A Comprehensive Chemical–Biological Investigation of the Moderately Toxic Plant Prospero autumnale: Insights into Its Bioactive Potential Using In Vitro and In Vivo Models

Prospero autumnale L. is a Mediterranean medicinal plant traditionally employed for inflammatory and neurological disorders. Nonetheless, its safety profile, toxicity, and application for treating inflammation and pain are yet to be comprehensively established. This investigation aimed to assess the bioactivity and toxicity of extracts derived from its aerial (AgP) and underground (UgP) parts. The phytochemical constituents of various P. autumnale extracts were analyzed using LC-MS/MS, and their phenolic content was quantified. The biological activities were evaluated through in vitro assays—including antioxidant, anti-inflammatory, acetylcholinesterase-inhibitory, and photoprotection assessments—and in vivo experiments, including evaluations of acute oral toxicity, anti-inflammatory, and analgesic effects. UgP extracts demonstrated significant antioxidant activity, with the methanolic extract exhibiting the highest reducing and superoxide scavenging capacities. Dichloromethane and ethyl acetate extracts performed exceptionally well in ABTS and DPPH assays. The aqueous extract from AgP exhibited noteworthy anti-inflammatory and analgesic effects, surpassing diclofenac in vitro and demonstrating efficacy in vivo. It also showed considerable acetylcholinesterase inhibition, while the ethyl acetate extract displayed high photoprotective potential. The acute toxicity was moderate (LD50: 300–400 mg/kg), indicating dose-dependent risks. LC-MS/MS analysis revealed diverse phenolics potentially contributing to both therapeutic and adverse effects. This research enhances the medicinal prospects of P. autumnale, provides new perspectives on plant utilization, and suggests its potential as a natural anti-inflammatory agent. However, due to moderate toxicity and dose-dependent effects, cautious application is advised. These findings underscore the importance of toxicological evaluation alongside bioactivity screening in ethnopharmacology to ensure safety.