Anxiolytic, Anticonvulsant Effects, and Cytotoxic Analysis of Methanolic Extracts from the Pulp and Seeds of Annona squamosa L.

Introduction: Generalized anxiety disorder is a neurological condition that can lead to cardiovascular disease and type II diabetes mellitus. Physical activity and the consumption of foods rich in antioxidant compounds are strategies that substantially improve anxiety. Purpose: This study aimed to assess the cytotoxic potential of methanolic extracts obtained from the pulp and seeds of Annona squamosa using different analytical approaches, in addition to investigating their anxiolytic and anticonvulsant effects in adult zebrafish. Methods: For toxicity in Artemia salina, eggs were incubated and, after hatching, the larvae were exposed to concentrations of extracts (0.06-5 μg/mL) for 24 hours. Toxicity in Drosophila melanogaster involved exposure to different concentrations of extracts (125-2000 μg/mL) with mortality readings every 3, 6, 12, 24, and 48 hours. Locomotor damage was assessed using the negative geotaxis test. In Danio rerio, anxiolytic activity was investigated through open field, light/dark, and PTZ-induced convulsion tests, where fish were exposed to oral extracts at different concentrations (40, 200, and 400 mg/kg). GABAergic neuromodulation was analyzed using benzodiazepine antagonists. Results: The extracts demonstrated high cytotoxicity against A. salina, with LD50 values of 2.24 μg/mL for the pulp extract and 1.64 μg/mL for the seed extract. In D. melanogaster, toxic effects were limited, with mild toxicity observed for the seed extract at high concentrations. In the zebrafish model, the seed extract showed toxicity at doses higher than 200 mg/kg, while the pulp extract was non-toxic. In terms of locomotor activity, both extracts affected the locomotor behavior of fish and D. melanogaster, with the seed exhibiting more pronounced effects. In the anxiolytic test, the extracts displayed effects similar to diazepam, modulating behavior via GABAergic neurotransmission. Finally, the seed extract demonstrated anticonvulsant activity in zebrafish, particularly in the acute and chronic stages of PTZ-induced seizures, indicating specific therapeutic potential. Discussion: The pulp and seed extracts have different toxicological profiles, with greater toxicity associated with the seed extract, especially at high concentrations. The changes in locomotor behavior and the anxiolytic effects observed in both models suggest that they act on neuromodulatory pathways, possibly mediated by the GABAergic system. The anticonvulsant activity of the seed extract in the acute and chronic stages of PTZ-induced seizures in zebrafish points to a potential therapeutic use in neurological disorders. Conclusion: The study revealed that the methanolic extract from the pulp of A. squamosa exhibited a safe anxiolytic effect, while the seed extract showed both anxiolytic and anticonvulsant effects, albeit with acute toxicity. New formulations are suggested to reduce the toxicity of the seeds and explore the plant’s therapeutic potential.