Purification and Molecular Docking Study of Angiotensin-I Converting Enzyme (ACE) Inhibitory Peptide from Alcalase Hydrolysate of Hazelnut (Corylus heterophylla Fisch) Protein

Although a number of bioactive peptides are capable of angiotensin I-converting enzyme (ACE) inhibitory effects, little is known regarding the mechanism of hazelnut peptides using molecular simulation. In the present study, gel filtration chromatography, reverse phase-high performance liquid chromatography, and liquid chromatography-electrospray ionization-tandem mass (LCESI-MS/MS) were employed for purifying and identifying the ACE inhibitory peptides from hazelnut. To understand the mode of action of these peptides, the interaction between the inhibitory peptides and ACE was investigated. The results identified novel ACE inhibitory peptides Asp-Asp-Glu-Leu-ArgGln-Ala (DDELRQA), Asp-Asp-Glu-Leu-Arg-Ala-Ala (DDELRAA), and AspGly-Glu-Leu-Arg-Glu (DGELRE). The binding free energies of DDELRQA, DDELRAA, and DGELRE for ACE were −10.2, −9.0, and −8.8 kcal/mol, respectively. This study proves the high stability of ACE inhibitory peptides derived from hazelnut against different temperature and pH of processing.


Introduction
Hypertension is a major chronic disease affecting 30% of the adult population in the world [1]. Although there are many causes of hypertension, it is well recog-nized that angiotensin I-converting enzyme (ACE) plays an important role in the fluid and salt balance in mammals, as well as the rennin-angiotensin and the kallikrein-kinin systems for the regulation of blood pressure [2] [3]. Many synthetic ACE inhibitors including captopril, enalapril and lisinopril and others have been used to prevent hypertension in clinical [4]. However, these synthetic ACE inhibitors have been suspected to threaten health by causing cough, taste disturbances and renal impairment [5]. There, thus, is a growing interest in finding ACE inhibitors from natural products [6]. Food-derived peptides with antihypertensive properties have received great interest during the past 30 years [7]. Various food proteins, derived from plant protein and animal protein had ACE inhibitory activity in vitro [8].
The hazel species Corylus. heterophylla Fisch is a nut tree that belongs to the Betulaceae family, which is the most well-known species of wild hazelnuts.
Moreover, the wild hazelnuts (species Corylus. heterophylla Fisch) are considered a rich source of many important compounds (polyphenols, protein, fat acid, carbohydrates, dietary fibre, aminophenols and microelements) useful for human diet health, which mainly distributed in Changbai Mountain, Jilin Province, in northeast of China, for centuries; its nuts have been harvested for oil extraction and as a food source. It is an economically important species, and its production accounts for nearly three quarters of the total output of the Chinese domestic market. The hazelnut dregs (containing around 58% of protein) are by-products of edible oil production were lost. In our previous work, we have analyzed the immunomodulatory effects of hazelnut hydrolysed peptides isolated and identified in the hazelnut dregs, by-products (35% -45% of waste production) from the hazelnut industries [9]. Recently, the use of hazelnut protein hydrolysates has been the subject of several research works, because of their biological activities, such as antioxidant, anti-inflammatory and antimutagenic properties [10]. For understanding the ACE inhibitory activities of protein hydrolysates from wild hazelnuts, the role of individual peptides in the hydrolysate should be examined. Therefore, the aim of the present study was to isolate and identify the ACE inhibitory peptides from hazelnut using gel filtration chromatography and high performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). Recently, molecular docking has been reported to be a useful tool for analyzing the interactions between ACE and its inhibitors with a high degree of accuracy and versatility. Previous studies have used molecular docking to elucidate the interactions between small molecules and proteins with high accuracy. Accordingly, molecular interactions between the purified peptides and ACE were elucidated. Thus, this study can provide the theoretical basis and technological support for industrial production of ACE inhibitory peptides.

