This work is a literature overview on angiotensin-converting enzyme (ACE) inhibitory/antihypertensive peptides in food protein sources. The following aspects related to peptides with the above-mentioned bioactivity are discussed: (i) mechanism of action of ACE, (ii) the structural character of ACE inhibitors/antihypertensive peptide sequences determined by different methods, including quantitative structure–activity relationship studies, (iii) their food sources, (iv) absorption of peptides, (v) in vitro and in vivo approaches involved in the production and potential release of peptide ACE inhibitors as well as in silico methods applied in research concerning peptides.
There is scientific evidence that diet has a direct relationship to cardiovascular diseases (CVD) (Mallikarjun Gouda and others 2006; Alissa and Ferns 2012). One of the major public CVD worldwide is hypertension, which usually occurs with other metabolic disorders such as obesity, prediabetes, and atherosclerosis (Tavares and others 2012). Data reported by the WHO and presented by Jimsheena and Gowda (2011) have shown that by 2020 CVD will become the leading cause of death and disability. The main factors related to the growth of this problem are excessive consumption of high-energy foods as well as reduced physical activity of consumers (Iwaniak and Dziuba 2009). Lin and others (2012) revealed that hypertension is called a “silent killer” because hypertensive patients (HP) are often asymptomatic for years. Thus, the ability of some molecules to inhibit angiotensin-converting enzyme (ACE) was considered to be a useful therapeutic approach to treat hypertension (Lin and others 2012). High blood pressure (BP) can be regulated medically by the administration of antihypertensive drugs (Tavares and others 2012). ACE inhibitory drugs were the 1st medications applied to lower BP in HP. Currently, the following drugs (synthetic ACE inhibitors) are available on the market: benazepril, captopril, enalapril, fosinopril, lisinopril, zestril, moexipril, perindopril, quinapril, accupril, ramipril, and trandolapril (Sweitzer 2003). According to the literature, ramipril was introduced in the early 1980s. This drug is known as a long-effective medication compared to the 1st-generation ACE inhibitory drug captopril (Pipkin 2002).
Apart from medical treatment of hypertension, diet may be also considered as a factor reducing the risk of CVD (Reddy and Katan 2004). Although antihypertensive drugs are available on the market, nutritionists claim that peptides which lower BP found in food are safer than “traditional” drugs and can be used as preventive agents (Vermeirssen and others 2004b). Numerous food components have been used for many years without any negative side effects (Vermeirssen and others 2004a).
The concept of viewing food as a remedy is ascribed to Hippocrates who, 2500 y ago, proclaimed: “Let food be your medicine, and medicine be your food.” This approach to food as a potential preventive agent has contributed to the growth of its market value, which places nutraceuticals between pharmaceuticals and medical foods. It is consistent with the idea that the special food components we consume must show biological activity (Ramaa and others 2006). This activity should be manifested by control, treatment, and/or stimulation of life processes (Ramaa and others 2006). Fernandez and others (2004) emphasize the role of ACE inhibitory peptides as a target for drug design resulting from the function of ACE in cardiovascular and renal diseases. To date, ACE inhibitors derived from food proteins are the best-known group of bioactive peptides (Iwaniak and Minkiewicz 2008) and their biofunctionality, make them a promising alternative when choosing between a synthetic drug and naturally-originating bioactive food components (Jimsheena and Gowda 2011).
CVD, including hypertension, belong to the most common problems of civilization. The BP-reducing ability of peptides has made them the aim of studies by many researchers (Guang and Philips 2009). Recent progress in the knowledge concerning bioactive peptides (for example, ACE inhibitors) from foods has been enhanced by the development of research methods. Udenigwe and Howard (2013) highlight the role of bioinformatics and proteomics as integral tools to develop research on bioactive peptides from proteins derived from meat. Such circumstances have encouraged us to describe the ACE inhibitory peptides derived from food proteins.
ACE and Its Role in Hypertension
ACE (EC 22.214.171.124) is a hypertension-responsible glycoprotein present both in biological fluids and many tissues (Guang and Philips 2009). The 3-dimensional structure of this glycoprotein shows that ACE is a zinc metallopeptidase (De Leo and others 2009) which can adopt 2 different forms: somatic and testicular (Ni and others 2012a). In recent years, a 3rd form of ACE was identified, defined as homolog ACE (ACEH) (Meisel and others 2006).
Somatic ACE consists of N- and C-terminal domains containing the active site and enabling the catalysis of angiotensin I (Ang I) to angiotensin II (Ang II). In testicular ACE, the C-terminal domain is involved in the hydrolysis of Ang I to Ang II. In ACEH, the ACE conversion is possible due to the active N-domain. Moreover, ACEH does not hydrolyze bradykinin (Meisel and others 2006).
The simplified scheme of conversion of Ang I to Ang II is presented in the Figure 1. There are 2 specific reactions catalyzed by this metallopeptidase. In the 1st reaction, ACE leads to the transformation of an inactive decapeptide, called angiotensin I (Ang I) into 2 products: C-terminal dipeptide HL as well the vasoconstrictor and salt-retaining angiotensin II (octapeptide) which plays a key role in BP regulation. The conversion of Ang I to Ang II causes vasoconstriction, which leads to a BP increase (Bhuyan and Mugesh 2011). The 2nd reaction (Figure 1) shows the simplified mechanism of the action of ACE on bradykinin, a nonapeptide (RPPGFSPFR) known to be a vasodilatory hormone. ACE cleaves the C-terminal dipeptide with the sequence FR from bradykinin, which causes the formation of an inactive heptapeptide called bradykinin 1-7 (RPPGFSP) (Bhuyan and Mugesh 2011; Iwaniak 2011).
Hypertension is one of the CVD. It is diagnosed when systolic blood pressure (SBP) is above 140 mm Hg and/or diastolic blood pressure (DBP) is above 90 mm Hg (Chen and others 2009). Depending on the values of SBP and DBP, Chen and others (2009) distinguished 4 types of hypertension: prehypertension (SBP: 129 to 139/DBP: 80 to 90 mm Hg), stage 1 hypertension (SBP: 140 to 159/DBP: 90 to 99 mm Hg), stage 2 hypertension (SBP: ≥ 160/DBP: ≥ 100 mm Hg), and stage 3 hypertension (SBP: ≥ 180/DBP: ≥ 110 mm Hg). Another classification of high BP problems relies on their etiology. Essential hypertension concerns unknown causes of its occurrence or several factors affecting the organism, including age, body mass, heredity, and lifestyle. Secondary hypertension occurs as a symptom of other medical dysfunctions such as renal disease, narrow arteries, and adrenal cortical disorders (Perez and Musini 2008; Chen and others 2009). It can be concluded that modification of lifestyle, by paying attention to physical activity and the kind of foods consumed (including those containing the ACE inhibitors) is one of the aims of prevention (medicinal chemistry), especially in the treatment of essential hypertension (Matsui and Tanaka 2010).
Different metabolic pathways can be responsible for BP regulation by means of an ACE (Meisel and others 2006). This mode of ACE action is related to the following systems (S): renin-angiotensin (RAS), renin-chymase (RCS), kinin-nitric oxide (KNOS), neutral endopeptidase (NEPS). RAS is also known as RAAS or the renin-angiotensin-aldosterone system (Ondetti and Cushman 1982). Among these systems of BP regulation by ACE, the RAS is considered to be the major one. It occurs in the blood circulatory system as well as in body organs such as the aorta, kidneys, lungs, and the brain (Matsui and Tanaka 2010). In the RAS system, renin (EC 126.96.36.199) is first released from prorenin due to the action of kallikrein (EC 188.8.131.52) (Ondetti and Cushman 1982; Beldent and others 1993). Renin then cleaves angiotensinogen to release the angiotensin I (Ang I). The hydrolysis of Ang I to Ang II (angiotensin II) proceeds as shown in Figure 1 (see reaction 1). According to Phillips (1983), Ang II is responsible for BP regulation. Apart from this, Ang II is a controller of the following body functions: activity of gonadotrophic-hormone-releasing-hormone (GHRH) and pituitary hormones in the reproductive cycle and pregnancy as well as neurotransmitter interactions (Phillips 1983). Hypertension caused by the presence of Ang II depends on the receptors located in the kidneys. In addition, angiotensin II can damage body organs due to elevated BP, which appears as a consequence of heart and kidney failure. This is related to the direct impact of Ang II on body tissues (Kumar and others 2009). For this reason, Meisel and others (2006) emphasize the beneficial role of angiotensin receptor blockers as well as ACE inhibitory activity peptides in BP reduction and the treatment of related diseases such as myocardial infarction, atherosclerosis, and diabetic nephropathy.
The RCS, which is located in human heart tissue, and chymase (EC 184.108.40.206) are responsible for the transformation of Ang I to Ang II. Moreover, chymase does not hydrolyze bradykinin (see Figure 1). The action of bradykinin is related to KNOS. One of the kinins (called kallidin) is degraded to bradykinin, a vasodilatory peptide. The vasodilatory effect of bradykinin is related to the conversion of L-arginine to nitric oxide (NO) by the action of nitric oxide synthase (NOS; EC 220.127.116.11). Vasodilation is inhibited by ACE by release of Phe-Arg (FR) from bradykinin (Figure 1) causing vasoconstriction (Tom and others 2003). Neutral endopeptidase (NEP, EC 18.104.22.168) is an enzyme involved in a number of actions causing vasoconstriction (Meisel and others 2006). An example of NEP action is the hydrolysis of bradykinin to inactive fragments and natriuretic peptides, known as vasodilators. NEP can also hydrolyze Ang I and Ang II to Ang 1-7 with vasodilating activity (Meisel and others 2006).
Food Peptides with an ACE Inhibitory Activity—Their Structural Nature
The identification of specific molecular properties which determine a peptide's biological activity can be an attribute considered in the production of nutraceuticals and functional foods (Dziuba and others 2005). Peptides known as ACE inhibitors belong to a group called “bioactive substances” (De Leo and others 2009). In food science, to be considered as a “bioactive substance” the analyzed compound must meet 2 criteria: (a) apart from the defined nutritional value of food product, an active substance encrypted in it has to show a measurable biological effect after excessive consumption; (b) the biological effect must be beneficial for health (Möller and others 2008).
ACE inhibitors are the most-studied food peptides and display different biological functions (Erdmann and others 2008). This trend can also be observed by analyzing databases of biopeptides. For example, the BIOPEP database currently displays the sequences of peptides with 44 various bioactivities, including ACE-inhibitory activity (Minkiewicz and others 2008). BIOPEP contains (as of June 2013) 2609 peptide sequences and 556 of them are ACE inhibitors (BIOPEP database, http://www.uwm.edu.pl/biochemia). The EROP-Moscow database (Zamyatnin and others 2006) (http://erop.inbi.ras.ru) recently contained (as of June 2013) 10229 peptides, including 313 ACE inhibitors, annotated within the category “enzyme inhibitors.” This category recently contained 581 peptides.
