Caffeic Acid–Chicoric Acid (1:1) Mixture Exerts Antioxidant Effects and Regulates Intestinal Health in Oxazolone-Induced Zebrafish

Caffeic acid (CaA) and chicoric acid (ChA) each exhibit significant antioxidant activity when used alone, yet their combined effects on antioxidant capacity and intestinal health in zebrafish remain unclear. This study used isobolographic analysis to identify a 1:1 ratio of CaA to ChA as optimal for synergistic antioxidant activity, with its ABTS and DPPH IC50 (21.65 μg/mL, 69.66 μg/mL) outperforming single monomers in vitro. In an oxazolone (Oxa)-induced zebrafish intestinal oxidative stress model, the CaA and ChA (CaA–ChA) mixture exerted antioxidant effects by upregulating the mRNA expression levels of HTR2A, Akt, Nrf2, and downstream antioxidant enzyme genes including SOD, CAT, and GPx in the zebrafish intestine, while downregulating Keap1 mRNA expression. Intestinal microbiota analysis revealed that the CaA–ChA mixture could positively regulate the intestinal microecological structure, characterized by targeted enrichment of the beneficial bacterium Cetobacterium and inhibition of the proliferation of potential pathogenic bacteria, including Bosea and Mycobacterium. Correlation analysis confirmed that the abundances of these key genera were closely associated with the expression of signaling pathway markers, suggesting that the microbiota–signaling pathway crosstalk was involved in the regulation of antioxidant processes. In conclusion, the CaA–ChA mixture (1:1) exerts a protective effect against intestinal oxidative stress, with the potential involvement of dual gut microbiota modulation and the HTR2A/Akt/Nrf2/Keap1 pathway. These findings provide experimental and theoretical support for the combined antioxidative application of CaA and ChA.