Safety Evaluation of Hot Water and Ethanol Extracts of Crescentia cujete L. Bark in Zebrafish Embryo Model for Potential Functional Ingredient Applications

Abstract

The exploration of natural plant sources for functional ingredients has gained significant attention in food science, with emphasis on bioactive compounds that can enhance human health. Crescentia cujete L. (calabash tree) is traditionally valued in folk medicine, yet the safety of its bark extracts for functional food applications remains underexplored. This study evaluated the safety performance of hot water and ethanol extracts of C. cujete bark using the zebrafish embryo toxicity (ZFET) model, an established in vivo tool for early toxicological screening. Fertilised zebrafish embryos were exposed to graded concentrations of each extract from 0 to 120 hr post-fertilisation (hpf), and endpoints including survival, hatching, heart rate, and morphological development were monitored. The hot water extract demonstrated a significantly higher median lethal concentration (LC₅₀) of 9.69 mg/mL, while the ethanol extract exhibited a markedly lower LC₅₀ value of 0.42 mg/mL (p<0.001), suggesting higher relative safety of the aqueous preparation. Dose-dependent reductions in survival and hatching rates were observed, with ethanol extracts producing more severe developmental delays and cardiac abnormalities. Heart rate disturbances, pericardial edema, and axial malformations were most prominent at concentrations near the ethanol LC₅₀ value, whereas the hot water extract induced only mild effects at higher concentrations. No significant changes in heart rate were observed with the hot-water extract, whereas significant reductions were observed at higher concentrations of the 50% ethanol extract (p<0.05). These findings indicate that the extraction solvent significantly influences the safety profile of C. cujete bark. The hot water extract demonstrated lower developmental toxicity in the zebrafish embryo model and warrants further investigation through phytochemical characterisation and mammalian toxicity studies. By establishing the first zebrafish-based safety evaluation of C. cujete bark, this study highlights the importance of integrating toxicological validation in the development of novel plant-derived functional ingredients.