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- Study of the bioaccessibility, biodisponibility and cytotoxicity of Salvia divinorum: method development and in vitro evaluationPublication . Calado, Sara Agostinho; Alba, Maria Eugénia Gallardo; Duarte, Ana Paula Coelho; Pires, Bruno Miguel PinheiroSalvia divinorum is a psychoactive plant traditionally used in spiritual and medicinal contexts in Mexico, whose main active constituent is salvinorin A (SA). This diterpene exhibits a unique pharmacological profile, acting as a potent and selective agonist of the ?-opioid receptor, which distinguishes it from classical psychedelic substances. Due to these properties, the plant has attracted attention both as a recreational drug, particularly among young people in Europe and the United States, and within scientific research, given its potential therapeutic applications in the management of pain, depression, anxiety, and addiction. However, adverse effects and the absence of clear regulation raise toxicological concerns. The present study aimed to investigate the bioaccessibility and bioavailability of SA, with particular focus on absorption through the oral mucosa, one of the common routes of consumption. To this end, an in vitro model simulating the oral digestive process was developed, employing artificial saliva. Plant extraction was carried out from dried S. divinorum leaves, subjected to maceration and subsequent incubation with artificial saliva at 37 °C under agitation for 3 hours. The resulting extracts were analysed by highperformance liquid chromatography coupled to diode array detector (HPLC-DAD), confirming the presence of SA, but not its metabolite salvinorin B (SB). In parallel, human tongue carcinoma cells (UPCI:SCC154) were used to assess both permeability and cytotoxicity of the extract. The MTT assay revealed a dose- and timedependent effect, with reduced cell viability after prolonged exposures. The bioaccessibility assay further demonstrated that oral digestion markedly decreases the fraction of SA available for systemic absorption. The results confirmed that SA is effectively absorbed through the oral mucosa, accounting for the rapid and intense effects reported in the plant’s consumption. Nevertheless, a significant cytotoxic impact was also observed in cellular models, underscoring the need for caution in repeated or chronic use. From an analytical perspective, HPLC-DAD proved to be a robust technique for quantifying SA in complex matrices. In conclusion, S. divinorum combines considerable pharmacological potential with risks associated with uncontrolled use. The evidence gathered in this study highlights the need for further clinical investigations to evaluate safety and efficacy, as well as the development of formulations that could enable therapeutic applications of SA while minimising its adverse effects.