Structure Characterization and Antihypertensive Effect of an Antioxidant Peptide Purified from Alcalase Hydrolysate of Velvet Antler

Velvet antler collected from a farmed elk at 75 days after casting was obtained from Daesungsan Deer Farm (Daegwallyeong, Korea). Sliced fresh velvet antler was freeze-dried and ground into a fine powder.

To verify the antihypertensive effect of antioxidant peptide derived from velvet antler, separation and purification of antioxidant peptide were performed according to a previously reported method (Ding et al., 2019). Firstly, 10 g of the ground dried velvet antler powder was added into 1 L of distilled water. Alcalase enzyme (food-grade commercial proteases, Novo Nordisk, Bagsvaerd, Denmark) was then added with a substrate to enzyme ratio of 100:1. Enzymatic hydrolysis was conducted under optimal conditions (pH 8.0, 50°C) for 24 h, after which the hydrolysate was boiled at 100°C for 10 min to inactivate the enzyme. The hydrolysate was clarified by centrifugation at 3,000×g for 20 min to remove any unhydrolyzed residue. The supernatant of alcalase hydrolysate was filtered and adjusted to pH 7.0. The enzymatic hydrolysate was fractionated using ultrafiltration membranes (MWCO: 5 kDa) with a Millipore’s Lab scale TFF system (Millipore Corporation, Bedford, MA, USA) at 4°C. Then, <5 kDa fraction was obtained and lyophilized. The fraction (500 mg) was separated with ultrafiltration membranes and dissolved with 5 mL of filtered distilled water. This fraction was loaded onto a Sephadex G-25 column (2.5×100 cm) pre-equilibrated with filtered distilled water. Elution was then carried out with filtered distilled water at a flow rate of 1.5 mL/min. Absorbance of each fraction was measured at 220 nm and sub-fractions were collected. Lastly, the target fraction was then subjected to reverse-phase high performance liquid chromatography on an Atlantis T3 column (3 μm, 3.0×150 mm, Waters, New York, NY, USA) with a linear gradient of acetonitrile (0%–100% v/v, 30 min) at a flow rate of 1.0 mL/min. Elution peaks were detected at 220 nm. Finally, the antioxidant peptide was collected and lyophilized followed by identification of its amino acid sequences.

Molecular weight (MW) and amino acid sequences of the antioxidant peptide purified from velvet antler were determined using a MicroQ-TOFIII mass spectrometer (Bruker Daltonics, Bremen, Germany) coupled with an electrospray ionization (ESI) source. The purified peptide was dissolved in distilled water and infused into the ESI source. Its MW was determined by singly charged (M+H) state analysis in mass spectrum.

ACE inhibition activity of antioxidant peptide was measured using an ACE kit-WST (Dojindo Molecular Technologies, Kumamoto, Japan) in accordance with the manufacturer’s protocols. This ACE kit-WST assay is safe and simple. It provides highly reproducible data.

In order to assess the interaction between Asp-Asn-Arg-Tyr-Tyr (DNRYY) and ACE, molecular docking analysis were carried out using Accelrys Discovery Studio 3.5 (Accelrys, San Diego, CA, USA), according to a previously reported procedure (Kang et al., 2020). The crystal structure of ACE (PDB code: 1O86) was obtained from the Protein Data Bank (PDB, http://www.rcsb.org) and then molecular docking of DNRYY into the active site of the ACE was performed. The molecular docking runs were performed with the CDOCKER and Calculate Binding Energies tool. Docking of DNRYY to ACE was performed as follows: (1) a 2D structure was converted into a 3D structure; (2) proteins were prepared, and the binding site was defined; and (3) docking of compounds was performed using CDOCKER tool. The binding energy of the produced candidate compound-protein complexes were calculated using Calculate Binding Energies tool.

Anti-hypertensive effect in animal model was carried out in accordance with a previously reported method (Ko et al., 2012). Male SHRs (10 weeks of age) were purchased from SLC (Shizuoka, Japan). SHRs with tail systolic blood pressure (SBP) over 180 mmHg were randomly divided into three groups (n=6): Control group (saline); sardine peptide treated group (positive control); and the purified peptide treated group. Saline, sardine peptide, and the purified peptide dissolved in 1 mL saline were orally administered to SHRs at a dose of 50 mg/kg. SBP was measured with a tail-cuff method at 0, 3, 6, and 9 h post-administration using a CODA^TM blood pressure monitor (Kent Scientific, Torrington, CT, USA). The experimental protocol was approved by the Laboratory Animal Administration Committee of Jeju National University (Approval NO. 2017-0017). The experiment was conducted according to Animal Experimentation Guidelines of the University.

