Identification of Rubisco anxiolytic-like peptides (rALPs) by comprehensive analysis of spinach green leaf protein digest

Highlights: • Spinach Rubisco digested by gastrointestinal proteases showed anxiolytic-like effects. • rALPs were identified by comprehensive peptide analysis of the Rubisco digest. • rALPs exhibited potent anxiolytic-like effects after oral administration in mice. • rALP-1/rALP-1(1–7) and rALP-2 acted via 5-HT_1A and δ-opioid systems, respectively. Rubisco, an enzyme for photosynthetic carbon dioxide fixation, is a major green leaf protein and known as the most abundant protein on the Earth. We found that Rubisco digested mimicking gastrointestinal enzymatic conditions exhibited anxiolytic-like effects after oral administration in mice. Based on a comprehensive peptide analysis of the digest using nanoLC-Orbitrap-MS and the structure-activity relationship of known anxiolytic-like peptides, we identified SYLPPLTT, SYLPPLT and YHIEPV [termed Rubisco anxiolytic-like peptide (rALP)-1, rALP-1(1–7) and rALP-2, respectively], which exhibited potent anxiolytic-like effects after oral administration. The anxiolytic-like effects of rALP-1/rALP-1(1–7) were blocked by a serotonin 5-HT_1A receptor antagonist, whereas rALP-2-induced effects were inhibited by a δ-opioid receptor antagonist. In conclusion, novel Rubisco-derived anxiolytic-like peptides, rALP-1/rALP-1(1–7) and rALP-2, act via independent neural pathways.