Novel Plant-Protein (Quinoa) Derived Bioactive Peptides with Potential Anti-Hypercholesterolemic Activities: Identification, Characterization and Molecular Docking of Bioactive Peptides

Hypercholesterolemia remains a serious global public health concern. Previously, synthetic anti-hypercholesterolemic drugs were used for ameliorating this condition; however, long-term usage presented several side-effects. In this regard, natural products as an adjunct therapy has emerged in recent times. This study aimed to produce novel bioactive peptides with anti-hypercholesterolemic activity (cholesterol esterase (CEase) and pancreatic lipase (PL)) from quinoa protein hydrolysates (QPHs) using three enzymatic hydrolysis methods (chymotrypsin, protease and bromelain) at 2-h hydrolysis intervals (2, 4, and 6 h). Chymotrypsin-generated hydrolysates showed higher CEase (IC50: 0.51 mg/mL at 2 h) and PL (IC50: 0.78 mg/mL at 6 h) inhibitory potential in comparison to other derived hydrolysates and intact quinoa proteins. Peptide profiling by LC-MS QTOF and in silico interaction with target enzymes showed that only four derived bioactive peptides from QPHs could bind in the active site of CEase, whereas twelve peptides could bind in the active site of PL. Peptides QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, and ASNLDNPSPEGTVM were identified to be potential CEase inhibitors, and FSAGGLP, QHPHGLGALCAAPPST, KIVLDSDDPLFGGF, MFVPVPH, and HVQGHPALPGVPAHW were identified as potential PL inhibitors on the basis of the maximum number of reactive residues in these bioactive peptides. In conclusion, QPHs can be considered as an alternative therapy for the treatment of hypercholesterolemia.


Introduction
The rise in diet-related metabolic disorders such as hyperlipidemic or hypercholesterolemia, cardiovascular diseases, hypertension, and other related metabolic diseases has been identified as the major cause of mortality globally, and thus has become a serious public health issue. The consumption of processed food and animal products has been associated with the occurrences of these health issues. Cholesterol esters are normally found in the human diet, which the human intestine cannot sufficiently absorb, thereby producing free fatty acids and cholesterol that elevate the level of blood cholesterol. It is a known fact that rising levels of cholesterol and triglycerides in the blood serum results in hyperlipidemia or hypercholesterolemia [1]. According to [2], the molecular mechanism for understanding the physiological development of hyperlipidemia is quite difficult. However, Quinoa seeds used in this study were purchased from the local market (Al-Ain, United Arab Emirates). Enzymes such as protease (Type XIV; ≥3.5 units/mg solid derived from Streptomyces griseus), α-chymotrypsin (EC 3.4.21.1 from bovine pancreas; C4129 40 units/mg), and bromelain (B4882: 3-7 units/mg solid; EC 3.4.22.33 from pineapple stem), HPLC grade reagents (acetonitrile and methanol), and other chemicals such as formic acid p-nitrophenyl butyrate, o-phthaldialdehyde, trizma base, β-mercaptoethanol, SDS, and sodium tetra-borate were procured from Sigma Aldrich (St. Louis, MO, USA). Other analytical grade reagents used were purchased from the UAE (BDH Middle East, Dubai, United Arab Emirates).

Preparation of Quinoa Protein Isolate
Firstly, quinoa seeds were oven dried (40 • C for 3 h), dry-blended (IKA A11, Guangzhou, China), and passed through a screen (3 µm pore size) to obtain a fine powder. The methodology described by [6] as adopted from [14] was used to obtain quinoa protein isolate (QPI). In sum, defatted quinoa flour was dispersed in 0.015 mol L −1 NaOH (1:10 ratio) maintained at a pH of 10-11 and was stirred for 2 h on an electric shaker (24 ± 2 • C), and the resulting slurry was stored at 4 • C overnight. The slurry was homogenized for consistency and centrifuged twice at 4 • C at a speed of 10,000× g for 20 min after overnight storage. Whatman No. 1 paper was used to filter the obtained supernatant, and the pH of the filtrate reduced to 4.5 using the pH drop method with 1.0 mol L −1 HCl solution to precipitate quinoa protein. Afterwards, precipitated proteins were separated by centrifuging at 4 • C for 10 min at the speed of 10,000× g, and were subsequently washed twice with deionized water. The pH of the resultant pellets was adjusted to pH 7.0 (with 1.0 mol L −1 NaOH) and stored at −20 • C until further use. The Kjeldahl method was adopted to determine the protein content of quinoa protein isolate (QPI) slurries, which was found to be 44%.

Quinoa Protein Hydrolysate Production
The methodology as described by [6] was used in the production of quinoa protein hydrolysates (QPHs). The obtained QPI slurry was thoroughly homogenized (T 25 digital ULTRA-TURRAX ® homogenizer IKA ® -Werke GmbH, Staufen, Germany). The protein content of the slurry was set to 4% with deionized water, and the content was divided into four batches for treatment with bromelain, chymotrypsin, and protease, respectively, while the last part was kept as control. Then, 1 mol/L NaOH and 5N HCl were used to adjust the pH of the treated protein slurries for the bromelain enzyme to 7.0, the chymotrypsin enzyme to 7.8, and the protease enzyme to 8.0, respectively. Furthermore, enzyme concentration was pre-calculated, suspended in 1 mL of distilled water resulting in a 1% enzyme/substrate ratio, and subsequently added to the QPI slurry. The samples were then distributed into 50 mL falcon tubes in triplicate for each period of hydrolysis and incubated (water bath) for up to 6 h at the speed of 100 ramps/min at a controlled temperature of 50 • C. Samples were taken every 2 h from all enzymatic incubations, and the reaction was inactivated at 95 • C for 600 s. Generated QPHs were centrifuged at 4 • C at a speed of 10,000× g for 10 min, and supernatants were collected and stored at −20 • C for further analysis. All of the analyses were performed within two weeks of the production of hydrolysates.

