Integrated quantum chemical and in vitro investigation of Capsanthin antioxidant activity: Mechanism (HAT), cultivar variability, enhanced bioavailability, and key gene expression in peppers

Capsanthin, the main bioactive component in red peppers, has potent antioxidant properties; however, its biosynthetic regulation and antioxidant mechanisms require further elucidation. In this study, we investigated 10 pepper cultivars through integrated quantum chemical calculations and in vitro assays. Theoretical calculations revealed that the antioxidant mechanism of capsanthin involves hydrogen atom transfer. Microwave-assisted extraction coupled with macroporous resin purification revealed significant interspecific variation in the capsanthin content. In vitro evaluation showed concentration-dependent antioxidant activity, with Bayan exhibiting the strongest capacity. The antioxidant properties and bioavailability of chili peppers increased after in vitro digestion. The results of the gene expression analysis revealed LUT5 as the most highly expressed gene across cultivars, followed by CrtR and Ccs . Enzyme activity assays revealed that the activity of CRTR-β consistently exceeded that of CCS, which was correlated with gene expression levels. • The stable conformation of capsanthin was optimized using Gaussian software and DFT methods • Thermodynamic calculations and comparison with Vc confirmed HAT as the dominant antioxidant mechanism of capsanthin • Bayan demonstrated the strongest concentration-dependent antioxidant activity in the in vitro evaluation. • Ccs, LUT5, and CrtR are key biosynthetic genes for capsanthin in peppers