The large number of ACE inhibitors with known sequences became the aim of studies concerning the role of their structural characteristics in ACE inhibition. Although the full mechanism of interaction between an inhibitor and the ACE is not known, it is highly possible that the peptide (inhibitor) interacts with the C-domain of ACE (Meisel and others 2006). There are also some structural features which characterize the peptides with ACE inhibitory activity. They were discovered through laboratory experiments (Cheung and others 1980; Soubrier an others 1988) and/or chemometric techniques as part of the field of science called chemoinformatics (Massart and others 2003) which, according to the definition introduced by Desiere and others (2002), is a part of bioinformatics. The range of application of chemometric techniques in contemporary life sciences is wide and it concerns issues requiring the analysis of many data sets (Massart and others 2003). The analysis of peptide activity based on structure is defined as quantitative structure–activity relationship (QSAR). This chemometric technique has been successfully applied in food chemistry (Martinez-Mayorga and Medina-Franco 2009), including the field of study regarding the structure-activity of ACE inhibitors (Pripp and others 2004; Nakai and others 2005).
Taking into consideration the results of analytical and chemometric experiments, some structural characteristics of ACE inhibitors have been established. To generate the inhibition, ACE prefers peptides built up from specific amino acid residues typical to N- and C-ends of ACE inhibitors. Hydrophobic amino acids, especially those with aliphatic chains such as Gly, Ile, Leu, and Val are characteristic for the N-end of a peptide. Amino acids with cyclic or aromatic rings (Pro, Tyr, Trp) are found at the C-end of the ACE inhibitors (FitzGerald and others 2004; Vermeirssen and others 2004b).
Many peptides derived from food proteins contain Pro at the C-end. This rule concerns mostly short-length peptides (Iwaniak and Dziuba 2009). The occurrence of C-terminal proline, as well as N-terminal branched-side aliphatic amino acids, affected the ACE inhibitory activity. Such N- and C-terminal amino acid compositions of a dipeptide favored the hydrophobic interactions with ACE (Pripp and Ardö 2007). The observations concerning the impact of the physicochemical properties on N- and C-terminal amino acids on the bioactivity of peptides were consistent with earlier research conducted by Hellberg and others (1991) and Pripp (2005). Pripp and others (2005) interpreted the QSAR model of dipeptide ACE inhibitors and suggested that molecular size and hydrophobicity of C-terminal position of a dipeptide follows the electronic properties and hydrophobicity of the N-terminal position. Based on literature data on QSAR design of ACE inhibitory peptides, Pripp and others (2005) reported that, in general, highly active peptide should be composed of large, hydrophobic, aromatic amino acid with a polar functional group in the C-terminus. Iwaniak (2011) applied PCA (principal component analysis) to analyze 98 dipeptide ACE inhibitors collected in the BIOPEP database. To analyze the correlations between the structure of dipeptides and their activity, the indices characterizing physico-chemical properties of the 20 most common amino acids were used. The above-mentioned indices were available in the AAindex database (Kawashima and others 2008). The results of Iwaniak (2011) showed that the highest positive correlations were related to components concerning the bulkiness of the C-terminal amino acid. It is consistent with the conclusion made by Wu and Aluko (2007) who emphasized the impact of physicochemical attributes of amino acids such as hydrophobicity, bulkiness, and electronic properties on the bioactivity of peptides.
The research conducted by Wu and others (2006a) led to establish some structural attributes characterizing di- and tripeptide ACE inhibitors. Dipeptides were composed of amino acids with bulky and hydrophobic side chains. In the case of tripeptides, the 1st residue was usually aromatic, the 2nd was positively charged, and the 3rd residue (C-terminal) was hydrophobic.
Peptides with longer chains than those ones built up from 2 or 3 amino acids possess basic amino acids (Arg, Lys) at the C-end. Fitzgerald and others (2004) showed that positively charged guanidine and ε-amine groups derived from Arg and Lys, respectively, significantly contribute to the increased activity of an ACE inhibitor. Replacement of Arg caused a total loss of activity. It can be concluded that the action of ACE inhibitors requires an interaction between the inhibitory molecule and anionic binding site which differs from the ACE catalytic site (FitzGerald and others 2004). The partial least squares method applied by Wu and others (2006b) allowed to systemize the structural requirements for ACE inhibitors with the length of the chains ranging from 2 to 10 amino acids. The results of the above-mentioned studies showed that the C-terminal amino acid determines the potency of ACE inhibitory peptides composed of 4 to 10 residues. In the case of tetrapeptides, the most favorable amino acids (starting from C-terminus) are Tyr and Cys. Residues such as His, Trp, and Met are usually found in the 2nd position of a tetrapeptide. In the 3rd position of a peptide chain, Ile, Leu, Val, and Met is located and Trp is placed as the 4th residue (Wu and others 2006b).
Some of the sequences of ACE inhibitors possess glutamic acid at the C-end. It can be explained by the capability of the peptide to chelate zinc by glutamic acid, which is a component of the ACE active center (Fitzgerald and others 2004). Apart from the role of amino acid hydrophobicity on the peptides’ activity, Nakai and Li-Chan (1993) highlighted the importance of the following structural parameters on polypeptide functionality: charge density, disulfide bonds, and hydrogen bonds. In the case of peptides produced synthetically, other authors suggest the significance of the secondary structure on their functional properties (Darewicz and Dziuba 2001).
Activity of ACE Inhibitors Derived from Food
Lower BP is possible due to the inhibition of ACE by certain peptides defined as ACE inhibitors or antihypertensive peptides. The use of term “ACE inhibitor” or “antihypertensive peptide” is related to activity of the peptide in vitro and/or in vivo (Suetsuna and Chen 2001). Some of the ACE inhibitors show their activity in vitro but they are inactive in vivo. This loss of activity can be related to degradation of a peptide due to the action of intracellular peptidases or enzymes existing in the digestive tract or blood serum. Another reason for the loss of activity by peptides is their modification in the liver (Yamada and others 2002).
Antihypertensive activity of long-chain ACE inhibitors can result from the process of their hydrolysis into shorter, active fragments. Shorter fragments, such as di- or tripeptides, are absorbed from the intestine and then directly interact with the appropriate receptors (Yamada and others 2002).
There are ACE inhibitors which show this effect in vivo and less actively or not at all in vitro. An example of a peptide with a known ACE inhibitory effect in vitro is fragment 142-148 of β-lactoglobulin. During the process of simulated human digestion in the presence of serum proteinases and peptidases, the fragment 142-148 was not active as a hypotensive agent (Walsh and others 2004). Another example of peptides which are more potent in vivo than in vitro are the sequences LKPNM and LKP originating from fish protein (Fujita and Yoshikawa 1999). The antihypertensive effect of the above-mentioned sequences was compared with captopril. It was found that the activity of these peptides relative to the drug was 66% (LKPNM) and 91% (LKP). In turn, the in vitro ACE-inhibitory activities of the peptides LKPNM and LKP were 0.92% and 7.73%, respectively, compared to captopril (IC50 = 0.022 μM) (Fujita and Yoshikawa 1999). Based on these observations, Fujita and Yoshikawa (1999) suggested that the higher activity of peptides in vivo may result from their higher tissue affinities and from being eliminated more slowly from the body than a synthetic drug (captopril).
According to Yamada and others (2002), a possible reason for the differences between the activity of peptides in vitro and in vivo is the mechanism of BP regulation differing from the ACE mode of action. For example, it can be manifested by the interaction of the peptide with opioid receptors present in the nervous, hormonal, immunological, and intestinal systems of mammals (Teschemacher and others 1997). Researchers have focused on determining whether the differences between the activities of identical sequences of ACE inhibitors both in vitro and in vivo are the result of the existence of 2 domains in the catalytic site of an ACE (Meisel and others 2006).
The activity of ACE inhibitors can be determined by different analytical methods (Iwaniak and Dziuba 2009). The most popular tests to determine the peptide's ACE inhibitory activity in vitro are based on spectrophotometric, fluorometric, colorimetric, and radiochemical methods as well as chromatography techniques (Murray and others 2004; Murray and FitzGerald 2007). The measure of activity is usually given as the IC50, defined as the inhibitor concentration necessary to lower the ACE activity to 50% of the initial value (Donkor and others 2005). Another measure of the ACE inhibitory activity is the inhibition index, or the percentage inhibition achieved by a defined ACE inhibitory peptide concentration (Murray and FizGerald 2007). According to Pripp (2005), the experimental IC50 values commonly occur in the literature and they are also widely used in the research concerning the QSAR of ACE inhibitors. It was found that the same peptide can be characterized by different values of IC50. These differences result from the different analytical assays applied to measure the ACE inhibitory activity as well as the different laboratory conditions used by researchers around the world. Thus, Murray and FitzGerald (2007) attempted to specify the accurate conditions (for example, the number of ACE activity units used in an assay) which were necessary to calculate the IC50. The practical consequence of the lack of a “unified” procedure to calculate the activity of the peptides may have caused the lack of precision of QSAR models (Pripp 2005), which was confirmed by Katritzky and others (2005) who found that the creation of the QSAR models based on linear regression depended on the quality of the experimental data.
The ACE inhibitory activity assay relies on a reaction of an ACE with a synthetic specific substrate, resulting the release of a particular product. The ACE activity can be quantified spectrophotometrically by monitoring the release of product from the substrate. The most popular synthetic substrates to determine the dose-biological response of an ACE inhibitor are: HHL (hippuryl-L-histidyl-L-leucine) and FAPGG (furanacryloyl-L-phenylalanyl-glycylglycine). In the former, ACE releases hippuric acid (HA) and in the reaction with FAPGG the products are: FAP (furanacryloyl-L-phenylalanine) and GG (glycylglycine). The products (that is, HA and FAP) are quantified spectrophotometrically at 228 nm and 340 nm, respectively. If the reaction mixture contains the peptide (ACE inhibitor) the release of product (such as HA) is inhibited, which can affect the absorbance values. The absorbance value of the sample containing the ACE inhibitor (peptide) will be smaller compared to one without a peptide in the sample (Lahogue and others 2009; Iwaniak 2011).
In the fluorometric method, ACE is responsible for the catalysis of internally fluorescent quenched substrate Abz-Gly-Phe(NO)2-Phe (o-aminobenzoylglycl-ρ-nitro-L-phenylalanyl-L-proline). The released product is o-aminobenzoylglycine (Abz-Gly), and can be fluorometrically determined at 400 nm. The presence of an inhibitor in a reaction mixture will reduce the quantity of Abz-Gly, which will be observed by decreasing fluorescence values (Sentandreu and Toldrá 2006).