Degree of Hydrolysis (DH%)
The previously published o-phthaldialdehyde (OPA) methodology of [15] was used for the determination of DH values.

Degree o f hydrolysis(DH%)
Herein, h tot = total number of peptide bonds per protein equivalent; and h = number of hydrolyzed bonds, and calculated using h = (SerineNH2 − β)/α. Where h tot , β, and The PL-inhibitory activity of generated hydrolysates was measured using the methodology of [13]. Here, PL (20 µL) and p-nitrophenyl butyrate (25 µL) were mixed with each sample (50 µL) in a sodium chloride: sodium phosphate buffer (pH 7.2, 100 mM), and then incubated in a 96-well microplate reader. The quantity of the resultant reaction was modified with the above buffer to 150 µL and incubated (37 • C for 30 min). After the incubation period, the released p-nitrophenyl for each sample was measured on a plate reader (Epoch 2, BioTek, Winooski, VT, USA) at 405 nm. The PL-inhibitory activity was calculated using the equation below Here: A = the absorbance of control; B = the absorbance of control reaction blank; C = the absorbance of sample, and D = the absorbance of the sample blank. The IC 50 values were determined by plotting the percentage inhibition against the concentration of the test compound and expressed in mg (eqv protein)/mL.

Determination of Cholesterol Esterase Inhibitory Activity of QPHs
The CEase-inhibitory activity was determined as per the procedure documented by [13]. Briefly, QPHs (25 µL), substrate (50 µL) containing 5 mM p-nitrophenyl butyrate in sodium phosphate (100 mM): NaCl buffer (pH 7.2, 100 mM) was placed in a 96-well microtiter plate. Subsequently, the mixtures were incubated with 50 µL of porcine pancreatic CEase (5 µg/mL) at 37 • C for 180 min. After that, the p-nitrophenol released from the enzymatic hydrolysis of p-nitrophenyl butyrate was read at 405 nm. The percentage CEase inhibition was determined using Equation (2) above. The IC 50 values were determined by plotting the percentage inhibition against the concentration of the test compound and expressed as mg (eqv protein)/mL. For the identification of peptides in selected hydrolysates, QC-6 (chymotrypsingenerated at 6 h hydrolysis), LC-MS Q T-O-F was conducted as described previously by [16]. The identification of peptides was performed as follows: Advance Bio Peptide Map, C18 column (2.1 × 100 mm, 2.7 µm particles; Agilent, Santa Clara, CA, USA) was used for the peptide separation whereas the LC-MS Q T-O-F (model 6520, Agilent, Santa Clara, CA, USA) was used for the analysis. The mobile phases used were: (A) deionized water containing 0.1% formic acid, and (B) acetonitrile containing 0.1% (v/v) formic acid with a flow rate of 15 µL/min. The mobile phase gradient of the HPLC system was as follows: (a) 0-5 min, 10% B; (b) 5-115 min, 10-95% B, (c) 115-120 min, 95% B, (d) 120-135 min, 95-10% B, and (e) 140-150 min, 10% B. The electrospray ionization-quadrupole-time-of flight system (ESI-QTOF) condition was: (a) mass range: 70-2000 m/z; (b) collision energy: 6V/100 Da (offset-2); (c) flow rate: 15 µL/min; (d) ion spray sources: 3.5 kV; (e) drying gas: Nitrogen at a temperature of 350 • C with a flow rate of 10 L/min; (f) nebulizer pressure: 3 psig; (g) fragmentor voltage: 110 V; and (h) fragmentation mode: collision induced dissociation (CID). Data generated from the mass spectrometry approach were analyzed using PEAKS studio version 6.0 [17]. The precursor was selected on the basis of a minimum charge of 2 and a maximum charge of 10. From the generated peptides, only peptides with average local confidence (ALC) > 70% were chosen for further analysis. Potential bioactive peptides were screened using the PeptideRanker web server (http: //bioware.ucd.ie/, accessed on 26 July 2022) [18]. Peptides showing a score of more than 0.5 were regarded as potentially biologically active and subjected to further in silico analysis.

