(E)-en-8,10-diynamide, N-isobutylundeca-3(E)-en-8,10-diynamide, and N-isobutyl-2(E),4(Z)-undecadiene-8,10-diynamide, are reported for the first time in H. longipes roots. Multivariate analyses (PCA and OPLS-DA) revealed significant stage-dependent metabolic variation, particularly in affinin. The lack of correlation between valine decarboxylase activity and affinin levels suggests additional regulatory steps in its biosynthesis. Vasodilatory activity increased during development, reaching maximum effect during fructification and defoliation stages, with no significant differences between them. These findings highlight the impact of phenological stage on alkamide production and bioactivity, providing a basis for optimizing cultivation and harvest timing.