Mechanisms of food protein-derivedantihypertensive peptides other than ACEinhibition

Chibuike C. Udenigwe *, Aishwarya MohanHealth and Bioproducts Research Laboratory, Department of Environmental Sciences, Faculty of Agriculture,Dalhousie University, Truro, NS, Canada B2N 5E3

A B S T R A C T

Hypertension is an important risk factor for developing cardiovascular disease and a major

contributor to global mortality. Cryptic bioactive peptides released from food proteins have

been widely pursued for the management of hypertension, mostly based on their per-

ceived activity in inhibiting angiotensin I-converting enzyme of the physiological blood

pressure-regulating renin angiotensin system (RAS) pathway. However, there is consider-

able evidence that food protein-derived peptides can interact with other RAS steps and related

pathways in the vascular system, potentially contributing to blood pressure reduction. This

contribution highlights these plausible antihypertensive mechanisms induced by food

peptides other than ACE inhibition including renin and endothelin system inhibition, an-

giotensin receptor and calcium channel blocking, and arginine–nitric oxide pathway-

mediated effects. It appears that the antihypertensive peptides and hydrolysates may employ

concerted mechanisms at the protein and gene levels in regulating elevated blood pres-

sure during hypertension, suggesting the need for comprehensive characterization of their

interaction with the cardiovascular system.

© 2014 Elsevier Ltd. All rights reserved.

A R T I C L E I N F O

Article history:

Received 21 January 2014

Received in revised form 6 March

2014

Accepted 7 March 2014

Available online

Keywords:

Antihypertensive peptides

Renin

Angiotensin II receptor blockers

Calcium channel blockers

Arginine–nitric oxide

Endothelin converting enzyme

1. Introduction

Cardiovascular disease (CVD) is a leading cause of global mor-tality contributing to 48% of deaths due to non-communicablediseases, ahead of cancer and chronic respiratory diseases(World Health Organization, 2012). Elevated blood pressureduring hypertension was identified to be strongly associatedwith CVD, and currently estimated to contribute to 13% of totalglobal deaths, as well as 51 and 45% of deaths due to strokeand coronary heart disease, respectively (World Health Orga-nization, 2012). Therefore, hypertension is considered a keytarget for controlling CVD-related mortality and improvingglobal health (Sharp, Aarsland, Day, Sonnesyn, & Ballard, 2011;

World Health Organization, 2012). In fact, changes in systolic(SBP) and diastolic blood pressure (DBP) by −5 and −2.5 mmHgduring hypertension were associated with 12 and 20% reduc-tion in the risks of developing coronary artery disease andstroke, respectively, regardless of intervention mode (Law, Morris,& Wald, 2009). Physiologically, the renin–angiotensin system(RAS) is the primary pathway for regulating blood pressure andvascular tone (Daien et al., 2012). The RAS pathway is initi-ated in the kidney when blood pressure falls with the conver-sion of prorenin zymogen to the active form, renin. Thereafter,renin is released into the blood stream where it functions incleaving the N-terminal region of angiotensinogen to releasea decapeptide, angiotensin (AT)-I. This process occurs slowlyserving as the first and rate-limiting step that determines the

* Corresponding author. Tel.: +1 (902) 893-6625; fax: +1 (902) 893-1404.E-mail address: cudenigwe@dal.ca (C.C. Udenigwe).

http://dx.doi.org/10.1016/j.jff.2014.03.0021756-4646/© 2014 Elsevier Ltd. All rights reserved.

