Predicted Release and Analysis of Novel ACE-I, Renin, and DPP-IV Inhibitory Peptides from Common Oat (Avena sativa) Protein Hydrolysates Using in Silico Analysis

The renin-angiotensin-aldosterone system (RAAS) plays an important role in regulating hypertension by controlling vasoconstriction and intravascular fluid volume. RAAS itself is largely regulated by the actions of renin (EC 3.4.23.15) and the angiotensin-I-converting enzyme (ACE-I; EC 3.4.15.1). The enzyme dipeptidyl peptidase-IV (DPP-IV; EC 3.4.14.5) also plays a role in the development of type-2 diabetes. The inhibition of the renin, ACE-I, and DPP-IV enzymes has therefore become a key therapeutic target for the treatment of hypertension and diabetes. The aim of this study was to assess the bioactivity of different oat (Avena sativa) protein isolates and their ability to inhibit the renin, ACE-I, and DPP-IV enzymes. In silico analysis was carried out to predictthe likelihood of bioactive inhibitory peptides occurring from oat protein hydrolysates following in silico hydrolysis with the proteases papain and ficin. Nine peptides, including FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA, FLLA, and FEPL were subsequently chemically synthesised, and their bioactivities were confirmed using in vitro bioassays. The isolated oat proteins derived from seven different oat varieties were found to inhibit the ACE-I enzyme by between 86.5 ± 10.7% and 96.5 ± 25.8%, renin by between 40.5 ± 21.5% and 70.9 ± 7.6%, and DPP-IV by between 3.7 ± 3.9% and 46.2 ± 28.8%. The activity of the synthesised peptides was also determined.


Introduction
High blood pressure is the single largest risk factor attributed to deaths worldwide. It is responsible for 12.8% of deaths, and affects all countries and income groups [1]. Furthermore, high systolic blood pressure is globally attributable to 51% of strokes, 45% of ischaemic heart disease, and between 37% (Southeast Asia) and 54% (European countries) of cardiovascular deaths [1]. Hypertension is therefore a considerable problem in our society, not only placing a great burden on our healthcare system, but also

Bioactivity Prediction In Silico
The peptides that resulted from oat protein hydrolysates were ranked for bioactivity according to their PeptideRanker score and known inhibitory peptide characteristics (Table 2), as previously described [36]. PeptideRanker, available at http://bioware.ucd.ie/~compass/biowareweb/Server_ pages/peptideranker.php [37], is a server that predicts how likely a peptide is to be bioactive based on an N-to-1 neural network algorithm [37]. PeptideRanker predicts how likely peptides are to be bioactive, but does not indicate the targets for which they are most suitable. A literature search was therefore carried out to identify the characteristics of peptides that have been shown to increase the likelihood of inhibition with the enzyme targets in this study (Table 2). Table 2. Characteristic criteria used to identify tripeptides with predicted renin, angiotensin-I-converting enzyme (ACE-I), and dipeptidyl peptidase-IV (DPP-IV) inhibition activity. Additional in silico analysis was carried out to predict water solubility, resistance to gastrointestinal digestion, toxicity, and allergenicity ( Figure 1). Solubility in water was predicted using PepCalc, which is available at http://pepcalc.com. Resistance to digestion was predicted using PeptideCutter, which is available at http://web.expasy.org/peptide_cutter/ [36] with the enzymes' chymotrypsin-low specificity, chymotrypsin-high specificity, pepsin (pH 1.3), pepsin (pH > 2), and trypsin. Toxicity was scanned with default settings using ToxinPred, which is available at http://www.imtech.res.in/raghava/toxinpred/ multi_submit.php [36]. Allergenicity was predicted using AllerTOP, which is available at http://www. pharmfac.net/allertop/ [36] (Figure 1).

Renin Inhibition Assay
Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for renin inhibition activity. The Renin Inhibition Screening Assay (Cambridge BioSciences, Cambridge, UK) was carried out as per the manufacturer's instructions. Briefly, 10 µL of each sample inhibitor at a concentration of 1 mg/mL dimethyl sulfoxide (DMSO) was added to 20 µL renin substrate, 150 µL assay buffer, and 10 µL renin, in triplicate. The samples were incubated at 37 • C for 15 min, and read with excitation wavelengths of 340 nm and emission wavelengths of 500 nm. Fluorescence was read using a FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany). The percentage inhibition was calculated using the following equation: % Renin inhibition = 100% Initial activity − Inhibitor × 100/100% Initial activity (1)

