Screening of angiotensin-I converting enzyme inhibitory peptides derived from soft-shelled turtle yolk using two orthogonal bioassay-guided fractionations

- Orthogonal bioassay-guided fractionation.
- A novel ACEI peptide from SSTY.
- In vitro, in vivo and in silico ACEI study.

Two orthogonal bioassay-guided fractionations were simultaneously applied to screen angiotensin-I converting enzyme inhibitory (ACEI) peptides from soft-shelled turtle yolk (SSTY) hydrolysate. Peptides derived from thermolysin digest of SSTY were fractionated separately using reversed phased (RP) and strong cation exchange (SCX) chromatography, then the inhibitory activities of resulting fractions were examined using ACEI assay. The peptides in the most active RP and SCX fractions were characterized by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to give 11 and 35 non-redundant peptides, respectively. AKLPSW, simultaneously found in the active RP and SCX fractions, was identified as the most effective ACEI peptide. This identity of AKLPSW was further confirmed using synthetic peptide and its IC50 value was determined as 15.3 ± 0.9 μM. The kinetic study revealed that AKLPSW is a noncompetitive inhibitor and its interaction towards ACE was proposed using molecular docking. Furthermore, the antihypertensive effect of AKLPSW was demonstrated using spontaneously hypertensive rats.