Anxiolytic and Memory Protective Effects of Withanolide D Isolated From Acnistus arborescens in Adult Zebrafish

This study evaluated the anxiolytic and memory-protective effects of withanolide D in adult zebrafish (Danio rerio). The compound was administered at doses of 4, 20, and 40 mg/kg and showed no signs of toxicity after 96 h of observation at any of the doses tested. In the open field test, withanolide D reduced locomotor activity, indicating a sedative-like effect. Nevertheless, in the light/dark test, doses of 20 and 40 mg/kg produced an anxiolytic-like response, suggesting that anxiolytic and motor effects may partially overlap depending on dose. The anxiolytic effect observed at the minimum effective dose (20 mg/kg) was reversed by flumazenil (FMZ), supporting the involvement of a benzodiazepine-sensitive GABA_A modulatory domain. Furthermore, withanolide D, at a dose of 4 mg/kg, prevented ethanol-induced memory impairment in the inhibitory avoidance task, suggesting a protective effect on memory consolidation. Molecular docking analyses revealed favorable interactions of withanolide D at the extracellular α1γ2 interface of the GABA_A receptor, supporting a putative allosteric interaction in a functionally related modulatory region rather than definitive occupation of the classical diazepam site. Consistently, normal mode analysis (NMA) showed that withanolide D increases receptor mobility compared to diazepam, with RMSF values reaching up to 0.98 Å, indicating enhanced structural flexibility and dynamic modulation of the protein. Thus, these findings suggest that withanolide D has therapeutic potential for the treatment of anxiety, in addition to providing protective effects on memory.