Effects of the Agrobacterium rhizogenes rolC Gene Insertion on Secondary Metabolites Profile and In Vitro Biological Activity of Acmella oleracea (L.) R.K. Jansen

This study investigates the transformation of <i>Acmella oleracea</i> with the <i>Agrobacterium rhizogenes rolC</i> gene and evaluates its impact on phytochemical composition and biological activity. A total of 480 plant nodes were subjected to <i>Agrobacterium</i>-mediated transformation, leading to the regeneration of 35 putative transgenic plants. Molecular analysis confirmed the presence of the <i>rolC</i> transgene in 17 clones, of which four (C123, C127, C129, and C132) exhibited <i>rolC</i> mRNA expression. Phytochemical profiling of hydroalcoholic extracts of aerial parts (AP) and roots (R) revealed significant differences (<i>p</i> ≤ 0.05) between transgenic and non-transgenic plants (CTR). Compared to non-transgenic plants, transgenic AP exhibited lower total phenolic content but retained or increased flavonoid concentrations, particularly flavan-3-ols, whereas R extracts consistently showed reduced secondary metabolite levels. LC-DAD-ESI-MS analysis identified a diverse metabolite profile, with AP being notably rich in flavonoids (48.65%) and alkylamides (32.43%), including spilanthol. Functional assessments across antioxidant and anti-inflammatory assays demonstrated that R extracts exhibited stronger bioactivity compared to AP extracts, as indicated by lower IC₅₀ values (0.004-2.18 mg/mL for R vs. 0.007-7.24 mg/mL for AP). However, iron-chelating capacity was higher in AP extracts, correlating with flavonoid concentration. Hierarchical clustering confirmed that transgenic lines C123 and C127 most closely resembled the control, while C129 and C132 displayed distinct metabolic profiles. These findings highlight <i>rolC</i>'s role in modulating secondary metabolite synthesis, influencing both the phytochemical composition and functional properties of <i>A. oleracea</i> extracts.