Production, Analysis and in Vivo Antihypertensive Evaluation of Novel Angiotensin-I-converting Enzyme Inhibitory Peptides from Porcine Brain

Abstract

This study investigated the angiotensin I-converting enzyme (ACE)-inhibitory activity and antihypertensive effect of alcalase-, flavourzyme-, and protease N-digested porcine brain hydrolysates (PBHs) on spontaneously hypertensive rats (SHRs) after their oral administration. In addition, we investigated whether a peptide fraction with a molecular weight of less than 3 000 Da inhibits ACE in vitro and lowers arterial blood pressure. The results revealed that PBHs obtained after 4 h of hydrolysis with protease N (PBP4H) yielded peptides enriched with ACE-inhibitory activity (86.29 ± 5.76%). Furthermore, PBP4H had the lowest ACE-inhibitory activity IC₅₀ value (1.945 mg/mL). Oral administration of PBP4H-100 (100 mg/kg bw/day) and low-molecular-weight peptide fractions, PBP4H < 3 000 Da (50 mg/kg bw/d) reduced the systolic blood pressure (−30 mmHg) in SHRs. Moreover, the long-term administration of PBP4H-100 and PBP4H < 3 000 Da significantly suppressed hypertension. In addition, results of the plasma ACE activity was positively related with the SBP lowering effects of PBP4Hs. Overall, these results reveal the antihypertensive effect of PBH and as a functional supplement for suppressing hypertension.

Introduction

Hypertension is one of the major risk factors for cardiovascular diseases, stroke, myocardial infarction, and end-stage renal disease (Chobanian et al., 2003; Zhang et al., 2006). In 2008, worldwide, approximately 40% of adults aged 25 and above had been diagnosed with hypertension; the number of people with the condition rose from 600 million in 1980 to 1 billion in 2008. Of these, complications of hypertension account for 9.4 million deaths worldwide every year (World Health Organization, 2013). Therefore, studying functional foods that prevent hypertension is crucial (Saiga et al., 2008). Angiotensin I-converting enzyme (ACE; peptidyl dipeptidase; E.C. 3.4.15.1) is a membrane-bound metalloprotease playing a crucial physiological role in the regulation of blood pressure in the renin-angiotensin system (RAS) (Shi et al., 2010). ACE converts decapeptide, Angiotensin I into the potent vasoconstrictor octapeptide, Angiotensin II and catalyzes the degradation of bradykinin, a vasodilator peptide. The inhibition of ACE activity reduces the Angiotenisn II concentration, which subsequently and concomitantly reduces blood pressure. Thus, ACE inhibition might provide new insights into the development of antihypertensive drugs. Currently, several synthetic ACE inhibitors such as captopril, enalapril, and lisinopril are used in clinical application, however, some undesirable side effects such as cough, taste disturbance, renal impairment, and angioneurotic edema may occur (Liu et al., 2010).

Food-derived peptides exhibit several health benefits including antimicrobial properties, blood pressure-lowering effects, immunomodulatory effects, cholesterol-lowering abilities, antithrombotic properties, antioxidant function, and opioid activities (Hartman and Meisei, 2007; Jamdar et al., 2010). Functional peptides derived from dietary sources are considered milder and safer than synthetic drugs (Lee et al., 2010). Furthermore, such peptides generally have multifunctional properties and are easily absorbed (Yu et al., 2006). Functional peptides usually contain 3–20 amino acid residues, and their activity is based on their amino acid composition and sequence (Pihlanto, 2000). Several researchers have focused on the production and isolation of ACE-inhibitory peptides from various natural sources. ACE-inhibitory peptides can be produced from various animal materials such as bovine casein (Jiang et al., 2010; Miguel et al., 2009), meat protein (Ahhmed and Muguruna, 2010), fish protein (Fujita and Yoshikawa, 1999; Lee et al., 2010; Wu et al., 2015), egg white (Liu et al., 2010), milk (Seppo et al., 2003; Quiros et al., 2007), chicken skin (Onuh et al., 2015), chicken collagen (Saiga et al., 2008), and chicken leg bone (Cheng et al., 2008). Some of these animal materials have been reported to exhibit an antihypertensive activity in vivo by reducing systolic blood pressure (SBP) in humans (Seppo et al., 2003) and spontaneously hypertensive rats (SHRs) (Onuh et al., 2015). The SHRs model has been reported to be the most widely used of the hypertensive animal models because some of the pathophysiological processes resemble that of human essential hypertension (Onuh et al., 2016).

Porcine brain, obtained as a byproduct from the head of swine, is abundant in proteins. It have been traditionally valued as an effective Chinese medicine and food material. More than 5 million porcine are slaughtered annually in Taiwan, however, most porcine brains (more than 600 tons) are consumed only in part, and most of the remainder is converted to animal feed or discarded; this may cause environmental pollution. Animal brains have potential for use in medical and functional food products. The function of porcine brain peptides only studied on antioxidant activities (Zou et al., 2016), and has not been thoroughly investigated for ACE-inhibitory activities. To the best of our knowledge, this is the first attempt to produce protein hydrolysates, which exhibit an antihypertensive activity from porcine brain. Various enzymes have been used to produce hydrolysates from protein sources, Alcalase from bacterial and fungal sources have been utilized to generate bioactive peptides (Pihlanto, 2000), and Flavourzyme is used to produce ACE inhibitory peptides. Therefore, in this study, we choose Alcalase, Flavourzyme and Protease N, and to be evaluated their effectiveness in degrading the porcine brain proteins for ACE-inhibitory activity. Some research results indicated that bioactive peptides from food protein hydrolysates decreased blood pressure and enhanced blood flow by interfering with the RAS (Girgih et al., 2014; Onuh et al., 2016). The aim of this study is to analyze the in vitro potential of ACE-inhibitory capacity and the possible antihypertensive effect of different porcine brain hydrplysates on hypertensive rats. The ability of a peptides fraction of low molecular mass (<3 000 Da) was also in vitro analyzed and to lower the arterial blood pressure in vivo.

