Article pubs.acs.org/JAFC
Naturally Occurring Angiotensin I‑Converting Enzyme Inhibitory Peptide from a Fertilized Egg and Its Inhibitory Mechanism Xiang Duan,†,‡ Fengfeng Wu,†,‡ Mei Li,†,‡ Na Yang,†,‡ Chunsen Wu,†,‡ Yamei Jin,†,‡ Jingjing Yang,† Zhengyu Jin,†,‡ and Xueming Xu*,†,‡ †
State Key Laboratory of Food Science and Technology, and ‡School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi Jiangsu 214122, People’s Republic of China ABSTRACT: This study was performed to investigate the angiotensin I-converting enzyme (ACE) inhibitory activity of peptides derived from fertilized eggs and elucidate the inhibition mechanism of these peptides. During incubation, ACE inhibitory activity of the peptides remained stable before day 12 and then increased markedly on day 15. Two ACE inhibitory peptides, VGVIKAVDKKAGGAGKVT and HLFGPPGKKDPV, were purified from peptides on day 15 by consecutive chromatography. Because HLFGPPGKKDPV possessed a higher ACE inhibitory activity (IC50 = 125 μM), an antihypertensive effect of this peptide was further evaluated in vivo. The result showed that this peptide had an antihypertensive effect in spontaneously hypertensive rats (SHRs) at a dosage of 10 mg/kg. Furthermore, Lineweaver−Burk plots suggested that HLFGPPGKKDPV played as a non-competitive inhibitor against ACE, as supported by docking simulation. These data indicated that a fertilized egg has potential as antihypertensive components in functional foods and nutraceuticals. KEYWORDS: ACE inhibitory peptide, fertilized egg, inhibitory pattern, molecular docking, spontaneously hypertensive rat
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INTRODUCTION Hypertension is one of the major risk factors for the development of cardiovascular diseases, stroke, and the end stage of renal disease.1 Treatment of hypertension is an effective way to reduce the risk of these diseases.2 The renin− angiotensin system (RAS) plays a key role in maintaining blood pressure homeostasis.3 Angiotensin I-converting enzyme (ACE) is a key enzyme in RAS; it raises blood pressure by converting the inactive decapeptide angiotensin I to the potent vasoconstrictor octapeptide angiotensin II as well as inactivating the vasodilating nonapeptide bradykinin.4 Therefore, inhibition of ACE is considered as an effective approach to controlling hypertension.1 Since the discovery of ACE inhibitors in snake venom,5 many chemical ACE inhibitors, such as captopril, enalapril, alacepril, and lisinopril, were designed.6 However, these chemical ACE inhibitors are known to have various side effects, such as coughing, skin rashes, and angioedema.7,8 Therefore, many studies have been initiated to develop novel, safe, and natural ACE inhibitors from natural sources.9,10 In recent years, several ACE inhibitory peptides have been isolated from a wide range of food sources, such as gelatin,11 casein,12 tuna,13 corn,14 soy beans,15 egg white,16 and dried bonito.17 For a better understanding of inhibitory properties of peptides, the structure−activity relationship should be clarified. Terashima et al.18 investigated a series of peptides derived from an ACE inhibitory peptide (VTVNPYKWLP) and concluded that the Pro residue in the C-terminal end strongly enhanced ACE inhibition activity. In addition, the length of peptides also played a key role in ACE inhibitory ability.19 Recently, Wu et al.20 have contructed a database consisting of 168 dipeptides and 140 tripeptides to study the quantitative structure−activity relationships of ACE inhibitory peptides. It showed that amino acid residues with bulky side chains as well as hydrophobic side © 2014 American Chemical Society
chains were preferred for dipeptides, while for tripeptides, the most favorable residues for C terminus, middle position, and N terminus were aromatic amino acids, positively charged amino acids, and hydrophobic amino acids, respectively. However, these structure−activity correlation studies were mainly based on amino acid sequence analysis. In recent years, the crystal structures of ACE and its inhibitors have been analyzed to elucidate the relationship between the inhibition mechanism and the molecular structure of inhibitors.21−23 These studies provided a new method to analyze the structure−activity relationship of ACE inhibitory peptides. Traditionally, fertilized eggs are widely considered as natural dietary supplements in many Asian countries.24 During chick embryonic development, RAS plays a principal role in regulation of blood pressure.25 Savary et al.25 reported that ACE was produced at a very early stage and its activity increased during incubation. Whether naturally occurring peptides derived from fertilized eggs have ACE inhibitory effect is still unknown. To our knowledge, this is the first research to investigate ACE inhibitory activity and the structure−activity relationship of the peptides from fertilized eggs. Because the fertilized eggs that are eaten by Asian consumers are commonly 12-day-old eggs,26 the fertilized eggs before day 15 of incubation were chosen for investigation in this paper. The objectives of the present study were to (1) determine ACE inhibitory activity of peptides derived from fertilized eggs and (2) investigate the inhibition mechanism of a purified ACE inhibitory peptide. Received: Revised: Accepted: Published: 5500
