Identification of Ginsentide-like Peptides from Cacao Beans with Oxidative Stress Protection

Chocolate, derived from Theobroma cacao beans, is valued for its antioxidative benefits. However, little is known about cacao-derived peptides that counter oxidative stress. Ginsentides found in Panax ginseng are cysteine-rich peptides that exhibit the "cure-all" health benefits of ginseng by coordinating multiple physiological systems to reduce cellular stress and damage. Here, we report the discovery and characterization of ginsentide-like peptides from T. cacao beans and chocolate. By combining database searches, isolation, and mass spectrometry analysis, we identified a panel of 33-residue ginsentide-like peptides, termed cocotides tC1-tC12, which are also rich in methionine. We isolated, purified, sequenced, and chemically replicated cocotide tC1 for detailed characterization. We showed that the highly compact tC1 is resistant to proteolytic degradation, and its fluoresence-labeled form shows cell-penetrating property. Modeling by AlphaFold3 revealed that cocotide tC1 and ginsentide share a similar structural fold, but tC1 contains a metal-ion-binding site composed of Met and Asn. We used N-terminal biotinylated tC1 and affinity-enrichment mass spectrometry to identify ferrochelatase as one of its intracellular targets. Functional studies demonstrated that cocotide tC1 is a Fe³⁺-binding peptide and a strong antioxidant. Tandem-mass-tag-based quantitative proteomics analysis showed that cocotide tC1 affects proteins associated with mitochondrial functions, oxidative stress, cell proliferation and repair, protein ubiquitination, and DNA damage in HaCaT keratinocytes. Altogether, our findings indicate that cocotides are ginsentide-like peptides with the potential to maintain cellular homeostasis and protect against oxidative damage in skin keratinocytes.