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speed of blood pressure regulation by the RAS pathway. AT-Icirculates in the blood until its C-terminal dipeptide residueis cleaved by angiotensin I-converting enzyme (ACE) to forman octapeptide AT-II, which acts as a potent vasoconstrictor.AT-II binds its cell surface receptors to trigger a process thatresults in the secretion of aldosterone from adrenal glandsleading to increased reabsorption of salt and water, and in-crease in blood pressure by arterial constriction (Chen et al.,2009; Segall, Covic, & Goldsmith, 2007). Food protein-derivedpeptides have been widely pursued for controlling elevatedblood pressure during hypertension towards better cardiovas-cular health (Martínez-Maqueda, Miralles, Recio, & Hernández-Ledesma, 2012). Moreover, peptides have also shown bioactivitiesthat can potentially ameliorate other modifiable CVD risk factorssuch as type 2 diabetes (Power, Nongonierma, Jakeman, &Fitzgerald, 2014) and high blood lipid levels (Howard &Udenigwe, 2013), and non-traditional risk factors such as oxi-dative stress (Samaranayaka & Li-Chan, 2011).

2. Angiotensin converting enzyme (ACE), thetraditional hypertension target for foodprotein-derived peptides

ACE directly catalyzes the RAS reaction step that produces thepotent vasoconstrictor and can also hydrolyze and inactivatebradykinin leading to attenuation of the vasodilatory func-tion of bradykinin (Segall et al., 2007). Consequently, ACE haslong been investigated as a major physiological target for de-veloping antihypertensive drugs and natural products. Al-though ACE inhibiting drugs have been clinically prescribed forreducing blood pressure, they appear to pose many side effectsthat discourage their use by hypertensive patients. ACE-inhibiting natural products have been vigorously pursued duringthe last two decades as agents for lowering blood pressureduring hypertension. Accordingly, a plethora of peptide se-quences enzymatically released from food proteins are fre-quently reported to inhibit ACE activity in vitro, and many alsomodulate the elevated blood pressure associated with hyper-tension in animal and human models (Hong et al., 2008;Martínez-Maqueda et al., 2012; Möller, Scholz-Ahrens, Roos, &Schrezenmeir, 2008; Udenigwe, Ejike, Quansah, & Eze, 2011).Based on the current literature, physiological antihyperten-sive activities of peptides are often attributed to ACE inhibi-tion, and even when weak in vitro activities are observed, thesepeptides are considered ‘pro-drugs’ that serve as precursorsof ACE inhibitors during physiological proteolysis. AT-II can alsobe produced from AT-I through ACE-independent pathways suchas the chymase-catalyzed reaction. Therefore, ACE inhibitionmay not be completely responsible for attenuating hyperten-sion. In fact, antihypertensive effects can be mediated via pro-cesses other than the RAS system. For instance, subcutaneousadministration of opioid-like milk-derived peptides α-lactorphin(Tyr-Gly-Leu-Phe) induced hypotensive effects in hyperten-sive and normal rats through a process mediated by opioidreceptors (Nurminen et al., 2000). Therefore, food protein-derived peptides may be acting via multiple mechanisms re-sulting in pronounced lowering of blood pressure. Emergingevidence indicates that bioactive peptides can also interact with

RAS-related renin, AT-II receptor, arginine–nitric oxide pathway,endothelin system, and Ca2+ channels (Fig. 1, Table 1), whichmay mediate their physiological antihypertensive effects in ad-dition to ACE inhibition.

