Enhanced In Vivo Antinociceptive Activity and GC–MS Chemical Profiling of In Vitro-Grown Acmella radicans Plants Elicited with Rhizopus microsporus

Pain remains a major global health challenge, driving the search for safer analgesics from medicinal plants. Although Acmella radicans is traditionally used for pain relief, the effects of fungal elicitation on its chemical profile and antinociceptive activity remain unknown. Therefore, this study evaluated the effects of Rhizopus microsporus elicitation on the chemical profile and in vivo antinociceptive activity of lipophilic fractions from in vitro-grown A. radicans. In vitro-grown plants were biotically elicited with aqueous mycelial extracts of R. microsporus. Lipophilic fractions obtained from ethanolic extracts were analyzed by GC–MS and evaluated for antinociceptive activity using the mouse formalin test. Elicitation with a 0.5% mycelial extract (48 h fungal culture, 48 h exposure) increased ethanol extract yield to 18.8%, compared with 15.3% in non-elicited in vitro plants and 6.1% in wild plants. GC–MS analysis tentatively identified 62 metabolites and indicated marked changes in the lipophilic chemical profile, including lower relative abundance of spilanthol and higher relative abundances of phenethyl alkamides, stigmasterol, β-sitosterol, and 2,4-di-tert-butylphenol, along with the tentative identification of (2E,4E)-N-(2-phenylethyl)deca-2,4-dienamide, a compound not previously reported in the genus Acmella. LFEP showed the highest efficacy among the plant fractions, reducing neurogenic pain by 60.3% versus 88.7% with tramadol, while its significant effect in the inflammatory phase approached that of diclofenac (54.1% inhibition), with tramadol producing the strongest inhibition (81.0%). Fungal elicitation modified the lipophilic chemical profile of A. radicans and enhanced its in vivo antinociceptive activity, supporting its application as a biotechnological strategy to improve the pharmacological potential of medicinal plant in vitro cultures.