November 20, 2013 May 19, 2014 May 27, 2014 May 27, 2014 dx.doi.org/10.1021/jf501368a | J. Agric. Food Chem. 2014, 62, 5500−5506
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source. Samples were loaded onto a reversed-phase C18 column with a diameter of 0.15 mm × 10 cm and particle size of 5 μm. Separation of the peptides was performed using a gradient going from 5 to 90% acetonitrile in 0.1% formic acid over 40 min. The MS/MS raw data were acquired using Thermo-Xcalibur data acquisition, and the acquired data were searched against the NCBI chicken-specific database using SEQUEST software (Bioworks 3.1 package, Thermo Electron). Peptides were searched using the following parameters: peptide tolerance, 2.0 amu; enzyme, trypsin (KR/P); enzyme limits, fully enzymatic (cleaves at both ends); and maximum missed cleavages, 2. The filtering criterion were set as ΔCn ≥ 0.1, and Xcorr ≥ 1.9 with charge state 1+, Xcorr ≥ 2.2 with charge state 2+, and Xcorr ≥ 3.75 with charge state 3+ were used as a cutoff for peptide identification. To investigate the antihypertensive activity and inhibition pattern of peptides, the solid-phase technique was used to synthesize the purified peptides on a peptide synthesizer (Symphony, Protein Technologies, Inc., Tucson, AZ). [(9-Fluorenylmethyl)oxy]carbonyl (FMOC) amino acids were successively coupled in the presence of 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). ACE inhibitory activity of the synthesized peptides were investigated according to the above-described method. The IC50 value was the concentration of the sample that inhibited 50% of the ACE activity. Antihypertensive Action in Spontaneously Hypertensive Rats. Conscious SHRs were housed individually in steel cages in a room kept at 25 °C with a 12 h light−dark cycle and fed a standard laboratory diet. Tap water was freely available. The purified peptide was dissolved in saline at a dose of 10 mg/kg of body weight and injected orally using a metal gastric zoned in SHR. Antihypertensive activity of P2 was evaluated by measuring the change of systolic blood pressure (SBP) at 1, 2, 3, 6, 9, 22, and 24 h after oral administration of 10 mg/kg of body weight. The lowering efficacy of the peptide on SBP was compared to that of captopril (10 mg/kg). Control rats were administrated with the same volume of saline solution. Following oral administration, SBP was measured by tail-cuff method with a CODA monitor (Kent Scientific Corporation, Torrington, CT) after warming up SHR in a chamber maintained at 37 °C for 10 min. Determination of the Inhibition Pattern on ACE. To determine the ACE inhibition pattern of the purified peptide, various substrate (HHL) concentrations (1, 2, and 5 mM) were incubated with the ACE solution in either the absence or presence of 100 or 200 μM purified peptide at 37 °C. ACE inhibitory activity was determined according to the above-described method. The ACE inhibition pattern was determined with Lineweaver−Burk plots.30 Molecular Docking. To verify the inhibition pattern of the purified peptide, a docking simulation was performed. The structure of the purified peptide was constructed and optimized with the polypeptide builder function of the public domain web server PEPFOLD, which predicted and optimized the conformation of side chains by calculating energy values. The conformation with the lowest energy was considered to display a fully native structure.30 In addition, the model for ACE used in this study was the crystal structure labeled 1O8A in the Protein Data Bank (PDB), which represented the human testis ACE (tACE). The conformation of the peptide−tACE complex was constructed and optimized with a protein-docking program, ZDOCK, which performed the three-dimensional (3D) search of the spatial degrees of freedom between the two molecules. The default parameters of ZDOCK were used, and the conformation with the highest ZDOCK score was identified as the best docking mode.31 Statistical Analysis. The experiments were run in triplicate. The results were expressed as the mean ± standard deviation and were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL). Differences between means were analyzed by an analysis of variance (ANOVA) test with the least significant difference (LSD) post-hoc analysis (p < 0.05).
