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In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE
Process Biochemistry 49 (2014) 898–904
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In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE Peng Li a , Jia Jia b , Ming Fang a , Lujia Zhang a , Mingrong Guo a , Jingli Xie a,∗ , Yuelan Xia a , Li Zhou a , Dongzhi Wei a a State Key Laboratory of Bioreactor Engineering, Department of Food Science and Engineering, East China University of Science and Technology, Shanghai 200237, PR China b Shandong Silk Textile Vocational College, Zibo 255300, Shandong, PR China
a r t i c l e
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Article history: Received 8 October 2013 Received in revised form 12 February 2014 Accepted 13 February 2014 Available online 22 February 2014 Keywords: ACE inhibitory peptide Molecular docking Pistacia vera L. Purification Spontaneously hypertensive rats (SHRs)
a b s t r a c t The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel’s hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC50 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC50 value of 126 M, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a considerable public health problem worldwide and poses a major risk factor for cardiovascular diseases, the number one cause of death in the world. The WHO estimated that by 2020, heart disease and stroke will have surpassed infectious diseases to become the leading cause of death [1]. On the other hand, health food and diet could be an aid to prevent the cardiovascular diseases, for instance, the antioxidant, vitamins, minerals and the bio-functional peptide from foods. The angiotensin Iconverting enzyme (ACE) inhibitory peptide, which can be released by gastrointestinal enzyme hydrolysis and regulate blood pressure through binding to ACE [2,3]. ACE is a membrane anchored dipeptidyl peptidase that hydrolyses angiotensin I to the potent vasoconstrictor angiotensin II and by abrogating a potent vasodilator bradykinin to its inactive fragments, which play a key role in the blood pressure homeostasis. Thus, inhibition of ACE is considered as the first line of therapy for treating hypertension [4]. The
∗ Corresponding author. Tel.: +86 21 64251803; fax: +86 21 64252563. E-mail address: [email protected] (J. Xie). http://dx.doi.org/10.1016/j.procbio.2014.02.007 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
first peptide inhibitors of ACE were reported from snake venom of Bothrops jararaca [5]. The drugs, captopril, lisinopril, enalapril for medical intervention are based on the snake venom peptide scaffold. Although these drugs show dramatically activity, however, along with various side effects such as coughing, skin rashes, and angioedema are concerned [6,7]. In recent years, ACE inhibitory peptides from food sources are promising natural bio-functional alternatives to the synthetic drugs and are currently the best known class of bioactive peptides. These specific sequences of bioactive peptides can be released through proteolysis either by food processing or by gastrointestinal digestion. Various ACE inhibitory peptides from enzymatic hydrolysates derived from different sources have been reported, such as the Atlantic salmon [8], areca nut [9], Chlorella ellipsoidea [10], kidney bean [11], brownstripe red snapper [12], soy bean [13], hard clam [14], wakame [15], insect protein [16], lupin [17], egg [18], peas [19], and so on. In recent years, computational biology was used to predict the interaction between protein and small molecular such as bioactive peptides, thus, computational (in silico) methods can be used for inhibitory mechanism study as an assistive tool and the design of novel enzyme inhibitors [20–23]. An increasing number of