Selection of Identified PL and CEase Inhibitory Peptides Using Peptide Ranker
The preliminary insight into the molecular mechanism of peptides for their PL and CEase inhibitory activities were explored through an in silico docking investigation using the online program Pepsite2, accessible at http://pepsite2.russellab.org (accessed on [16][17][18][19] August 2022) [19]. Herein, 3D structures of CEase (PDB code: 1AQL) and PL (PDB code: 1ETH) were introduced from the Protein Data Bank through http://www.rcsb.org/ pdb/ (accessed on 16-19 August 2022). Peptide inputs were then entered jointly with a protein receptor in PDB format. The best interaction of peptides with enzymes showing p-values < 0.05 and the interaction with key hotspots on enzymes playing a significant role in enzyme inhibition were taken into consideration for further analysis. The 3D structures of human and bovine CEase, PDB IDs 1F6W and 1AQL respectively, and PL (PDB IDs: 1LPB and 1ETH) were obtained from the Protein Data Bank (PDB) [20]. These proteins were processed and optimized prior to docking using the Schrödinger Suite's Protein Preparation Wizard (Schrödinger Suite 2021-1: Protein Preparation Wizard; Schrödinger LLC, New York, NY, USA). This workflow prepares a PDB structure for docking by adding and optimizing hydrogen bonds, assigning bond orders, simplifying multimeric complexes, creating disulfide bonds, deleting unwanted water molecules, adjusting ionization states, fixing disoriented groups, filling missing loops and sidechains and finally, energy minimizing and optimizing to produce a geometrically stable structure [21].

Active Site Identification and Grid Generation
The active sites of CEase (PDB IDs: 1F6 W and 1AQL) and PL (PDB IDs: 1LPB and 1ETH) that are directly involved in ligand-binding were identified from the literature The active site of lipase is located in the N-terminal domain and their catalytic activity is mediated by the catalytic triad Ser152, Asp176 and His263 Similarly, CEase also shares a comparable catalytic triad composed of Ser194, Asp320 and His435. For the docking analysis, a receptor grid suitable for peptide docking was generated for the minimized protein structures enveloping the active site residues. No constraints were applied when default parameters for van der Waals scaling factor (1.00) and charge cut-off (0.25) were used. The OPLS 2005 force field was used for structural representation [22].

Peptide Docking and Binding Free Energy Calculation
The Peptide Docking panel of Schrödinger Maestro [23] was employed to perform the docking of peptides to the protein structures. Using this tool, small peptides lower than 16 amino acids can be docked and scored with either Glide Score or molecular mechanicsgeneralized Born surface area (MM-GBSA) approach. Each peptide was docked to the protein receptor using Glide with increased sampling in several docking runs. Subsequently, OPLS molecular mechanics force field was used for pose optimization. After carrying out conformer clustering, ten representative peptide poses were chosen. Lastly, selected poses were re-scored and ranked based on GlideScore empirical scoring function [23,24].
The binding free energy of the best docked poses obtained after peptide docking were evaluated using the MM-GBSA approach. Binding free energy calculations were performed using Schrödinger Prime with OPLS 2005 force field combined with VSGB 2.0 implicit solvent model. The peptide was minimized, and the receptor was treated as rigid for the MM-GBSA calculations.

Statistical Analysis
All hydrolysates were produced in three batches representing three replicates. All analyses were carried out in triplicate. Data analysis was performed with SPSS 24.0 statistical software (SPSS INC., Chicago, IL, USA) using one-way analysis of variance. Means separation was conducted using Tukey's multiple range test, and a p-value of 5% was defined as statistically significant (p < 0.05).

Degree of Hydrolysis (DH%)
The DH and enzyme specificity are among the measurable factors to be controlled because they define the biological activities of the resulting peptides. Studying the hydrolysis of protein reveals the capability of an enzyme to degrade proteinaceous substrate, and this serves as a pointer for their proteolytic reaction [25]. Noteworthy, the protein profile obtained from intact quinoa (QPI), and quinoa protein hydrolysates (QPHs) from the three enzymes understudied after 2, 4, and 6 h of hydrolysis is depicted in Supplementary Figure  S1.
In this work, the effects of different enzymes (chymotrypsin, protease, and bromelain) at 2 h-interval reaction periods on the DH of quinoa protein hydrolysates (QPHs) were reported (Table 1). The results obtained showed a characteristic similar hydrolysis pattern among all the enzymes, where the DH values significantly increased with the progression of hydrolysis time up to 6 h (p < 0.05). These results are comparable with those obtained by [26], where they reported increased DH values of quinoa protein hydrolysates as a function of increased hydrolysis time. Moreover, varying DH values were demonstrated among the QPHs generated using the three enzymes of study. The DH levels in the range of 36.01 to 66.05%, and 45.05 to 63.3%, and 34.99 to 51.91% were obtained for chymotrypsin, protease, and bromelain generated QPHs, respectively.
Based on the enhanced DH values with progressive hydrolysis time, chymotrypsinproduced hydrolysates after 6 h (QC-6) displayed the highest value of 66.05%. However, it was not noted that the DH value obtained in protease-produced hydrolysates after 6 h (QP-6) was not significantly different from the DH value of QC-6h (p > 0.05). Bromelainproduced hydrolysates after 6 h (QB-6) showed an intermediate DH value of 51.91%, and the lowest protein hydrolysis was demonstrated in both bromelain-produced hydrolysates (QB-2) and chymotrypsin-produced (QC-2) after 2 h of bio-catalysis (p > 0.05). In general, chymotrypsin was found to be the most effective enzyme in hydrolyzing quinoa proteins as opposed to the other two proteolytic enzymes (bromelain and protease). The hydrolytic pattern of chymotrypsin indicates greater proteolytic efficiency of the enzyme, which may be attributed to the size and action of the released peptides. As a result, the abundance of small-sized peptides with enhanced effectiveness against metabolic markers could be produced from chymotrypsin at prolonged hydrolysis periods. This is consistent with previous studies that suggested that higher DH due to disintegration of more peptide bonds could be attributed to the higher activity of the enzyme [27].
The enzymatic hydrolysis of quinoa proteins using bromelain and chymotrypsin exhibited a rapid and steady increase in the DH values over a period of 6 h, an indication that the larger peptides that were released at a lower hydrolysis time were further disintegrated into smaller peptides as the hydrolysis time progressed. We hypothesize that a higher degree of hydrolysis due to the increased time of hydrolysis is often related to further enzyme activity on the reaction substrates. In contrast, the progression of hydrolysis-reaction from 2 h to 4 h (p > 0.05) of protease-generated hydrolysates did not show a very rapid increase in the displayed DH. However, after progressing the bio-catalysis reaction to 6 h, a significantly higher DH (p < 0.05) was recorded, as presented in Table 1. A similar hydrolysis reaction where increasing hydrolysis up to 4 h was found to be slower than between 4 to 6 h of hydrolysis [6].
The level of DH values obtained in this study remains comparable to those of other studies. For instance, the hydrolysis of quinoa proteins after 2 h of hydrolysis by pepsin pancreatin and papain was noticed to be in the range of 15-35% [7]. Similarly, 4 h of hydrolysis by Alcalase reported a DH value of 48% [14]. The variations noticed between DH produced by different enzymes after a similar time of hydrolysis could be ascribed to the specificity and substrate affinity of each enzyme towards the quinoa protein substrate [16].