The results are represented as mean±SD. Statistical analysis was done using one-way analysis of variance (ANOVA) complemented by Duncan’s multiple range test using SPSS software version 28 (IBM, Armonk, NY, USA). The level of statistical significance was set at p<0.05.

In our previous study, the IC₅₀ value of the purified peptide from Alcalase hydrolysate of velvet antler against peroxyl radical was 0.343 mg/mL, suggesting that the purified peptide could possess potent antioxidant activity (Ding et al., 2019). As previous reported, MW and structure of the bioactive peptides is closely related to ACE inhibitory activity (Lee and Hur, 2017). However, the MW and amino acid sequence of the purified peptide have not been determined yet. Thus, in this study, amino acid sequence and MW of the purified peptide were determined using a Q-TOF ESI mass spectrometer. As shown in Fig. 1, the purified antioxidant peptide was identified as a pentapeptide DNRYY. The MW of this pentapeptide was 730.31 Da.

Fig. 1. Identification of amino acid sequence (A) and molecular weight (B) of the antioxidant purified peptide from alcalase hydrolysate of velvet antler by Q-TOF ESI mass spectrometer. Q-TOF, quadrupole time-of-flight; ESI, electrospray ionization.

Download Original Figure

Antihypertensive effect is the most extensively studied bioactivity in relation to food-derived peptides. They main mechanism of action is based on the inhibition of the ACE that converts the inactive angiotensin I into the potent vasoconstricting angiotensin II (Miralles et al., 2018). Angiotensin II produced by the action of ACE also amplifies oxidative stress by inducing the formation of intracellular reactive oxygen species (Del Pino-García et al., 2017; Kang et al., 2020). Thus, ACE inhibition intensifies the antioxidant defense system in humans. Several food-derived bioactive peptides have been reported to possess both antioxidant and antihypertensive effects (Kang et al., 2020; Toldrá et al., 2020). Therefore, we investigated to provide evidence for the antihypertensive effect of an antioxidant peptide purified from velvet antler alcalase hydrolysate in the present study. In order to validate the antihypertensive effect of the purified antioxidant peptide (DNRYY), we evaluated the inhibitory activity of DNRYY against ACE. As shown in Fig. 2, DNRYY inhibited ACE activity in a dose dependent manner. At concentrations of 1.75, 3.5, 7, and 14 μM, it inhibited ACE activity by 23.56%, 48.11%, 69.83%, and 89.53%, respectively. IC₅₀ values of DNRYY against ACE was 3.72 μM. Findings of this study exhibited that DNRYY might be useful as an effective velvet antler-derived antihypertensive peptide. The structure of a peptide is an important factor for its ACE inhibitory activity (Lee et al., 2009; Lee et al., 2014). As reported previously, aromatic amino acids at the C-terminus, hydrophobic amino acid residues at the N-terminus, and positively charged amino acids at the middle in peptide structure play an important role in the ACE inhibition activity of peptides (Cushman and Cheung, 1971; Welderufael et al., 2012; Wu et al., 2016). Especially, it has been proven that peptides with C-terminal Tyr residues such as Val-Tyr, Met-Tyr, Ile-Tyr, and Arg-Val-Tyr show potent ACE inhibitory activities (Chen et al., 2009; Erdmann et al., 2006; Ko et al., 2012; Liu et al., 2021; Yu et al., 2019). In this study, the identified pentapeptide contained Tyr at the C-terminal, which might have contributed to its ACE inhibition activity. This study provides strong evidence for ACE inhibitory activity of antioxidant peptide purified from velvet antler alcalase hydrolysate. Results of preceding studies and the present study demonstrated that the purified peptide had both antioxidant and antihypertensive effects.

Fig. 2. ACE inhibitory activity of DNRYY. Inhibitory activity was measured using an ACE kit. Values are expressed as mean±SD of triplicate experiments. ^a–d Values with different letters are significantly different at p<0.05, as determined by Duncan’s multiple range test. ACE, angiotensin converting enzyme; DNRYY, Asp-Asn-Arg-Tyr-Tyr.