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researches are booming for the quantitative structure–activity relationship of ACE inhibitory peptide and the mechanism of peptide binding with ACE by using the molecular simulation on computer, and the common software are Discovery Studio, Autodock and Dock [24–26]. Pistachio (Pistacia vera L.) is a nutritional food, which is rich in protein, vitamins, minerals and oil, consuming around the world [27]. However, little study has been reported on the pistachio kernel’s anti-hypertension functionality related to ACE inhibition. An attempt was made in this study to discuss the ACE inhibitory activity of gastrointestinal enzymatic hydrolysates from the pistachio kernel in vitro and in vivo. Then a novel ACE inhibitory peptide was isolated and identified from the pepsin–trypsin hydrolysate (Pe–Tr–H) with highest ACE inhibitory activity. Extend study was conducted to explore the binding mechanism including hydrogen bond, hydrophobic interaction, electrostatic interactions, Van der Waals interaction force and total energy between peptide and ACE by docking experiments. 2. Materials and methods 2.1. Materials The pistachio (P. vera L.) was obtained from Paramount Farms Ltd. Co. (San QiaoKun valley, CA, USA). Pepsin, trypsin, ACE (EC 3.4.15.1) from rabbit lung and hippuryl–histidyl–leucine (HHL) as a substrate peptide of ACE were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other reagents used in this study were analytical grade chemicals. 2.2. Enzymatic hydrolysis Pistachios were shelled to obtain the kernel, and then were milled into powder in a food processor (JYL-B031, Joyoung, Jinan, China) to obtain pistachio kernel powder (PKP). PKP was mixed with distilled water (10%, w/v) and digested with pepsin (5000 U/g powder, pH 1.8) at 37 ◦ C for 4 h. The hydrolysis reaction was terminated by heating in a boiling water bath for 10 min, and then the pH was adjusted to 7.8. The mixture was further digested with trypsin (5000 U/g powder) at the same condition for another 6 h. After heating in a boiling water bath for 10 min, the hydrolyzed solution was then centrifuged at 9000g at 4 ◦ C for 25 min. The supernatant was collected and lyophilized as the pepsin–trypsin hydrolysate (Pe–Tr–H). The pepsin hydrolysate (Pe–H) and trypsin hydrolysate (Tr–H) were obtained at the same condition. 2.3. ACE inhibition measurement The ACE inhibitory activity was measured according to the method of Cushman and Cheung [28] with slight modifications. Ten milligram sample was dissolved in 1 ml distilled water and then diluted to seven different concentrations for ACE inhibitory measurements. Fifteen microliter of sample solution in certain concentration added with 15 l substrate HHL (8.3 mM Hip–His–Leu in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) was pre-incubated at 37 ◦ C for 5 min, and the reaction was initiated by adding 5 l of ACE solution (310 mU/ml) and incubated for 60 min at the same temperature. The reaction was terminated by the addition of 1.0 M HCl (200 l). Ten microliters of the reaction solution were injected directly onto a Thermo BDS-C18 column (3.0 mm × 250 mm, 5 m, Thermo Scientific Co. Ltd., USA). The mobile phase was 10% acetonitrile and 90% water with 0.1% trifluoroacetic acid (TFA). The flow rate was 0.7 ml/min and monitored at 228 nm to evaluate the ACE inhibition activity of hydrolysates. All
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determination was carried out at least in triplicate. The inhibition activity was calculated using the following equation:
ACE inhibition (%) = 1 Ainhibitor /Acontrol
× 100
where Ainhibitor and Acontrol are, respectively, the relative areas of the hippuric acid (HA) peak of the assay with inhibitor and of the control sample without inhibitor. The IC50 value was defined as the concentration of inhibitor that could inhibit 50% of the ACE activity. 2.4. SHRs and measurement of blood pressure In vivo testing was conducted by the methods of Matsui [3] with some modifications. The Spontaneously hypertensive rats (SHRs, 10-week-old, male, specific pathogen-free, 250–320 g body weight) with tail systolic blood pressure over 180 mmHg were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). SHRs were then housed individually in steel cages (6 SHRs per group) in a room kept at 22 ± 2 ◦ C with a 12 h light–dark cycle, and fed a standard laboratory diet. Tap water was freely available. The SHRs experiment was conducted by Shanghai Second Military Medical University (Shanghai, China). The study was approved by the Shanghai Second Military Medical University Animal Care and Use Ethics Committee, and the animals were cared for in accordance with the institutional ethical guidelines. PKP and gastrointestinal enzymatic hydrolysate powders were mixed in saline and were orally injected into SHR at a dose of 1 g per kg body weight, Control rats were administrated with the same volume of saline solution. After oral administration, SHRs were warming up in a chamber maintained at 37 ◦ C for 10 min. The systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were measured by tail-cuff method with a Softron BP system (Softron BP-98A, Tokyo, Japan) and all the results were measured three times. 