Untargeted metabolomics reveals phenology and cultivation-dependent signatures of vasodilatory activity in Heliopsis longipes leaves

Plant metabolomes are highly responsive to developmental and environmental cues; however, the extent to which these metabolomic shifts influence biological activity remains poorly understood in medicinal plants. Heliopsis longipes (chilcuague) is a Mexican endemic species whose roots contain affinin, a vasodilatory and antihypertensive alkamide. Despite representing the largest proportion of plant biomass, its leaves remain chemically and pharmacologically underexplored. This study integrated metabolomics, chemometrics, and network pharmacology to investigate how phenological stage and cultivation condition shape metabolomic signatures associated with vasodilatory activity in H. longipes leaves. Dichloromethane extracts were obtained from wild and greenhouse-grown plants collected at four phenological stages: vegetative, flowering, fruiting, and defoliation. Untargeted metabolomic fingerprinting was performed using GC-MS and FTIR-ATR, while vasodilatory activity was evaluated in isolated rat aorta. ASCA revealed that the interaction between cultivation condition and phenological stage was the dominant source of metabolomic variation, accounting for 38.2% and 41.6% of the variance in GC-MS and FTIR-ATR datasets, respectively. Wild fruiting-stage extracts exhibited the highest vasodilatory efficacy (E_max = 100%; EC₅₀ = 404.50 ± 61.30 µg/mL) and a distinct metabolomic signature characterized by differential accumulation of eudesmane-type sesquiterpenes, lignans, and phenylpropanoids. PLS regression (R²Cal = 0.93) demonstrated a strong association between vasodilatory activity and a multicomponent metabolomic signature rather than a single bioactive constituent. Lutein and β-carotene were identified for the first time at the metabolomic level in H. longipes leaves. Bioassay-guided fractionation yielded sesamin, which was consistently detected across all experimental conditions and proposed as a chemical marker. Network pharmacology suggested that the identified metabolites may collectively contribute to vasodilation through modulation of PI3K/AKT/eNOS/NO, NF-κB/MAPK, and Ca²⁺/PDE/cAMP-cGMP signaling pathways. These findings establish wild fruiting as the optimal harvesting stage and position H. longipes leaves as a sustainable source of vasodilatory metabolites with phytopharmaceutical potential.