MATERIALS AND METHODS
Materials. The fertilized eggs on days 3, 6, 9, 12, and 15 of the incubation period were prepared and lyophilized on the basis of our previous work.27 The spontaneously hypertensive rats (SHRs, 10 weeks old, male, specific pathogen-free, 180−240 g of body weight) with tail systolic blood pressure (SBP) over 180 mmHg were obtained from Shanghai Slaccas Laboratory Animal Co., Ltd. (Shanghai, China). All animal protocols in this study adhered to the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of the People’s Republic of China [(2006)398]) and were approved by the Ethics Committee of Jiangnan University, China (JN 28 2013). The testing chemicals including ACE (from rabbit lung) and substrate peptide hippuryl-L-histidyl-L-leucine (HHL) were purchased from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade (Shanghai Chemical Reagents Co., Shanghai, China). Preparation of Naturally Occurring Peptides. Peptides were prepared with the method described by Park et al.,28 with some modification. The lyophilized egg samples (1.0 g) were prepared in 100 mL of Milli-Q water. After centrifugation at 10000g for 20 min at 4 °C, the supernatant was microfiltered through a 0.45 μm membrane and then ultrafiltrated through a 3 kDa cellulose membrane (Amicon, Beverly, MA). The filtrates were lyophilized and then stored at −20 °C until use. Assay of ACE Inhibitory Activity. The ACE inhibitory activity assay was performed with the method described by Terashima et al.29 Peptides (50 μL, 10 mg/mL in deionized water) with 50 μL of ACE solution (25 mU/mL) were preincubated at 37 °C for 10 min, and the mixture was then incubated with 50 μL of substrate (8.3 mM HHL in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) for 60 min at the same temperature. The reaction was terminated by the addition of 1.0 M HCl (200 μL). The reaction mixture was filtered with Millex-LG (Nihon Millipore Co., Ltd., Tokyo, Japan), and 10 μL of the filtrates was injected into the high-performance liquid chromatography (HPLC) system (Shimadzu LC-10 AD, Shimadzu, Kyoto, Japan) equipped with a hydrophobic column (Cosmosil 5C18MS-II, 4.6 × 150 mm). An isocratic mobile phase composed of 80% Milli-Q water containing 0.1% (v/v) trifluoroacetic acid and 20% acetonitrile containing 0.1% (v/v) trifluoroacetic acid was used at a flow rate of 1.0 mL/min. Peak areas for hippuric acid (HA) generated by the ACE reaction was monitored at 228 nm with a detector (Shimadzu SPD-20A). ACE inhibitory activity was obtained by the following equation: ACE inhibitory activity (%) = [(Vm − Vin)/Vm] × 100, where Vm was the peak area without the sample solution and Vin was that with the sample solution. Purification of ACE Inhibitory Peptides. The ACE inhibitory peptides were purified according to the method described in our previous work.27 Briefly, on the basis of the ACE inhibition result, the peptides on day 15 showed the strongest inhibitory activity and, thus, were employed for purification of ACE inhibitory peptide. The lyophilized peptides on day 15 were loaded onto a Mono Q 5/50 GL ion-exchange column with the Ä KTA purifier system (Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with 20 mM Tris− HCl (pH 8.0) and eluted with a linear gradient of NaCl (0−1.0 M) in the same buffer at a flow rate of 2 mL/min. Each fraction was monitored at 280 nm, collected, and lyophilized for ACE inhibitory activity assay at a concentration of 1 