2.5. Purification and identification of ACE inhibitory peptide The Pe–Tr–H powder was first separated by UF membrane with 3 kDa molecular weight cut-off (Millipore Co., Beverly, USA). Two fractions with molecular weights of <3 kDa, and >3 kDa were collected to assay the ACE inhibition in vitro and then lyophilized for further use. The lyophilized powder was dissolved in distilled water and was applied to a Sephadex G-15 followed by G-10 column (1.0 cm × 80 cm; Pharmacia, Sweden), eluted with ultrapure water at a flow rate of 48 ml/h and were monitored at 215 nm by way of fast protein liquid chromatography (FPLC) (AKTA P-900; GE, USA). Fractions were collected automatically and the highest active fraction obtained was pooled and further purified using RP-HPLC on an Ultimate 3000 C18 semi-prep column (10 mm × 150 mm; DIONEX, USA). The mobile phase was 0.1% TFA in water (v/v) as eluent A and acetonitrile containing 0.1% TFA as eluent B. A linear gradient was conducted by the eluent B (5–50% in 60 min) at a flow rate of 1.5 ml/min and monitored at 215 nm. Fractions were collected based on the profile of the eluted peptides, and then lyophilized. Molecular weight of purified peptide from pistachio kernel’s hydrolysates was determined by electrospray ion trap mass spectrometry (LCQ Deca Model 5890, HP, USA), and the amino acid sequence was determined in positive ion mode by de novo sequencing method of MALDI–TOF/TOF system. The sample was dissolved in the water and acetonitrile (1:1), and Cyano-4-hydroxycinnamic acid (CHCA) was used as substrate. MALDI–TOF MS spectra were acquired with an AB Sciex 4800plus, MALDI–TOF/TOF MS equipped with a Nd:YAG laser (emitting at 355 nm, operated at 200 Hz). The spectra were recorded in the reflectron positive ion mode and externally calibrated with “TOF/TOF calibration” standard solution.
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The 4000 series Explorer and DATA Explorer (AB Sciex, America) were used for data acquisition and processing. The acceleration voltage was set to 20 kV and the extraction delay time used was 450 ns.
2.8. Statistical analysis for the results of experiments All results were presented as means ± standard deviation (S.D.). Significance of differences between two samples was determined by the Student t-test. A p value of less than 0.05 was taken as significant.
2.6. Synthesis of purified peptide The identified peptide was synthesized by the GL Biochem Co. Ltd. (Shanghai, China), so as to confirm its ACE inhibitory activity.
2.7. Molecular docking of peptide with human ACE The 3D structure of human ACE (1O8A) was derived from the Protein Data Bank. The energy minimized conformation of peptide was obtained by means of ChemOffice 2008. Molecular docking study borrowed ideas from Pan and Cao [29] with some modification. The docking was implemented using the Flexible Docking tool of DS 3.5 software with the cofactors (zinc and chloride ions) and the default pH setting of Discovery Studio 3.5 in docking was 7.0. The docking runs, centered at zinc, were carried out with a radius ˚ of 15 A.
Fig. 2. Elution profile with ACE inhibitory activity of Pe–Tr–H I on Sephadex G-15 and Sephadex G-10.
Fig. 1. Effects of signal oral administration on SHR’s SBP (a) and DBP (b). Saline was used as a control. Single oral administration was performed with a dose of 1 g/kg body weight. And SBP and DBP were measured 0, 1, 2, 4, 6 and 8 h after oral administration. PKP for pistachio kernel powder; Pe–H for pepsin hydrolysate; Tr–H for trypsin hydrolysate; Pe–Tr–H for pepsin–trypsin hydrolysate. * p < 0.05, ** p < 0.01 compared with control.
Fig. 3. Elution profile with ACE inhibitory activity of F-3 on RP-HPLC. (a) F-3 was separated into four fractions; (b) IC50 of four fractions.
