Toxicological profile and neuromodulatory effects of a secoiridoid-rich extra-virgin olive oil extract in a zebrafish model of chemically induced seizures

Background: 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. Purpose: This study aimed to evaluate the acute toxicity and neuromodulatory effects of this new EVOO extract in zebrafish ( Danio rerio) larvae. Methods 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. Results: The 96-h LC50 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 EVOO×PTZ 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 ). Conclusion: 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.