In Vivo Atlas of Neuroprotective Cyclic Dipeptides Derived from Food Gelatin Using Peptidomics and Feature-Based Molecular Networking

Food gelatin is widely used in the food industry, yet its <i>in vivo</i> active substances remain unclear. This study proposes a strategy for <i>in vivo</i> research on donkey-hide gelatin that integrates peptidomics, feature-based molecular networks (FBMN), and targeted spatiotemporal metabolomics to address the above issues. Peptidomics based on database searching revealed a time-dependent decrease in peptide numbers and an increase in oligopeptide ratios in the small intestine over 24 h, with oligopeptides increasing from 50 to 100%. FBMN visualization in serum allowed the first identification of cyclodipeptides (CDPs) originating from gelatin. Targeted metabolomics detected 35 CDPs in serum, with 12 proline/hydroxyproline-CDPs being particularly abundant. Spatiotemporal analysis revealed different distributions of CDPs in the gastrointestinal and serum, and they showed prolonged retention <i>in vivo</i>. Twelve proline/hydroxyproline-CDPs displayed neuroprotective effects in HT-22 cells, with cyclo(Hyp-Ala) showing the highest efficacy. This study presents a strategy for identifying exogenous bioactives in complex matrices, applicable to other food proteins.