Cannabis sativa Extract Modulates Gut Microbial Metabolism in Autism Spectrum Disorder and Neurotypical Microbiota: An Exploratory In Vitro Study

Background: Gut microbiota alterations have been implicated in Autism Spectrum Disorder (ASD), but the effects of cannabinoid-based interventions on microbial metabolism remain unclear. This study evaluated the effects of a standardized Cannabis sativa extract on microbial composition and metabolism, epithelial barrier integrity, and inflammatory responses using an in vitro colonic fermentation model. Methods: Fecal samples from 10 children with ASD and 10 neurotypical controls were pooled within groups to generate one ASD-derived (ASD group) and one neurotypical inoculum (NT-group). Each inoculum underwent 48 h anaerobic fermentation in three independent vessels per condition with 0.02, 0.10, or 0.50 mg/mL extract. Cytotoxicity was assessed in Caco-2 cells, microbial composition by 16S rRNA gene sequencing, and ammonia and short-chain fatty acids in both groups. Barrier integrity and inflammatory responses were evaluated only with ASD-derived fermentation supernatants in a Caco-2/THP-1 co-culture model. Results: The extract showed low cytotoxicity in Caco-2 cells across the concentrations evaluated. In the ASD group, treatment was associated with lower ammonia and acetate and higher butyrate concentrations, while propionate remained unchanged. Alpha and beta diversity were largely preserved, and epithelial barrier integrity was not significantly altered. Supernatants from the 0.50 mg/mL condition were associated with lower TNF-α concentrations, whereas IL-6, IL-8, and IL-10 did not differ among conditions. Conclusions: The standardized Cannabis sativa extract modulated microbial metabolism, reducing ammonia production and, in ASD microbiota, increasing butyrate while decreasing acetate, without major changes in microbial diversity. The highest concentration also reduced TNF-α production in the Caco-2/THP-1 model, with no significant effects on barrier integrity or other cytokines. These exploratory findings suggest a potential effect of the extract on microbial metabolic and inflammatory responses and should be considered hypothesis-generating.