Cholesterol Esterase Inhibitory Activity
QPHs were analyzed for their ability to inhibit CEase. CEase is a known polymeric enzyme existing in the bile that initiates dietary cholesterol esters' hydrolytic reaction, thereby releasing cholesterol and free fatty acids [28]. Inhibiting CEase can indirectly prevent cholesterol absorption by the human body by diminishing cholesterol's release from dietary lipids. The CEase inhibitory potential of generated QPHs and QPI were measured in terms of IC 50 (half maximal inhibitory concentrations), and the results are illustrated in Table 1. The low IC 50 value of hydrolysates connotes high CEase inhibitory activity. Upon hydrolysis, the QPHs derived from bromelain showed an increased CEase inhibition, with progression of the bio-catalysis reaction from 2 to 6 h (IC 50 values = 0.67 mg/mL to 0.64 mg/mL to 0.63 mg/mL, respectively). Therefore, bromelain-generated hydrolysates with high CEase inhibitory activity can be developed at longer hydrolysis times. On the other hand, the CEase inhibitory potential of chymotrypsin and protease derived hydrolysates decreased with the increased bio-catalysis reaction time from 2 to 6 h.
From this study, it was evident that chymotrypsin-generated QPHs showed the highest CEase inhibitory activity with IC 50 values of 0.51 mg/mL, 0.55 mg/mL, and 0.55 mg/mL after 2, 4, and 6 h of hydrolysis, respectively. However, there was no significant difference (p > 0.05) observed between hydrolysates generated at 4 h and 6 h hydrolysis time. Similar results were obtained by [6], where chymotrypsin exhibited higher activity compared to other enzymes used, and this implies that it is more effective in hydrolyzing quinoa protein. This was attributed to the broad specificity of chymotrypsin enzymes to generate small-sized peptide and free amino acids. In addition, the variation exhibited in the IC 50 values of hydrolysates with respect to different time periods could be explained with the ability of specific enzymes to either degrade or generate CEase inhibitory peptides during hydrolysis [29]. The protease-generated hydrolysates showed a lower CEase inhibitory activity in comparison to bromelain and chymotrypsin generated hydrolysates. A possible explanation could be that the cleavage sites that the enzyme binds to was not fully exposed, which consequently reduced the inhibition effect. However, the protease hydrolyzed under hydrostatic pressure processing showed an increased inhibition rate of up to 49.1% [30].
Unhydrolyzed quinoa protein, i.e., intact quinoa protein (QPI), showed the least CEase inhibitory activity (IC 50 value of 1.01 mg/mL). A study of the CEase-inhibitory activity of amaranth protein recently reported in our lab also showed a significant low CEase-inhibitory effect of amaranth protein isolate in comparison to IC 50 values recorded for hydrolysates [13]. The hydrolysate with the utmost CEase inhibitory IC 50 value of 0.51 mg/mL (chymotrypsin-derived for 2 h) was approximately two times lower than the IC 50 value (1.01 mg/mL) of QPI, indicating that derived hydrolysates can therefore inhibit CEase two-fold more than their intact protein counterpart. Furthermore, the results suggested an overall high CEase inhibitory activity of QPHs compared to intact quinoa protein, which is evident from the lower IC 50 values observed. This is similar to the study of Jafar, et al. [31] that reported the higher CEase inhibitory activity of protein hydrolysates derived from camel whey compared to intact protein from Camel whey.