Materials and Methods

Materials    Alcalase (≥2.4 U/g, P4860; Sigma, St. Louis, USA) is an endoprotease from Bacillus licheniformis. Flavourzyme (≥500 U/g, P6110, Sigma, St. Louis, USA) is an exoprotease and endoprotease complex from Aspergillus oryzae. Protease N (Amano Enzyme Inc., Nagoya, Japan). ACE from rabbit lungs, the ACE synthetic substrate hippuryl-L-histidyl-L-leucine (HHL) and furanacrylol-L-phenylalanyl glycylglycine (FAPGG) were purchased from Sigma-Aldrich Ltd. (Sigma, St. Louis, USA). All chemicals used were obtained from Merck Ltd. (Merck Millipore, Darmstadt, Germany) or Sigma-Aldrich Ltd. The solvents and other chemical reagents used were of reagent grade.

Preparation of PBHs for alcalase, flavourzyme, and protease N    According to the method described by Cheng et al. (2008), with slight modifications, porcine brains were obtained from a slaughterhouse in Pingtung, Taiwan, immediately ground with two volumes of water in a blender (TM21/2-4C; Vorwerk Semco, France), and heated to 100°C in a boiling water bath for 15 min. The porcine brains were digested for 10 h by using alcalase (enzyme-to-substrate ratio, 1:50) at pH 8.0 and 50°C, flavourzyme (enzyme-to-substrate ratio, 1:50) at pH 7.5 and 50°C, and protease N (enzyme-to-substrate ratio, 1:50) at pH 7.0 and 50°C, respectively (suspended buffer were 0.025 M KH₂PO₄ and 0.1 M NaOH). The enzymatic hydrolysis was stopped every 2 h by heating to 100°C for 10 min, and hydrolysates were collected. After incubation, the hydrolysates were centrifuged (10 000 g at 4°C) for 20 min, filtered through a filter with a 0.45-µM pore size, lyophilized (Body: FD-5N, Eyela, Japan; pump: G-100D, ULVAC, Japan), and stored at −80°C.

Analysis for peptide content and degree of hydrolysis    The peptide content was measured according to the method described by Church et al., (1983). The o-phthaldialdehyde (OPA) reagent was prepared daily by dissolving 40 mg of OPA in 1 mL of methanol and mixing with 25 mL of 100 mmol sodium tetraborate (borax) buffer (pH 8.3), 2.5 mL of 20% sodium dodecyl sulfate, and 100 mL of β-mercaptoethanol. The volume was adjusted to 50 mL. Furthermore, 20 mL of the hydrolysate was added to 1.5 mL of the OPA reagent and incubated for 2 min at room temperature. The absorbance was read at 340 nm by a spectrophotometer (Infinite F200 Pro, TECAN Trading AG, Switzerland). Gly-Leu was used as the standard. The degree of hydrolysis (DH) was estimated through the change in the peptide content and expressed as follows: % DH = [1 − (peptide content of the hydrolysate at 0 h)/(peptide content of the hydrolysate)] × 100.

Assessment of ACE-inhibitory activity in vitro    The assay ACE-inhibitory activity was performed using the method of Cushman and Cheung (1971), with slight modifications as described by Cheng et al., (2008). ACE, a dipeptidyl carboxypeptidase extracted from rabbit lungs (A6778, Sigma, Reagent grade, USA), was prepared using 300 mmol/L borax buffer (pH 8.3) (8 mU/50 µL). HHL (H4884, Sigma, Reagent grade) was used as a synthetic substrate. The sample (30 µL) was added to the ACE solution (50 µL), and the reaction was started by adding 50 µL of 5 mmol/L HHL. After incubation at 37°C for 30 min, the reaction was terminated by adding 380 µL of 1.0 N hydrochloric acid. The resulting hippuric acid was extracted using 1.5 mL of ethyl acetate. After centrifugation (3 600 g at 4°C for 5 min), 1 mL of the upper layer was transferred into a microcentrifuge tube and heated in a dry bath (Type 17600; Thermolyne, Dubuque, USA) at 100°C for 1 h. The hippuric acid was dissolved in 1.0 mL of deionized water, and the absorbance was read at 228 nm by using a spectrophotometer (Infinite F200 Pro). Inhibitory activity was calculated using the absorbance of hippuric acid liberated from HHL: ACE-inhibitory activity (%) = {1 − [(absorbance of the sample at 228 nm − absorbance of the sample blank at 228 nm)/(absorbance of the control at 228 nm − absorbance of the control blank at 228 nm)]} × 100. The IC₅₀ value was defined as the concentration of peptides required to inhibit 50% of the ACE activity.

Separation and purification of ACE-inhibitory peptides from PBHs    According to the method of Miguel et al., (2009), with slight modifications, peptides exhibiting the highest ACE-inhibitory activity in PBHs were divided into two groups by using an ultrafiltration membrane: those with a molecular mass of less than 3 000 Da and those with a molecular mass of more than 3 000 Da (Millipore Stirred Cells, Merck Millipore).