A colorimetric assay to determine the ACE inhibitory activity is based on the further reaction of hippuric acid with 2,4,6-trichloro-S-triazine or TNBS (2,4,6-trinitrobenezensulfonic acid) and reading the absorbance at 382 and 415 nm, respectively. However, the above-mentioned assay is not suitable for peptide ACE inhibitors because of the reaction of colorimetric reagents with amine groups of peptides (Hayakari and others 1978; Cinq-Mars 2006). Jimsheena and Gowda (2009) modified the colorimetric method of ACE inhibitory activity assay by complexing pyridine and benzene sulfonyl chloride (BSC) with HA formerly released from HHL. The complex of pyridine and BSC with HA gave a yellow color and was read at 410nm. The method proposed by Jimshheena and Gowda (2009) was recommended as a high-throughput procedure for screening ACE inhibitors because it was simple, sensitive, less time-consuming and required no solvent extraction.
Ryan and others (1980) described the ACE assay involving acylated tripeptides mostly containing 3H-atom emitting γ-isotopes. These tripeptides are substrates for an ACE action yielding to obtain dipeptide and acyl amino acid. The 3H-labeled product can be separated by a partition between the organic solvent and the aquenous solution. The amount of product is measured by scintillation count in the organic environment (Jimsheena 2010).
Using chromatography techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC) in ACE inhibitory activity assays is based on the separation of HHL from HA. The application of RP-HPLC shortens the assay duration due to elimination of the process of hippuric acid extraction into ethyl acetate, as was stated in the method developed by Cushman and Cheung (1971).
In vivo, the activity of peptides, including the ACE inhibitors which lower BP, is based on tests on HP or spontaneously hypertensive rats (SHR). Both HP and SHR are first fed with the extract or food product containing a specific dose of peptide and the DBP and SBP are then measured, taking the particular time intervals into consideration (Vermeirssen and others 2004a). Tavares and others (2012) found that studies involving the SHR are the best experimental methods of research concerning the antihypertensive peptides. The main reason for this is that rats exhibit vascular reactivity and renal functions similar to humans.
The discrepancy between the ACE inhibition in vitro and the BP-lowering effect in vivo provided a basis to classify these peptides into 3 categories (Ryan and others 2011; Zeng and others 2013). The 1st group of ACE inhibitors was defined as “true inhibition-type peptides.” The activity of such peptides expressed by IC50 is not affected by preincubation by ACE. Examples of such peptides are the sequences IY and IKW (Li and others 2004). In the 2nd group, called “substrate type,” peptides are hydrolyzed by ACE and display lack of or weak inhibitory activity. For instance, the ACE inhibitory activity (IC50) of the peptide FKGRYYP was 0.55 mol·dm−3. This value increased (activity was weakened) to 34.0 mol·dm−3 after the hydrolysis of FKGRYYP by ACE into FKG, RY, and YP (Li and others 2004).
The 3rd category, called “pro-drug type” inhibitors, includes peptides which are precursors of “true inhibition-type peptides,” which are released by ACE or digestive tract proteases. An example of a sequence belonging to this category was the peptide LKPNM given by Li and others (2004) which had an IC50 value of 2.40 mol·dm−3. LKPNM was then hydrolyzed by ACE and produced an LKP peptide with an ACE inhibitory activity approximately 8 times exceeding this of the precursor peptide (IC50 = 0.32 mol·dm−3). Another example of a “pro-drug-type” inhibitor is a peptide with the sequence IVGRPRHQG. Its hydrolysis with trypsin (EC 22.214.171.124) led to obtaining 2 peptides: IVGRPR and HQG. They were antihypertensive in SHR after oral intake, whereas the parent sequence (IVGRPRHQG) lost its activity after intravenous administration (Li and others 2004). This showed that peptides defined as “true inhibition-type” or “pro-drug type” can cause a decrease in the SBP in SHR (Ryan and others 2011).
ACE Inhibitory Peptides Encrypted in Food Protein Sequences
Many of the literature data on ACE inhibitors have been published since 2011. For example, the input query “ACE inhibitors” in the Scopus database (Scopus, http://www.scopus.com) revealed 1290 articles about peptides with the above-mentioned bioactivity (last entry: July 2013). Other keywords in the Scopus database such as “ACE inhibitory peptides,” and “bioactive peptides” and, more precisely, “food proteins as a source of bioactive peptides,” showed 230, 141, and 11 publications about peptides with an ACE inhibitory activity, respectively. These numbers indicate that ACE inhibitory/antihypertensive peptides are an important subject of scientific interest. It also means that even maximum efforts placed on an elucidation of the role of ACE inhibitors originating from food proteins in the prevention of CVD do not guarantee a full and detailed description of all peptide sequences identified in their precursors.
ACE inhibitors can be found in food proteins of various origins. Peptides possessing ACE inhibitory activity, including the antihypertensive effect confirmed on humans and SHR, have been detected in milk, eggs, fish, meat, and plants (Jauhiainen and Korpela 2007; Erdmann and others 2008; Korhonen 2009). Below, we describe the ACE inhibitory/antihypertensive peptides found in major food protein sources.
Milk and Dairy Products
Milk proteins still have a leading role as a source of ACE inhibitors and/or bioactive peptides in general (Iwaniak and Dziuba 2009). The most commonly known peptides of milk origin are the sequences VPP and IPP (Pripp 2008). They were found in β-casein and κ-casein, respectively. The effect of these 2 peptides was confirmed in vivo in SHR fed with sour milk (Li and others 2004). VPP and IPP peptides are now the ingredients of nutraceutical antihypertensive drinks such as the Japanese “Calpis” (Silva and Malcata 2005) and Finnish “Evolus” (Tidona and others 2009). It was found that the BP of HP who were administered 95 mL of Calpis daily for a period of 4 to 8 wk was significantly lowered (Silva and Malcata 2005). The volume of 95 mL was equivalent to the 1.2 to 1.6 mg of antihypertensive peptides and lowered systolic pressure by 14.9 mm Hg and diastolic pressure by 8.8 mm Hg (Darewicz and others 2011). The milk drink Evolus®, produced by use of Lactobacillus helveticus LBK-16H strain, revealed a significant antihypertensive effect in humans. The daily dose of the drink was 150 mL (FitzGerald and others 2004). Examples of other commercial products based on milk proteins which lower BP in humans are: Casein DP (Japan) and BioZate (USA). The former contains a fragment of αs1-casein (FFVAPFPEVFGK) and the latter is a product of whey protein hydrolysis (Haque and Chand 2008).
β-Casein contains a fragment called a “strategic zone.” It is a part of a protein chain between 60 and 70 amino acid residue and possesses ACE inhibitory, opioid, and immunostimulating bioactivities (Haque and Chand 2008). ACE inhibitory activity was detected in fragments 177-183 and 193-202 of β-casein and 23-24, 23-27, and 194-199 of αs1-casein released upon the action of trypsin (EC 126.96.36.199). Quirós and others (2007) isolated 2 ACE inhibitors from β-casein fermented by Enterococcus faecalis. They were the sequences LHLPLP (fragment 133-138) and LVYPFPGPIPNSLPQNIPP (fragment 58-76). Casein sequences EMPFPK and YPVEPFTE derived from bovine γ-casein were able to potentiate bradykinin activity, which was displayed on isolated guinea pig ileum (Silva and Malcata 2005).
Some ACE inhibitors may function as immunomodulators (Pagelow and Werner 1986). The sequence TTMPLW found in the tryptic hydrolysate of αs1-casein was able to inhibit ACE and displayed an immunomodulating effect (Migliore-Samour and others 1989). Certain casein peptides, known as casomorphin 7 (YPFPGPI) and 10 (YQQPVLGPVR), were found to function as ACE inhibitors and immunostimulators (Kayser and Meisel 1996). FitzGerald and others (2004) collected from the literature the more potent in vitro ACE inhibitory sequences found in caseins and whey proteins. Peptides derived from caseins are called casokinins and those present in whey proteins are named lactokinins. Potent casokinins were characterized by the following sequences: VAP (αs1–CN; f25-27; IC50 = 2.0 μM·dm−3), FALPQY (αs2–CN; f174-179; IC50 = 4.3 μM·dm−3), IPP (β–CN; f74-76; IC50 = 5.0 μM·dm−3), VTSTAV (κ–CN; f185-190; IC50 = 52.0 μM·dm−3). The primary structures of lactokinins, known as potent ACE inhibitors were: WLAHK (α-La; f104-108; IC50 = 77.0 μM·dm−3), WLAHK (α-La; f104-108; IC50 = 77.0 μM·dm−3), ALPMHIR (β-Lg; f142-148; IC50 = 42.6 μM·dm−3), ALKAWSVAR (bovine serum albumin; f208-216; IC50 = 3.0 μM·dm−3) (FitzGerald and others 2004).
According to Zhao and Li (2009), the activity of casein hydrolysates to inhibit ACE can be improved by alcalase-catalyzed hydrolysis coupled with plastein reaction. This is the result of a decrease of free amine group amount and the formation of a greater number of peptides in the reaction mixture (Zhao and Li 2009).
Peptides known as lactorphins, obtained by proteolysis of α-lactalbumin and β-lactoglobulin using gastric and pancreatic enzymes, showed a lowered BP effect. Jäkälä and Vapaatalo (2010) indicated that α-lactorphin (YGLF) possessed a transient dose-dependent blood-pressure-reducing effect. This effect was abolished by a naloxone known as an opioid receptor antagonist. The sequences YGLF and YLFF (called β-lactorphin) in vitro improved the function of arteries. β-Lactosin B (ALPM), a peptide derived from β-lactoglobulin, was able to reduce the BP in SHR (Jäkälä and Vapaatalo 2010).
An example of a peptide with di-functional activity is YQEPVLGPVRGPFPIIV, obtained from bovine casein by Lactobacillus casei Shirota. This peptide plays the role of an ACE inhibitor and displays an antithrombotic effect which had been unknown in the past (Rojas-Ronquillo and others 2012).
Bovine lactoferrin is known as a protein with multifunctional (antimicrobial, anticancer, immunomodulating) properties (Darewicz and others 2011; Artym 2012). This protein was also found to be a precursor of novel 38 hypotensive peptides produced by enzymatic hydrolysis with pepsin. Lactoferrin-derived peptides such as LIWKL, RPYL, and LNNSRAP inhibited ACE activity in vitro and their IC50 values were 0.47, 56.5, and 105.3 μM, respectively. These peptides acted as antihypertensive agents to SHR. The peptide LIWKL had the most remarkable antihypertensive activity compared to RPYL and LNNSRAP and the effect of BP reduction remained significant for up to 24 h postadministration (Ruiz-Giménez and others 2012).