Pancreatic Lipase (PL) Inhibitory Activity
Pancreatic lipase is the primary enzyme that contributes largely to the digestion of dietary lipids, and the only effective way to adjust lipid absorption is by inhibiting the enzyme [32]. The pancreatic lipase inhibitory activity IC 50 values for QPHs (bromelain, chymotrypsin, protease) and un-hydrolyzed quinoa protein are depicted in Table 1. Results from this study showed that the PL-inhibitory IC 50 values varied from 0.90 to 10.4 mg/mL, implying a substantial ability to inhibit PL activity. The PL-inhibitory activity of all the hydrolysates (0.90-3.32 mg/mL) exhibited significantly higher inhibition than the QPI, indicating that hydrolysis efficiently increased the inhibitory activity of quinoa protein. Hydrolysate QC-6 demonstrated the highest PL-inhibitory activity (0.78 mg/mL), followed by QB-6, recording IC 50 values of 0.90 mg/mL, respectively. All chymotrypsin and bromelain generated hydrolysates showed higher PL-inhibitory activities than the protease generated hydrolysates. The different inhibitory activity observed among hydrolysates generated from different enzymes (bromelain, chymotrypsin and proteases) could be explained with the specificity and varying degree of hydrolysis among other factors of the individual enzyme [5].
On the other hand, a significant effect (p < 0.05) was observed in the pancreatic lipase inhibitory activity of QPHs upon hydrolysis with different enzymes at different hydrolysis times. A significant increase in the PL inhibitory activity of QPHs was observed with the increase in hydrolysis time progression, which is shown with lower IC 50 values. For instance, chymotrypsin derived hydrolysate with the most potent PL-inhibitory activity IC 50 values decreased from 2.90 to 0.78 mg/mL as the hydrolysis time increased. In the past, various studies have demonstrated an increase in the enzyme inhibitory potential of protein hydrolysates with the increase in the duration of hydrolysis. This implies that the progressive time of hydrolysis would sufficiently release peptides that might bind the active site of pancreatic lipase, thus inhibiting lipase enzymes. In addition, hydrolysates generated from chymotrypsin showed the maximum inhibitory activity in comparison to bromelain and proteases. This result upholds the potent characteristic of chymotrypsin generated hydrolysates, as reported by [6,16].
Furthermore, overall, QPHs showed the highest pancreatic lipase inhibitory activity in comparison to unhydrolyzed samples, and this accounted for an approximately 110% reduction in the IC 50 value. This result agrees with the findings of Mudgil, et al. [33], who reported an increased PL-inhibitory activity of milk proteins after a bio-catalysis reaction using bromelain, chymotrypsin, alcalase and papain. Similarly, [34] had previously shown the potential of quinoa as a coadjutant therapeutic agent of cardiovascular diseases. The authors also reported quinoa's ability to minimize lipid profile and glucose levels that are initiated by fructose which often leads to most of the well-known detrimental effects in humans. The use of derived hydrolysates from quinoa seeds comprising grains greater than 10% was reported to significantly lessen high levels of plasma and liver total cholesterol in nourished mice [35]. Consequently, QPHs can substitute synthetic drugs as anti-hyperlipidemia therapeutic agents, having shown excellent potential towards PL-inhibition.