Evaluation of the blood-pressure lowering effect in SHRs    The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC Approval No. 99-20) at National Chung Hsing University. The rats were handled and sacrificed in accordance with the guidebook for the care and use of laboratory animals (The Chinese-Taipei Society of Laboratory Animal Sciences). Sixty male SHRs (6 weeks old and weighting 200 ± 50 g at the beginning) were purchased from the National Laboratory Animal Center, Taipei, Taiwan. These rats were housed in a room under conditions of controlled temperature (24 ± 2°C), relative humidity (50 ± 10%), and 12-h light-dark cycle (lighting from 07:00 to 19:00). A laboratory diet (MF-18; Oriental Yeast Co., Tokyo, Japan) and an overhead water tap were available ad libitum throughout 9 weeks for this experiment. After a week of acclimation, the SHR rats were randomly divided into six groups, with 10 animals in each group. The method used was based on that developed by Cheng et al. (2008) and Lee et al. (2010). PBHs exhibiting the highest ACE-inhibitory activity were dissolved in 1 mL of deionized water and orally administered (10, 50, and 100 mg/kg bw/day) to the rats by using a metal gastric zonde. Captopril (D-2-methyl-3-mercapto-propionyl-L-proline, China Chemical and Pharmaceutical Co., Ltd., Taipei, Taiwan; 1.5 mg/kg bw/day) was administered as a positive control. Control SHR rats were orally administrated the same volume of water only. The weight gain of the rats was recorded weekly, and both SBP and heartbeat were measured every 2 weeks by using the tail-cuff method and an indirect blood pressure meter (BP-98-A, Softron, Tokyo, Japan) after warming the tail to 39°C, controlled by using a thermostat, for 10 min. The blood pressure of each rat was calculated as the average of three individual measurements. At the end of the experiment (15th week of life), the rats were fasted for 8 h before being sacrificed to weigh their hearts. The rats were sacrificed by first inducing anesthesia through administering isoflurane (VIP3000, Veterinary Anesthesia Vaporizer, Matrx). Blood was collected and plasma was obtained from the collected blood samples by centrifuging at 5 000 g for 10 min and stored at −80°C for analyses.

Determination of plasma ACE activity    The plasma ACE activity was determined according to a previously reported spectrophotometric method (Girgih et al., 2014). A 1 mL aliquot of 0.5 mM FAPGG (dissolved in 50 mM Tris-HCl buffer containing 0.3 M NaCl, pH 7.5) was mixed with 20 µL plasma or ACE (final enzyme concentrations were 0.0313, 0.0625, 0.125, 0.25, 0.5 U/mL), and 200 µL of 50 mM Tris-HCl buffer. Rate of decrease in absorbance was monitored at 345 nm and recorded for 2 min at 23°C. The result was expressed as ΔA/min and plotted against ACE enzyme concentration to obtain a standard curve. ACE activity (U/mL) of the plasma was obtained by linear regression using the standard curve.

Identification of PBH peptides <3 000 Da obtained after 4 h of hydrolysis with protease N    The peptide sequences of the resulting fractions were analyzed in Biotechnology Center, National Chung Hsing University through LC-MS/MS (Triple TOF 6600; Applied Biosystems/QSTAR Elite; Thermo Electron/Finnigan LTQ XL) for peptide sequencing.

Statistical analysis    The statistic analysis in this study was conducted according to the General Linear Models Procedure of Statistical Analysis System (Version 9.1.3 for Windows). The differences in average values were compared using one way ANOVA with Tukey's Honestly Significant Difference (HSD) test calculator for comparing multiple treatments. A p value of less than 0.05 was considered to be significant.

Results and Discussion

Peptide content and DH    To produce ACE-inhibitory peptides, porcine brain proteins were separately hydrolyzed using various commercial digestive enzymes. In this experiment, we used three proteolytic enzymes, namely alcalase, flavourzyme, and protease N, and evaluated their effectiveness in degrading the porcine brain proteins for ACE-inhibitory activity. Extensively hydrolyzed proteins may be bitter in taste due to the exposure and accumulation of hydrophobic side chains of amino acids of low-molecular-weight peptides (Panyam and Kilara, 1996). Alcalase is an endoprotease from Bacillus licheniformis, which breaks peptide bonds from non-terminal amino acids while used to produce protein hydrolysis (Ambigaipalan et al, 2015), whereas Protease N is a C-terminal proteinase from Bacillus subtilis while used to produce protein hydrolysates. In addition, Flavourzyme is an exoprotease and endoprotease complex enzyme that breaks the N-terminal of peptide chains, which used for debittering and generation of hydrolysates (Villanueva et al., 1999).

Figure 1 presents the changes in the peptide content during the porcine brain protein hydrolysis. The enzymatic hydrolysis increased the peptide content with time. The peptide content markedly increased at the beginning of the reactions, especially between 0 and 2 h. The increase in the peptide content in alcalase and flavourzyme N hydrolysates slowed down after 4–6 h, whereas that in the peptide content in protease N hydrolysates continued to increase and finally reached 25.65 ± 2.36 mg/mL. The protease N hydrolysates exhibited the highest peptide content, which had reached 26.81 ± 4.33 mg/mL after hydrolysis for 8 h. The final peptide content was significantly higher in protease N hydrolysates than in alcalase (16.23 ± 3.54 mg/mL) and flavourzyme (16.63 ± 3.26 mg/mL) hydrolysates (p < 0.05).

Fig. 1.

Changes in the peptide content during the hydrolysis of the porcine brain protein with various enzymes.

Data represent mean ± SEM (n = 3).