mg/mL. The lyophilized fraction having the highest activity was further dissolved in phosphate buffer (0.05 M sodium phosphate and 0.15 M NaCl at pH 7.2) and subjected to a Superdex Peptide 10/300GL column at a flow rate of 0.5 mL/min. Each peak was collected and lyophilized, and ACE inhibitory activity was also investigated at a concentration of 1 mg/mL. The most active fraction was lyophilized for further research. Identification and Synthesis of the Purified Peptide. The fraction with the strongest inhibitory activity was dissolved in 0.1% formic acid in deionized water for liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis. LC−MS/MS analysis was performed using a LTQ mass spectrometer (Thermo Electron, Bremen, Germany) coupled with a a nanoelectrospray ionization
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RESULTS AND DISCUSSION Change in ACE Inhibitory Activity of Peptides. During incubation, the inhibitory activity remained stable up to day 12 5501
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ACE inhibitory activity and, thus, were employed for isolation of ACE inhibitory peptide. A Mono Q 5/50 GL column was used to fractionate the peptides, and the fractions were pooled to obtain fractions 1−6 (lower panel of Figure 2a). Although ACE inhibitory activity was observed in all of these fractions, fraction 2 exhibited the highest inhibitory activity (upper panel of Figure 2a). Thereafter, fraction 2 was eluted on a Superdex Peptide 10/300GL column, and six peaks were monitored (lower panel of Figure 2b). Among these peaks, fraction C expressed strong ACE inhibitory activity (upper panel of Figure 2b). Therefore, fraction C was subjected to linear trap quadrupole (LTQ) MS/MS analysis to identify its amino acid sequence. On the basis of mass spectrometric analysis, two peptides (named P1 and P2) were identified (Figure 3). Because the amino acid sequence of P2 has been reported in our previous work,27 the mass spectrum of P2 was cited from ref 27. The two peptides were composed of 18 and 12 amino acid residues, and the amino acid sequences were VGVIKAVDKKAGGAGKVT (molecular weight of 1698.03 Da) and HLFGPPGKKDPV (molecular weight of 1291.51 Da), respectively. To confirm ACE inhibitory activity of the two peptides, P1 and P2 were synthesized with a peptide synthesizer and their inhibitory activity was measured. The results showed that the half maximal inhibitory concentration (IC50) of P2 was 125 μM, while P1 exhibited lower activity (IC50 > 250 μM). Therefore, P2 was selected for further research. Interestingly, our previous work indicated that HLFGPPGKKDPV also possessed antioxidant activity against the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical,27 which is in accordance with the previous study reporting that peptides with high ACE inhibitory activity usually possessed a high level of DPPH radical scavenging activity at the same time.33 Actually, since Hou et al.34 suggested that some antioxidant peptides had effects on ACE, many multifunctional peptides have been
and then dramatically increased on day 15, reaching 78.25% at 10 mg/mL (Figure 1). During chick embryonic development,
Figure 1. Change in ACE inhibitory activity of peptides derived from fertilized eggs at different developmental stages. The sample concentration was 10 mg/mL for measurement of ACE inhibitory activity. Different letters (a and b) above columns indicate significant differences (p < 0.05).