Selection and Identification of Cholesterol Esterase Inhibitory Peptides from Selected QPHs
Based on the overall higher CEase and PL inhibitory activities of chymotrypsin generated hydrolysates obtained at the highest hydrolysis time of 6 h (QC-6), it was further chosen for peptide identification by LC-MS Q T-O-F. Additionally, the ABTS radical scavenging and anti-hemolytic activities shown by this specific hydrolysate reported in a previously published work by [6] made it an exciting hydrolysate to characterize its existing bioactive peptides. The identified peptides were classified as bioactive peptides by their scoring of >0.80 on a web server known as Peptide Ranker and further exploration into their in silico interaction with target enzymes using the Pepsite 2 web server [19].
In total, 136 peptides were identified (Supplementary Table S1), however, only 35 peptides were shortlisted to be biologically active based on a Peptide Ranker score greater than 0.8 (Table 2), and these peptides were subjected to an in-silico mode for further structureactivity analysis. The potential active binding sites of the identified peptides on the binding sites of CEase enzyme were accessed based on a statistical significance of 5% ( Table 2). The significant variation (p < 0.05) implies that peptides derived from QC-6 significantly bound to the hotspot sites of CEase, resulting in non-competitive inhibition due to the modification in protein interaction networks or loss of enzyme activity that blocked its catalytic binding sites and substrate [19]. The results obtained from PepSite 2 showed that the peptides FFE, DFTF, DFLM, ML, CDCP, CYTF, QHPHGLGALCAAPPST, LR, RR, HVQGHPALPGVPAHW, AGLR, FTVM, LLPYH, ASNLDNPSPEGTVM, and HMLH had a significant (p < 0.05) binding effect on CEase (Table 2). However, QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, and ASNLDNPSPEGTVM among the listed peptides were observed to be capable of binding the active sites of CEase. The most potent identified peptides HVQGHPALPGVPAHW and QHPHGLGALCAAPPST that could bind up to 13 and 14 bound residues were selected for mass spectrometry analysis. Two to sixteen amino acid residues were observed in the thirty-five peptides recorded (Table 2). This number is within the range of anti-hypercholesterolemic peptides reported by [36] for β-lactoglobulin peptides. Previous studies have suggested that peptides that can bind to more than eight residues have the ability to effectively inhibit the activity of antidiabetic and antihypertensive properties [37]. This connotes that the identified peptides (QHPHGL-GALCAAPPST, HVQGHPALPGVPAHW, ASNLDNPSPEGTVM) from this study have the potential of inhibiting the activity of CEase.   According to [38], the active binding sites of CEase, which is vital for its catalytic function, contain an oxyanion hole (glycine and alanine), the catalytic triad (histidine and serine), and esteratic site. Some generated bioactive peptides from QC-6 derived hydrolysates could bind to part of the active site containing the catalytic triad, specifically His435 and Ser194. For instance, QHPHGLGALCAAPPST, HVQGHPALPGVPAHW, ASNLDNPSPEGTVM, HVASGAGPW, and KPGGTAGSALPRPAHW could bind up to fourteen, thirteen, thirteen, nine, and six bound residues of CEase, respectively. This result is consistent with previous studies where peptides that have shown higher inhibition potential to His435 and Phe324 residues are categorized as CEase inhibitors because they play a major role in the binding of cholesterol [29]. Similarly, [32], stated that peptides derived from Camel milk whey hydrolysates that could bind to residues His435 and Phe325 are important CEase inhibitors. Likewise, peptide HVASGAGPW could only bind to residue Phe324, which also suggests a potential CEase inhibitor.
Furthermore, [29] had earlier predicted the binding site residues of nine inhibitors of CEase using molecular docking to human and bovine CEase. The observation from this peptide sequencing indicated that the identified peptide sequence was able to form a hydrogen bonding with Gly107 in human CEase and formed a hydrophobic interaction with Ala108 in both human and bovine CEase. In addition, the Trp227 and Phe 324 of bovine CEase was observed to form cation-π interactions and π-stacking with the Phe1 residue of the peptide. Similarly, here Ala108, Phe324, and Trp227 were the major residues with active hotspots, except for Gly107 residues with no active site. This suggests that the most potent peptides QHPHGLGALCAAPPST, HVQGHPALPGVPAHW and ASNLDNPSPEGTVM could bind Ala108, Ser194, Ala195, Trp227, Phe324 and His435 in the active site of CEase, which could suggest an excellent bioactive peptide with prospects of inhibiting CEase, thus limiting dietary cholesterol absorption, which is anticipated for a CEase inhibitor.
Moreover, the CEase inhibitory capacity can further be explained with existing hydrophobic amino acids, and residue near the C-terminal and N-terminal end in the sequenced peptides. A previous study reported the major role the hydrophobicity of peptides plays in the anti-hypercholesterolemic activity of peptides, which is mainly the binding of bile acids. Identified peptides with anti-hypercholesterolemic ability inhibit bile acids absorption in the ileum; as a result, they reduce the level of blood cholesterol concentration [30]. Hydrophobic amino acids, namely alanine, leucine, phenylalanine, proline, etc. have been directly associated with the hypercholesterolemic-lowering activity of peptides. Similarly, peptides with specific hydrophobic amino acids at their terminal ends have demonstrated antidiabetic inhibitory potential. In the present study, aliphatic (Leu and Ala) and aromatic (Phe, Tyr and Trp) hydrophobic amino acids were identified, which are desirable at the C-terminal of the peptide. A previous study identified peptide FDGEVK derived from β-lactoglobulin tryptic hydrolysate as a potential anti-hypercholesterolemic peptide due to the presence of two such C-terminal amino acids [36]. Therefore, bioactive peptides derived from quinoa proteins could potentially inhibit CEase enzymes.

Identification of PL Inhibitory Peptides
Peptides with PL inhibitory potentials are shown in Table 3. From this study, results show that the peptides ML, MLLL, QHPHGLGALCAAPPST, LPLLR, MFVPVPH, HVQGHPALPGVPAHW, FTVM, LLPYH, MVLP, and HMLH derived from QC-6 showed a significant (p < 0.05) binding effect to PL. In this study, the number of peptides derived from QC-6 was 35, and the number of amino acids ranged from 2 to 16. This value is comparable to the range reported by [33] for PL-inhibiting peptides derived from Camel milk protein hydrolysates and the range reported by [39] for pinto beans. However, this was lower than the amino acid value of twenty-three derived from Cumin seeds [40]. The results indicated two peptides (QHPHGLGALCAAPPST and HVQGHPALPGVPAHW) with the most significant (p < 0.05) potent PL-inhibitory potential, binding up to 14 and 13 important amino acids, suggesting superior linkages to the target enzyme (Table 3).  Furthermore, these peptides have also demonstrated CEase inhibitory potential, as explained above, implying that these peptides have valuable anti-hypercholesterolemic properties. Other peptides (FSAGGLP, MFVPVPH, and ASNLDNPSPEGTVM) have shown a similar potent inhibitory potential binding up to 11, 11, and 12 important amino acids of PL, respectively. These peptides could be considered as potential PL inhibitors due to their maximum interaction with the binding sites of PL enzyme. Similarly, some peptides showed a high number of binding sites, thus possessing promising PL-inhibitory activity. Peptide HMCH could interact with nine bound residues, four peptides (LPLLR, FTVM, LLPYH, and MVLP) with eight bound residues, and peptide DFLM interacting with six bound residues. Among all the peptides that significantly (p < 0.05) bound to the active sites, only peptide ML interacted with a lower number of hotspots (four sites), and can be regarded as a poor PL inhibitor.
Furthermore, all six of the most potent peptides that interacted with maximum bound residues could bind Phe78, Ser153, Phe216, His264, and His152 from the hotspot sites of PL. The other peptides (HMCH, LPLLR, FTVM, LLPYH, and MVLP, DFLM) were capable of binding to similar active sites. Similar binding spots for PL inhibitory activity from cumin seeds have been reported by [40], and camel whey protein hydrolysates by [29]. Moreover, the peptides DFLM, KIVLDSDDPLFGGF, LPLLR, FTVM, LLPYH, MVLP, ASNLDNPSPEGTVM, and HMLH showed the ability to bind to similar hotspots (Phe78, Ser153, His152, Phe216, and His264). Furthermore, peptides showed the potential to indirectly inhibit PL by binding Try115, Leu154, Gly155, Ala179, and Pro181, which do not belong to the characteristic catalytic triad of pancreatic lipase.
It was observed that peptides that have the potential to bind a higher number of active sites of lipase were the hydrophobic amino acids. A majority of the peptides that exhibited the potential of binding a high number of binding sites of lipase consisted of hydrophobic amino acid residues such as proline and leucine. This is comparable to a previous study that showed proline and leucine to be the most active residue bound of lipase inhibiting peptides derived from pinto beans [39]. In addition, hydrogen bonding can also influence hydrophilic amino acid to bind generated bioactive peptides inhibitors to PL [40]. The PL inhibitory method of derived peptides from seed proteins has been reportedly measured against the mechanism of a synthetic lipase inhibitor (Orlistat), and a similar PL inhibitory mechanism observed between them [39,40]. Thus, novel QPHs derived from chymotrypsin at 6 h have PL-inhibitory peptides with the potential to bind active sites of lipase enzyme. This study provides novel PL inhibitory peptdies derived from quinoa that could be health-promoting in combating hypercholesterolemia.

