Bioassay-guided Isolation of Antibacterial Compounds from Ethanolic Extract of Alstonia scholaris (L.) R. Br. Leaves and Developmental Toxicity Evaluation in Zebrafish (Danio rerio) Embryos.

Aquaculture faces severe threats from bacterial disease outbreaks, creating an urgent need for safe, and sustainable alternatives to synthetic antibiotics and harmful chemicals. Alstonia scholaris (L.) R. Br. (Apocynaceae), widely recognized in traditional medicine for its therapeutic potential, has received limited attention in aquaculture. This study evaluated the antimicrobial potency of A. scholaris leaf extracts against key fish pathogens. The ethanolic extract showed promising broad-spectrum antibacterial potency and further bioassay-guided fractionation of it targeting Pseudomonas fluorescens led to putative identification of oleanolic acid as the principal bioactive constituent. The isolated compound exhibited broad-spectrum antibacterial activities against P. fluorescens, Streptococcus agalactiae, and Streptococcus iniae. Safety evaluation using zebrafish embryos demonstrated that the ethanolic extract caused no mortality at or below 125 µg/mL and was found non-teratogenic, while, the isolated compound exhibited no embryotoxicity up to 300 µg/mL concentration. This study reports the first identification of oleanolic acid from the ethanolic extract of A. scholaris leaves using bioassay-guided fractionation targeting P. fluorescens. These findings highlight the potential of the A. scholaris leaves ethanolic extract and its isolated active compound as safe, effective, and sustainable antimicrobial alternatives for bacterial disease management in aquaculture.