Zhang_2026_Chem.Biol.Interact_432_112014

Isofraxidin, a hydroxylated coumarin occurring in traditional medicinal plants such as Eleutherococcus senticosus, has been widely investigated for anti-inflammatory, antioxidant and neuroprotective actions in adult models, yet its developmental safety is unclear. Wild-type AB zebrafish embryos were exposed to isofraxidin from 6 to 96 hpf. Survival was monitored to calculate the 96 hpf LC(50), and developmental endpoints (malformations, body length, heart rate and yolk sac area) were assessed at 96 hpf. Neurobehavior was quantified by spontaneous tail coiling and locomotor tracking. Neurodevelopment was evaluated using brain ventricle morphology, neuronal fluorescence in Tg(elavl3:EGFP) larvae, acetylcholinesterase activity, and qRT-PCR of neurodevelopment-related genes. Network toxicology was applied to predict intersecting targets and enriched pathways, followed by validation of oxidative stress (ROS, SOD, CAT, GSH and MDA) and apoptosis (acridine orange staining and apoptosis-related transcripts). Isofraxidin caused dose dependent developmental toxicity, reducing survival and increasing malformations at higher concentrations. Sublethal exposure produced robust neurobehavioral deficits, with decreased tail coiling and reduced locomotor activity. Concordantly, isofraxidin impaired brain ventricle development, reduced neuronal fluorescence, decreased AChE activity, and dysregulated neurodevelopmental gene expression. Mechanistically, network toxicology highlighted oxidative stress and apoptosis-associated pathways; experimentally, isofraxidin increased ROS accumulation, suppressed antioxidant defenses (lower SOD, CAT and GSH), elevated lipid peroxidation (higher MDA), and enhanced apoptotic signals in the brain region. These findings demonstrate developmental neurotoxicity of isofraxidin in zebrafish and support oxidative stress and apoptosis as a key mechanism, emphasizing the need to evaluate early-life safety ethnopharmacological bioactives.