3. Renin, an emerging target for developingantihypertensive food peptides

3.1. The structure and function of renin

Renin catalyzes the first and rate-limiting step of the RASpathway and its inhibition can suppress upstream AT-I pro-duction thereby reducing the concentration of circulating AT-II. Since angiotensinogen is the only known physiological reninsubstrate, inhibition of the RAS enzyme is believed to be spe-cific with limited possibility of interacting with other physi-ological processes and developing side effects. Renin belongsto the aspartate protease (AP) family that includes pepsin,chymosin, cathepsin D, β-secretase and plasmepsins (Parr,Keates, Bryksa, Ogawa, & Yada, 2007). APs are characterized bythe presence of two aspartate (usually Asp32 and Asp215) resi-dues in their catalytic sites, which play key roles in cleavingpolypeptide substrates (Rahuel et al., 2000). Renin is releasedinto the blood stream by kidney cortex juxtaglomerular cellsin response to low blood pressure or sodium level (Persson,2003). The renal cells constitutively produce prorenin, a 406-amino acid residue zymogen that contains a signal peptide (f1–23) and a prosegment (f24–66). Prorenin can be converted tomature renin through irreversible proteolytic cleavage of theprosegment by enzymes such as proconvertase 1 and cathep-sin B, or by non-proteolytic activation where it binds the(pro)renin receptor, which induces a reversible unfolding re-sulting in the removal of the prosegment from the enzyme cata-lytic cleft (Danser & Deinum, 2005). In fact, when bound to itsreceptor, prorenin exhibits a portion of the proteolytic activ-ity of renin in converting angiotensinogen to AT-I at the cellsurface (Danser & Deinum, 2005). Therefore, physiological totalrenin activity, measured as plasma renin activity, can reliablyindicate risk of hypertension, and inhibition of renin activityby natural products can be explored for the management ofhypertension.

Fig. 1 – Prospective antihypertensive mechanisms of foodprotein-derived peptides. ECE, endothelin-convertingenzyme; ET-1, endothelin-1; ACE, angiotensin I-convertingenzyme.

46 j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 4 5 – 5 2

3.2. Renin inhibition

Pepstatin is a potent AP inhibitor and the first reported renininhibitor; however, the activity of the statine-containinghexapeptide was found to be orders of magnitude lower thanits inhibitory effect on pepsin (Fisher & Hollenberg, 2005).Peptide-like (type-I) renin inhibitors that mimic fragments ofangiotensinogen have been investigated as possible antihy-pertensive agents. Moreover, an endogenously expressed reninbinding protein (RnBP), later found to be N-acetyl-D-glucosamine2-epimerase, was reported to form a high molecular weightcomplex with renin (RnBP-renin heterodimer) thereby inhib-iting its activity (Takahashi, Kumagai, Shindo, Saito, &Kawamura, 2000). The selective binding was found to be me-diated by a leucine zipper (f195–216) in RnBP (Inoue, Takahashi,Fukui, & Miyake, 1991).The primary RnBP sequence in the renin-binding region can become the basis for designing potent renin-inhibiting peptides that may be identified and released fromfood proteins using bioinformatic tools. Several synthetic pep-tides displayed high renin-inhibitory activities at low concen-trations, but their large sizes and unfavourable lipophilicityare thought to be associated with low oral bioavailability andpoor physiological activity (Raheul et al., 2000; Staessen, Li, &Richart, 2006). Aliskiren is the only commercial clinically proventhird generation (structurally unrelated to peptides) syn-thetic renin inhibitor for managing hypertension; it func-tions by specifically binding renin at its large hydrophobiccatalytic cavity (Wood et al., 2003) and was found to be a moreeffective antihypertensive agent than ACE inhibitors (Verdecchiaet al., 2010). Recent clinical evidence suggests that aliskiren

may be harmful to patients with type 2 diabetes who are atrisk of developing cardiovascular and renal diseases (Parvinget al., 2012).

3.3. Food-derived peptides as direct renin inhibitors

Recent developments indicate that some food protein-derivedhydrolysates and peptides possess in vitro renin inhibitory ac-tivities. An earlier study reported that flaxseed protein hydro-lysates generated with several enzymes moderately inhibitedthe activity of human recombinant renin with IC50 values of1.22–2.81 mg/mL (Udenigwe, Lin, Hou, & Aluko, 2009). Subse-quent studies have reported a wide range of food protein hy-drolysates with moderate to high renin inhibitory activities(Ajibola, Fashakin, Fagbemi, & Aluko, 2013; Fitzgerald et al., 2012;He et al., 2013a; Onuh, Girgih, Aluko, & Aliani, 2013; Udenigwe& Aluko, 2012; Udenigwe, Li, & Aluko, 2012a), and a recent studydemonstrated that renin inhibition by protein hydrolysates canbe retained within a food (bread) matrix (Fitzgerald et al., 2014).There is currently a dearth of detailed structure–activity re-lationship (SAR) information, although it appears that renininhibition is dependent on hydrophobicity and molecular sizeof hydrolysates and peptide fractions (Alashi et al., 2014; Heet al., 2013b). Moreover, protease specificity was found to in-fluence bioactivity with Alcalase yielding the very active renininhibiting hydrolysates (Alashi et al., 2014; He et al., 2013b), al-though contradicting observation has been reported where pan-creatin exhibited better prospects (Alashi et al., 2014). Moreover,Corolase PP was used to hydrolyze macroalgae (Palmaria palmata)proteins to yield hydrolysates with higher renin-inhibitory