Dairy products are a well-documented source of bioactive peptides, which include the ACE inhibitors. They are found in ripening cheeses, sour milk, and fermented milk products such as dahi and yogurts. Examples of fragments with a defined ACE inhibitory activity identified in dairy products are presented in Table 1.
Table 1. Some examples of ACE inhibitory fragments found in dairy products. IC50 values (expressed in μM), annotated in the BIOPEP database or in references cited below, are given in parentheses
It was found that the activity of peptides present in ripening cheeses changes during the ripening process. As the ripening continues, the liberated peptides can be further degraded to inactive fragments, which affects the effect of ACE inhibition. For example, the ACE inhibition in medium-aged Gouda cheese was double that of long-ripened cheese (Choi and others 2012). Cheese whey protein hydrolyzed by proteinase K released some tripeptides able to lower BP in SHR after a single-dose administration. The strongest antihypertensive activity was assigned to the IPA sequence occurring in the cheese whey hydrolysate (Abubakar and others 1998).
The popularity of yogurt is still increasing due to its beneficial effect related to the peptides produced during fermentation, decreased immunogenicity, and lower lactose content (Adolfsson and others 2004). Papadimitriou and others (2007) identified some ACE inhibitory peptides in Greek yogurt, a type which has an increased solids content. They originated from β-, κ-, αs1-, and αs2-caseins and possessed the following sequences: KAVPQ, GVPKVK, GVPKVKE, SQPK, YQEP, TQTPVVVP, DKIHPFAQ, YPVEPFTE (β-CN), NQFLPYPY (κ-CN), RPKHPIKH (αs1-CN), and YQKA (αs2-CN). Some of these sequences shared a local sequence identity with fragments with reported antihypertensive effect. They were: DKIHPFAQ (DKIHPF; IC50 = 257 μM), DKIHPFAQ (DKIHP; IC50 = 113 or 578 μM or 234 μg·cm−3), TQTPVVVP (NIPPLTQTPV; IC50 = 173 μM), KAVPQ (SKVLPVVPQ; IC50 = 39 μM) and RPKHPIKH (RPKHPI; IC50 = 40.3 μM). The reported peptides with documented antihypertensive activity had the IC50 values given in parentheses and the fragments with identical sequences are listed in bold (Papadimitriou and others 2007).
Apart from the well-studied ACE inhibitory and antihypertensive peptides VPP and IPP identified in milk and yogurt, other sequences with the above-mentioned activity were also identified in casein. They were released from casein by pepsin (EC 188.8.131.52) and were characterized by the following primary structures: RYLGY, AYFYPEL (source: αs1-CN), and YQKFPQY (source: αs2-CN). They were able to inhibit ACE in the concentrations of 0.7, 6.6, and 20.1 μM, respectively. The peptide LHLPLP found in milk fermented with Enterococcus faecalis lowered BP in SHR (Jäkälä and Vapaatalo 2010).
The studies of Chen and others (2010) indicated that fermented mare's milk (koumiss) is also rich in ACE inhibitory peptides. Four novel amino acid sequences: YQDPRLGPTGELDPATQPIVAVHNPVIV, PKDLREN, LLLAHLL, and NHRNRMMDHVH were found in both untreated and digested koumiss with pepsin, trypsinase, and chymotrypsin. The sequence YQDPRLGPTGELDPATQPIVAVHNPVIV corresponded to β-casein of mare's milk and the other 3 peptides were not a part of any milk proteins. The peptides were classified as pro-drug type or a combination of pro-drug and true-inhibitor types. Based on these observations, koumiss can also be qualified as a drink with beneficial health effects (Chen and others 2010).
According to the literature, certain egg proteins are a source of peptides that play a role in BP control. This role is mainly assigned to the sequences derived from avian egg-white proteins (Kovacs-Nolan and others 2005). Some examples of ACE inhibitory/antihypertensive peptides found in egg proteins are present in Table 2.
Table 2. The exemplary egg protein fragments with confirmed ACE inhibitory and/or antihypertensive effect
Hoppe (2010) identified a peptide with confirmed ACE inhibitory activity (YAEERYPIL) in ovalbumin pepsin hydrolysate. YAEERYPIL, as well as 2 other sequences, RADHPFL and IVF, were released during the hydrolysis of egg white protein by pepsin (EC 184.108.40.206) after 3 h of incubation (Miguel and Aleixandre 2006; Miguel and others 2006). This showed that a single dose of RADHPFL and IVF contributed to a reduction of BP in SHR and did not affect the normotensive rats (Miguel and Aleixandre 2006).
Other peptides with the ACE inhibitory bioactivity or antihypertensive effects found in the enzymatic hydrolysates of egg white proteins had the sequences RADHPF (derived from ovalbumin and showing antihypertensive activity in SHR) and RVPSL (from ovotransferrin as the ACE inhibitor) (Hoppe 2010; Liu and others 2010). The peptides RADHPFL and RADHPF are called ovokinin and ovokinin 2-7, respectively. The further hydrolysis of ovokinin produced other peptides: RADHPF (ovokinin 2-7) and RADHP (from ovokinin 2-7). Their bioactivity, expressed by IC50 value, showed that these sequences had weaker ACE inhibitory activity (IC50 “RADHPF” ≥400 μM and IC50 “RADHP” = 260 μM) than ovokinin (IC50 “FRADHPFL” = 3.2 μM) (López-Fandiño and others 2007).
Several structural modifications of ovokinin-derived peptides were made to improve their bioavailability and oral activity. The modifications concerned the synthesis of several peptides similar to ovokinin 2-7. Two sequences (RPFHPF and RPLKPW) revealed 10 and 100 times greater activity, respectively, than ovokinin 2-7 after oral administration by SHR. Miguel and Aleixandre (2006) explained the stronger activity of these sequences by their resistance to digestive tract enzymes due to the substitution of amino acids. It was emphasized that both ovokinin 2-7 and its derivative RPLKPW sequence were not able to inhibit the ACE in vitro (Miguel and Aleixandre 2006). Yoshii and others (2001) hydrolyzed chicken egg yolk solutions with Newlase F from the genus Rhizopus as well as gastrointestinal enzymes such as pepsin (EC 220.127.116.11), trypsin (EC 18.104.22.168) and chymotrypsin (EC 22.214.171.124) and obtained several low-molecular-mass oligopeptides with an ability to inhibit ACE in vitro. The 50% ACE inhibitory concentrations ranged from 1.15 to 1.33 mg·mL−1 depending on the enzyme applied. Further research involving Wistar Kyoto rats showed that egg yolk oligopeptides lowered the rats’ BP (both systolic and diastolic) at the level of 10%. The optimal volume of oligopeptides dissolved in water and injected into SHR was 5 mL per kg body weight, which was equivalent to a concentration of 500 mg hydrolysate per kg body weight (Yoshii and others 2001).
Two novel ACE inhibitory peptides were identified in hen egg-white lysozyme (HEWL). They were identified as F2 and F9 peptides and their sequences were NTDGSTDYGILQINSR and VFGR, respectively. The values of the IC50 parameter for the F1 peptide were 4.9 μM and 22.1 μM for F2 (Memarpoor-Yazdi and others 2012). Three other HEWL peptides with confirmed ACE-inhibitory effect were found by Rao and others (2012). They were isolated by RP-HPLC and identified by UPLC-MS/MS and their sequences were the following: MKR (IC50 = 25.7 μM), RGY (IC50 = 61.9 μM), VAW (IC50 = 2.86 μM). In addition, the HEWL treated by gastrointestinal enzymes, pepsin (EC 126.96.36.199), α-chymotrypsin (EC 188.8.131.52), and trypsin (EC 184.108.40.206), showed activity against ACE expressed by IC50 = 15.6 μg·mL−1 (Rao and others 2012).
Studies by You and Wu (2011) indicated that the ACE inhibiting activity of peptides obtained from egg proteins depends on the enzymes applied for the hydrolysis. Hydrolysis of proteins from egg yolk and egg white with thermolysin and alcalase (non gastrointestinal tract enzymes) led to the release of more potent ACE inhibitors than those released from the same substrates but hydrolyzed by gastrointestinal enzymes such as pepsin and pancreatin. The activity of ACE inhibitory peptides corresponded to their amino acid composition, especially in the content of positively charged residues (You and Wu 2011).
Meat and Fish
Meat is known as a valuable source of high-quality proteins playing a role in metabolism. Thus, many researchers are currently studying meat proteins as a source of biopeptides. The special interest in meat results from the economic and environmental aspects of using renewable animal by-products as well as by introducing innovative meat-derived foods in human nutrition (Udenigwe and Howard 2013). According to Ryan and others (2011), ACE inhibiting peptides can be released from meat proteins by protelytic enzymes. Escudero and others (2012b) discovered that the peptide KAPVA, along with 2 other sequences, RPR and PTPVP (source: pork meat digest), showed significant antihypertensive activity to SHR after oral administration. Among these sequences, RPR possessed the greatest in vivo activity. Based on these results, pork meat could serve as a source of bioactive components which could be applied in the production of functional foods or nutraceuticals (Escudero and others 2012b).
Another example of peptides derived from porcine meat are 2 sequences of ACE inhibitors identified in the porcine myosin heavy chain after its hydrolysis with thermolysin (EC 220.127.116.11). They were defined as myopentapeptides A (MNPPK) and B (ITTNP) and corresponded to positions 79-83 and 306-310 in the protein chain, respectively (Ryan and others 2011). In vivo studies by Nakashima and others (2002) showed that these myopeptanopeptides lowered the BP at SHR. The maximum reduction of BP in SHR was 6 h following the administration of myopentapeptides A and B. Myopentapeptide A reduced SBP in SHR of 23.4 mm Hg and myopentapeptide B of 21.0 mm Hg. The dose of peptide was 1 mg per kg body weight (Nakashima and others 2002).
The porcine myosin light chain was found to be a precursor of other peptides with confirmed BP-lowering and/or ACE inhibitory effect. Feeding hypertensive rats the purified VKKVLGNP octapeptide (a product of myosin light chain hydrolysis with pepsin) caused a BP decrease after 3 h. The peptide dosage was 10 mg per kg body weight. It should be noted that after 9 h the BP of rats returned to the state before the administration of VKKVLGNP. The IC50 value of this peptide was 28.5 μM (Katayama and others 2007). A similar tendency to return to the initial BP 9 h following administration was shown by another myosin-derived peptide, KRVITY. This peptide was identified in a pepsin hydrolysate of porcine myosin B, but the reduction of SBP in SHR occurred after 6 h following oral administration (Muguruma and others 2009).