Download Original Figure

Molecular docking is an application wherein molecular modeling techniques are used to predict how a protein (enzyme) interacts with small molecules (ligands; Ko and Lee, 2021). The application of in silico molecular docking tools can simplify process and cut down costs for discovery of bioactive peptides, and illuminate the action mechanism (Fan et al., 2020; Zhao et al., 2019). In the present study, in order to understand potential molecular interactions between the velvet antler-derived pentapeptide (DNRYY) structure and specific amino acids at the binding site of ACE, computational molecular docking of the DNRYY into the binding site of ACE (PDB code: 1O86) was conducted using Discovery Studio 3.5 (DS). As shown in Fig. 3A, the complex of ACE-DNRYY binding sites was predicted by the 2D diagram. DNRYY formed five hydrogen bonds with amino acid residues in ACE: One with HOH2570, one with ARG522, one with ARG124, and two with GLU143. DNRYY also formed two pi interactions with HIS387 and TRP357 residues in ACE. Additionally, DNRYY interacted with the GLU403 (attractive charge) and ARG522 (salt bridge) amino acid residues through electrostatic interactions. These results suggest that DNRYY can interact with amino acids residues of ACE and inhibit ACE activity. Pi interactions and hydrogen bonds in ACE-ligand interactions are known to contribute to ACE inhibition activity (Fu et al., 2017; Ling et al., 2018; Tu et al., 2018). It has been reported that food-derived bioactive peptides have strong interactions with ACE by forming high numbers of pi interactions and hydrogen bonds with ACE amino acid residues, thus effectively inhibiting ACE (Chamata et al., 2020; Fan et al., 2020; Ko et al., 2017). The present study also showed that DNRYY could form high numbers of pi interactions and hydrogen bonds with ACE amino acid residues. Accordingly, our results suggest that the potent ACE inhibitory activity of DNRYY can be attributed to high numbers of hydrogen bonds and pi interactions at the ACE binding site. The docking of stabilized poses of ACE-DNRYY complex was obtained from DS 3.5 binding energy program. Binding energy value was –86.200 kcal/mol for CDOCKER interaction energy and –456.853 kcal/mol for binding energy. These results indicate that DNRYY has a good and stable binding affinity with ACE. Overall, the docking results provide strong evidence for the ACE inhibition activity of DNRYY.

Fig. 3. Specific interactions between DNRYY and ACE after automated docking of DNRYY to the ACE enzyme binding site. Predicted 3D structure of the ACE (Protein Data Bank; PDB code: 1O86)–DNRYY complex and 2D diagram (A) are shown. Binding energy values were obtained from Discovery Studio (DS) 3.0 binding energy calculation program (B). ACE, angiotensin converting enzyme; DNRYY, Asp-Asn-Arg-Tyr-Tyr.

Download Original Figure

ACE-inhibitory peptides derived from foods have gained interest as potential hypertension treatment agents. Several studies have reported that bioactive peptides exhibited significant blood pressure lowering effect by inhibiting ACE activity in vivo studies using SHRs (Lee and Hur, 2017; Miralles et al., 2018; Toldrá et al., 2020). Thus, to verify the in vivo antihypertensive effect of ACE inhibitory peptide (DNRYY) derived from velvet antler alcalase hydrolysate, the ACE inhibitory peptide was evaluated by measuring changes of SBP at 3, 6, and 9 h after oral administration of 50 mg/kg of body weight to SHRs. The SBP reduction effect of DNRYY was compared with effects of a sardine peptide, a commercially available antihypertensive peptide (derived from Sardinops melanostictus) used in blood pressure lowering functional food ingredient in Korea and Japan. As shown in Fig. 4, the saline administrated group exhibited slight changes in SBP in the range of 185–197 mmHg. However, at 3 h after oral administration of DNRYY, SBP was significantly decreased compared to that in the saline group. The DNRYY administrated group showed a similar SBP reduction to the sardine peptide administrated group with activities maintained until 6 hours. These results clearly show that oral administration of purified peptide (DNRYY) can decrease SBP significantly in SHRs. Previous many studies have revealed that food-derived peptides consisting of Tyr at the C-terminus, such as Val-Tyr, Met-Tyr, Arg-Val-Tyr, Ile-Tyr, and Val-Glu-Gly-Tyr show antihypertensive effects in SHRs through ACE inhibition activity (Ko et al., 2012; Matsui et al., 2004; Tanaka et al., 2006). Accordingly, the antihypertensive effect of DNRYY in SHRs can be attributed to its ACE inhibition activity. These results of this study confirmed that this ACE inhibitory peptide from velvet antler alcalase hydrolysate could effectively reduction of blood pressure.

Fig. 4. Change of SBP after oral administration of ACE inhibitory peptide (DNRYY) in SHR. Sardine peptide was used as a positive control. Single oral administration was performed at a dose of 50 mg/kg body weight. SBP was measured at 0, 3, 6, and 9 h after oral administration of DNRYY. Values are expressed as mean±SD of triplicate experiments. ^* p<0.05; ^** p<0.01, indicate significant differences compared with the only saline-treated control group. DNRYY, Asp-Asn-Arg-Tyr-Tyr; SBP, systolic blood pressure; ACE, angiotensin converting enzyme; SHR, spontaneously hypertensive rat.

Download Original Figure