ACE was produced during early embryogenesis, at a stage when blood circulation is not yet established.25 Girard has reported that the blood pressure increased rapidly and exponentially from 3 to 10 days of incubation; thereafter, the increase was slower.32 This trend is in good agreement with the result that the inhibitory activity of peptides was low before day 12 and then increased markedly up to day 15. The results indicate that the peptides may play a potential role in regulating blood pressure by inhibiting ACE during incubation. Purification and Identification of ACE Inhibitory Peptide. The peptides on day 15 exhibited the strongest
Figure 2. Purification profiles and ACE inhibitory activity of fractions derived from fertilized eggs on day 15. The sample concentration of each fraction was 1 mg/mL for measurement of ACE inhibitory activity. (a) Chromatography of peptides by a Mono Q 5/50 GL column (lower panel) and ACE inhibitory activity of each fraction (upper panel). (b) Chromatography of active fraction (fraction 2) by Superdex Peptide 10/300GL (lower panel) and ACE inhibitory activity of each fraction (upper panel). Different letters (a−e) above columns indicate significant differences (p < 0.05). 5502
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agreement with the reported ACE inhibitory peptide size. Previous studies have demonstrated a good correlation between certain amino acid residues and ACE inhibitory activity of peptides.21,43 Because the activity of ACE is to cleave the Cterminal dipeptide of oligopeptide substrates, the inhibitory activity of peptides is strongly influenced by their C-terminal tripeptide sequences.30 Rohrbach et al.44 demonstrated that the most potent ACE inhibitors contain hydrophobic amino acid residues at their three C-terminal positions. P2 contained six hydrophobic amino acid residues (L, F, P, and V), and two of them (P and V) were at the C-terminal tripeptide sequence. In addition, it was reported that most naturally occurring ACE inhibitory peptides contained P, K, or aromatic amino acid residues.41 P2 contained the two amino acid residues (P and K) and one aromatic amino acid residue (F), which may play an important role in its inhibitory activity. Antihypertensive Effect of the Purified Peptide on SHR. Antihypertensive activity of P2 was evaluated by measuring the change of SBP at 1, 2, 3, 6, 9, 22, and 24 h after oral administration. As shown in Figure 4, the SBP in the
Figure 3. Identification of the amino acid sequence of the purified peptides (a) P1 and (b) P2. MS/MS experiments were performed on a LC system coupled to a linear ion trap mass spectrometer model LTQ (LC−ESI−LTQ). The mass spectrum of P2 was cited from ref 27.
Figure 4. Changes in SBP of SHR after oral administration of P2. Captopril was used as a positive control. Single oral administration was performed with a dose of 10 mg/kg of body weight, and SBP was measured 0, 1, 2, 3, 6, and 9 h after administration. (∗) Significant difference from the control at p < 0.05.
purified from various food sources.35,36 These active peptides have considerable potential for use as multifunctional ingredients in functional foods to control chronic diseases. Basic Local Alignment Search Tool (BLAST) analysis revealed that HLFGPPGKKDPV corresponded to the fragment 302−313 of ovotransferrin.27 Ovotransferrin is an iron-binding glycoprotein and appears to be a multifunctional protein.37 Some ACE inhibitory peptides have been purified from ovotransferrin hydrolysates.38−41 Lee et al.41 have characterized an hexapeptide derived from ovotransferrin (KVREGT), possessing strong ACE inhibitory activity (IC50 = 9.1 μM). Lee et al.38 also isolated an ovotransferrin octapeptide (KVREGTTY), which had an IC50 value of 102.8 μM against ACE. In recent research,40 three novel tripeptides (IQW, IRW, and LKP) with high ACE inhibitory activity (IC50 lower than 3.0 μM) were purified from enzymatic hydrolysate of ovotransferrin. In this work, P2 had a relatively low activity compared to other reports. However, in contrast to those peptides obtained via enzymatic hydrolysis of proteins, P2 is the first naturally occurring ACE inhibitory peptide from a fertilized egg. ACE inhibitory peptides usually contain 2−12 amino acid residues.42 Thus, the molecular weight of P2 is in good
