Effects of Eriobotrya japonica (Thunb.) Lindl. Leaf Extract on Zebrafish Embryogenesis, Behavior, and Biochemical Pathways

Abstract

Eriobotrya japonica (Thunb.) Lindl. leaves are rich in polyphenolic compounds, yet their toxicological effects in aquatic models remain poorly understood. This study evaluated the impact of a hydroethanolic E. japonica leaf extract on zebrafish embryos through the use of morphological, behavioral, and biochemical parameters. The 96 h LC₅₀ was determined as 189.8 ± 4.5 mg/L, classifying the extract as practically non-toxic, according to OECD guidelines. Thereby, embryos were exposed for 90 h to 75 and 150 mg/L concentrations of the E. japonica leaf extract. While no significant effects were noted at the lowest concentration of 150 mg/L, significant developmental effects were observed, including reduced survival, delayed hatching, underdevelopment of the swim bladder, and retention of the yolk sac. These malformations were accompanied by marked behavioral impairments. Biochemical analysis revealed a concentration-dependent increase in superoxide dismutase (SOD) and catalase (CAT) activity, suggesting the activation of antioxidant defenses, despite no significant change in reactive oxygen species (ROS) levels. This indicates a potential compensatory redox response to a pro-oxidant signal. Additionally, the acetylcholinesterase (AChE) activity was significantly reduced at the highest concentration, which may have contributed to the observed neurobehavioral changes. While AChE inhibition is commonly associated with neurotoxicity, it is also a known therapeutic target in neurodegenerative diseases, suggesting concentration-dependent dual effects. In summary, the E. japonica leaf extract induced concentration-dependent developmental and behavioral effects in zebrafish embryos, while activating antioxidant responses without triggering oxidative damage. These findings highlight the extract's potential bioactivity and underscore the need for further studies to explore its safety and therapeutic relevance.

Keywords: Danio rerio; Eriobotrya japonica (loquat); behavior; biochemical pathways; embryogenesis.

Figure 1

Morphological analysis of zebrafish embryos after 96 h of exposure to extract from E. japonica leaves. Results on the percentage of malformations (a), size (b), eye area (c), swim bladder area (d), yolk area (e), head area (f), pericardial area (g), head–trunk angle (h), and representative images of malformations observed in embryos (i). The values 75 and 150 denote concentrations in mg/L; * indicates significant differences (p < 0.05), ** indicates significant differences (p < 0.01), and ns indicates no significant differences between groups (p > 0.05); b: swim bladder, y: yolk sac.

Figure 2

Determination of speed (a), distance moved (b), immobility (c), absolute turn angle (d), distance to center (e), and representative locomotion path of larvae exposed to the different E. japonica leaf extract concentrations (f). The values 75 and 150 denote concentrations in mg/L; * indicates significant differences (p < 0.05), ** indicates significant differences (p < 0.01), and ns indicates no significant differences between groups (p > 0.05).

Figure 3

Levels of indicators of oxidative stress—ROS (a), cellular damage—apoptosis (b), and mitochondrial stress, neurotoxicity, and cellular dysfunction—ΔΨm (c). The values 75 and 150 denote concentrations in mg/L; ns indicates no significant differences between groups (p > 0.05).

Figure 4

Biomarkers of the 1st line of antioxidant defense: SOD (a), CAT (b), and GPx (c) activity. Values are expressed as the mean ± standard deviation of 5 independent replicates. The values 75 and 150 denote concentrations in mg/L; ** indicates significant differences (p < 0.01), and ns indicates no significant differences between groups (p > 0.05).

Figure 5

Metabolic biomarkers: GR (a) and GST (b) activity. The values 75 and 150 denote concentrations in mg/L; ns indicates no significant differences between groups (p > 0.05).

Figure 6

Levels of biomarkers of first-line antioxidant—GSH (a), metabolic—GSSG (b), and redox indices—OSI (c). The values 75 and 150 denote concentrations in mg/L; ns indicates no significant differences between groups (p > 0.05).

Figure 7

General indicators of cell damage and oxidative stress: LPO (a), protein carbonyls (b), and DNAds (c). The values 75 and 150 denote concentrations in mg/L; ns indicates no significant differences between groups (p > 0.05).

Figure 8

Levels of indicators of cell damage and damage to the nervous system (neurotoxicity): LDH activity (a), AChE activity (b), and NO levels (c). The values 75 and 150 denote concentrations in mg/L; ** indicates significant differences (p < 0.01), and ns indicates no significant differences between groups (p > 0.05).