ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes Giovanna Boschin a,⇑ , Graziana Maria Scigliuolo b , Donatella Resta b , Anna Arnoldi a,b a Laboratory of Food Chemistry and Mass Spectrometry, Department of Pharmaceutical Sciences, Università degli Studi di Milano, via Mangiagalli 25, 20133 Milano, Italy b HPF Nutraceutics s.r.l., via Balzaretti 9, 20133 Milano, Italy article info Article history: Received 14 February 2013 Received in revised form 3 June 2013 Accepted 17 July 2013 Available online 24 July 2013 Keywords: Angiotensin converting enzyme (ACE) inhibitors Enzymatic protein hydrolysates Hypertension Hippuric acid Legumes Lupin Pepsin Protein isolates abstract The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhib- itory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC 50 values of 224 and 226 lg/ml, respectively. Considering the prom- ising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin pro- tein isolates and purified protein fractions were tested. The most active mixture, showing an IC 50 value of 138 lg/ml, was obtained hydrolysing a mixture of lupin a + b conglutin. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Cardiovascular diseases, such as atherosclerosis, coronary heart disease, stroke, and heart failure, are a major health concern, be- cause they are one of the leading causes of death in most industria- lised countries. One of the main risk factors is hypertension, defined by the World Health Organisation (WHO) as the exceeding of 90 mmHg for the diastolic arterial pressure, and 140 mmHg for the systolic pressure. Hypertension is commonly treated with blood pressure lower- ing drugs, in particular with the inhibitors of the angiotensin I con- verting enzyme (ACE; EC 3.4.15.1), which plays an important role in regulating blood pressure in the renin-angiotensin system. This enzyme catalyses the conversion of the biologically inactive angio- tensin I to the potent vasoconstrictor angiotensin II, and inactivates the potent vasodilator bradykinin (Skeggs, Kahn, & Shumway, 1956). Inhibitors bind tightly to the ACE active site, competing with angiotensin I for occupancy; as a consequence, ACE cannot convert angiotensin I to angiotensin II (Skeggs et al., 1956). Considering that synthetic ACE inhibitors, such as captopril, lis- inopril and enalapril, may produce side effects, such as coughing, taste disturbances and skin rashes, there is interest in natural inhibitors, and numerous studies are focused on the production and isolation of ACE-inhibitory peptides from different food pro- teins. Food derived ACE-inhibitory peptides can be introduced into functional foods or dietary supplements. An alternative solution may be the incorporation into functional foods of specific proteins that can release ACE-inhibitory peptides during digestion. In both cases, their integrity, absorption and bioavailability are relevant is- sues (Roberts, Burney, Black, & Zaloga, 1999; Vermeirssen, van Camp, & Verstraete, 2004). The first active peptides were obtained from milk proteins (Nakamura et al., 1995), but some have been isolated from plant seeds, such as soybean (Chen, Yang, Suetsuna, & Chao, 2004; Wu & Ding, 2002), pea (Aluko, 2008; Barbana & Boye, 2010; Roy, Boye, & Simpson, 2010), rice, sunflower, and wheat (Guang & Phillips, 2009). The present study aims to compare the ACE-inhibitory activity of peptides derived from the enzymatic digestion of lupin and other grain legume proteins using the same experimental protocol. In addition to lupin, peptides derived from some purified protein 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.07.076 Abbreviations: ACE, angiotensinconverting enzyme; BSA, bovin serum albumin; E/S, enzyme/substrate; HA, hippuric acid; HHL, hippuryl-histidyl-leucine; HL, histidyl-leucine; LPI, lupin protein isolate; SDS–PAGE, sodium dodecyl sulphate– polyacrylamide gel electrophoresis; TPE, total protein extract. ⇑ Corresponding author. Tel.: +39 02 50318210; fax: +39 02 50318204. E-mail address: giovanna.boschin@unimi.it (G. Boschin). Food Chemistry 145 (2014) 34–40 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem