In Vitro Analysis of Antioxidant Properties of Mint Oils

Feed use of antioxidants is critical in protecting animals from oxidative stress. Due to consumer resistance to synthetic antioxidants, there is growing interest of using alternative natural substance in animal feed industry. The current study aimed to evaluate the antioxidant properties of peppermint oil, spearmint oil, and scotch oil using in vitro tests and cell culture models. The general antioxidant capacity of mint oils was first evaluated through four chemical-based assays including DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay (DPPH), Trolox equivalent antioxidant capacity assay (TEAC), reducing power assay, and ferric reducing antioxidant power assay (FRAP) with the tested doses from 0 to 500 mg/mL for each mint oil. The effectiveness of mint oils in mitigating lipid peroxidation was subsequently analyzed at various doses (from 0 to 2000 µg/mL) using freshly harvested liver tissue from pigs. In addition, IPEC-J2, a porcine jejunum epithelial cell line, was employed as in vitro model to determine the effect of mint oils on intestinal oxidative damage of animals. IPEC-J2 cells were cultured with each mint oil (0, 5, 10, 25, 50, 100, and 200 µg/mL) to test cell viability and cellular antioxidant activity (CAA). The optimal dose (25 µg/mL) was chosen to perform intracellular antioxidant assay by analyzing the production of glutathione disulfide and total glutathione by H2O2 stimulated IPEC-J2 cells. All data were analyzed by PROC MIXED of SAS. CONTRAST statements were performed to assess linear or quadratic effects of mint oils given at various doses. Chemical-based assays indicated that all mint oils had antioxidant activity, while peppermint oil exhibited (P < 0.05) the lowest half maximal effective concentration in DPPH and TEAC assays and the highest reducing capacity in FRAP and reducing power assays. Lipid peroxidation was inhibited (linear, P < 0.001) by all mint oils in a dose-dependent manner, and the lowest lipid peroxidation was observed at 1,000 µg/mL of all mint oils. High dose of mint oils (200 µg/mL) caused cell death. The maximal CAA was observed at 5 µg/mL for peppermint oil, and 100 µg/mL for spearmint oil and scotch oil. The addition of 25 µg/mL of spearmint oil or scotch oil increased (P < 0.05) the production of glutathione from H2O2-treated IPEC-J2 cells suggesting a mechanism of enhancing endogenous antioxidant defense. In conclusion, all three mint oils have in vitro antioxidant effects with a greater response observed in peppermint oil. More research is warranted to evaluate the antioxidant capacity of 3 mint oils in vivo.