Purification of a Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Peptide with an Antihypertensive Effect from Loach (Misgurnus anguillicaudatus)

To isolate and characterize novel angiotensin I-converting enzyme (ACE) inhibitory peptide from loach (Misgurnus anguillicaudatus), six proteases, pepsin, alpha-chymotrypsin, bromelain, papain, alcalase, and Neutrase, were used to hydrolyze loath protein. The hydrolysate (LPH) generated by bromelain [ratio of enzyme to substrate, 3:1000 (w/w)] was found to have the highest ACE inhibitory activity (IC50, 613.2 +/- 8.3 mu g/mL). Therefore, it was treated by ultrafiltration to afford fraction of LPH-IV (MW < 2.5 kDa) with an IC50 of 231.2 +/- 3.8 mu g/mL, having higher activity than the other fractions. Then, LPH-IV was isolated and purified by consecutive purification steps of gel filtration chromatography and reverse-phase high-performance liquid chromatography to afford a purified peptide with an IC50 of 18.2 +/- 0.9 mu g/mL, an increase of 33.7-fold in ACE inhibitory activity as compared with that of LPH. The purified peptide was identified as Ala-His-Leu-Leu (452 Da) by Q-TOF mass spectrometry and amino acid analyzer. An antihypertensive effect in spontaneously hypertensive rats revealed that oral administration of LPH-IV could decrease systolic blood pressure significantly.