Wei and Chiang (2009) indicated that hydrolysates of porcine blood possessed an ACE inhibitory effect. Hydrolysis of blood plasma with a combination of different enzymes produced ACE inhibitors with different chain lengths. They were usually fragments containing from 2 to 12 amino acids. Ranging the hydrolysis duration from 2 to 10 h contributed to the production of peptides composed of 9 to 13 amino acid residues and molecular masses from 1002 to 1450 Da. The time of hydrolysis affected the ACE inhibition by a porcine blood hydrolysate. More than 6 h of this process lowered the activity of the hydrolysate against ACE (Wei and Chang 2009). Ren and others (2011) identified 3 following sequences in discolored porcine blood pepsin hydrolysate: WVPSV, YTVF, and VVYPW. They were responsible for an ACE inhibition and their median inhibitory concentrations were 0.368, 0.226, and 0.254 mg·cm−3, respectively. Feeding the SHR water solutions containing the above-mentioned peptides, at a dose of 10 mg per kg body weight, led to BP reduction 3 and 15 h after administration. After 3 h, the SBP of the hypertensive rats was reduced by 22.5 (WVPSV), 18.5 (YTVF), and 9.6 (VVYPW) mm Hg. Next, the monitoring of SBP (15 h later) showed the following BP reductions in SHR: 9.6 (WVPSV), 14.75 (YTVF), and 4.9 (VVYPW) mm Hg (Ren and others 2011).
Pepsin and pancreatin are usually involved in the proteolysis of meat-originating proteins. An example of such a peptide is the sequence KAPVA (IC50 approximately 46.6 μM) found in titin.
Some of the peptides known as ACE inhibitors were identified in collagen and troponin. As regards the latter protein, biopeptides with an ACE inhibitory activity were found in bovine, and chicken collagens. For example, the hydrolysis of bovine collagen with Alcalase®, pronase E, and collagenase, preceded by the processes of fractionation at each stage of hydrolysis by the above-mentioned enzymes, yielded 2 ACE inhibitors: GPV and GPL. Their calculated values of IC50 were 4.7 and 2.5 μM, respectively (Ryan and others 2011).
Protease from Aspergillus oryzae played a role in the release of some ACE inhibitors from chicken collagen. They were longer chain sequences rich in glycine, proline, and hydroxyl proline. The sequences derived from chicken collagen were able to inhibit ACE and possessed an antihypertensive effect. Feeding the SHR with collagen hydrolysates (3 g water or saline hydrolysate per kg body weight) caused a noticeable reduction in BP 2 h following their administration and the maximum reduction of BP (50 mm Hg) was observed after 6 h (Saiga and others 2008; Ryan and others 2011).
Beef is also a meat containing proteins which are a source of ACE-inhibitory peptides. The peptide VLAQYK (obtained from sarcoplasmic proteins of beef rump) was considered for testing in clinical trials as a component of functional food possessing properties capable of reducing the elevated BP. The calculated value of the IC50 VLAQYK sequence was 32.06 μM and this peptide was also found to act as an antihypertensive agent (Jang and Lee 2005). Antihypertensive activity of VLAQYK was observed on SHR fed for 8 wk with doses of 0.2, 0.5, and 1.0 g of a peptide per kilogram body weight (Jang and others 2004).
The sequence WYPAAP is another example of a peptide functioning as an ACE inhibitor and antihypertensive agent. It was identified in duck skin by-products subjected to proteolysis with 9 proteolytic enzyme preparations (alcalase, collagenase, flavourzyme, neutrase, papain (18.104.22.168), pepsin, Protamex, trypsin, and α-chymotrypsin). The purified peptide possessed activity against ACE (IC50 = 0.095 mg·mL−1) and intravenous injection of the WYPAAP to SHR at a dose of 1 mg·kg−1 body weight led to a significant reduction in BP and heart rate. The reduction of SBP and heart rate was noted 0.5 h after the injection and continued up to 6 h (Lee and others 2012).
Escudero and others (2012a) found that process of dry-curing Spanish ham contributed to the accumulation of small-size peptides generated during the process. It was showed that water-soluble extract of a Spanish dry-cured ham possessed an antihypertensive effect in SHR. The single doses of the extracts administered orally to SHR were: 4.56, 1.48, and 8.7 mg lyophilized sample·kg−1 body weight. Depending on the dose of the dry-cured ham extracts, different values of SBP reduction were noted. The values of SBP 6 h after administration were: 38.38 ± 5.84, 27.48 ± 5.11, and 23.56 ± 7.7 mm Hg, respectively (Escudero and others 2012a). Escudero and others (2013) found the sequence AAATP to be the most potent antihypertensive agent present in water soluble extract of Spanish dry-cured ham. The ACE inhibitory activity of AAATP was 100 μM. This peptide lowered SBP in SHR by 25.62 ± 4.5 mm Hg 8 h after oral administration (dose: 1 mg peptide·mL−1 distilled water) (Escudero and others 2013).
Since fish and shellfish proteins are structurally diversified, they are a substrate to produce peptides with multifunctional bioactivities (Harnedy and FitzGerald 2012). Fish protein hydrolysates have particularly interested food biotechnologists due to the availability of a highly balanced amino acid content, high protein content, and the presence of biologically active peptides, including ACE inhibitors/antihypertensive peptides (Chalamaiah and others 2012). Gehring and others (2009) reported that fish protein hydrolysates have been shown to inhibit ACE. Hydrolysates produced with an accompanying fraction consisting of soluble peptides from alkaline-processed catfish protein isolate have been proven to inhibit ACE. The ACE inhibition by the hydrolysates was estimated for 3 degree of hydrolysis (DH) levels: 5%, 15%, and 30%. The ACE inhibition varied depending on DH and the highest percentage of ACE inhibition (90.6%) was observed when the DH of hydrolysates was 5% (Gehring and others 2009).
Peptides with an ACE inhibitory activity were found in traditional Japanese food, called katsuobushi, a fermented, smoked skipjack tuna (Katsuwonus pelamis) (Vercruysse and others 2005). It was discovered that katsuobushi contains the ACE inhibitory sequence LKPNM (Ryan and others 2011). Hydrolysis of katsuobushi by various proteases exhibited ACE inhibitory activity in the digests. Among them, the strongest activity was detected in thermolysin (EC 22.214.171.124) digest (IC50 = 29 μg·cm−3), as well as a long-lasting antihypertensive activity was observed after oral administration in hypertensive and borderline subjects (Vercruysse and others 2005). The SBP decreased by 12.5 mm Hg in 30 hypertensive and borderline hypertensive humans (Ryan and others 2011). Fujita and others (2001) confirmed that 1.5 g katsuobushi daily had a potent antihypertensive effect on hypertensive humans. Thus, a thermolysin digest of katsuobushi was approved as a “food for special health use” (FOSHU) in Japan (Vercruysse and others 2005).
Actin of skipjack tuna was a source of 8 peptides with confirmed ACE inhibitory activity. For example, the sequence LKPNM (IC50 = 2.4 μM), classified as a pro-drug-type ACE inhibitor, was used as a substrate to produce a more potent peptide LKP (IC50 = 0.32 μM) in vivo. The above-mentioned sequences were administered intravenously to SHR and showed an antihypertensive effect. Oral administration of LKPNM and LKP to rats with hypertension revealed the maximum BP reducing effect after 6 and 4 h, respectively (Vercruysse and others 2005). Data collected by Ryan and others (2011) showed that administration of the same dose of both peptides (60 mg·kg−1 body weight) reduced SBP in SHR by 23 (LKPNM) and 18 (LKP) mm Hg.
Six dipeptides recognized as ACE inhibitors/antihypertensive agents were identified in the thermolysin hydrolysate of chum salmon. These included WA, VW, WM, MW, IW, and LW and they possessed the following IC50 values expressed in micromoles: 277.3, 2.5, 96.6, 9.9, 4.7, and 17.4, respectively. The maximum reduction of BP was 4 h following oral administration of the hydrolysate containing the above-mentioned peptides. The doses of a hydrolysate which had an impact on the maximal reduction of SBP in SHR were 500 and 2000 mg·kg−1 body weight. The reduction of SBP was 28 mm Hg (dose: 500 mg·kg−1) and 38 mm Hg (dose: 2000 mg·kg−1) (Ono and others 2003). Ewart and others (2009) used salmon rack proteins and hydrolyzed them by different enzymes (alcalase, flavourzyme, fungal protease concentrate, Protease GC106, Multifect Neutral, and Protease S-Amano) to produce peptides. Several peptides were found in a salmon protein hydrolysate (LAF, LTF, IIF, LAY, IAY, VFY, YAY, VLW, IAW, YAL, YNR). All of them showed activity against the ACE. The most abundant were the sequences VLW, VFY, and LAF. The tripeptides IAW, IIF, and VLW were the most potent (IC50 values of 9.5, 22.7, and 24 μM, respectively). Depending on the proteases applied to obtain salmon protein hydrolysate (SPH), the reduction of mean carotid BP in SHR ranged from 0% (Multifect Neutral) to 11% (Protease-S-Amano) compared to the control group. The estimated dose of SPH administered orally to SHR was 1500 mg·kg−1 body weight (Ewart and others 2009).
Other fish proteins from sardine, tuna, and Alaskan pollack, were found to be sources of ACE inhibitors. For instance, hydrolysis of sardine muscle with alcalase from Bacillus licheniformis produced the following sequences: MF, RY, MY, LY, YL, IY, VF, GRP, RFH, AKK, RVY, GWAP, KY, and VY. All of these were able to act as ACE inhibitors. In the case of the latter sequence (VY), the antihypertensive effect was confirmed in vivo (Matsufuji and others 1994).
Tuna dark muscle treated with various proteases such as alcalase, neutrase, pepsin, papain, α-chymotrypsin, and trypsin also revealed an ACE inhibitory/antihypertensive effect. Among the hydrolysates, the strongest bioactivity was found in pepsin hydrolysate due to the presence of the WPEAAELMMEVDP sequence. This peptide was classified as a non-competitive inhibitor and its calculated IC50 was 21.6 μM. Oral administration of the above-mentioned sequence to SHR showed its potential as an antihypertensive agent. The maximal BP reduction was revealed between 3 and 6 h following intake at a dose of 10 mg per kg body weight. The effect of BP reduction remained after 10 h and the difference in SBP between the control and hypertensive rats was 15 mm Hg (Qian and others 2007). Tuna was also a source of 2 other sequences defined as ACE inhibitors, PTHIKWGD (originating from tuna extract) and its analogue PTHIKW (Kohama and others 1988). Lee and others (2010) isolated a non-competitive inhibitor of ACE from enzymatic hydrolysate of tuna frame protein built up from 21 amino acid residues (GDLGKTTTVSNWSPPKYKDTP). This peptide was antihypertensive to SHR (maximal BP decrease: 21 ± 2 mm Hg; time interval: 6 h; dose 10 mg·kg−1 body weight) and its ACE inhibitory activity in vitro, expressed as IC50 value, was 11.28 μM (Lee and others 2010).
