Natural products modulate social behavior and gut microbiota in a valproic acid-induced zebrafish model of autism spectrum disorder

Natural products rich in honey- and plant-derived bioactive compounds are increasingly investigated as modulators of the gut-brain-microbiome axis, a pathway implicated in autism spectrum disorder (ASD)-related behavioral and gastrointestinal alterations. This study evaluated whether two novel formulations, a honey-based product (API) and a plant extract-based product (MOD), modulate exploratory, anxiety-like, and social behavior and intestinal microbiota composition in an adult valproic acid (VPA)-induced zebrafish model of ASD. Adult wild-type zebrafish were assigned to control, VPA (300 μM), API (500 mg/L), MOD (500 mg/L), VPA+API, and VPA+MOD groups. Behavioral outcomes were assessed using the novel tank test and a T-maze social behavior test, while intestinal microbiota was profiled by 16S rRNA gene sequencing using an Oxford Nanopore platform. VPA exposure induced mild locomotor and anxiety-like behavioral alterations, whereas API and MOD further modified exploratory and social behavioral readouts in both healthy and VPA-treated zebrafish. Both formulations increased the time spent near the social stimulus; however, this finding should be interpreted cautiously because treatment-associated hypolocomotion and altered exploratory activity may influence T-maze performance. Microbiota profiling revealed a Pseudomonadota-dominated community across all groups, with descriptive treatment-associated shifts in secondary phyla and diversity patterns, particularly in combined VPA+API and VPA+MOD groups. However, no significant differences in alpha diversity, beta diversity, or genus-level abundance remained after multiple-testing correction. These findings suggest that API and MOD may modulate behavioral outcomes in adult zebrafish exposed to VPA, while their effects on gut microbiota remain exploratory and require validation in larger, adequately powered studies.