Table 1 – Sources, identity and prospective hypotensive mechanisms of food protein-derived bioactive peptides otherthan ACE inhibition.

Source Peptide Mechanism Reference

Pea Ile-Arg, Lys-Phe, Glu-Phe, Leu-Arg, Asn-Arg,Phe-Thr

Renin inhibition Li & Aluko, 2010; Udenigweet al., 2012b

Synthetic Ile-Trp, Leu-Trp Renin inhibition Udenigwe et al., 2012bMacroalgae Ile-Arg-Leu-Ile-Ile-Val-Leu-Met-Pro-Ile-Leu-Met-Ala Renin inhibition Fitzgerald et al., 2012Rapeseed Gly-His-Ser, Arg-Ala-Leu-Pro, Leu-Tyr, Thr-Phe Renin inhibition He et al., 2013b, 2013cFlaxseed Arginine-rich peptide fractions KCl-2 and KCl-F1 Potential nitric oxide precursor Doyen et al., 2014; Udenigwe

et al., 2012bMilk (lactoferrin) Leu-Ile-Trp-Lys-Leu, Arg-Pro-Tyr-Leu,

Arg-Arg-Trp-Gln-Trp-Arg, 3 kDa peptide fractionAT-II receptor blockers Fernández-Musoles et al., 2014

Milk (α-lactalbumin,β-lactoglobulin)

α-lactorphin (Tyr-Gly-Leu-Phe), β-lactorphin(Tyr-Leu-Leu-Phe)

Opioid receptor-mediatednitric oxide production

Nurminen et al., 2000; Sipolaet al., 2002

Amaranth Glutelin-derived peptides Nitric oxide production viabradykinin-mediated pathway

Barba de la Rosa et al., 2010

Egg (ovalbumin) Ile-Val-Phe, Arg-Ala-Asp-His-Pro-Phe-Leu,Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu

Bradykinin B1receptor-mediated nitric oxideproduction

Miguel et al., 2007

Synthetic His-Arg-Trp, Trp-His, severaltryptophan-containing peptides

Voltage-dependent L-type Ca2+

channel blockersTanaka et al., 2008, 2009

Milk (β-lactoglobulin) Ala-Leu-Pro-Met-His-Ile-Arg Modulation of endothelin-1release

Maes et al., 2004

Milk (lactoferrin) Gly-Ile-Leu-Arg-Pro-Tyr,Arg-Glu-Pro-Tyr-Phe-Gly-Tyr

Endothelin-converting enzyme(ECE) inhibition

Fernández-Musoles et al.,2013b

LfcinB f17–32, f17–31, f20–25, f19–25, f18–25,f17–25, f17–24, f17–22a

Inhibition of ECE andECE-dependentendothelin-1-inducedvasoconstriction

Fernández-Musoles et al., 2010

a N- and C-terminal of peptides were acetylated and amidated, respectively.

47j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 4 5 – 5 2

activity than those produced with Alcalase and Flavourzyme(Harnedy & FitzGerald, 2013). Therefore, the efficiency of pro-teases in producing renin-inhibiting peptides seems to dependon the nature of the protein precursors. Furthermore, simu-lated food protein hydrolysis with gastrointestinal enzymes hasalso resulted in products with renin inhibitory activities (Girgih,Udenigwe, Li, Adebiyi, & Aluko, 2011; Onuh et al., 2013), sug-gesting the possibility of deriving antihypertensive effects whendietary proteins are consumed.