The extent and progress of proteolysis in the porcine brains were quantified by measuring the DH in peptide content extracts. Figure 2 illustrates the increase in DH during hydrolysis for up to 10 h. Compared with no hydrolysis, the DH significantly increased after 2 h of hydrolysis (p < 0.05). After proteolytic digestion, the DH increased to 57.81 ± 11.2%, 54.56 ± 7.61%, and 78.58 ± 4.48% in alcalase-, flavourzyme-, and protease N-digested PBHs, respectively, after 2 h of hydrolysis. After 6 h of hydrolysis, the DH markedly increased to 73.91 ± 1.77%, 54.68 ± 9.84%, and 76.13 ± 1.63% in the alcalase-, flavourzyme-, and protease N-digested PBHs, respectively. Furthermore, the DH slowly increased throughout the hydrolysis. After 10 h, the DH was tripled and reached 71.02 ± 5.67%, 71.72 ± 6.21%, and 82.35 ± 0.98% in the alcalase-, flavourzyme-, and protease N-digested PBHs, respectively. These findings indicate that significant proportions of peptides were released during hydrolysis, which are consistent with the findings of previous studies. The hydrolysis with different digestive enzymes exhibited a significant increase in the peptide content and low-molecular-weight peptides (Cheng et al., 2008; Lee et al., 2010).

Fig. 2.

Changes in the degree of hydrolysis during the hydrolysis of the porcine brain protein with various enzymes.

Data represent mean ± SEM (n = 3).

Inhibition of ACE activity in vitro    Table 1 lists the ACE-inhibitory activities of the porcine brains hydrolyzed with various enzymes. The PBHs inhibited less than 20% of the activity in 0 h, whereas further enzymatic treatment for 2 h doubled their activity to 66.56% (flavourzyme), 86.24% (protease N), and 77.85% (alcalase). Various peptides derived from blood proteins possess ACE-inhibitory activity. Mito et al. (1996) isolated four peptides from the hemoglobin of swine blood cells digested with alcalase, and they reported that these peptides inhibited ACE activity with an IC₅₀ of 5.8, 7.4, 2.1, and 1.9 µM, respectively. Yu et al. (2006) isolated peptides from porcine hemoglobin digested with pepsin, trypsin, and papain, and they indicated that the peptides digested with pepsin (porcine stomach mucosa) had the highest ACE-inhibitory activity (IC₅₀ = 1.19 mg/mL). In the current study, among the various enzymatic treatments, PBHs obtained after 4 h of hydrolysis with protease N (PBP4H) exhibited the highest ACE-inhibitory activity (86.29 ± 5.76%). In addition, the ACE-inhibitory activity IC₅₀ value was lowest for PBP4H (1.945 ± 0.005 mg/mL), which was selected for further study (Table 2).

Table 1. ACE-inhibitory activity of hydrolysates derived from the porcine brains protein digested with various enzymes

Time (h)	Alcalase	Flavourzyme	Protease N
0	15.52±8.01bx	17.21±6.58bx	16.26±0.92cx
2	77.85±1.16ax	66.56±5.26ay	86.24±5.60ax
4	74.37±8.20ax	64.82±3.66ax	86.29±5.76ax
6	80.33±1.70ax	57.85±1.41az	71.04±4.78by
8	78.41±3.41ax	62.95±6.36ay	68.06±7.20by
10	73.86±2.37ax	61.19±8.26ay	78.31±3.39abx

¹ Values are means ± standard deviation of three replicate analyses.

^a-c Means with different superscript letters in the same column are significantly different (p < 0.05).

^x-z Means with different superscript letters in the same row are significantly different (p < 0.05).

Table 2. IC₅₀ value for ACE-inhibitory activity of hydrolysates derived from the porcine brain protein digested with various enzymes

	IC501 (mg/mL)
Time (h)	Alcalase	Flavourzyme	Protease N
0	18.652±0.015ax	18.652±0.028ax	18.652±0.069ax
2	7.032±0.009by	15.501±0.008ax	1.985±0.009cz
4	4.623±0.006cy	11.328±0.005bx	1.945±0.005cz
6	2.042±0.007cy	9.321±0.005cx	3.289±0.007cy
8	6.614±0.007bz	13.625±0.007bx	9.325±0.004by
10	4.037±0.008cz	13.441±0.006bx	7.035±0.006by

¹  Values are means ± standard deviation of three replicate analyses.

a-c Means with different superscript letters in the same column are significantly different (p < 0.05).

x-z Means with different superscript letters in the same row are significantly different (p < 0.05).

To measure the contribution of small peptides to ACE-inhibitory activity, PBP4H were fractionated using an ultrafiltration membrane with a molecular-weight cutoff of 3000 Da. The magnitudes of the ACE-inhibitory activity of different peptides were compared (Table 3). The ACE-inhibitory activity IC₅₀ value associated with the low-molecular-weight fraction (< 3 000 Da) was the lowest (0.951 ± 0.009 mg/mL). This result is in agreement with that of another study (Saiga et al., 2008), indicating that peptide fractions with lower molecular weights have higher activities.

Table 3. ACE-inhibitory activity of each porcine brain hydrolysate

Sample	PBP4H	low fraction (<3000 Da)	high fraction (>3000 Da)
IC50 (mg/mL)a	1.945 ± 0.005	0.951 ± 0.009	2.028 ± 0.002

Data represent mean ± SEM (n = 3).

^a  The concentration of the peptide required to inhibit 50% of the ACE activity.

ACE activity increases blood pressure by producing the vasoconstrictor peptide angiotensin II and by degrading the vasodilator peptide bradykinin. Therefore, ACE inhibitors are used as therapeutic agents against hypertension. Peptides derived from food proteins exhibit ACE-inhibitory activity (Hartman and Meisel, 2007) and are considered milder and safer than synthetic drugs. In general, peptide fractions with a molecular weight equal to or lower than 6 000 Da were separated using an ultrafiltration membrane for easier handling and higher digestivity (Saiga et al., 2008). In addition, these peptides were observed to transfer into blood 2 h after ingestion (Iwai et al., 2005), and peptides with a lower molecular weight had low allergenicity.

