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).