Molecular Docking of Shortlisted Peptides in the Active Site of Human and Bovine CEase
To elucidate the binding pose and to identify the underlying intermolecular interactions of potential quinoa derived peptides in the active site of human and bovine CEase, molecular docking was performed. Table 4     CEase is a member of the alpha/beta hydrolase fold family. In the human CEase protein, the active site region includes an oxyanion hole comprising Gly107-Ala108 and Ala195 residues, and a catalytic triad containing Ser194-His435-Asp320 residues; both regions are essential for the functional activity of the protein. The hydroxyl group of Ser194 functions as a nucleophile that facilitates the hydrolytic action [41]. Apart from this, two acidic amino acids, Asp434 and Glu437, are positioned close to the active site region which renders a negative charge to the whole catalytic domain of the protein.
Among the docked peptides, the top docked peptide HVASGAGPW fits perfectly in the active site and is stabilized by hydrogen and hydrophobic bonds. Interestingly, Gly107, Ala108, and Ala195 residues, of the oxyanion hole, and His435, from the catalytic triad, were involved in the strong binding of this peptide with human CEase. The peptide AHCGGLPY showed interactions with several residues surrounding the active site including Ala108. Similarly, other top-docked quinoa peptides such as LYNDWDLR, MFVPVPH and FSAG-GLP have shown interactions with the residues involved in the active site ( Figure 1). These interactions can further block the catalytic activity of human CEase, supporting their potential as human CEase inhibitors. Like human CEase, bovine CEase shares similar residues (Ser194-His435-Asp320) in the catalytic triad and oxyanion hole of the molecule [42]. Sequence analysis revealed that the residue identity of human CEase and bovine CEase is 80%, and the overall three-dimensional structure of the active site region closely resembles human CEase [43]. The docked peptide AHCGGLPY exhibited a GlideScore of −10.05 kcal/mol and an MM-GBSA-based binding free energy of −72.14 kcal/mol. The active site residues Ala108 and Ser194 formed hydrogen and hydrophobic interactions with this peptide. Following this, the peptides MFVPVPH, FSAGGLP, HVASGAGPW and LPLLR also exhibited good GlideScores and MM-GBSA values (Table 4; Figure 1). The top peptides identified were observed to interact with the critical residues involved in the active site of the enzyme, which directly contributes to the inhibitory activity of the peptides and ligand interaction diagrams, which are represented in Figure 2.

Molecular Docking of Identified Peptides in the Active Site of Human and Porcine PL
The binding pose and intermolecular interaction of quinoa peptides with PL was elucidated using molecular docking simulations. GlideScore and calculated binding energy, based on MM-GBSA, for the best-docked poses as well as intermolecular interactions observed in the docked protein-peptide complex are provided in Table 5. Similar to several esterases and lipases, the PL active site is positioned in the N-terminal domain, which has a catalytic triad (Ser152, Asp176, and His263), and access to this site is controlled by a surface loop. Movement of the lid induces conformational changes in the protein structure that eventually leads to oxyanion hole unmasking that is involved in the interfacial binding region [44].