Materials
Hazelnut protein was obtained from the College of Food Science and Engineer-

Preparation of Hazelnut Protein Hydrolysates
Hazelnut protein hydrolysates were prepared according to the method described

Determination of ACE Inhibitory Activity
The ACE inhibitory activity of peptides was evaluated using the method described by da Cruz et al. [11].

Gel Filtration Chromatography
The ACE inhibitory peptides were purified according to the method described by Sangsawad et al. [12], with slight modifications. The fraction showing the highest ACE inhibitory activity was redissolved in distilled water at a concentration of 30 mg/mL and subjected to further purification using the Sephadex G-15 column (1 × 50 cm), which was eluted using distilled water at a flow rate of 1 mL/min. The absorbance of the fractions was measured at 220 nm. The fractions exhibiting the highest ACE inhibitory activity were pooled. Each pooled fraction was analyzed for the ACE inhibitory activity.

RP-HPLC
Following gel filtration chromatography, the fraction exhibiting the highest ACE inhibitory activity was further purified by RP-HPLC using the Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA, USA) with a Diamonsil C18 HPLC column (4.6 × 250 mm, 5 μm; loading quantity, 80 μL). The column was eluted using a linear gradient of acetonitrile (5% -60%) supplemented with 0.1% trifluoroacetic acid, at a flow rate of 0.5 mL/min. The fractions, corresponding to the elution peaks, were collected and lyophilized immediately for determining their ACE inhibitory activities. Next, fraction with highest activity was pooled using Waters 2545 QGM semi-preparative HPLC (Waters Corporation, Milford Massachusetts, USA) with a SunFire Prep C18 column (10 × 250 mm, 5 μm), which was eluted using a linear gradient of acetonitrile (5% -60%; supplemented with 0.1% trifluoroacetic acid) at a flow rate of 5 mL/min and a loading quantity of 2 mL. Absorbance of the eluent was measured at 215 nm.
Next, the amino acid sequence of the peptide fraction exhibiting the highest ACE inhibitory activity was determined.

Identification of Hazelnut Peptide
Based on the previous experiments, the amino acid sequence of the peptides in the fraction showing the highest ACE inhibitory activity was identified as de-

Molecular Docking
The

Stability of ACE Inhibitory Peptides
The

Statistical Analysis
Analysis of variance was performed using the SPSS version 13.0 software (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicates. Data are represented as the mean ± standard deviation. The results were considered to be statistically significant at P < 0.05.

Purification of Hazelnut Peptides
High degree of hydrolysis can be used for obtaining low-molecular-weight peptides. Thus, alcalase was used for the preparation of ACE inhibitory peptides due to its broad substrate specificity and high degree of hydrolysis. After hydrolysis with alcalase, the hydrolysate was subjected to gel filtration chromatography, and RP-HPLC, which separated the ACE inhibitory peptides (from the bioactive fractions) based on differences in molecular weight, and hydrophobicity. As shown in Figure 1(a), following Sephadex G-15 chromatography, two major fractions were obtained. The fractions were labeled A1 and A2. Next, they were pooled, lyophilized, and subjected to the ACE inhibition assay. The highest ACE inhibitory activity (72.31% ± 2.47%, P < 0.05) was observed for fraction A1.
Since the Sephadex G-15 column was used, these results indicated that the majority of peptides present in fraction A1 were high-molecular-mass peptides. These results were similar to those reported by Connolly, O'Keeffe, Piggott, Nongonierma and FitzGerald (2015), who demonstrated that the low-molecularmass peptides were not always associated with a high ACE inhibitory activity. Therefore, the results of the present study suggested that molecular mass may not be the only physicochemical characteristic associated with ACE inhibition. It has also been reported that certain high-molecular-mass peptides may act as substrates for ACE, thereby interfering with the hydrolysis of the synthetic substrate (HHL) used in the assay.
Using gel filtration chromatography, the A1 fraction, which showed the highest ACE inhibitory activity was further purified, and subjected to RP-HPLC.
RP-HPLC is widely used as the final step in peptide purification and possesses several advantages such as high sensitivity, resolution, and column efficiency.