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3. Results and discussion 3.1. Effect of signal oral administration on SHRs The antihypertensive effect of PKP, Pe–H, Tr–H and Pe–Tr–H were evaluated by measuring the changes of SBP (Fig. 1a) and DBP (Fig. 1b) in SHR at 0, 1, 2, 4, 6, and 8 h after the oral administration of 1 g/kg body weight. The control group was injected with the same volume of saline. The Results indicated that PKP could slightly low SBP and DBP at first 6 h and keep stable from 6 h to 8 h
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after the oral signal administration. The Pe–H showed a smoothly decreasing in SBP for around 10 mmHg at 8 h and 9 mmHg in DBP at 6 h. The Tr–H and Pe–Tr–H exhibited a similar progress. The SBPs and DBPs were dramatically decreased by both hydrolsyates at 1 h after oral administration, and then continuously dropped around 22 mmHg and 16 mmHg till 4 h, respectively, and such situations were maintained to 8 h (Fig. 1). Oral administration of gastrointestinal enzymatic hydrolysates displayed a higher in vivo antihypertensive activity, suggesting that the intake of hydrolysates could achieve more efficient blood
Fig. 4. The peptide obtained from hydrolysate was identified by MALDI–TOF/TOF mass spectrometer.
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pressure drop than the intake of whole kernel powder. The PKP could be digested by the gastrointestinal enzymes in vivo to form the ACE inhibitory peptides. However the effect of hydrolysis was not as optimal as those of the gastrointestinal enzymatic hydrolysis in vitro, which resulted in the comparative lower antihypertensive effects of PKP than those of the gastrointestinal enzymatic hydrolysates. The results were in coincidence with the works of Kim [2] and Matsui [3]. Moreover, the hydrolysates were continuously hydrolyzed in digestive tract, which could generate more active peptides although some previous peptides would be degraded. Accordingly, the gastrointestinal enzymatic hydrolysates released from pistachio kernel exhibited anti-hypertension effects in vivo, which indicate the pistachio kernel protein could be utilized for production of ACE inhibitor. The Pe–Tr–H displayed a higher anti-hypertension activity among the hydrolysates, thus, it was used for further investigation. 3.2. Separation of ACE inhibitory peptide from pepsin–trypsin hydrolysate of pistachio kernel The ACE inhibitory activity of Pe–Tr–H was measured as 0.87 ± 0.05 mg/ml IC50 . Then Pe–Tr–H was separated into two molecular weight (MW) groups, Pe–Tr–H I (MW < 3 kDa) and Pe–Tr–H II (MW > 3 kDa) by UF membrane cut-off of 3 kDa. Pe–Tr–H I exhibited higher ACE inhibitory activity with IC50 value of 0.58 ± 0.02 mg/ml than that of the Pe–Tr–H II with IC50 value of 1.15 ± 0.06 mg/ml. The activity of Pe–Tr–H I was stronger than that of the hydrolysate (<3 kDa) we obtained from a marine animal, Phascolosoma esculenta [30] and weaker than walnut’s hydrolysate (<3 kDa) reported by Liu [31]. The fraction Pe–Tr–H I with lower molecular weight, yet exhibited higher ACE inhibitory activity, might because that peptides with lower molecular weight were thought to be much easier to reach the active site of ACE to realize the inhibitory activity [4,30–36]. Then, Pe–Tr–H I was selected for the further purification of the ACE inhibitory peptide. As shown in Fig. 2, the Pe–Tr–H I was separated into four fractions by tandem size exclusion chromatography Sephadex G-15 and Sephadex G10 columns, among which the fraction F-3 showed a higher ACE inhibitory activity with IC50 of 0.26 ± 0.009 mg/ml. The fraction F-3 was eluted into four portions through RP-HPLC (Fig. 3a), the fraction P-2 exhibited the potent ACE inhibitory activity with IC50 value of 0.074 ± 0.007 mg/ml (Fig. 3b). The ACE inhibitory activity of Pe–Tr–H was improved more than 11 times after three steps of purification (Table 1). 3.3. Identification of the ACE inhibitory peptide and synthesis The fraction P-2 was then analyzed by ESI–MS for molecular weight (MW) determination and MALDI–TOF/TOF for the characterization of peptide (Fig. 4). A pentapeptide ACKEP with molecular weight 547.2 Da was identified. The peptide was synthesized to confirm the ACE inhibitory activity, and the IC50 value of synthesized peptide was 126 M. Some previous studies have been discussed, that biological activities of protein hydrolysates are related to amino acid composition, sequence and configuration of peptides [2,15,33,37,38]. ACE was implied to prefer inhibitors containing hydrophobic amino acid residues at the C-terminal pos-