Molecular Docking of Identified Peptides in the Active Site of Human and Porcine PL
The binding pose and intermolecular interaction of quinoa peptides with PL was elu cidated using molecular docking simulations. GlideScore and calculated binding energy based on MM-GBSA, for the best-docked poses as well as intermolecular interactions ob served in the docked protein-peptide complex are provided in Table 5. Similar to severa esterases and lipases, the PL active site is positioned in the N-terminal domain, which has a catalytic triad (Ser152, Asp176, and His263), and access to this site is controlled by a surface loop. Movement of the lid induces conformational changes in the protein structure that eventually leads to oxyanion hole unmasking that is involved in the interfacial bind ing region [44].
The binding mode of the best-docked peptide, HVASGAGPW, within the active site of PL was analyzed. Results indicated that the peptide docked to the enzyme recorded a GlideScore of −11.74 kcal/mol and an MM-GBSA binding energy of −46.02 kcal/mol. The The binding mode of the best-docked peptide, HVASGAGPW, within the active site of PL was analyzed. Results indicated that the peptide docked to the enzyme recorded a GlideScore of −11.74 kcal/mol and an MM-GBSA binding energy of −46.02 kcal/mol. The other top docked peptides AHCGGLPY, FSAGGLP, LLPYH and MFVPVPH also exhibited good GlideScores and binding energies (Table 5). It was observed that peptide AHCGGLPY recorded a GlideScore of −11.73 kcal/mol, and −61.96 kcal/mol for the calculated binding energy. FSAGGLP recorded −9.72 kcal/mol and −79.39 kcal/mol for the GlideScore and MM-GBSA binding energy, respectively. The GlideScores for peptides LLPYH and MFVPVPH were −9.72 kcal/mol and −8.82 kcal/mol, respectively, and the MM-GBSA binding energies were −77.18 kcal/mol and −70.12 kcal/mol, respectively.  All of the reported peptides exhibited interactions with critical amino acids in the catalytic binding pockets of the enzyme by forming several hydrogen and hydrophobic interactions (Figure 3). Similar to human PL, the active site of porcine PL comprises the catalytic domain, consisting of Ser153, Asp177, and His264. Similar to human PL, the peptides HVASGAGPW, AHCGGLPY, MFVPVPH and FSAGGLP exhibited good GlideScores and binding energies. Apart from these peptides, LPLLR and CYTF also docked well in the active site of the protein. It was observed that the peptide HVASGAGPW was welldocked in the catalytic region by exhibiting −10.19 kcal/mol and −80.44 kcal/mol for the GlideScore and binding energy, respectively. Followed by this, the peptides AHCGGLPY and MFVPVPH had GlideScores of −10.95 and −8.58 kcal/mol and a binding energy of −53.03 and −67.89 kcal/mol. The peptides were stabilized primarily by electrostatic and hydrophobic interactions, with residues in the active site of the enzyme (Figures 3 and 4). −80.44 kcal/mol for the GlideScore and binding energy, respectively. Followed by this, the peptides AHCGGLPY and MFVPVPH had GlideScores of −10.95 and −8.58 kcal/mol and a binding energy of −53.03 and −67.89 kcal/mol. The peptides were stabilized primarily by electrostatic and hydrophobic interactions, with residues in the active site of the enzyme (Figures 3 and 4).

Conclusions
This study investigated the CEase-and PL-inhibitory potential of QPHs as a functional ingredient for producing nutraceuticals that can be used to manage hypercholester-

Conclusions
This study investigated the CEase-and PL-inhibitory potential of QPHs as a functional ingredient for producing nutraceuticals that can be used to manage hypercholesterolemia. This current study explored the generation of hydrolysates from quinoa proteins using three enzymatic approaches (chymotrypsin, proteases, and bromelain). The three enzymes explored produced hydrolysates with higher inhibitory activities towards CEase and PL compared to unhydrolyzed quinoa proteins. Nonetheless, chymotrypsin-generated hydrolysates at 6 h hydrolysis time exhibited higher CEase and PL inhibitory activity as confirmed by lower IC 50 values. Findings showed that QPHs effectively inhibited the two enzymatic markers responsible for hypercholesterolemia by disrupting the enzymesubstrate interactions at the enzyme hotspot site. Novel peptide ASNLDNPSPEGTVM was found to have the best potential to be a CEase inhibitor, while FSAGGLP, KIVLDS-DDPLFGGF, and MFVPVPH were found to be the potential PL inhibitors. The peptides QHPHGLGALCAAPPST and HVQGHPALPGVPAHW we observed to be potential inhibitors against both CEase and PL. In silico data showed that Gly107, Ala108, Ala195, and His435 were involved in the strong binding of these peptide with human CEase. Thus, peptides generated from quinoa seed protein may have a valuable application in ameliorating hypercholesterolemia based on predictions using software tools and molecular docking studies. However, further studies using in vitro, in vivo and cell line assays should be carried out to validate the potential anti-hypercholesterolemic activities observed in this work.
Supplementary Materials: The following supporting materials can be downloaded at: https://www. mdpi.com/article/10.3390/foods12061327/s1, Table S1: List of peptides identified in chymotrypsin hydrolysed quinoa protein hydrolysate QC6; Figure S1: Electrophoretic patters of quinoa proteins and quinoa protein hydrolysates. Funding: Authors would like to thank UAE University for funding this study through Asian University Alliance (AUA) grant (12F002).

Data Availability Statement:
The data presented in this study are available in the supporting materials.

Conflicts of Interest:
The authors declare that they have no conflict of interest with regard to the preparation of this article and that they have no affiliations with or involvement in any entity or organization with any financial or non-financial interest in the subject matter discussed in this manuscript.