The presented findings suggest that ACE inhibition is mainly attributable to peptide components with a molecular mass of less than 3 000 Da. Therefore, ultrafiltration through membranes with a molecular mass cutoff of 3 000 Da can be used to obtain a filtrate enriched in ACE-inhibitory peptides. This method has been previously proposed in other studies (Miguel et al., 2009; Quiros et al., 2007; Saiga et al., 2008).

Antihypertensive effect in SHRs in vivo    The antihypertensive activity of PBP4H was evaluated by measuring the change in SBP at 0, 2, 4, 6, 8, 10, and 12 h after its oral administration at 10, 50, and 100 mg/kg of body weight. Captopril, a potent ACE inhibitor with an IC₅₀ value of 0.002 µM (Fujita and Yoshikawa, 1999), was used as a positive control, and the control group was administered the same volume of reverse osmosis water. As presented in Fig. 3, the SBP observed in the quiescent state of the SHRs was 182 ± 5 mmHg. Compared with the control SHRs, the SBP decreased 4 h after the administration of PBP4H-50 (50 mg/kg bw/day), PBP4H-100 (100 mg/kg bw/day), and a low-molecular-weight PBH fraction (PBP4H < 3 000 Da, 50 mg/kg bw/day) to the SHRs. The lowest SBP was observed 6 h after the administration of PBP4H < 3 000 Da and captopril (30 and 31 mmHg, respectively; Figure 3). However, no significant difference was observed between the PBP4H-10-treated SHRs (10 mg/kg bw/d) and the control SHRs (p > 0.05). After the oral administration of PBP4H and captopril, the SBP markedly decreased and activities were maintained for 10 h; nevertheless, the antihypertensive effect of these products was transient and reverted 12 h after their administration. After 12 h, the SBP values derived were similar to the initial SBP values; therefore, investigating the influence of the long-term administration of PBP4H on antihypertensive effects is crucial.

Fig. 3.

Changes in systolic blood pressure (SBP) after the oral administration of porcine brain hydrolysates obtained after 4 h of hydrolysis with protease N (PBP4H) in spontaneously hypertensive rats (SHRs). Distilled water was used as the control and captopril (1.5 mg/kg bw/day) was used as the positive control. PBP4H doses of 10, 50, and 100 mg/kg bw/day were orally administered separately, and SBP was measured 0, 2, 4, 6, 8, and 10 h after the oral administration of PBP4H. Data represents the significance difference from the control at *p < 0.05 (n = 10, 7 weeks old male SHRs).

As shown in Fig. 4, the measured blood pressure decreased in SHRs that received low-molecular-weight PBH fractions (PBP4H < 3 000, 50 mg/kg bw/day) and PBP4H-100 (100 mg/kg bw/d) for 2 weeks, and it significantly decreased 2 weeks after the administration (p < 0.05). The SBP increased in all SHRs with age. Moreover, the SBP in SHRs that were continually administered protease N-digested PBHs tended to be significantly lower than that in the control SHRs at 2-8 weeks (p < 0.05); compared with the control SHRs, the SBP markedly decreased (31.2 ± 5.1 mmHg) in SHRs that received PBP4H < 3 000 Da at 8 weeks. At 4 weeks, compared with that in the control SHRs (190.4 ± 8.2 mmHg), the SBP significantly decreased in SHRs treated with PBP4H < 3 000 Da (170.5 ± 4.5 mmHg) and captopril (171.2 ± 5.3 mmHg; p < 0.05). At the end of the experiment (8 weeks), the SBP in SHRs treated with PBP4H-100, PBP4H < 3 000 Da, and captopril was maintained under 185 mmHg, whereas that in the control SHRs increased to 210.2 ± 5.8 mmHg. These findings also corroborated with the in vitro results on potential antihypertensive properties of PBP4H.

Fig. 4.

Changes in SBP after the long-term oral administration of PBP4H in SHRs. Distilled water was used as the control and captopril (1.5 mg/kg bw/day) was used as the positive control. PBP4H-100 and PBP4H < 3000 Da were orally administered at doses of 100 and 50 mg/kg bw/day, respectively, and the SBP was measured 0, 2, 4, 6, and 8 weeks after the oral administration. Data represents the significant difference from the control at *p < 0.05 (n=10, 7 weeks old male SHRs in 0 week experiment).

Plasma ACE activity of SHR    Plasma ACE activity of SHR after the long-term oral administration of PBP4H-100, PBP4H< 3 000, captopril and control is shown in Fig. 5. The captopril treatment had the most significant (p < 0.05) plasma ACE activity reduction effect (−0.125 U/mL) followed by the PBP4H<3 000 and PBP4H-100, however, plasma ACE activity for both two PBP4H treatment were significant ( p < 0.05) lower (-0.082 and -0.055 U/mL for the PP4H<3 000 and PBP4H-100, respectively) than the control group. Results of the plasma ACE activity was positively related with the SBP lowering effects of PBP4Hs suggesting that the treatment groups with the maximum SBP reduction also showed the maximum reduction of plasma ACE activity for SHR. This is consistent with the results obtained from resent researches that suggested plasma ACE activity could be a very good biomarker of hypertension in SHR. (Girgih et al., 2014; Onuh et al., 2016)

Fig. 5.

Plasma angiotensin I converting enzyme (ACE) activity of SHRs after the long-term oral administration of PBP4H. Distilled water was used as the control and captopril (1.5 mg/kg bw/day) was used as the positive control. PBP4H-100 and PBP4H < 3000 Da were orally administered at doses of 100 and 50 mg/kg bw/day, respectively. Bars with different letters have mean values that are significantly different (p < 0.05). Values are means (n=10 rats) ±SD.

Purification of PBP4H < 3 000 Da    In the current study, the amino acid sequences of resulting peptides and data of their molecular masses are presented in Table 4. We observed that low-molecular-weight fractions obtained from the porcine brains through an ultrafiltration membrane having a molecular-weight cutoff of 3 000 Da exhibited an antihypertensive effect on the SHRs. Among the fractions, we identified five peptides with ACE-inhibitory activity. These five fractions were respectively composed of Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys, Leu-Pro-Glu-Phe-Pro, Glu-Asp-Glu-Ser-Pro-Thr-Lys, and Glu-Glu-Ala-Gly-Gln-Val-Ala-Pro. Furthermore, the activity levels exhibited by these fractions were higher or similar to those reported in other studies. (IC₅₀ = 210.2 ± 3.5, 590.8 ± 2.4, 105.6 ± 4.2, 358.1 ± 1.6, and 108.4 ± 5.2 µg/mL, respectively).

Table 4. Sequences and mass spectrometry of ACE-inhibitory low-molecular -weight peptides from porcine brain hydrolysates obtained after 4 h of hydrolysis with protease N (PBP4H < 3000 Da)

Peptide no.	sequence	homology	IC50 (µg/mL)a
1	Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp	Porcine GAPDH	210.2±3.5
2	Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys	Porcine tropin	590.8±2.4
3	Leu-Pro-Glu-Phe-Pro		105.6±4.2
4	Glu-Asp-Glu-Ser-Pro-Thr-Lys		358.1±1.6
5	Glu-Glu-Ala-Gly-Gln-Val-Ala-Pro		108.4±5.2

Data represent mean ± SEM (n=3).

a  Concentration of peptide required to inhibit 50% of the ACE activity.

Although the structure-activity relationship of ACE-inhibitory peptides has yet to be established, these peptides exhibit common features. The ACE-inhibitory peptides usually contain 2–12 amino acids (Jiang et al., 2010). Regarding ACE-inhibitory peptides, the most potent and specific peptide inhibitors have similar structures, and ACE activity is strongly influenced by the C-terminal tripeptide sequence of these peptides (Lee et al., 2010). Tripeptides with tryptophan, tyrosine, proline, or phenylalanine and a hydrophobic amino acid at the C terminal exhibit high ACE-inhibitory activity because of the interaction among the three subsites at the active site of ACE (Pihlanto, 2000). In addition, the presence of the positive charge of Lys and Arg at the C-terminal residue may contribute to the inhibitory potency (Jiang et al., 2010).

Several peptides derived from the enzymatic hydrolysates of various natural sources exhibit ACE-inhibitory and antihypertensive activities in SHRs. The identified ACE-inhibitory peptides are Leu-Gly-Phe-Pro-Thr-Thr-Lys-Thr-Tyr-Phe-Pro-His-Phe (IC₅₀ = 4.92 µM) and Val-Val-Tyr-Pro-Trp (IC₅₀ = 6.02 µM) from the porcine hemoglobin (Yu et al., 2006); Arg-Val-Cys-Leu-Pro (IC₅₀ = 175 µM) from lizard fish (Wu et al., 2015); Val-Lys-Ala-Gly-Phe (IC₅₀ = 6.1 µM) and peptide (IC₅₀ = 3.9 mg/mL) from porcine actin (Muguruma et al., 2009) and porcine meat (Ahhmed and Muguruna, 2010). Although several studies have investigated ACE-inhibitory peptides derived from food proteins, only a few have investigated those derived from pig.

In the present study, Pro-Ala-Asn-Ile-Lys-Trp-Gly-Asp, Arg-Met-Leu-Gly-Gln-Thr-Pro-Thr-Lys, Leu-Pro-Glu-Phe-Pro, Glu-Asp-Glu-Ser-Pro-Thr-Lys, and Glu-Glu-Ala-Gly-Gln-Val-Ala-Pro from PBP4H < 3 000 Da contained hydrophobic amino acids, which may have contributed to ACE-inhibitory activity. Moreover, a single oral administration of low-molecular-weight PBHs significantly lowered the blood pressure in the SHRs (Fig. 2), and a long-term oral administration even suppressed hypertension in these rats (Fig. 3). Therefore, ACE-inhibitory peptides or potent inhibitory peptides in PBHs are considered to exhibit hypotensive activity through gastrointestinal digestion and absorption.

Lifestyle modifications and diet therapy are two of the most effective strategies to improve hypertension (Hermansen, 2000). However, this does not imply that hypertensive drugs can be completely replaced. In numerous cases, particularly in the prevention of hypertension or initial treatment of patients with mild hypertension, food-derived ACE-inhibitory peptides can be used as an additional treatment. To exert an antihypertensive effect after oral ingestion, functional peptides that inhibit ACE must reach the cardiovascular system in an active form; therefore, they must remain active during digestion by human proteases for them to be transported through the intestinal wall into the blood (Vermeirssen et al., 2004).

Our study results reveal that PBP4H is digested by gastric juices or proteases in the small intestine and the digested small peptides are subsequently absorbed in the intestine (Lee et al., 2010). Matsui et al. (2004) reported an accumulation of the ACE-inhibitory peptide (Val-Tyr) in tissue of orally treated SHRs. Val-Tyr accumulated in the kidney, lungs, and abdominal aorta and reduced ACE activity and angiotensin II levels. Furthermore, Matsui et al. (2003) reported that Val-Tyr acts on the human renin-angiotensin system through the retardation of angiotensin II formation-related enzyme activity. Therefore, additional studies are required to identify the mechanisms underlying the antihypertensive effect exhibited by low-molecular-weight fractions (PBP4H < 3000 Da) in SHRs.

