Squamocin as the Principal Toxic Constituent of Annona squamosa Seeds: Toxicity-Guided Isolation, Developmental and Neurotoxicity, and Metabolic Perturbations.

The seeds of Annona squamosa are widely used in traditional medicine and food, yet their safety profile remains unclear and the principal toxic constituent is unknown. This study aimed to identify the main toxic constituent and elucidate its toxicological mechanisms. A toxicity-guided isolation strategy using zebrafish larvae was employed, combined with morphological, behavioral, biochemical, metabolomic, and transcriptional analyses. Bioassay-directed fractionation identified squamocin, an annonaceous acetogenin, as the principal toxic constituent-the first reported acute toxicant from A. squamosa. Squamocin exerted potent multi-organ toxicity at nanomolar concentrations (LC10 = 62 nM; LC50 = 75 nM), including lethality, developmental teratogenicity, and craniofacial cartilage dysplasia, without substantial growth retardation. Neurotoxicity was dose-dependent: locomotor suppression at 20 nM and motor incoordination at 40 nM. Metabolomics revealed perturbations in amino acid and glycan metabolism, and transcriptional analysis confirmed altered expression of stress-related genes (pipox, myd88, and rigi). These findings establish squamocin as the major toxicant in A. squamosa seeds and provide a mechanistic framework for its broad toxicological spectrum, informing safer use of this material.