Following post-hydrolysis processing, Li and Aluko (2010)identified dipeptides Ile-Arg, Lys-Phe and Glu-Phe fromthermolysin-hydrolyzed pea proteins, which moderately in-hibited renin activity with IC50 values of 9.2, 17.8 and 22.6 mM,respectively. Moreover, other dipeptides identified in the peaprotein hydrolysate inhibited 20–49% renin activity at 3.2 mM(Udenigwe et al., 2012a). Subsequently, a quantitative SAR (QSAR)study using the peptide dataset suggested that the presenceof N-terminal aliphatic (e.g. leucine, isoleucine, valine) andC-terminal bulky amino acid residues (e.g. phenylalanine, tryp-tophan) contribute to higher renin inhibitory activity of di-peptides (Udenigwe et al., 2012a). Based on the QSAR study,dipeptide Ile-Trp was predicted as a strong renin inhibitor and,although not directly derived from food proteins, the peptideinhibited renin activity by 70% at 3.2 mM with IC50 2.3 mM.Moreover, dipeptides Leu-Tyr and Thr-Phe were reported toinhibit renin activity with IC50 values of 1.8 and 3.7 mM, re-spectively (He et al., 2013a), and their structures mostly agreedwith the proposed requirements for renin inhibition. In oneinstance, switching the position of amino acids residues fromThr-Phe to Phe-Thr resulted in a substantial decrease in ac-tivity, indicating the important contribution of the C-terminalbulky hydrophobic moiety to renin inhibition (He et al., 2013a;Udenigwe et al., 2012a). However, a highly hydrophilic tripep-tide Gly-His-Ser non-competitively inhibited renin activity withIC50 1.09 mM and Ki 1.016 mg/mL (approximately 3.3 mM) (He,Malomo, Girgih, Ju, & Auko, 2013c). This suggests possible al-losteric regulation since the peptide may have interacted withsites at the surface of the enzyme due to its hydrophilicity.Moreover, cationic tetramer Arg-Ala-Leu-Pro displayed the bestrenin inhibitory activity of the reported food protein-derivedpeptides with IC50 0.97 mM (He et al., 2013a). These observa-tions suggest the need for detailed SAR studies to identifyrequisite contributors to bioactivity other than those re-ported for dipeptides. In addition, a 13-amino acid residuepeptide, Ile-Arg-Leu-Ile-Ile-Val-Leu-Met-Pro-Ile-Leu-Met-Ala,identified in papain-hydrolyzed P. palmata proteins wasreported to inhibit renin activity with IC50 value of 3.3 mM(Fitzgerald et al., 2012), although the peptide appears meta-bolically unstable due to its large size and multiple proteo-lytic cleavage sites. Taken together, there is currently aknowledge gap on detailed SAR, which is needed for the designof potent renin inhibiting food protein-derived peptides forphysiological blood pressure reduction.

Furthermore, renin inhibiting peptide and protein hydro-lysates have induced decreases in SBP by 12–30 mmHg whenorally administered to spontaneously hypertensive rats (Girgihet al., 2011; He et al., 2013a, 2013b, 2013c; Li et al., 2011). As ex-pected, the purified renin-inhibiting peptides exerted their an-tihypertensive activities at a lower dosage (30 mg/kg bodyweight) over a shorter period (2–4 h) compared to crude protein