Conclusion

PBP4H yielded peptides enriched with ACE-inhibitory activity (86.29 ± 5.76%). Furthermore, PBP4H had the lowest ACE-inhibitory activity IC₅₀ value (1.945 mg/mL). The sequence of the purified low-molecular-weight fraction peptide (PBP4H < 3 000 Da) and their IC₅₀ values were analyzed in this study. Oral administration of PBP4H-100 (100 mg/kg bw/day) and PBP4H < 3 000 Da (50 mg/kg bw/d) reduced the SBP (−30 mmHg) in SHRs. Moreover, long-term administration of PBP4H-100 and PBP4H < 3 000 Da significantly suppressed hypertension. In addition, results of the plasma ACE activity was positively related with the SBP lowering effects of PBP4Hs. Overall, these results reveal the antihypertensive effect of PBHs and the potential application of these peptides as a functional supplement for suppressing hypertension. It could also enhance value-added utilization of porcine brain, which is currently mainly disposed as a waste product by the porcine industry while protecting the environment.

Acknowledgments    This research was supported by the Ministry of Science and Technology, Executive Yuan, R. O. C. Taiwan (Protocol No: NSC 100—2313—B—005—026—MY2).

References

•  Ahhmed,  A. M. and  Muguruna  M. (2010). A review of meat protein hydrolysates and hypertension. Meat Sci., 86, 110-118.
•  Ambigaipalan,  P.,  Al-Khalifa,  S., and  Shahidi,  F. (2015). Antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities of data seed protein hydeolysates prepared using Alcalase, Flzvourzyme and Thermolysin. J. Funct. Foods, 18, 1125-1137.
•  Cushman,  D. W. and  Cheung,  H.S. (1971). Spectrophotometric assay and properties of the angiotensin-converting enzyme of rabbit lung. Biochem. Pharmacol., 20, 1637-1648.
•  Church,  F. C.,  Swaisgood,  E. H.,  Porter,  D. H., and  Catignani,  G. L. (1983). Spectrophotometric assay using o-phthaldialdeyhde for determination of proteolysis in milk and isolated milk protein. J. Dairy Sci., 66, 1219-1227.
•  Chobanian,  A.V.,  Bakris,  G.L., and  Black,  H.R. (2003). The seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. J. Am. Med. Assoc., 289, 2560-2572.
•  Cheng,  F. Y.,  Wan,  T, C.,  Liu,  Y. T.,  Lai,  K. M.,  Lin,  L. C., and  Sakata,  R. (2008). A study of in vivo antihypertensive properties of enzymatic hydrolysate from chicken leg bone protein. Anim. Sci. J., 79, 614-619.
•  Fujita,  H. and  Yoshikawa,  M. (1999). LKPNM: A prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacol., 44, 123-127.
•  Girgih,  A. T.,  Alashi,  A. M.,  He,  R.,  Malomo,  S. A.,  Raj,  P.,  Netticadan,  T., and  Aluko,  R. E. (2014). Hemp seed meal protein hydrolysate reduces oxidative stress factors in spontaneously hypertensive rats. Nutrients, 6, 5652-56663
•  Hartman,  R. and  Meisei,  H. (2007). Food derived peptides with biological activity: From research to food applications. Curr. Opin. Biotech., 18, 163-169.
•  Hermansen,  K. (2000). Diet, blood pressure and hypertension. Brit. J. Nutr., 83, S1, S113-S119.
•  Iwai,  K.,  Hasegawa,  T.,  Taguchi,  Y.,  Morimatsu,  F.,  Sato,  K.,  Nakumura,  Y.,  Higashi,  A.,  Kido,  Y.,  Nakabo,  Y., and  Ohtsuki,  K. (2005). Identification of food-derived collagen peptides in human blood after oral ingestion of gelatin hydrolysate. J. Agric. Food Chem., 53, 6531-6536.
•  Jamdar,  S. N.,  Rajalakshmi,  V.,  Pednekar,  M. D.,  Juan  F.,  Yardi,  V., and  Sharma  A. (2010). Influence of degree of hydrolysis on functional properties, antioxidant activity and ACE inhibitory activity of peanut protein hydrolysate. Food Chem., 121, 178-184.
•  Jiang,  Z.,  Tian,  B.,  Brodkorb,  A., and  Huo,  G. (2010). Production, analysis and in vivo evaluation of novel angiotensin-I-converting enzyme inhibitory peptides from bovine casein. Food Chem., 123, 779-786.
•  Lee,  S. H.,  Qian,  Z. J., and  Kim,  S. K. (2010). A novel angiotensin I converting enzyme inhibitory peptide from tuna frame protein hydrolysate and its antihypertensive effect in spontaneously hypertensive rats. Food Chem., 118, 96-102.
•  Liu,  J. B.,  Yu,  Z. P.,  Zhao,  W. Z.,  Lin,  S. Y.,  Wang,  E. L.,  Zhang,  Y.,  Hao,  H.,  Wang,  Z. Z., and  Chen  F. (2010). Isolation and identification of angiotensin-converting enzyme inhibitory peptides from egg white protein hydrolysates. Food Chem., 122, 1159-1163.
•  Matsui,  T.,  Hayashi,  A.,  Tamaya,  K.,  Matsumoto,  K.,  Kawasaki,  T., and  Murakami,  K. (2003). Depressor effect induced by dipeptide, Val-Tyr, in hypertensive transgenic mice is due, in part, to the suppression of human circulating renin-angiotensin system. Clin. Exp. Pharmacol. P., 30, 262-265.