The peptide GPL isolated from purified Alaskan pollack skin gelatin hydrolysate became a standard to synthesize other sequences to study their activity against ACE. By solid-phase synthesis, novel ACE inhibitors containing Gly, Leu, and Pro were studied. It was confirmed that the following di- and tripeptides were able to inhibit ACE: GPL (2.65 μM), GP (252.63 μM), PL (337.32 μM), PGL (13.93 μM), LGP (0.72 μM), GLP (1.62 μM), PLG (4.74 μM), and LPG (5.73 μM). Numbers in brackets mean the concentrations corresponding to their half-maximal activity (IC50) (Byun and Kim 2002).
ACE inhibitory peptides can be found outside fish marine sources, such as giant jellyfish (Nemopilema nomurai). It is regarded as a traditional food in East Asian countries to treat asthma and hypertension. Peptide YI was identified in papain hydrolysate of jellyfish and its IC50 value was 6.56 μM (Kim and others 2011). Other marine sources are also of great interest in obtaining peptides helpful in the prevention of hypertension. Some examples of marine organisms, including their ACE inhibitors (in brackets) with a confirmed effect in vitro are: wakame (YNKL, IW), shrimp (IFVPAF), sea cucumbers (MEGAQEAQGD), microalgae (VECYGPNRPQF), and bonito (IRPVE) (Ngo and others 2012).
ACE inhibitory/antihypertensive potential was discovered in marine macroalgae hydrolysates. ACE inhibitory activity was associated with the following sequences of macroalgae: GKY, SVY, SKTY (Hizikia fusiformis), VY, IY, AW, VW, IW, LW (Undaria pinnatifida), and IY, MKY, AKYSY, LRY (Porphyra yezoensis). Antihypertensive activity was observed in peptides derived from Undaria pinnatifida and Porphyra yezoensis (Harnedy and FitzGerald 2011). Porphyra yezoensis is the precursor of nori oligopeptide (NOR), in which the sequence AKYSY is encrypted. The optimal dose of this peptide in NOP, which lowered BP in SHR, was 0.2 mg·kg−1 body weight (Saito and Hagino 2005). The examples of peptides derived from wakame (Undaria pinnatifida) were the sequences: YH, KY, FY, and IY. All of them were administered orally to SHR at a dose of 50 mg·kg−1 body weight. The significant reduction of SBP was the following: 50 mm Hg (YH, time: 3 h), 45 mm Hg (KY; time: 6 h), 46 mm Hg (FY, time: 3 h), and 33 mm Hg (IY, time: 3 h) (Suetsuna and others 2004). Sato and others (2002) reported that following sequences derived from wakame lowered SBP in SHR 3 h after administration: VY (8.2 mm Hg), IY (3.9 mm Hg), FY (4.3 mm Hg), IW (1.3 mm Hg), AW (4.8 mm Hg), LW (1.1 mm Hg), VW (5.2 mm Hg). All 7 peptides were administered to SHR at a dose of 1 mg·kg−1 body weight (Sato and others 2002).
Guang and Phillips (2009) reported that many various plants were sources of ACE inhibitors. These sources include the following plants: soybean, mung bean, sunflower, rice, corn, wheat, buckwheat, broccoli, mushroom, garlic, spinach, and grapes. These examples of plants, known as sources of ACE inhibitory/antihypertensive peptides, are briefly described below.
Soybeans and soy-based foods are known for their high quality protein. Soybean components, including proteins, are able to reduce cholesterol levels as well as chronic disease risks such as diabetes, obesity, and vascular diseases (Allison and others 2003). An example of a soybean commercial product known for its antihypertensive effect is douchi. It is a Chinese fermented food and has been applied as a medicine for millennia. The fermentation of douchi with Aspergillus egypticus revealed its ACE inhibitory/antihypertensive activity due to the presence of one peptide containing phenylalanine, isoleucine, and glycine in the ratio of 1:2:5 (Zhang and others 2006).
Enzymatic hydrolysates of soybean glycinin were recognized as sources of ACE inhibitors. Among them, the protease P glycinin hydrolysate was potent due to the identified peptide VLIVP (Wang and others 2008b). Hydrolysis of soybean glycinin by acid proteinase from Monascus purpureus led to the release of 2 peptides identified as SPYP (IC50 = 850 μM) and WL (IC50 = 65 μM). According to Kuba and others (2005), the further hydrolysis of SPYP peptide in vitro by pepsin, chymotrypsin and trypsin enhanced its ACE inhibitory activity. Two ACE inhibitors obtained from β-conglycinin were identified as LAIPVNKP and LPHF. Their IC50 values were 70 and 670 μM, respectively (Kumar and others 2010).
Peptic digests of soybean revealed activity against ACE. The activity of hydrolysates resulted from the presence of peptides identified by Edman's procedure. The sequences of ACE inhibitors, including their IC50 values (in brackets), were: IA (153 μM), YLAGNQ (14 μM), FFL (37 μM), IYLL (42 μM) and VMDKPQG (39 μM). Their antihypertensive activity was confirmed on SHR which was fed by saline solutions of peptidic fraction powders (0.9% w/v; 2.0 g per kg body weight). A significant reduction of SBP in SHR (17.5 mm Hg) was observed after 2 h and continued for 6 h following oral administration (Chen and others 2002).
Rapeseed is a plant that has been known by human civilization for about 3000 y. It contains components which may prevent hypertension (Hashmi and others 2010). Pedroche and others (2004) measured the ACE inhibiting activity of a rapeseed hydrolysate obtained by hydrolysis of rapeseed protein isolate with an alcalase. The rapeseed protein isolate possessed the ability to inhibit ACE by 23%. After 10 min of hydrolysis, the activity against ACE was doubled (Pedroche and others 2004). Kinetic studies of Mäkinen and others (2012) led to compare captopril with the ACE inhibitory activity of peptide fractions derived from alcalase hydrolysates of rapeseed. It was found that hydrolysates of rapeseed possessed moderate Ki ranges (0.2 to 0.3 mg·cm−3). Ki calculated for captopril was 0.0067 mg·cm−3. This indicates that the drug (captopril) had the stronger affinity to an ACE active site than the peptide fractions (Mäkinen and others 2012).
As was reported by Aluko (2008), the primary structures of many pea and mung bean proteins contain fragments used to form products useful in the treatment and prevention of human diseases. The results published by Li and others (2005) showed that alcalase hydrolysate of mung bean protein was able to inhibit the ACE (IC50 = 0.62 mg·cm−3). Another study carried out by Li and others (2006) indicated that mung-bean protein is a good precursor of ACE inhibitors such as KDYRL, VTPALR, KLPAGTLF, and alcalase hydrolysates of this plant have the potential to be utilized to produce functional foods with antihypertensive activity.
Sunflower proteins have unique functional properties which may expand the range of food uses for concentrated seed proteins (Sosulski and Fleming 1977). According to Megías and others (2009) extensive hydrolysis of sunflower protein by pepsin and pancreatin generated the release of ACE inhibitory peptide FVNPQAGS (IC50 = 6.9 μM).
Li and others (2007) also hydrolyzed rice protein with alcalase for 2 h and obtained a peptide described as a potent ACE inhibitor (IC50 = 18.2 μM). Its sequence was TQVY and the antihypertensive effects on SHR were also confirmed. Both the hydrolysate and purified peptide affected the BP in SHR. The dose of hydrolysate at 600 mg per kg body weight reduced SBP (25.6 mm Hg) after 6 h. The maximum antihypertensive effect of TQVY (40 mm Hg), at a dose of 30 mg per kg body weight, occurred at the same time as that of a hydrolysate (Li and others 2007).
Although corn is mainly viewed as an animal feed, its biochemical structure, low cost, and high abundance make it an interesting subject for the food and nonfood industries. Further studies are required, especially for corn gluten and its hydrolysates, due to the presence of amino acids involved in metabolic processes (Bong and others 2010). A corn gluten hydrolysate prepared by Pescalase (a serine protease from Bacillus licheniformis) was a source of a novel ACE inhibitor whose presence has not been confirmed in any proteins. This was the peptide PSGQYY, possessing an IC50 value of 0.1 mM. A dose of 30 mg per kg body weight lowered the BP in SHR. The maximum reduction of BP was 28 mm Hg and appeared in hypertensive rats 15 min after the administration (Suh and others 1999). A maize endosperm protein, called α-zein, was found as the precursor of peptides acting as ACE inhibitors. Hydrolysis of α-zein led to the release of tripeptides such as: LKP, LSP, LQP, LAY with IC50 values of 0.27, 1.7, 1.9, and 3.9 μM, respectively. Additionally, intravenous injection of peptide LKP (30 mg per kg body weight) to SHR led to a reduction in BP by 15 mm Hg (Miyoshi and others 1991).
Wheat is one of the major cereals in the world and wheat germ contains proteins which are a source of several essential amino acids (Hu and others 2012). Matsui and others (2010) reported the antihypertensive effect of wheat germ hydrolysate due to the presence of tripeptide IVY as a potent ACE inhibitor. The antihypertensive effect of IVY was tested on mice. Their mean arterial pressure gradually decreased after 5 mg per kg of IVY injection and gave a maximum reduction (19.2 mm Hg), after 8 min, which was held for 20 min. Moreover, the peptide IVY formed a subsequent ACE inhibitor (VY) as a result of its further metabolism through the aminopeptidase action in plasma. Thus, according to Matsui and others (2010), the intake of IVY as a physiologically functional food would act as a BP reductant through its own antihypertensive effect and VY release after absorption.
Nogata and others (2009) discovered that wheat milling by-products are also a source of ACE inhibitors acquired due to their autolysis. Six sequences of ACE inhibitors were identified: LQP, IQP, LKP, VY, IY, and TF. Maximal activity was reached under the following conditions: pH 3.2, temperature 40°C, time 12 h.
Common buckwheat (Fagopyrum esculentum) and tartary buckwheat (Fagopyrum tartaricum) are regarded as traditional foodstuffs available worldwide (Tsai and others 2012). Because little is known of the ACE inhibitory activity of the other parts of the buckwheat plant, Tsai and others (2012) studied the bioactivities of different parts and varieties of buckwheat. It was discovered that 50% ethanolic extracts of buckwheat hulls showed the greatest ACE inhibitory activity (IC50 = 30 μg·cm−3). Other extracts also characterized the activity to inhibit ACE, such as deionized-water extracts of groats which probably resulted from the presence of water-soluble peptides. An example of an ACE inhibitory sequence derived from Fagopyrum esculentum Moench is a tripeptide GPP with an IC50 = 6.25 μg protein·cm−3 (Kumar and others 2010).
Broccoli is known as a plant possessing chemoprotective properties (Abdulah and others 2009). Lee and others (2006) discovered that this plant can also be a food with an ability to inhibit ACE, because of the presence of the tripeptide YPK with an IC50 = 10.5 μg protein·cm−3 and identified in broccoli extracts by using column chromatography methods.
