ACEI peptides research

Food-derived ACEIp

ACEI peptides derived from proteins of food sources have attracted great attention as antihypertensive agents. The major difference between these ACEIp and synthetic ACEI is that the first do not cause significant BP lowering effects on normotensive subjects, avoiding acute hypotensive effects. Emerging evidence suggests that these food-derived peptides can act through other mechanisms besides ACE inhibition, such as the upregulation of ACE2 (an ACE homologue that counterbalances the detrimental effect of elevated ACE), endothelial function improvement, and reduced vascular oxidation and inflammation (Wu et al., 2017). Based on these findings, ACEIp have been proposed for the initial treatment of mildly hypertensive patients or as complementary treatment in hypertensive patients (Rosales-Mendoza et al., 2013).
Food-derived ACEIp have great potential as food additives/ingredients for novel functional foods/nutraceuticals, and as pharmaceutical ingredients for novel drug formulations. ACEIp have been identified in enzymatic hydrolysates of different food proteins, such as milk (Seppo et al., 2003; Miguel et al., 2006; Ruiz-Gimenez et al., 2012), egg (Asoodeh et al., 2012; Duan et al., 2014), plants (Marczak et al., 2006; Jakubczyk and Baraniak, 2014; Orona-Tamayo et al., 2015; Rayaprolu et al., 2015; Vásquez-Villanueva et al., 2015; Wu et al., 2016), meat (Castellano et al., 2013), fungi (Tran et al., 2014; Geng et al., 2015), and marine source proteins (Fujita and Yoshikawa, 1999; Balti et al., 2015; Ghanbari et al., 2015), and currently constitute the most well-known class of bioactive peptides. ACEIp can be enzymatically released from their precursor proteins during food processing and gastrointestinal digestion. An extensive overview of ACEIp isolated from food sources is given in Supplementary Table S2, while examples of commercially available nutraceuticals or food ingredients containing ACEIp are presented in Table 1. These nutraceuticals are normally formulated in the form of beverages, capsules or powders that can be directly consumed or used as ingredients for further food or pharmaceutical applications. The main food sources of ACEIp are fermented milk, bonito and other traditional foods with empirical health benefits, consumed by humans long before the concepts of ‘functional food’ and ‘bioactivity’ even existed. Hence, the most well-known natural ACEIp-containing functional foods are fermented milk-derived, including milk-derived beverages and sour milk tablets (Table 1). ACEIp from marine sources, such as bonito, sardine and seaweeds, are also present in food ingredients and functional foods in a broad variety of formulations, including BP-lowering capsules and tablets (Table 1). These ACEIp-containing products can be naturally obtained through fermentation by specific microorganisms, or artificially throughin vitro hydrolysis with gastrointestinal and commercial proteases (Hartmann and Meisel, 2007; Pihlanto and Mäkinen, 2013; Hayes and Tiwari, 2015)

Molecular determinants of ACEI peptides

The discovery of novel ACEIp was traditionally based on the analysis of whole food protein hydrolysates through wet chemistry techniques. However, this workflow has since significantly evolved and in silico methods play an increasingly important role in ACEIp research.In silico methods can be coupled to in vitro techniques in the screening and characterization of novel ACEIp (Sun et al., 2017). Particularly, the analysis of their ACE inhibitory activity based on the peptide primary structure, termed quantitative structure–activity relationship, has been widely used (Wu et al., 2006).
Considering results from analytical and chemometric studies, some rules were established concerning the primary structure of ACEIp. Potent ACEIp are generally short chain peptides (2-12 amino acids in length), although some larger inhibitory peptides were identified in fertilized egg (Duan et al., 2014), milk fermented with Enterococcus faecalis or the Lactobacillus casei strain Shirota (Quiros et al., 2007; Rojas-Ronquillo et al., 2012), koumiss (Chen et al., 2010), tuna (Lee et al., 2010), bonito (Hasan et al., 2006) and rotifer (Lee et al., 2009). Chain composition and amino acid position of ACEIp also play a role on the ACE inhibition potential. The N-terminal of potent ACEIp generally contains hydrophobic amino acids, especially those with aliphatic chains such as Gly, Ile, Leu, and Val (Iwaniak et al., 2014). The C-terminal tripeptide sequence of ACEIp (and ACE substrates) strongly influences ACEI activity, since ACE cleaves the C-terminal dipeptide of oligopeptide substrates with a wide specificity. Hydrophobic amino acid residues with aromatic or branched side chains at each of the C-terminal tripeptide positions are common features among potent inhibitors (Soares de Castro and Sato, 2015). In general, peptides showing higher activity against ACE have Pro, Tyr, Phe or Trp at their C-terminus (Norris and FitzGerald, 2013). Indeed, many potent food-derived ACEIp contain Pro residues at one or more positions in the C-terminal tripeptide region (Iwaniak et al., 2014). This rule concerns most particularly short-length peptides. Even though a similar pattern has been observed for long chain milk-derived peptides, their activity was generally not influenced by C-terminal Pro (Aluko, 2015),
The relationship between the peptides secondary structure and ACEI activity has also been analyzed (Yu et al., 2011). The elucidation of the crystal structure of a complex between human ACE and an inhibitor (Natesh et al., 2003) has provided a platform for analyzing ACEIp inhibitory mechanisms by molecular docking and molecular dynamics (MD) simulation (Xie et al., 2015). Molecular docking, a fast technique, has been coupled with accurate but time-consuming MD techniques in the discovery of novel inhibitory peptides and their mechanism of interaction with ACE at the molecular level (Alonso et al., 2006).
Finally, online in silico tools as ToxinPred and PeptideCutter can also be applied to predict the toxicity and enzymatic digestion of novel ACEIp (Sun et al., 2017).