•  Matsui,  T.,  Imamura,  M.,  Oka,  H.,  Osajima,  K.,  Kimoto,  K., and  Kawasaki,  T. (2004). Tissue distribution of antihypertensive dipeptide, Val-Tyr, after its single oral administration to spontaneously hypertensive rats. J. Pept. Sci., 10, 535-545.
•  Miguel,  M.,  Contreras,  M. M.,  Recio,  I., and  Aleixandre,  A. (2009). ACE-inhibitory and antihypertensive properties of a bovine casein hydrolysate. Food Chem., 112, 211-214.
•  Mito,  K.,  Fujii,  M.,  Kuwahara,  M.,  Matsumura,  N.,  Shimizu,  T.,  Sugano,  S., and  Karaki,  H. (1996). Antihypertensive effect of angiotensin-I converting enzyme inhibitory peptides derived from hemoglobin. Eur. J. Pharmacol., 304, 93-98.
•  Muguruma,  M.,  Ahhmed,  M. A.,  Katayama,  K.,  Kawahara,  S.,  Maruyama,  M., and  Nakamura,  T. (2009). Identification of pro-drug type ACE inhibitory peptide sourced from porcine myosin B: Evaluation of its antihypertensive effects in vivo. Food Chem., 114, 516-522.
•  Onuh,  J. O.,  Girgih,  A. T.,  Malomo,  S. A.,  Aluko,  R. E., and  Aliani,  M. (2015). Kinetics of in vitro renin and angiotensin converting enzyme inhibition by chicken skin protein hydrolysates and their blood pressure lowering effects in spontaneously hypertensive rats. J. Funct. Foods, 14, 133-143.
•  Onuh,  J. O.,  Girgih,  A. T.,  Nwachukwu,  I.,  Ievari-Shariati,  S.,  Raj,  P.,  Netticadan,  T.,  Aluko,  R. E., and  Aliani,  M. (2016). A metabolomics approach for investigating urinary and plasma changes in spontaneously hypertensive rats (SHR) fed with chicken skin protein hydrolysate diets. J. Funct. Foods, 22, 20-33.
•  Panyam,  D. and  Kilara  A. (1996). Enhancing the functionality of food proteins by enzymatic modification. Trends Food Sci. Tech., 7, 120-125.
•  Pihlanto,  L. (2000). Bioactive peptides derived from bovine whey proteins: Opioid and ace-inhibitory. Trends Food Sci. Tech., 11, 347-356.
•  Quiros,  A.,  Ramos,  M.,  Muguerza,  B.,  Delgado,  M. A.,  Miguel,  M., and  Aleixandre,  A. (2007). Identification of novel antihypertensive peptides in milk fermented with Enterococus faecalis. Int. Dairy J., 17, 33-41.
•  Saiga,  A.,  Iwai,  K.,  Hayakawa,  T.,  Takahata,  Y.,  Kitamura,  S.,  Nishimura  T., and  Morimatsu  F. (2008). Angiotensin I-converting enzyme-inhibitory peptides obtained from chicken collagen hydrolysate. J. Agric. Food Chem., 56, 9586-9591.
•  Seppo,  L.,  Jauhiainen,  T.,  Poussa,  T., and  Korpela,  R. (2003). A fermented milk high in bioactive peptides has a blood pressure-lowering effect in hypertensive subjects. Am. J. Clin. Nutr., 77, 326-330.
•  Shi,  L.,  Mao,  C.,  Xu,  Z., and  Zhang,  L. (2010). Angiotensin-converting enzymes and drug discovery in cardiovascular disases. Drug Discov. Today, 15, 332-341.
•  Vermeirssen,  V.,  Camp,  J. V., and  Verstraete,  W. (2004). Bioavailability of angiotensin I converting enzyme inhibitory peptides. Brit. J. Nutr., 92, 357-366.
•  Villanueva,  A.,  Vioque,  J.,  Clemente,  A.,  Bautista,  J., and  Millan  F. (1999). Production of an extensive sunflower protein hydrolysate by sequential hydrolysis with endo- and exo-proteases. Grasas Aceites, 50, 472-476.
• World Health Organization. (2013). A global brief on hypertension: silent killer, global public health crisis. World Health Organization, Geneva, Switzerland.
•  Wu,  S.,  Feng,  X.,  Lan,  X.,  Xu,  Y., and  Liao,  D. (2015). Purification and identification of Angiotensin-I Converting Enzyme (ACE) inhibitory peptide from lizard fish (Saurida elongata) hydrolysate. J. Funct. Foods, 13, 295-299.
•  Yu,  Y.,  Hu,  J.,  Miyaguchi,  Y.,  Bai,  X.,  Du,  Y., and  Lin,  B. (2006). Isolation and characterization of angiotensin-I converting enzyme inhibitory peptides derived from porcine hemoglobin. Peptides, 27, 2950-2956.
•  Zhang,  Y.,  Lee,  E. T.,  Devereux,  R. B.,  Yeh,  L.,  Best,  L. G., and  Fabsitz,  R. R. (2006). Prehypertension, diabets, and cardiovascular disease risk in a population based sample: The strong heart study. Hypertension, 47, 410-414.
•  Zou,  Y.,  Wang,  W.,  Li,  Q.,  Chen,  Y.,  Zheng,  D.,  Zou,  Y.,  Zhang,  M.,  Zhao,  T.,  Mao,  G.,  Feng,  W.,  Wu,  X., and  Yang,  L. (2016). Physicochemical, functional properties and antioxidant activities of porcine cerebral hydrolysate peptides produced by ultrasound processing. Process Biochem, 51, 431-443.