Extra-virgin olive oil (EVOO) is a rich source of bioactive compounds, including the secoiridoids oleocanthal (OLC) and oleacein (OLE), which exhibit antioxidant, anti-inflammatory, and neuroprotective activities. However, their direct impact on neuronal hyperexcitability and seizure susceptibility has not yet been specifically investigated in vivo . A sustainable extraction approach based on natural deep eutectic solvents (NaDESs) has recently enabled the production of a novel EVOO-derived extract enriched in OLC and OLE, whose toxicological and functional properties remain uncharacterized.

This study aimed to evaluate the acute toxicity and neuromodulatory effects of this new EVOO extract in zebrafish ( Danio rerio) larvae.

Embryos were exposed to a concentration range following OECD guideline No. 236 to define acute lethal endpoints. Non-toxic, sub-lethal concentrations (0.1, 0.25, and 0.5 mg/L), evaluated against vehicle-matched controls (NaDES and NaDES+PTZ), were selected for functional assessment in the pentylenetetrazole (PTZ) induced-seizure model. Behavioral responses were quantified across four light–dark locomotor tracking recording runs, integrated activity and burst count, with within-run comparisons and EVOO×PTZ interaction analyses. Transcriptional markers associated with neuronal activation ( fosab , bdnf ) and candidate neuromodulatory pathways ( serta , oxtra , oxtrb , nos1 ) were assessed.

The 96-h   was estimated at 4.230 mg/L (95% CI: 3.539–5.066 mg/L). Acute PTZ challenge induced marked hyperlocomotion accompanied by widespread transcriptional upregulation of all analyzed gene targets. NaDES itself altered baseline behavior. Pre-treatment with the EVOO extract reduced integrated activity and burst count in within-run comparisons, although   interactions indicated context-dependent effects without a simple monotonic pattern. These behavioral effects aligned with a coordinated downregulation of PTZ-induced immediate-early ( fosab , bdnf ) and pathway-related gene transcripts ( serta , oxtra , oxtrb , nos1 ).

In summary, EVOO-NaDES extract pre-treatment modulates PTZ-associated locomotor responses alongside correlated changes in selected transcriptional markers. Rather than establishing definitive functional mechanisms, this study provides a hypothesis-generating basis for candidate molecular targets, while the active vehicle and lack of electrophysiological validation preclude attributing these effects strictly to antiseizure activity.