Characterization of novel angiotensin-I converting enzyme inhibitory peptides derived from Taiwan red quinoa ( Chenopodium formosanum Koidz .) seed proteins using two sequential bioassay-guided fractionations

Taiwan red quinoa ( Chenopodium formosanum Koidz .) is a pseudo-cereal crop native to Taiwan with a rich protein content that can potentially be a bioactive peptide precursor, such as angiotensin-I converting enzyme inhibitory (ACEI) peptides. Taiwan red quinoa seed protein (TRQSP) thermolysin hydrolysate showed a relatively potent ACE IC 50 value of 58.5 µg/mL. After two sequential bioassay-guided fractionations, fraction F4.3 showed the best ACEI activity (89.3%). Liquid chromatography-tandem high-resolution mass spectrometry (LC-HRMS) incorporated with de novo peptide sequencing and database searching were performed to identify the peptide sequences from fraction F4.3. Based on the in silico study, three potential ACEI peptides (LGAVPPRY, IARDSAAVF, and VYLAELHF) were synthesized to identify their ACEI activity. The ACE IC 50 values of LGAVPPRY (LY8), IARDSAAVF (IF9), and VYLAELHF (VF8) were calculated as 29.3, 56.4, and 115.7 µM, respectively. LY8 was a non-competitive inhibitor, while IF9 and VF8 were competitive ACE inhibitors, as confirmed by the inhibition mechanism and simulation of molecular docking studies. The ACEI pre-incubation assay suggested that LY8 was a true inhibitor type; in the meantime, IF9 and VF8 were pro-drug inhibitor types. To our best knowledge, this is the first study to screen ACEI thermolytic peptides derived from Taiwan red quinoa seed protein.