quiescent state of SHR was 188 ± 4.4 mmHg. After oral administration of P2 and captopril, SBP clearly decreased and the activities were maintained for 9 h. The maximal decrements in SBP of P2 and captopril treatment groups were 21.1 and 27.1 mmHg at 6 h, respectively. The blood pressure has been gradually recovered in 22 h for all groups. In addition, there was no significant change in the heart rate (HR) after administration, which suggested that the administration of P2 did not have a bad effect on the circulatory system of SHRs. There have been many studies on ACE inhibitory peptides derived from egg proteins.45−47 Some of these peptides have been shown to possess hypotensive activity in SHRs.46,47 However, there is little information on the hypotensive activities of naturally occurring peptides from fertilized eggs. The depressor ability of P2 (21.1 mmHg) is higher than that of 20 mg/kg of ovokinin (13.5 mmHg), which was a heptapeptide derived from ovalbumin.46 It is well-known that small peptides, such as di- or tripeptides are easily absorbed in their intact forms in the intestine.3 Even though P2 has relatively high molecular weight compared to these small peptides, the result 5503
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showed that P2 exerted a substantial effect on reduction of SBP in SHR. This meant that this peptide could be directly absorbed or undergo further hydrolysis in the digestive tract to release smaller size true inhibitors. Inhibition Pattern on ACE. Various substrate (HHL) concentrations (1, 2, and 5 mM) were incubated with ACE solution in either the absence or presence of 100 or 200 μM P2. As shown in Figure 5, the three straight lines intersected at one
Figure 5. Lineweaver−Burk plot of ACE activity of P2: (■) control, (●) 100 μM the P2, and (▲) 200 μM P2.
point on the 1/S axis, which meant that this peptide was a noncompetitive inhibitor.30 Non-competitive inhibitors generally bind to non-catalytic sites of enzymes. They combine with enzyme molecules to produce a dead-end complex, regardless of whether a substrate molecule is bound or not.3 In addition, a decrease in the Vmax value was seen after incubating ACE with P2 (3.10, 2.01, and 1.39 mM/min for the absence and presence of 100 or 200 μM P2, respectively), whereas there was little effect on Km (11.3, 12.5, and 13.9 μM for the absence and presence of 100 or 200 μM P2, respectively). This is in agreement with previous research reporting that noncompetitive inhibition would lead to a decreased Vmax, with minimal effects on Km.48 In recent years, several noncompetitive inhibitory peptides have been purified from some food sources.30,49 However, the relationship between the inhibition pattern and structure of these peptides has not been fully researched.30 Molecular Docking. The conformation with the highest ZDOCK score is shown in Figure 6a. It was observed that P2 (green) was located on the opposite side of the ACE (gray) active site, which included the zinc ion (red). Previous research reported that non-competitive ACE inhibitors cannot bind at the active site but at the other exclusive of the ACE active site.3 Thus, P2 was further identified as a non-competitive inhibitor. The interaction force between P2 and tACE is shown in Figure 6b. With Pymol software, the theoretical calculation indicated that the peptide formed hydrogen bonds with Asn-66, Lys-118, Arg-124, Tyr-394, Glu-403, and Arg-522 of tACE. His1 donated its protons to Glu-403 and Tyr-394 to form hydrogen bonds, respectively. Gly-4 and Lys-9 in P2 accepted hydrogen bonds from Asn-66 and Arg-522, separately. Asp-10 and Pro-11 accepted protons from Arg-124 and Lys-118 to form hydrogen bonds, respectively. These interactions kept P2 away from the active site of tACE, and only Lys-9 formed a
Figure 6. Docking simulation of P2 binding to tACE and captopril− tACE complex. The zinc ion (red ball) was present in the active site of tACE. (a) Docking simulation of P2 (green) binding to tACE (gray). (b) Interaction between P2 (shown as sticks) and the residues of tACE (shown as lines). (c) Overlap of captopril (white) in the crystal structure of the captopril−tACE complex with P2 (green).