Many compounds have been extracted from mushrooms, and their potential classifies them as pharmaceutical substances. In traditional Chinese medicine, mushroom extracts were studied for the treatment of human diseases such as cancer, high cholesterol level, high BP, and diabetes (Rai and others 2005). Abdullah and others (2011) evaluated the ACE inhibitory potential of 11 hot- or cold-water extracts of culinary-medicinal mushroom species. Their IC50 values ranged from 0.050 (Pleurotus florida) to 0.890 (Agrocybe sp.) mg·cm−3. Studies carried out by Lee and others (2004) and Choi and others (2001) showed the possibility of applying the water extracts of edible mushrooms to produce 2 ACE inhibitors: GEP from Tricholoma giganteum and VIEKYP from Grifola frondosa. The IC50 of the 1st peptide was 0.31 mg·cm−3. Moreover, GEP lowered the BP at SHR, after a dose of 1mg per 1 kg body weight. The SBP reduction was 36 mm Hg and was achieved 2 h after administration (Lee and others 2004). In the case of VIEKYP, the purified inhibitor was a competitive inhibitor to ACE and inhibitory activity was maintained even after digestion by intestinal proteases (Choi and others 2001).
Experiments carried out on rats, dogs, and humans confirmed that the use of fresh garlic or its powder and/or extract had the ability to reduce BP (Suetsuna 1998). Suetsuna (1998) identified 7 dipeptides in aqueous extracts of Allium sativum (garlic) which were responsible for its activity against the ACE as well as its antihypertensive effect on SHR. These were SY, GY, FY, NY, SF, GF, and NF and had IC50 values of 66.3, 72.1, 3.74, 32.6, 130.2, 277.9, and 46.3 μM, respectively. The changes of SBP were measured in SHR at different time intervals: 0, 1, 2, 3, 4, and 6 h after oral administration of a single dose (200 mg per kg body weight). The maximal reduction of BP depended on the sequence and time after oral intake (DF, GF, and SF: 1 h; DY: 3 h; SY, GY, and FY: 4 h) (Suetsuna 1998).
Yang and others (2003) discovered that peptides MRWRD, MRW, LRIPVA, and IAYKPAG found in pepsin-pancreatin digests of spinach RuBisCo (ribulose-1,5-bisphosphate carboxylase, E.C. 126.96.36.199) inhibited the ACE. The activities of peptides expressed as IC50 (μM) were 2.1, 0.6, 0.38, and 4.2, respectively. The antihypertensive effect of the above-mentioned ACE inhibitors tested on SHR depended on the dose of the peptide injected to SHR. For example, 20 mg·kg−1 body weight of MRW (oral administration) maximally reduced the SBP (20 mm Hg) after 2 h and MRWRD caused maximal BP reduction (13.5 mm Hg) 4 h after the administration of 30 mg·kg−1 body mass. The maximal antihypertensive activity of IAYKPAG was observed 4 h after administration (dose: 100 mg per kg body weight, reduction of SBP: 15 mm Hg). In the case of IAYKPAG, it was worth noting that the shorter fragments of this sequence (such as IAYKP, IAY, and KP) showed a hypotensive character. The peptide LRIPVA was a potent ACE inhibitor (Yang and others 2003). Zhao and others (2008) reported that the activity of peptides to inhibit ACE originating from food proteins usually tends to be weakened in old SHR (25-wk-old and more). The peptide MRW isolated from spinach was able to lower BP in both older and younger rats (Zhao and others 2008).
Apart from the description of plant-derived ACE inhibitory/ antihypertensive peptides, there are many of them which are not discussed in this paper. The literature examples of other plant origin peptides (as well as their ACE inhibitory/antihypertensive sequences) found in other sources than those described in this chapter are given in Table 3.
Table 3. The example list of ACE inhibitory and/or antihypertensive peptides originating from food sources. IC50 values (expressed in μM), annotated in the BIOPEP database or in references cited below, are given in parentheses
1In bold: peptides with confirmed antihypertensive bioactivity.
a,b,c,d: literature cited to different peptide sequences derived from the same source.
Absorption of ACE Inhibitory/Antihypertensive Peptides
Approximately 90% of the absorption of peptides in the gastrointestinal tract takes place in the region of the small intestine (Regazzo 2010). Once the peptide passes through the small intestine, it reaches the large intestine. Then, most of the absorption takes place in the half of the colon defined as the “absorbing colon” (Garcia-Redondo and others 2010).
Korhonen and Pihlanto (2006) found that the antihypertensive bioactivity of peptides after oral ingestion can be achieved after their absorption in an intact form and their resistance is degraded by plasma peptidases. It was proven that some casein antihypertensive peptides containing C-terminal proline are resistant to proteolysis enzymes and thus they can be absorbed while still active (Mizuno and others 2004).
According to Woodley (1982), over 40 different enzymes have contact with orally administered protein during its passage through the small intestine. Once enzymatic release and activation of bioactive peptides occurs, they function as exogenous regulators interacting with the target sites of an organism (Yamamoto 1997). Peptide absorption, as well as the bioactivity of peptides, depends on their mechanism of transport (Roberts and others 1999). For example, short chains (di- and tripeptides) are absorbed across the brush border membrane due to the action of specific peptide transport system and then reveal their biological activity (Guang and Phillips 2009). The low-molecular-weight nutrients, such as di- and tripeptides, as well as glucose and amino acids, are mainly transported via respective transporter proteins (transporters) naturally occurring in the absorptive cells of plasma membranes. It is one of the 4 most important pathways of the intestinal nutrient transport and is called “transporter-mediated transport” (Shimizu 1999).
One of the active systems of transcellular transport of peptides is peptide transporter 1 (PepT1), belonging to the proton-dependent oligopeptide transporter (POT) family. The PepT1-mediated transport is degradative due to the rapid hydrolysis of di- and tripeptides by peptidases located in cytosol. PEPT1 facilitates the transfer from enterocytes to the bloodstream of short peptides, resistant to hydrolysis (Satake and others 2002; Segura-Campos and others 2011).
In the case of larger molecules (proteins and/or peptides), which cannot be absorbed via PepT1, their transport is made possible by transcytosis (Heyman and others 1990).
The other form of transport of low-molecular-weight nutrients, including oligopeptides, is paracellular transport. Such mechanism of transport is now thought to be more important than previously and it relies on passive diffusion between cells of substances not possessing specific transporters. The permeability between the cells is regulated by tight junctions (TJ), and modulation of the TJ facilitates this form of transport (Shimizu 1999). The components of TJ are proteins called occludins (ZO-1 and ZO-2) and cingulin. They are associated with intracellular filaments and cytoskeletal components. Thus, the permeability of TJ is physiologically regulated by intracellular messengers as well as by food components. The efficiency of absorption of oligopeptides improves when the paracellular permeability is increased by modulation of the TJ structure (Shimizu 1999).
To investigate the intestinal transport, the Caco-2 monolayer is usually applied as a model. Such a model was used for 2 dipeptide sequences, AF and IF, originated from soy sauce. It was discovered that AF and IF could be transported across the Caco-2 cell monolayers and showed ACE inhibitory activity. Moreover, it was indicated that IF had greater affinity toward the transport as compared with AF (Zhu and others 2008).
Gastrointestinal simulation of digestion of a peptide with antihypertensive activity (LHLPLP) obtained a shorter fragment (HLPLP), which was then transported by cellular peptidases to the intestinal epithelium. Formation of HLPLP during incubation in the Caco-2 cell chamber led to the appearance of these sequences after 3 min and its concentration increased during 60 min of incubation. The presence of orally-administered HLPLP was found in human plasma, which shows the intestinal absorption of this peptide in humans.
Sun and others (2009) examined the transepithelial transport of an antihypertensive sequence KVLPVP by Caco-2 monolayers. The obtained results indicated that paracellular transport diffusion was involved in the transport of an intact peptide (Sun and others 2009).
Two tripeptides known for their antihypertensive bioactivity (IPP and VPP) were detected in human plasma in picomolar concentrations. Their absorption increased after ingestion in the form of a yogurt beverage (Hernández-Ledesma and others 2011). Paracellular transport was found to be the main form of transport of VPP (Shimizu and Son 2007), since the transport mediated by PepT1 carrier, led to a rapid hydrolysis of the peptide (Regazzo 2010).
The factor determining the transport rate of longer peptide sequences depended on their susceptibility to brush border proteolytic enzymes. For instance, the peptide ALPMHIR (lactokinin) was transported intact and its concentration was too low to show ACE inhibitory effects, which indicated it had been cleaved by aminopeptidases (Regazzo 2010).
To conclude, the problem of absorption of ACE inhibitors is still an open question due to the differences occurring between the forms of transport of the individual peptides (Brandsch and others 2008) and Caco-2 models are found to be a helpful, but not ideal, tool to predict their oral bioavailability (Thwaites and others 1995).
The establishment of a detection model for the identification of mechanisms, by which peptides show antihypertensive bioactivity in vivo, is the most challenging task for scientists. The model of simulated gastrointestinal tract digestion of proteins and peptides mimics their digestion in humans (Jao and others 2012). Thus, Jao and others (2012) elucidated the importance of research concerning the implementation of strategies relying on increasing the resistance to digestive enzymes and the cellular permeability of antihypertensive peptides. Other authors have highlighted the importance of the preservation of the peptides’ bioactivity during digestion processes by peptide modification via chemical reactions, for example, lipidation or physical methods such as microencapsulation (Segura-Campos and others 2011).
Production of ACE Inhibitory/Antihypertensive Peptides
The market for functional food products which have an impact on health has expanded in recent years. It has led the food industry to introduce marketing strategies which embrace consumer expectations with health-promoting foodstuffs (Tidona and others 2009) Thus, the production of peptides connected to certain antihypertensive bioactivity involving 2 aspects, such as low cost and convenient formulations, is a worthy challenge that will help to exploit the beneficial and therapeutic effects of peptides (Rosales-Mendoza and others 2013).
According to Tavares and others (2012), bioactive peptides, including ACE inhibitors, may generally be obtained from food protein by hydrolysis. The enzymatic release of peptides from parent proteins is possible during: (a) the process of protein digestion in the GI tract, (b) food processing, and (c) proteolysis by enzymes of microorganism and/or plant origin (Hajirostamloo 2010). In the case of enzymatic digestion of proteins in the gastrointestinal tract, the bioactivity of released peptides depends on the possibility to reach their receptors intact, which is a key factor in their transport to peripheral organs, and subsequent absorption into the intestinal epithelium (Vermeirssen and others 2004a).