hydrolysates, which showed similar SBP lowering effect 6–8 hafter administering 100–200 mg/kg body weight. These prom-ising effects may not be attributed solely to renin inhibitionas most of the peptides were also observed to inhibit ACE ac-tivity, or modulate RAS gene expression. In fact, long-termdietary intake of hemp seed protein hydrolysate was re-cently found to suppress both plasma renin and ACE activi-ties with concomitant blood pressure reduction inspontaneously hypertensive rats (Girgih, Alashi, He, Malomo,& Aluko, 2013). Furthermore, egg-derived pentapeptide RVPSLdecreased renin mRNA expression (and those of other RAS com-ponents including ACE and AT-II receptor) in the kidney of spon-taneously hypertensive rats that received 50 mg/kg body weightof the peptide for 4 weeks (Yu, Yin, Zhao, Chen, & Liu, 2014).These activities resulted in decreased serum levels of AT-II, reninand aldosterone, and pronounced blood pressure reductioncomparable to the activity of a fivefold lower dosage of captopril,an antihypertensive drug. In contrast, long-term administra-tion of ACE-inhibiting lactoferrin hydrolysate induced eleva-tion of plasma renin activity, possibly due to the loss of negativefeedback regulation by AT-II, which was decreased by the treat-ment (Fernández-Musoles, Manzanares, Burguete, En Alborch,& Salom, 2013a). Moreover, weakly active or inactive renin in-hibiting peptides may also display physiological blood pres-sure lowering activity. For instance, a pea protein hydrolysatethat inhibited only 19% renin activity at 1 mg/mL exhibitedpotent antihypertensive effect in a kidney disease rat modeland hypertensive humans, and was found to downregulate renalexpression of renin mRNA in the rat model (Li et al., 2011). Thisindicates that antihypertensive food protein-derived pep-tides may be acting via a concerted mechanism at the proteinand gene levels, and may also involve other RAS steps andrelated biochemical pathways (Fig. 2).

4. Food protein-derived peptides as AT-IIreceptor blockers

Considering that prorenin exhibits renin-like activity whenbound to its cell surface receptor (Danser & Deinum, 2005), theinhibition of renin (and ACE) activities may not completely sup-press AT-II production. Therefore, AT-II receptor blockers (ARBs)or antagonists are considered effective antihypertensive agentsespecially for patients with sensitivity to ACE inhibitors, sinceARBs do not elicit the side effects associated with the inhibi-tion of bradykinin degradation. Selective blocking of AT1 re-ceptors results in the suppression of AT-II-inducedvasoconstriction irrespective of mode of AT-II production;losartan was the first synthetic ARB and several ARBs are cur-rently available in the market. Yu et al., (2014) reported thategg peptide Arg-Val-Pro-Ser-Leu decreased renal AT-II recep-tor mRNA expression, although it is not known if the peptidedirectly interacted with the gene, or altered transcription factorsor upstream activators leading to the observed effect. More-over, emerging evidence indicates that some food protein-derived peptides can directly block AT-II receptor, expandingtheir prospective antihypertensive mechanisms other than ACEinhibition. Milk lactoferrin-derived peptides Leu-Ile-Trp-Lys-Leu, Arg-Pro-Tyr-Leu, Arg-Arg-Trp-Gln-Trp-Arg (LfcinB20–25) and

48 j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 4 5 – 5 2

a 3-kDa hydrolysate fraction were found to reduce AT-II-induced contraction of isolated rabbit carotid arterial seg-ments by 21–44% (Fernández-Musoles et al., 2014). Based onthe study, Arg-Pro-Tyr-Leu showed the best effect and aradioligand receptor binding assay demonstrated that the cat-ionic peptide dose-dependently inhibited AT-II binding to humanand rabbit AT-specific (AT1) receptors. These effects were re-ported to be selective for AT-II receptor as the endothelin (ET)-1receptor-mediated vasoconstriction was not inhibited by thepeptide (Fernández-Musoles et al., 2014). Based on the prom-ising results, further work is needed to characterize the peptide–receptor interactions and possibly develop SAR models for futuredesign of food protein-derived ARBs.