ACE-I Inhibition Assay
Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for ACE-I inhibition. The bioassay (ACE Kit-WST, Dojindo Laboratories, Kumamoto, Japan) was carried out according to the manufacturer's instructions. First, 20 µL of each sample inhibitor at a concentration of 1 mg/mL ddH 2 O was added to 20 µL substrate and 20 µL enzyme working solution in triplicate. Samples were incubated at 37 • C for 1 h. Each well then had 200 µL indicator working solution added, followed by a further incubation at room temperature for 10 min. Absorbance at 450 nm was read using a FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany). The percentage inhibition was calculated using the following equation: % ACE-I inhibition = 100% Initial activity − Inhibitor × 100/100% Initial activity (2)

DPP-IV Inhibition Assay
Protein isolates from all seven oat varieties and selected synthesised peptides were tested in vitro for DPP-IV inhibition. The bioassay (DPP-IV Inhibitor Screening Assay Kit, Cayman Chemical, Ann Arbor, MI, USA) was carried out as per the manufacturer's instructions. First, 10 µL of each sample inhibitor at a concentration of 1 mg/mL assay buffer was added to 30 µL diluted assay buffer, 10 µL diluted DPP-IV, and 50 µL substrate solution, in triplicate. Samples were incubated at 37 • C for 30 min. Fluorescence was read with excitation wavelengths of 355 nm and emission wavelengths of 460 nm using a FLUOstar Omega microplate reader (BMG LABTECH GmbH, Offenburg, Germany). The percentage inhibition was calculated using the following equation: % DPP-IV inhibition = 100% Initial activity − Inhibitor × 100/100% Initial activity (3)

In Silico Bioactivity Prediction
In silico analysis of oat protein isolates identified a number of bioactive peptides that had previously been reported in the BIOPEP database (Table 3). These peptides were not selected for chemical synthesis. Based on the known characteristics of renin, ACE-I and DPP-IV inhibitory peptides (Table 2), novel peptides were identified for in vitro analysis in this study. The peptides chosen had the amino acid sequences FFG, IFFFL, PFL, WWK, WCY, FPIL, CPA, FLLA, and FEPL ( Table 4). The tables predicted to have the greatest bioactivities are shown in Table A1 (Appendix A). Bioactive peptides need to survive degradation by gastrointestinal digestion, reach their target intact, and maintain bioavailability in order to exert a beneficial physiological effect [44]. The novel peptides were therefore analysed in silico for predicted solubility, resistance to digestion, toxicity, and allergenicity ( Table 4). Most of the peptides were expected to be poorly soluble due to their high hydrophobic residue content. Most of the peptides were also expected to be broken down by gastrointestinal digestive enzymes, although this could be overcome by methods such as encapsulation [45]. The peptides were also expected to be non-toxic and non-allergenic (Table 4).

Renin Inhibition
Oat protein isolates displayed renin inhibition values ranging between 40.5% (±2.16%) and 70.9% (±7.7%) (Figure 2). Barra oat had the highest levels of renin inhibition, with 70.9% (±7.7%) inhibition, followed by Vodka oat, which had 66.1% (±22.1%) inhibition. There was significantly lower renin inhibition activity from synthesised peptides compared with that of the oat protein isolates.  However, renin inhibition was not observed with the synthesised peptides. Only the peptide IFFFL was found to inhibit renin by 17.1% (±2.6%) when tested at a concentration of 1 mg/mL compared with the control.

ACE-I Inhibition
The crude oat protein extracts inhibited ACE-I by between 86.6% (±10.7%) and 96.5% (±25.8%). These results were therefore comparable with that of the positive control captopril, which was found to inhibit ACE-I by 97.7% (±23.2%) when tested at a concentration of 1 mg/mL (Figure 2). Protein  However, renin inhibition was not observed with the synthesised peptides. Only the peptide IFFFL was found to inhibit renin by 17.1% (±2.6%) when tested at a concentration of 1 mg/mL compared with the control.

