Date seed-and licorice-derived nanoemulsions inhibit clinostomid metacercariae recovered from naturally infected Nile tilapia (Oreochromis niloticus): First report.

Herein, we present the development and comprehensive evaluation of green nanoemulsions derived from date seeds (DNE) and licorice (LNE) as antiparasitic agents targeting clinostomiasis in freshwater fish. Focusing on Clinostomum (C.) complanatum and Euclinostomum (E.) heterostomum-parasites that pose significant health risks to humans and cause substantial economic losses in aquaculture-these nanoemulsions were characterized using transmission electron microscopy, and dynamic light scattering. The analyses confirmed their uniform spherical morphology, nanoscale size, and robust colloidal stability. Molecular docking analyses revealed strong binding affinities between key phytoconstituents and essential parasitic enzymes, with glycyrrhizin exhibiting the highest affinity (-9.28 kcal/mol) for Euclinostomum cytochrome c oxidase. Key constituents in DNE included caffeic acid, epicatechin, ferulic acid, and 5-O-caffeoylshikimic acid; LNE featured glycyrrhizin among other phenolics. Both DNE and LNE induced marked, concentration- and time-dependent mortality in excysted metacercariae, with species-specific potency: LNE was more effective against C. complanatum (LC₅₀ = 0.032 %) while DNE showed superior activity against E. heterostomum (LC₅₀ = 0.004 %). Comet assays further revealed significant, dose-dependent DNA damage in both parasite species, with DNE causing more pronounced genotoxicity in E. heterostomum and LNE inducing greater DNA fragmentation in C. complanatum. These findings highlight the potential of plant-derived nanoemulsions as sustainable alternatives for parasite control in aquaculture, offering targeted, species-specific efficacy while minimizing ecological risks and reducing reliance on synthetic chemotherapeutants. The integration of such green nanotechnological approaches represents a promising strategy for effective disease management and the advancement of sustainable aquaculture practices. These resultshighlight the date seed and licorice-based nanoemulsions as promising in vitro antiparasitic candidates which justify further in vivo confirmation.