Toxicological Evaluation of Zingiber purpureum Extract and Its Effects on Gut Microbiota in Zebrafish

Zingiber purpureum Roscoe (Phlai) is renowned in Southeast Asia for its anti-inflammatory, antioxidant, and antibacterial activities, yet its safety and effects on gut health in vivo are not well characterized. This study investigated the biological effects of ethanolic-soluble Phlai extract (ESPE) in zebrafish (Danio rerio), focusing on chemical marker standardization in phenylbutanoids and cucurminoids, developmental toxicity, gut microbiota investigation, and tissue histopathology. Zebrafish embryos were exposed to ESPE to assess toxicity while adult females received an ESPE-enriched diet containing 20 mg ESPE/g diet (2% w/w) for 14 days. Post-treatment analyses included intestinal and multi-organ histology, and 16S rRNA sequencing for microbiome profiling. HPLC analysis confirmed that ESPE contained 9.91% (w/w) of (E)-1-(3,4-dimethoxyphenyl)butadiene (DMPBD) and 0.49% (w/w) curcumin. The 96-h LC50 for embryos was 45.54 µg/mL. In adults, ESPE improved gut health, as shown by greater mucosal thickness, increased goblet cells, and enhanced enterocyte organization. Microbiome analysis showed that overall gut microbial alpha diversity and beta diversity did not differ significantly between groups. However, exploratory differential abundance analysis identified differences in the relative abundance of specific genera in the ESPE-treated group, including Crenobacter, Cellvibrio, Pseudaeromonas, and Starkeya. Muscle tissue demonstrated healthy myofibril regeneration without fibrosis. ESPE was well tolerated, enhanced intestinal function, and without systemic toxicity. These findings support the potential of ESPE as a bioactive extract for further investigation of intestinal health and safety in translational pharmacology applications.