coordination bond with the zinc ion that located in the active site. The zinc ion is the key ion in the active site of ACE; it plays a crucial role in opening up the catalytic site.50 In the docking simulation, the zinc ion was attracted by Lys-9 of P2 through a coordination bond. Therefore, the zinc ion might be forced away from the active site, and thus, the substrate HHL bonded to ACE could not be catalyzed to produce HA.30 To further elucidate the inhibition pattern of P2, the docking simulation of P2 and the captopril−ACE complex (PDB code 1UZF) was performed (Figure 6c). Captopril is a specific 5504
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competitive ACE inhibitor.22 According to the crystal structure of the captopril−ACE complex, captopril (white) formed hydrogen bonds with key residues in ACE active site and occupied the key positions, which would be occupied by substrate HHL, such as the S1′ and S2′ subsites.30 This is consistent with the competitive inhibition model. Contrarily, P2 (green) located away from the ACE active site (S1′ and S2′), and only Lys-9 interacted with the zinc ion (red). Therefore, we concluded that P2 could not compete with substrate for the active site, indicating that P2 is a noncompetitive ACE inhibitor. In summary, ACE inhibitory activity of peptides derived from fertilized eggs was investigated. During incubation, ACE inhibitory activity of peptides remained stable before day 12 and then markedly increased on day 15. The peptides on day 15 were then employed for isolation of ACE inhibitory peptide. An identified peptide (HLFGPPGKKDPV) exhibited potent ACE inhibitory activity with an IC50 value of 125 μM and acted as a non-competitive inhibitor against ACE. An antihypertensive effect in SHR revealed that oral administration of the peptide could decrease SBP significantly. These results showed that fertilized eggs could be seen as potential candidates to develop nutraceuticals against hypertension and its related disease.
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(4) Zhao, Y.; Li, B.; Dong, S.; Liu, Z.; Zhao, X.; Wang, J.; Zeng, M. A novel ACE inhibitory peptide isolated from Acaudina molpadioidea hydrolysate. Peptides 2009, 30, 1028−1033. (5) Ferreira, S. H.; Bartelt, D. C.; Greene, L. J. Isolation of bradykinin-potentiating peptides from Bothrops jararaca venom. Biochemistry 1970, 9, 2583−2593. (6) Vercruysse, L.; Van Camp, J.; Smagghe, G. ACE inhibitory peptides derived from enzymatic hydrolysates of animal muscle protein: A review. J. Agric. Food Chem. 2005, 53, 8106−8115. (7) Antonios, T. F. T.; MacGregor, G. A. Angiotensin converting enzyme inhibitor in hypertension: Potential problems. J. Hypertens. 1995, 13, S11−S16. (8) Vermeirssen, V.; Van Camp, J.; Verstraete, W. Optimisation and validation of an angiotensin-converting enzyme inhibition assay for screening of bioactive peptides. J. Biochem. Biophys. Methods 2002, 51, 75−87. (9) Mine, Y.; Shahidi, F. Nutraceutical Proteins and Peptides in Health and Disease; CRC Press: Boca Raton, FL, 2005. (10) Kovacs-Nolan, J.; Phillips, M.; Mine, Y. Advances in the value of eggs and egg components for human health. J. Agric. Food Chem. 2005, 53, 8421−8431. (11) Oshima, G.; Shimabukuro, H.; Nagasawa, K. Peptide inhibitors of angiotensin I-converting enzyme in digest of gelatin by bacterial collagenase. Biochim. Biophys. Acta 1979, 566, 128−137. (12) Maruyama, S.; Suzuki, H. A peptide inhibitor of angiotensin Iconverting enzyme in the tryptic hydrolysate on casein. Agric. Biol. Chem. 1982, 46, 1393−1394. (13) Kohama, Y.; Matsumoto, S.; Oka, H.; Teramoto, T.; Okabe, M.; Miura, T. Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochem. Biophys. Res. Commun. 1988, 155, 332−337. (14) Miyoshi, S.; Ishikawa, H.; Kaneko, T.; Fukui, F.; Tanaka, H.; Maruyama, S. Structure and activity of angiotensin I-converting enzyme inhibitors in a α-zein hydrolysate. Agric. Biol. Chem. 1999, 55, 1313−1318. (15) Kuba, M.; Tana, C.; Twata, S.; Yasuda, M. Production of angiotensin I-converting enzyme inhibitory peptides from soybean protein with Monascus purpureus acid proteinase. Process Biochem. 2005, 40, 2191−2196. (16) Mine, Y.; Kovacs-Nolan, J. New insights in biologically active proteins and peptides derived from hen egg. World Poultry Sci. J. 2006, 62, 87−96. (17) Yokoyama, K.; Chiba, H.; Yoshikawa, M. Peptide inhibitors for angiotensin I-converting enzyme from thermolysin digest of dried bonito. Biosci., Biotechnol., Biochem. 1992, 56, 1541−1545. (18) Terashima, M.; Baba, T.; Ikemoto, N.; Katayama, M.; Morimoto, T.; Matsumura, S. Novel angiotensin-converting enzyme (ACE) inhibitory peptides derived from boneless chicken leg meat. J. Agric. Food Chem. 2010, 58, 7432−7436. (19) Jimsheena, V. K.; Gowda, L. R. Angiotensin I-converting enzyme (ACE) inhibitory peptides derived from arachin by simulated gastric digestion. Food Chem. 2011, 125, 561−569. (20) Wu, J.; Aluko, R. E.; Nakai, S. Structural requirements of angiotensin I-converting enzyme inhibitory peptides: quantitative structure−activity relationship study of di- and tripeptides. J. Agric. Food Chem. 2006, 54, 732−738. (21) Natesh, R.; Schwager, S. L. U.; Evans, H. R.; Sturrock, E. D.; Acharya, K. R. Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin Iconverting enzyme. Biochemistry 2004, 43, 8718−8724. (22) Natesh, R.; Schwager, S. L. U.; Sturrock, E. D.; Acharya, K. R. Crystal structure of the human angiotensin-converting enzyme− lisinopril complex. Nature 2003, 421, 551−554. (23) Watermeyer, J. M.; Kroger, W. L.; O’ Neill, H. G.; Sewell, B. T.; Sturrock, E. D. Probing the basis of domain-dependent inhibition using novel ketone inhibitors of angiotensin-converting enzyme? Biochemistry 2008, 47, 5942−5950. (24) Duan, X.; Li, M.; Wu, F.; Yang, N.; Nikoo, M.; Jin, Z.; Xu, X. Post fertilization changes in nutritional composition and protein conformation of hen egg. J. Agric. Food Chem. 2013, 61, 12092−12100.
AUTHOR INFORMATION
Corresponding Author
*Telephone/Fax: +86-510-85917100. E-mail: xmxu@jiangnan. edu.cn. Funding
This work was supported by the National “Twelfth Five-Year” Plan for Science and Technology Support of China (2012BAD37B06), the Doctor Candidate Foundation of Jiangnan University (JUDCF13005), and the Graduate Student Innovation Project of Jiangsu Province (CXLX13_751). Notes
The authors declare no competing financial interest.
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ABBREVIATIONS USED ACE, angiotensin I-converting enzyme; ANOVA, analysis of variance; BLAST, Basic Local Alignment Search Tool; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FMOC, [(9-fluorenylmethyl)oxy]carbonyl; HA, hippuric acid; HBTU, 2-(1H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; HHL, hippuryl-L-histidyl-L-leucine; HPLC, high-performance liquid chromatography; HR, heart rate; RAS, renin−angiotensin system; SBP, systolic blood pressure; SHR, spontaneously hypertensive rat; tACE, testis ACE
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REFERENCES
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Journal of Agricultural and Food Chemistry
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dx.doi.org/10.1021/jf501368a | J. Agric. Food Chem. 2014, 62, 5500−5506