In vitro and physiological ACE inhibitory activity

Following predictions by in silico techniques, the ACE inhibitory activity of novel ACEIp needs to be confirmed. In vitroinhibitory activity assays generally rely on spectrophotometric, fluorometric, colorimetric and radiochemical methods, as well as chromatography techniques (Murray and FitzGerald, 2007). The measure of activity is usually given as the IC50, defined as the concentration of peptide required to inhibit ACE activity by 50% (Donkor et al., 2005). However, in vitro ACE inhibitory activity does not always conduce to a BP lowering effect, and ACE inhibitory activity assays must be performed in vivo . These assays generally consist in measuring the BP response in spontaneously hypertensive rats (SHR), following intravenous or intraperitoneal injection, or oral administration, of ACEIp or related extracts.
Data from the ACE in vitro inhibition and the in vivoBP-lowering effect have provided a basis to classify ACEIp into 3 categories (Ryan et al., 2011): 1) Inhibitor-type ACEI peptides resistant to cleavage by ACE, hence their activity is not significantly altered by binding to ACE; 2) Substrate-type ACEI peptides, showing a decrease in ACEI activity due to their cleavage by ACE upon binding; 3) Prodrug-type ACEI peptides, larger peptides that are converted to potent ACE inhibitors following hydrolysis by ACE or gastrointestinal proteases.
The susceptibility to cellular peptidases is another important factor determining the physiological activity of ACEIp. Some peptides may be deactivated by their susceptibility to degradation by gastrointestinal, intestinal and kidney brush borders, serum and blood proteinases and peptidases during transport to the target organ(s). Conversely, oligopeptide sequences containing encrypted ACEIp may be activated and their ACEI activity increased, following in vivo proteinase and peptidase activities (Tai et al., 2018). The intestinal absorption and bioavailability of peptidic ACEI fragments is also of paramount importance. ACEI should be able to cross the intestinal wall and sequentially enter the blood circulation in order to exert an antihypertensive effect. The Caco-2 cell (a human epithelial colorectal adenocarcinoma cell line) has been generally used as a model to investigate the stability of ACEIp during absorption/transepithelial transport. Usually, di-/tripeptides are absorbed across the brush border membrane in their intact forms, via specific peptide transporter systems, while others can be prone to protease degradation (Shen and Matsui, 2017).

Formulation/delivery strategies

During storage and/or food processing, the ACEIp activity may decrease due to partial degradation or destabilization by proteases, extreme conditions (e.g. of pH, temperature, oxygen) and/or by unwanted interaction with other components (cations, lipids, proteins, etc.). Protective strategies have been employed to maintain the bioavailability and antihypertensive activity of ACEIp directly included in food/nutraceuticals. Encapsulation solutions aiming at maintaining peptide activity during their shelf life or even upon consumption include the use of liposomes, chitosan particles, among others.
Encapsulation in food-grade liposomes, which can retain both hydrophilic and hydrophobic peptides, can protect and help the transport of bioactive peptides ( Mozafari et al., 2008; Malheiros et al., 2010). Liposomes may even increase nutritional value if derived e.g. from natural soy lecithin or partially purified phosphatidylcholine, with high content in polyunsaturated fatty acid and low tocopherol content (Taladrid et al., 2017). Peptide-loaded liposomes may be easily introduced into functional foods via a film matrix, which helps avoiding unwanted strong flavors or peptide interactions with other constituents (da Silva Malheiros et al., 2010). Encapsulation of ACEIp into food grade chitosan nanoparticles (CNP) ensures safe oral administration and decreases gastrointestinal enzymatic degradation. Indeed, ACEIp stabilized by CNP were shown to more efficiently reduce BP for extended time periods in SHR (Auwal et al., 2017). Other encapsulation methods may also improve the bioavailability and efficacy of the ACEIp (Huang et al., 2017).
Finally, additives such as skim milk, sugars and sugar alcohols, soybean (Glycine max ) casein, casein hydrolysate, and others help to mask the ACEIp’s bitterness, and improve their applicability in functional foods/nutraceuticals (Iwaniak et al., 2016a; Iwaniak et al., 2016b; Pooja et al., 2017).