SIMULTANEOUS DETERMINATION OF 8 SMALL ANTIHYPERTENSIVE PEPTIDES WITH TYROSINE AT THE C-TERMINAL IN LAMINARIA JAPONICA HYDROLYSATES BY RP-HPLC METHOD

A simple and efficient RP high-performance liquid chromatography method was developed for simultaneous analysis of eight small antihypertensive peptides with tyrosine at the C-terminal in Laminaria japonica hydrolysates (LHs). The chromatography was performed on an Xterra MS-C18 column at 45C, with an aqueous mobile phase (0.1% trifluoroacetic acid, pH 2.8) acetonitrile in a linear gradient at the flow rate of 0.7 mL/min, and detected at 218 nm. The detection limits ranged from 0.0624 to 0.2365 mu M. Correlation coefficients were greater than 0.9997. The recovery ranged from 92.17 to 120.08%. The small peptides were successfully determined by this method in LHs and the structures were confirmed by ESI-MS/MS, and the structure-activity relationships were discussed. The results demonstrated that the proposed method could be used to determine the peptides with tyrosine at the C-terminal, and to characterize a widespread detection mode for small peptide analysis in easy operation and low cost. PRACTICAL APPLICATIONS This study is the first to report that marine Laminaria japonica protein hydrolyzed by combined enzymes of the alcalase, trypsin and papain displayed high angiotensin-converting enzyme (ACE) inhibitory activity. An effective and efficient reverse phase (RP) high-performance liquid chromatography method was developed for simultaneous analysis of eight small antihypertensive peptides with tyrosine at the C-terminal. Adopting the proposed method, Laminaria japonica hydrolysates and the ACE inhibitory activities of small peptides will be successfully analyzed. The results also demonstrated that Laminaria japonica proteins would be a good source of hypotensive peptides.