5. Food peptides and the arginine–nitricoxide pathway

The RAS pathway works in concert with the kinin–nitric oxidesystem in regulating blood pressure (Fig. 2). In the vascular en-dothelium, bradykinin mediates a signalling process that resultsin the activation of Ca2+/calmodulin-dependent endothelial nitricoxide synthase (eNOS) which catalyzes the conversion of ar-ginine to nitric oxide and citrulline. Nitric oxide is a potent va-sodilator and can also react with homocysteine to formS-nitroso-homocysteine, which can also induce vasodilation(Perna, Ingrosso, & De Santo, 2003). Therefore, higher level ofarginine in the vascular endothelium can potentially inducevasodilation, and can be explored for hypertension therapy.This

concept formed the basis of two studies that produced arginine-rich flaxseed peptide fractions, which produced more pro-nounced and rapid lowering of SBP in spontaneouslyhypertensive rats, compared to the amino acid form of argi-nine (Doyen, Udenigwe, Mitchell, Marette, & Aluko, 2014;Udenigwe et al., 2012b). Since the arginine-rich peptides alsoexhibited moderate in vitro ACE and renin inhibitory activi-ties, it is also possible that more than one mechanism was in-volved in producing the antihypertensive effects. Ex vivo studiesare needed to ascertain if arginine supplementation would con-tribute to the nitric oxide pool within the vascular endothe-lium. Nitric oxide has also been suggested to mediate theendothelium-dependent vasorelaxation effects of opioid-likemilk peptides α-lactorphin (Tyr-Gly-Leu-Phe) and β-lactorphin(Tyr-Leu-Leu-Phe), via opioid receptors, in isolated mesen-teric arterial rings of spontaneously hypertensive rats(Nurminen et al., 2000; Sipola et al., 2002). In another study,amaranth trypsin-digested glutelins were reported to inducenitric oxide production in coronary endothelial cells, and cor-responding nitric oxide-induced vasodilation in isolated rataortic rings (Barba de la Rosa et al., 2010). In this case, the hy-drolysates induced the activation of eNOS by phosphoryla-tion at Ser-1177 residue consistent with the process mediatedby bradykinin in the vascular endothelium. Thus, the ob-served activity was suggested to be due to ACE inhibition,bradykinin accumulation and its subsequent binding to the B2receptor (Barba de la Rosa et al., 2010), amongst otherpossible mechanisms, although arginine may not havebeen involved since amaranth glutelin is particularly low inarginine.

Fig. 2 – The renin–angiotensin system and kinin–arginine–nitric oxide system pathways indicating key targets forfood-derived peptides; eNOS, endothelial nitric oxide synthase; ACE, angiotensin I-converting enzyme.

49j o u rna l o f f un c t i ona l f o od s 8C ( 2 0 1 4 ) 4 5 – 5 2

6. Endothelin-converting enzyme inhibitionby food peptides

Endothelin-converting enzyme (ECE) plays a vital role in bloodpressure regulation particularly in cleaving big endothelin toform endothelin (ET)-1, which binds its selective (ETA) and non-selective (ETB) receptors to induce a wide range of physiologi-cal effects, including vasoconstriction (Kedzierski & Yanagisawa,2001). The components of the ET system pathway are ex-pressed in several tissues, and ECE inhibitors and ET recep-tor agonists are gaining particular interest as targets forantihypertensive therapy (Kedzierski & Yanagisawa, 2001). Pep-tides derived from food proteins have been reported to inter-act with the ET system. Okitsu, Morita, Kakitani, Okada, andYokogoshi (1995) reported that pepsin digests of bonito pyrolicappendix and beef exhibited considerable ECE inhibitory ac-tivities, which were completely lost on further hydrolysis withPronase. This indicates that peptides were responsible for theobserved bioactivity as opposed to constituent amino acids ofthe hydrolysates released with Pronase. Moreover, a bovineβ-lactoglobulin-derived cationic peptide Ala-Leu-Pro-Met-His-Ile-Arg was found to suppress the release of ET-1 in culturedendothelial cells possibly via ACE inhibition, AT-II reduction andbradykinin accumulation (via a process mediated by B2 recep-tors), which have all been associated with decreased releaseof ET-1 (Maes et al., 2004). Although no direct relationship withECE was reported for the whey-derived peptide, eight bovinelactoferricin B-derived peptides were shown to inhibit in vitrorelease of ET-1 from big ET by ECE with concomitant suppres-sion of ECE-dependent ET-1-induced vasoconstriction(Fernández-Musoles et al., 2010). This indicates a direct ECEinhibitory activity by peptides. Moreover, the same group iden-tified ECE-inhibiting peptides Gly-Ile-Leu-Arg-Pro-Tyr and Arg-Glu-Pro-Tyr-Phe-Gly-Tyr from ECE/ACE inhibitingantihypertensive lactoferrin hydrolysates (Fernández-Musoleset al., 2013b).There is currently no reported relationship betweenACE and ECE inhibition, although some peptides exhibit thedual effects, which can potentially lead to pronounced bloodpressure lowering effects during hypertension.