Identification of Hazelnut Peptides
The B5 fraction, which exhibited the highest ACE inhibitory activity, was analyzed using LC-ESI-MS/MS, in order to identify the constituent peptides that may be potentially responsible for ACE inhibition. A search of potential ACE inhibitory activity that can be derived from the identified peptides was carried out with the BIOPEP database in which the identified peptides were compared to previously reported sequences. As shown in Figure 2, three novel peptides were screened de novo from fraction B5, namely Asp-Asp-Glu-Leu-Arg-Gln-Ala  According to previous reports on the relationship between structure and activity of ACE inhibitory peptides, the C-terminal end of the peptide is a major contributor to the activity, and the nature of the C1-C4 amino acids seems to control the inhibitory potential [7]. Many ACE inhibitory peptides exhibit some common structural properties, including abundant hydrophobic amino acids, . This may be due to the presence of hydrophilic amino acid residues, which affect the ACE inhibitory activity by restricting the entry of the peptide to the enzyme active site. Hence, the structure-activity relationship of ACE inhibitory peptides needs to be further explored. Recently, the quantitative structure-activity relationship (QSAR) models for ACE inhibitory peptides have been studied to analyze structure information. However, significant time and resources will be required to build models.

Inhibition Mechanism of Hazelnut Peptides
Understanding the molecular interactions between ACE and the ACE inhibitory peptides will help in designing and synthesizing more efficient peptide inhibitors. To elucidate the molecular interactions between the purified hazelnut peptides and ACE, molecular docking studies were performed. The results predicted the preferred orientations of the three peptides required for the formation of a stable complex with ACE.
The molecular docking results of synthetic peptides and ACE were shown in    Ala354 and Glu384 of ACE active center played an important role in the ACE active center, which forming hydrogen bonds with inhibitor [15]. It is beneficial to increase the affinity of the peptide with the ACE molecule to higher inhibitory activity. In addition, Zn 2+ is an important ligand ion of ACE active center, and electrostatic interaction with Zn 2+ may directly lead to inactivation of ACE molecules [16]. In future, flexible docking may be adopted in calculating the change of distance and energy between the residue and Zn 2+ .

Stability of ACE Inhibitory Peptides against Heat Treatment and in Vitro Digestion
ACE inhibitory peptides could exert in vivo antihypertensive effect if they reach  [17]. So, after oral ingestion, peptides need to resist complete hydrolysis by gastrointestinal enzymes and brush border peptidases, and be able to pass through the intestinal wall with preserving their biological activity [18]. Peptides can be degraded in this process and their biological activity can be activated or inactivated. As Figure 4(a) showed ACE peptide retained its inhibitory activity even after severe thermal treatments at 100˚C. In addition, inhibitory activity remained constant over a wide pH range of 2 -10 ( Figure 4(b)), indicating that peptides were both thermo and pH stable. In addition, activity of hazelnut peptide decreased when treated with Cu 2+ , Ca 2+ and high concentration of NaCl, but other metal ion could not affect their inhibition.   [20]. These results indicated that orally administered ACE inhibitory peptides may not keep their sequence integrity in the stomach, but break into new smaller bioactive peptides that could reach the blood stream.

Conclusion
Novel ACE inhibitory peptides from hazelnut protein hydrolysates, namely Asp-Asp-Glu-Leu-Arg-Gln-Ala (DDELRQA), Asp-Asp-Glu-Leu-Arg-Ala-Ala (DDELRAA), and Asp-Gly-Glu-Leu-Arg-Glu (DGELRE) were successfully purified and identified using gel filtration chromatography, RP-HPLC, and LC-ESI-MS/MS. This screening method may be used as a valuable tool for identifying novel food-derived ACE inhibitory peptides. Furthermore, molecular docking results indicated that the interaction of peptides with certain amino acids in the ACE active site markedly contributed towards stabilizing the docking complex. The strong inhibition of ACE exhibited by DDELRQA may be attributed to the formation of hydrogen bond interactions. The above results provided novel insights and helped in improving our current understanding of the interaction between ACE and its inhibitors.