As regards the release of ACE-inhibiting peptides during the process of protein digestion in the GI tract, various attempts have been made to simulate gastrointestinal digestion to release ACE inhibitors from various sources (Hernández-Ledesma and others 2011). The aim of the simulation of digestion processes is to mimic the digestion of protein(s) in the human body as well as to analyze the potential of peptide release which takes place after the normal consumption of food proteins (Udenigwe and Aluko 2012). The idea of GI tract simulation of protein digestion was successfully applied to identify some ACE inhibitors in pepsin and pancreatin hydrolysates of human milk and infant formulas (Hernández-Ledesma and others 2007).
The following enzymes, which are commercially available, have been used to produce ACE inhibitors from food proteins: trypsin, subtilisin, chymotrypsin, thermolysin, pepsin, proteinase K, papain, and plasmin, as well as the enzymes derived from fungal and bacterial sources (Hajirostamloo 2010; Yamamoto 2010). Among these enzymes, trypsin is the most widely applied to yield biologically active peptides in vitro (Möller and others 2008) and has been used to produce most of the ACE inhibitors from bovine αs2-casein as well as bovine, ovine, and caprine κ-casein macropeptides (Korhonen and Pihlanto 2006).
The results of hydrolysis of protein in vitro and in vivo can be altered by the factors defining the nature of substrate and enzyme, for example, the location of enzyme and substrate in different intra- and extracellular areas, the impact of inhibitors on the enzyme(s), and the adaptability of the substrate to access the peptide bond or other environmental predispositions (Rawlings 2009). Moreover, enzymes on their own are costly, which increases the total cost of biopeptide production. An alternative way to reduce the cost of processing is to use proteases of microbial origin. Lactic acid bacteria (LAB) proteases and Neutrase, Subtilisin, Orientase, Protex 7L, Protamex 1.5 have been used in the large-scale production of biologically active peptides (Zambrowicz and others 2013). The proteolytic systems of LAB, such as Lactococcus lactis, Lactobacillus helveticus, and Lactobacillus delbrueckii ssp. bulgaricus, are already well-characterized. They are built up from a cell-wall proteinase and several specific intracellular enzymes such as endopeptidases, aminopeptidases, dipeptidases as well as tripeptidases (Korhonen and Pihlanto 2006). It has been reported on the basis of in vitro experiment, that proteinases from Lactobacillus lactis and Lactobacillus helveticus CP790 release potent ACE-inhibiting peptides. For example, 2 peptides known for their antihypertensive effect (VPP and IPP) were identified in skim milk fermented with Lactobacillus helveticus CP790 and Saccharomyces cerevisiae (FitzGerald and others 2004). ACE inhibitory peptides were found in yogurt-type fermented foods produced using Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus lactis ssp. cremoris (Gobetti and others 2000) and also in ripening cheeses (Sieber and others 2010). It has been observed that selected strains of Lactobacillus helveticus are widely applied in cheese manufacturing due to their high proteolytic activity as well as their ability to release the antihypertensive lactotripeptides VPP and IPP (Kenny and others 2003). It should be noted that extended ripening times cause the ACE inhibitors to be degraded (Murray and FitzGerald 2007).
As was mentioned above, ACE inhibitory peptides usually contain proline. Due to this fact, they can be produced by proline-specific microorganism peptidases. An example of such a microorganism is Lactobacillus rhamnosus which possesses a proteolytic system, including proline-specific aminopeptidases (Moslehishad and others 2013). Moslehishad and others (2013) showed that Lactobacillus rhamnosus PTCC 1637 produced potent ACE inhibitors from bovine and camel milk proteins. The highest accumulation of these peptides was observed in caseins rich in proline residues.
Many laboratories are developing technologies to produce high-yield peptide products with potent bioactivity. Thus, ACE inhibitors obtained via protein proteolysis and identified in a hydrolysate are being further processed. Food processing includes the properties of peptides, including ACE inhibitors such as size, net charge, and hydrophobicity which may affect when enhancing the bioactivity of the potential product (Udenigwe and Aluko 2012).
Membrane and chromatography techniques are especially being applied to obtain fractions containing low-molecular-weight ACE inhibitors and to define their molecular ranges. Membrane separation technologies are common in the separation of proteins and, in some cases, they cannot be directly applied in peptide separation due to the size of an active sequence. It may be composed from 2 to 20 amino acids and many peptides, including ACE inhibitors, may show multiple activity. This means that separation of a single, short peptide with a specific activity is difficult (Bazinet and Firdaous 2009). As reported by Bazinet and Firdaous (2009), ultrafiltration (UF) has been the membrane technique used to enrich peptides with a specific molecular weight. This was confirmed in work carried out by Pihlanto and others (2008) concerning the application of UF to produce ACE inhibitors from potato protein hydrolysates.
Different chromatography techniques are an alternative to peptide fractionation. The correct selection of chromatography techniques depends on the physicochemical properties of a peptide. For example, chromatography using selective ion-exchange columns is useful to fractionate peptides possessing a characteristic net charge, while RP-HPLC was found to be helpful in detecting peptides with a hydrophobic/hydrophilic character (Udenigwe and Aluko 2012). Normal-phase HPLC is a preferential technique to separate more hydrophilic peptides (Wang and Gonzalez De Meija 2005). Another chromatographic technique, affinity chromatography, was successfully applied by Megias and others (2006) to purify ACE inhibitors from food proteins by immobilization of ACE on a glyoxyl-agarose and immobilized copper.
Protein hydrolysis combined with techniques to separate and purify released biopeptides (including ACE inhibitors) is summarized in Figure 2. Recent developments have shown the suitability of computational methods in the production of peptides. The bioinformatic approach includes the selection of protein sequence, prediction of potential products of its proteolysis, prediction of ACE inhibitory/antihypertensive activity of products, optimization of hydrolysis to produce peptides with the highest activity, prediction of protein/peptide structure and bioactive peptide properties, and QSAR modeling (Carrasco-Castilla and others 2012). According to Carrasco-Castilla and others (2012), the involvement of the computational methodologies prior to laboratory experiments on peptides is helpful in optimizing their production as well as research into peptides of special interest. Additionally, the combination of computer tools with experimental work in the process of biopeptide production has contributed to a reduction of costs and facilitates their manufacturing (Carrasco-Castilla and others 2012).
The proposed scheme of a potential procedure to produce an ACE inhibitor is applicable both on a laboratory and industrial scale. Although the various stages (such as selection of protein, hydrolysis, and ACE inhibition assay) are the same in both cases, the methods of separation and purification of peptides may differ because of a lack of unified technology. However, over the years, progress in the development and introduction of new techniques has been observed (Korhonen and Pihlanto 2007).
Antihypertensive peptides can also be produced with the help of genetic engineering methods. The following examples of antihypertensive sequences, such as HHL, HVLPVP, FFVAPFPEVFGK, and GHIATFQER, were expressed in Escherichia coli (Jeong and others 2007). Rao and others (2009) designed and expressed an antihypertensive multimer peptide (AHPM) which was the precursor of 11 peptides known for their antihypertensive effect. Simulated digestion released highly active fragments of AHPM (VWIS, VW, RIY, IY, LW, IKW, LKPNM, LKP, RPLKPW, NMAINPSK, IPP) which demonstrated that the strategy involving the expression of peptide in the form of a fusion protein or a tandem gene was useful to obtain low-cost functionally active peptides. Although having advantageous results, it is worth noting that the expressed products may harm the host organism, affect high-level gene expression, and are controversial as being foods described as genetically modified organisms (Hernández-Ledesma and others 2011).
Knowledge of the structure-activity relationships of peptides may also contribute to the development of synthesis methods suitable in peptide production. For example, Ma and others (2011) synthesized the peptide KVLPVP and its mimic sequences KVLPVY and KVLPRF using the solid-phase method. The sequence KVLPVP was originally known as a high-ACE-inhibitory casein peptide.
The advantage of synthesis methods in peptide production is the possibility to replace specific amino acids in a peptide sequence, leading to modification of its biological function. For example, the antihypertensive activity of hen egg ovokinin 2-7 improved after the replacement of the C-terminal phenylalanine by tryptophan (Yamada and others 2002).
Due to hydrolysis in the gastrointestinal tract, some of the orally-administered peptides may lose their activity. The alternative to “save” the bioactivity of the peptides is their microencapsulation (Chen and others 2003). Chen and others (2003) confirmed on SHR the antihypertensive effect of 2 microencapsulated synthetic ACE inhibitors, LKP and LRP. Oral administration of the sequence Leu-Lys-Pro of a saline solution containing 0.18 mmol of LKP per kilogram body weight) significantly reduced the SBP in SHR by 45 mm Hg and showed a prolonged duration, demonstrating the significant protective effect of encapsulation by liposome formation (Chen and others 2003).
The field of production of bioactive peptides is an important one for both scientists and manufacturers. The task includes several aspects: development and enrichment of the product while retaining the peptides in a form which guarantees bioactivity (Korhonen and Pihlanto 2003), optimization and design of healthy products through the introduction and development of new technologies (Tidona and others 2009), involvement of bioinformatics-aided tools to speed up and optimize bioactive food production (Carrasco-Castilla and others 2012), and the protection of consumers from harmful products by establishing strict legal procedures (Korhonen and Pihlanto 2006). Another aspect which should be taken into consideration when producing ACE inhibitors is sharing non desirable bioactivity. For example, some ACE-inhibiting peptides have a bitter taste, thus their application in food products may be problematic (Darewicz and others 2011).
Food is not only considered as a source of energy and nutrients necessary to maintain the proper functions of a body. Researchers are currently trying to discover new features of food components which may help to prevent many “diseases of civilization.” The diversity of protein sequences which exist in nature can produce many peptides suitable for the regulation of BP. The common efforts of researchers, food manufacturers, and nutritionists are focused on the protein sources of ACE inhibitors, improvement of their bioactivity, and formulating them as commercial foods beneficial for our health. Such food is not able to replace drugs in acute hypertension, but may be useful in prevention. The BP-reducing effect of peptides in foods is not as strong as the effect of drugs. Most food peptides showed a lower ACE inhibitory activity than drugs. Their low concentration in foods may be another effect limiting their activity.
Contemporary research methods such as bioinformatics and proteomic tools can also be applied in research on peptides from a variety of food sources and the proposed methodology is useful in arranging proteolytic processes, identification of potentially released peptides, prediction of their bioactivity, and the QSAR approach.
This work was supported by Univ. of Warmia and Mazury funds (project nr. 529-0712-0809). The authors do not declare a conflict of interest.
Iwaniak prepared the literature overview concerning the basis of hypertension and the structure of ACE inhibitors, including the QSAR approach, the mechanism of ACE action, and was engaged in drafting the manuscript. Minkiewicz collected the sequences of peptides and prepared the chapters concerning the food protein sources of ACE inhibitory/antihypertensive peptides. Darewicz elaborated the chapters dedicated to the absorption of peptides and their production.