7. Calcium channel-blocking peptides

Blocking of voltage-dependent calcium channels (VDCC) canreduce Ca2+ influx into vascular muscle cells and suppress va-soconstriction. Therefore, Ca2+ channel blockers can be usedto lower blood pressure during hypertension. A number of pep-tides have demonstrated the ability to interact with VDCC.Tanaka, Tokuyasu, Matsui, and Matsumoto (2008) reported thattryptophan-containing dipeptides (Trp-His, His-Trp,Trp-Leu andTrp-Val) produced vasodilation effect in isolated rat thoracicaortic rings. Trp-His was later found to inhibit intracellular Ca2+

increase normally associated with AT-II stimulation in vascu-lar smooth muscle cells, similar to the L-type VDCC blockingeffect of dihydropyridine (Wang, Watanabe, Kobayashi, Tanaka,& Matsui, 2010). Based on the structure of the tryptophan/histidine-containing peptides, several tripeptides were latershown the induce vasodilation with His-Arg-Trp exhibiting the

most effective activity with EC50 1.2 mM (Tanaka, Watanabe,Wang, Matsumoto, & Matsui, 2009). The study suggested thatN-terminal imidazole (histidine), C-terminal indole groups (tryp-tophan) and a basic amino acid in the middle position are im-portant structural requirements for VDCC blocking effect. It isimportant to note that a mixture of amino acids tryptophanand histidine did not lower intracellular Ca2+ in the vascularsmooth muscle cells, which confirms the role of the peptidebond in order to observe the endothelium-independent vaso-dilation (Wang et al., 2010). Although the studies used syn-thetic tryptophan-containing peptides, not directly derived fromfood, the active sequences are encrypted in various degreeswithin food protein primary structure, and their liberationduring enzymatic hydrolysis should be monitored especiallyfor elucidating the prospective mechanisms of antihyperten-sive protein hydrolysates.

8. Conclusion

Based on recent literature, it can be concluded that food protein-derived enzymatic hydrolysates and peptides may employmultiple mechanisms in exerting their physiological antihy-pertensive effects other than ACE inhibition. In vitro, cellularand animal studies have demonstrated that food peptides caninhibit renin and the endothelin system, serve as sources ofarginine for nitric oxide production, and also block angioten-sin receptors and calcium channels. These mechanisms werereported for various peptides, but it would be necessary to evalu-ate the possible multifunctional mechanisms of each antihy-pertensive peptide when characterizing in vivo effects. Moreover,these mechanisms can possibly explain the hypotensive effectsof peptides that lack ACE inhibitory activity in vitro. Detailedinformation on the structural requirements of peptides for ac-tivity against these emerging targets, in addition to the plethoraof literature information on ACE inhibition, will allow for thedesign of potent food protein-derived peptides for physiologi-cal blood pressure lowering during hypertension. These effortswill potentially contribute to the development of peptide-based functional foods with established functional mecha-nisms for managing CVD and related mortality.

Acknowledgement

Natural Sciences and Engineering Research Council of Canada(NSERC) is acknowledged for providing funding to CCU througha Discovery Grant.

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