Protective effect of the ethanol extract of C. cyrtophyllum leaves (EE) against CuSO₄ toxicity. (A) Mortality rates of zebrafish larvae after treatment with 20 µM CuSO₄ and ethanol extracts of C. cyrtophyllum (EE) at different doses. (B) Representative photographs of zebrafish larvae treated with 20 µM CuSO₄ and ethanol extracts of C. cyrtophyllum (EE) at different doses. The data are presented as mean ± S.E. for three different experiments performed in triplicate. ### p < 0.001, ** p < 0.01, and *** p < 0.001 compared to the CuSO₄ alone group.

Effect of the ethanol extract of C. cyrtophyllum leaves (EE) on Cu²⁺ chelation ability. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and *** p < 0.001 compared to the control group.

Inhibitory effect of ethanol extract (EE) from leaves of C. cyrtophyllum Turcz on CuSO₄-stimulated ROS production in zebrafish larvae. The zebrafish were exposed to different concentrations of EE (5, 20, and 40 µg/mL) or 100 µM quercetin for 1 h and then exposed to 10 µM CuSO₄ for 20 min to induce oxidative stress. After 20 min, larvae were incubated with H2DCFDA (20 µg/mL) for 1 h. Fluorescent probes were used to detect and quantify oxidative stress by measuring the fluorescent intensity (FI) in larvae using Autovision software and a BD pathway 855 system (BD Biosciences). (A) Fluorescence intensity obtained from individual zebrafish larvae. The data are presented as medians for six different larvae, * p < 0.05 and ** p < 0.01 compared to the CuSO₄ alone Group. (B) Representative fluorescence micrographs of ROS production.

Effect of ethanol extract (EE) of C. cyrtophyllum Turcz leaves on expression of antioxidant genes: sod (A); gpx4 (B); hsp70 (C), and gadd45bb (D). The zebrafish larvae were exposed to EE for 1 h and to CuSO₄ 10 µM for 4 h. After 4 h, larvae were collected for qPCR analysis. The relative gene expressions are presented as the ratio of the quantity of candidate gene/average quantity of housekeeping genes. A pool of 20 larvae per group (n = 3) was used. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, and *** p < 0.001 compared to the CuSO₄ alone group.

Effect of ethanol extract (EE) of C. cyrtophyllum Turcz leaves on the expression of genes in the eicosanoid pathway (pla2, cox-2, and c3a.). The zebrafish larvae were exposed to EE for 1 h and CuSO₄ 10 µM for 4 and 24 h. After 4 or 24 h, larvae were collected for qPCR analysis. The relative gene expressions are presented as the ratio of the quantity of candidate gene/average quantity of housekeeping genes. A pool of 20 larvae per group (n = 3) was used. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, and *** p < 0.001 compared to the CuSO₄ alone group.

Relative expression of genes involved in immune responses (il-1ß and il-8) of zebrafish larvae after exposure to CuSO₄ and treatment with the ethanol extract (EE) of C. cyrtophyllum Turcz at different doses. The zebrafish larvae were exposed to EE for 1 h and to CuSO₄ 10 µM for 4 and 24 h. After 4 or 24 h, larvae were collected for qPCR analysis. The relative gene expressions are presented as the ratio of the quantity of candidate gene/average quantity of housekeeping genes. A pool of 20 larvae per group (n = 3) was used. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, *** p < 0.001 compared to the CuSO₄ alone group.

Relative expression of genes involved in immune responses (tnf-α and nf-ƙb) of zebrafish larvae after exposure to CuSO₄ and treatment with the ethanol extract (EE) of C. cyrtophyllum Turcz at different doses. The zebrafish larvae were exposed to EE for 1 h and to CuSO₄ 10 µM for 4 and 24 h. After 4 or 24 h, larvae were collected for qPCR analysis. The relative gene expressions are presented as the ratio of the quantity of candidate gene/average quantity of housekeeping genes. A pool of 20 larvae per group (n = 3) was used. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, and *** p < 0.001 compared to the CuSO₄ alone group.

Relative expression of genes involved in immune responses (mpo) of zebrafish larvae after exposure to CuSO₄ and treatment with the ethanol extract (EE) of C. cyrtophyllum Turcz. The data are presented as mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, *** p < 0.001 compared to the CuSO₄ alone group.

Relative expression of anti-inflammatory gene (il-10) of zebrafish larvae after exposure to CuSO₄ and treatment with the ethanol extract (EE) of C. cyrtophyllum Turcz at different doses. The zebrafish larvae were exposed to EE for 1 h and to CuSO₄ 10 µM for 4 and 24 h. After 4 or 24 h, larvae were collected for qPCR analysis. The relative gene expressions are presented as the ratio of the quantity of candidate gene/average quantity of housekeeping genes. A pool of 20 larvae per group (n = 3) was used. Each bar represents the mean ± S.E. for three different experiments performed in triplicate. * p < 0.05, ** and ### p < 0.01, and *** p < 0.001 compared to the CuSO₄ alone group.