ACE-I Inhibition
The crude oat protein extracts inhibited ACE-I by between 86.6% (±10.7%) and 96.5% (±25.8%). These results were therefore comparable with that of the positive control captopril, which was found to inhibit ACE-I by 97.7% (±23.2%) when tested at a concentration of 1 mg/mL (Figure 2). Protein extracted from Barra oat varieties displayed the highest ACE-I inhibition values, and inhibited the enzyme by 96.5% (±25.8%) when assayed at a concentration of 1 mg/mL compared with the positive control.
The highest levels of ACE-I inhibition with the synthesised peptides were observed for the peptides WCY (97.8 ± 21.7%), FLLA (97 ± 16.2%), and WWK (95.3 ± 14.2%) (Figure 3). The lowest levels of inhibition were seen with IFFFL (53 ± 41.2%) and FEPL (48.9 ± 7.8%), contradicting previous findings of the beneficial effect of leucine at the C-terminus of an ACE-I inhibiting bioactive peptide [40]. However, the poor activity from IFFFL and FEPL could also be due to the larger peptide size as is the case with the peptide PFL, which also has a C-terminal leucine, and displayed higher levels of ACE-I inhibition (81.4 ± 33.8%). Alternatively, the lower ACE-I inhibitory values observed for FEPL could also be due to the presence of proline at the penultimate position within the peptide, which has been suggested to reduce the binding affinity of peptides with the ACE-I enzyme [46].
Peptides containing the sequence Xaa-Pro (where Xaa represents any amino acid, and proline is present at the second residue from the N-terminus) have been found to be effective DPP-IV inhibitors [43]. The peptides CPA (22.2 ± 4.8%) and FPIL (13.1 ± 3.2%) were the only chemically synthesised peptides that were found to inhibit DPP-IV at a concentration of 1 mg/mL.

Discussion
Functional foods in the form of bioactive peptides offer additional physiological benefits beyond their basic nutritional value. Bioactive peptides that inhibit the enzymes within RAAS, such as renin and ACE-I, are used as alternatives to antihypertensive pharmaceutical drugs [7]. Similarly, DPP-IV inhibitory bioactive peptides have also been shown to effectively prevent the onset of type-2
Peptides containing the sequence Xaa-Pro (where Xaa represents any amino acid, and proline is present at the second residue from the N-terminus) have been found to be effective DPP-IV inhibitors [43]. The peptides CPA (22.2 ± 4.8%) and FPIL (13.1 ± 3.2%) were the only chemically synthesised peptides that were found to inhibit DPP-IV at a concentration of 1 mg/mL.

Discussion
Functional foods in the form of bioactive peptides offer additional physiological benefits beyond their basic nutritional value. Bioactive peptides that inhibit the enzymes within RAAS, such as renin and ACE-I, are used as alternatives to antihypertensive pharmaceutical drugs [7]. Similarly, DPP-IV inhibitory bioactive peptides have also been shown to effectively prevent the onset of type-2 diabetes by preventing the cleavage of the glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) incretins [43]. Bioactive peptides have been identified from a variety of food sources, including milk proteins, seaweed, and meat [21,28,30], as well as a number of grains, including rice, soybean, wheat, and barley [22][23][24]54]. This study determined the ACE-I, renin, and DPP-IV inhibitory activities of oat protein isolates and peptides synthesised in vitro.
While there are pharmaceutical therapies for hypertension and type-2 diabetes, they are often accompanied by adverse side effects, such as a dry cough, anaphylaxis, renal impairment, hyperkalaemia, and inflammation-related pain [55][56][57]. Pharmaceuticals have several reported side effects, but are active at lower concentrations than food-derived bioactive peptides. However, peptides consumed with IC 50 values of 100-500 µM have been shown to be active in vivo and inhibit ACE-I, renin, and DPP-IV enzymes [58,59]. Furthermore, functional foods with bioactivities can be a beneficial alternative to synthetic drugs for individuals who have borderline disease states and do not warrant the prescription of pharmaceutical drugs [6].
Oat protein isolates were found to inhibit renin by between 40.5% (±2.16%) and 70.9% (±7.7%) ( Figure 2) in vitro when assayed at a concentration of 1 mg/mL protein, but the selected synthesised peptides did not inhibit renin to the same degree ( Figure 3). This demonstrates that it is necessary to carry out in vitro work and characterise all of the peptides present in a hydrolysate. The observed activities could also be due to other compounds present in the protein isolate, such as phenolic compounds, and phenolic compounds may still be present despite the use of dialysis to concentrate the protein fraction. The active peptide(s) could have been previously identified (Table 4) or were not chosen for synthesis. The characteristics of renin inhibitory peptides are not as well defined as other bioactive peptide targets (such as ACE-I inhibitory peptides, for example) due to the notably poor potency that has been observed with renin peptide inhibitors [60]. The peptides IFFFL, FLLA, and WWK ( Figure 3) were chosen for chemical synthesis based on previously published literature by Udenigwe and colleagues (2012), who observed that the bulky amino acids at the N-terminus aided in renin inhibition (Table 3).
The oat protein isolates generated in this work were all found to inhibit the enzyme ACE-I by between 86.6% (±10.7%) and 96.5% (±25.8%) (Figure 2). This was significantly greater than a similar study assessing crude protein extracts derived from barley [54]. Unlike the renin assay, there were comparable levels of activity in the selected synthesised peptides and the oat protein extracts (Figure 4). This suggests that the correct peptides were selected for chemical synthesis, which is largely because the mechanism of the action of ACE-I inhibitory peptides is better understood.
The Barra oat protein isolate had the highest activity with 46.3% (±28.8%) inhibition, followed by Rhapsody oat (25.4 ± 1.8%) and Selwyn oat (20.7 ± 3.1%). Similarly, the selected synthesised peptides also had relatively poor DPP-IV inhibition ( Figure 5). DPP-IV is a proline-specific endopeptidase that cleaves dipeptides from the N-terminus [61]. Of the nine peptides synthesised (Table 4), those containing a proline residue were therefore selected for testing DPP-IV inhibition.
In silico methods were used heavily in this study to evaluate the potential of oat protein to generate bioactive peptides, as well as predict generated bioactive peptides following hydrolysis with the food-grade proteases papain and ficin. Similar bioinformatic techniques have already been described in other studies [36], which highlighted their value in reducing time and expense for the preliminary screening of novel sources of bioactive peptides. The preparatory in silico screening of potential bioactive peptides for characteristics such as allergenicity, toxicity, solubility, and degradation are additional important uses for such online software and tools, especially when presenting these peptides for possible human consumption.
peptides also had relatively poor DPP-IV inhibition ( Figure 5). DPP-IV is a proline-specific endopeptidase that cleaves dipeptides from the N-terminus [61]. Of the nine peptides synthesised (Table 4), those containing a proline residue were therefore selected for testing DPP-IV inhibition.
In silico methods were used heavily in this study to evaluate the potential of oat protein to generate bioactive peptides, as well as predict generated bioactive peptides following hydrolysis with the food-grade proteases papain and ficin. Similar bioinformatic techniques have already been described in other studies [36], which highlighted their value in reducing time and expense for the preliminary screening of novel sources of bioactive peptides. The preparatory in silico screening of potential bioactive peptides for characteristics such as allergenicity, toxicity, solubility, and degradation are additional important uses for such online software and tools, especially when presenting these peptides for possible human consumption.

