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Fig. 3 Evaluation of anti-inflammatory activity of N3 by assessing the COX-2 protein expression using immunohistochemistry staining in zebrafish larvae. A) Immunohistochemistry staining of COX-2 protein in zebrafish larvae head region. B) Protein expression in % is quantified by staining intensity using ImageJ software. Experimental groups (n = 15/group) included zebrafish larvae treated with N3 at 50 µM, N3 at 100 µM, PTZ (6 mM), positive control (PC) Valproic acid at 100 µM, and control group. Each assay was conducted in triplicate with three independent experiments. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. Significant differences between tested samples and the negative control are indicated by ****p < 0.0001, and ns-not significant