Conclusions
There has been little work carried out to evaluate the potential of the common oat (Avena sativa) as a potential source of bioactive peptides. Bioinformatics techniques were used to predict whether oats were indeed a rich source of bioactive peptides following in silico hydrolysis with the proteases papain and ficin on the main storage proteins. Oat protein isolates displayed the highest inhibition bioactivity against the target ACE-I (86.6-96.5%), with lower inhibition levels observed with renin (40.5-70.9%) and DPP-IV (3.7-46.3%). Following the chemical synthesis of nine novel peptides, in vitro bioassays gave mixed results as to their efficacy in inhibiting the injurious enzyme targets ACE-I (48.9-97.8%), renin (0-17.1%), and DPP-IV (0-22.2%). The in silico methods utilised in this study correctly identified ACE-I inhibitory peptides, beyond that of renin and DPP-IV inhibitory peptides. This could reflect a more specific use of these in silico methods until the characteristics of Figure 5. DPP-IV inhibition of several synthesised peptides that were identified in silico from the hydrolysis of oat proteins with papain or ficin, which are both proteases. The activity of peptides was compared with that of a sitagliptin inhibitor (positive control). Peptides were assayed at a concentration of 1 mg/mL. Values are the mean of triplicate samples.

Conclusions
There has been little work carried out to evaluate the potential of the common oat (Avena sativa) as a potential source of bioactive peptides. Bioinformatics techniques were used to predict whether oats were indeed a rich source of bioactive peptides following in silico hydrolysis with the proteases papain and ficin on the main storage proteins. Oat protein isolates displayed the highest inhibition bioactivity against the target ACE-I (86.6-96.5%), with lower inhibition levels observed with renin (40.5-70.9%) and DPP-IV (3.7-46.3%). Following the chemical synthesis of nine novel peptides, in vitro bioassays gave mixed results as to their efficacy in inhibiting the injurious enzyme targets ACE-I (48.9-97.8%), renin (0-17.1%), and DPP-IV (0-22.2%). The in silico methods utilised in this study correctly identified ACE-I inhibitory peptides, beyond that of renin and DPP-IV inhibitory peptides. This could reflect a more specific use of these in silico methods until the characteristics of renin and DPP-IV inhibitory peptides are better understood.

Conflicts of Interest:
The authors declare no conflict of interest. Table A1. Top 15 novel peptide hydrolysates identified following in silico hydrolysis of oat proteins with proteases papain and ficin that were predicted to have high bioactivity for the inhibition of ACE-I and DPP-IV. * Peptides that were chosen for chemical synthesis and tested with in vitro assays.