Fig. 5. The structural comparison of lisinopril, enalapril and ACKEP. The same construction of the proline residue at the C-terminal was marked with red box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
itions [39], and the existence of the proline residue at the C-terminal most likely strengthens the inhibitory activity of peptides to ACE [40]. As shown in Fig. 5, the peptide ACKEP has same C-terminal proline residue as lisinopril and enalapril, which plays a key role in binding with ACE and strengthening the inhibitory activity of these peptides. In the deeper speculate, maybe the imidazole ring of proline residue is easy to combine with the amino acid residues which in the active center of ACE [40]. 3.4. In silico simulation In order to explore the structural–functional mechanism between ACKEP and ACE, the computation of molecular docking was made. The most stabilized pose of the ACKEP bond with ACE was obtained, and their 3D and 2D structures were exhibited in Fig. 6a and b, respectively. The inhibitor combined with the residues of ACE through the main interaction forces of hydrogen bonds, hydrophobic, Van der Waals and electrostatic force. The hydrogen bonds interaction force played a main role. The molecular docking of ACKEP on the ACE binding site revealed that ACKEP was encompassed by a hydrophobic pocket which formed by the electron cloud of hydrophobic interactions (Fig. 6c), what was in accordance with some recent reports [41,42]. The ACKEP
Table 1 Purification of ACE inhibitory peptide from Pe–Tr–H. Purification steps
Sample
IC50 (mg/ml)
Enhanced fold of activity
Pepsin and Trypsin hydrolysate UF Sephadex G-15 and G-10 C18 semi-prep column
Pe–Tr–H Pe–Tr–H I F-3 P-2
0.87 ± 0.05 0.58 ± 0.02 0.26 ± 0.009 0.074 ± 0.007
1.0 1.5 3.4 11.8
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Fig. 6. 3D and 2D structures of ACKEP binding with ACE. (a) General overview of theoretical docking poses (gray) at the ACE catalytic site. (b) ACKEP interaction with ACE residues in 2D, atoms in green means Van der Waals interaction force, and pink atoms means electrostatic interaction force and gray atom means zinc. (c) Details of ACE (gray) and ACKEP (green) interaction after automated docking. Dotted line in black stand for hydrogen bonds, and difference density map (blue clouds) was the electron cloud of hydrophobic interactions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
makes contact via Van der Waals interactions with His353, His513, Val351, Val518, Phe391, Phe512, Glu384, Glu403, Ala354, Trp357, Tyr360, Asn406, Pro407, Gly404 of ACE, as well the electrostatic interactions with Ser355, Lys368, His383, Ala356, Asn70, His387, Tyr523, His410, Arg522, Asp358, Tyr394, Arg402 of ACE (Fig. 6b). The interaction energy of ACKEP binding with ACE was summarized in Table 2. The O25 and H67 of the ACKEP formed hydrogen bonds with Ala356 (7.27 kJ/mol) and Asn70 (−29.18 kJ/mol) of ACE, respectively. According to the total energy, those two hydrogen bonds between ACE and ACKEP were not stable, thus they were not significant for the ACE inhibitory activity. On the other hand, the O5 of ACKEP formed two hydrogen bonds with Arg522
(−507.37 kJ/mol), also the H60 formed hydrogen bonds with Asp358 (−168.83 kJ/mol), which contributed significantly to stability especially. ACE contains the zinc binding motifs, His383, E411, His387, and a downstream Glu384 residue [43,44]. Molecular docking showed that the ACKEP makes contact via Van der Waals interactions with Glu384 as well as the electrostatic interactions with His383 and His387, resulting ACKEP may prevent ACE from binding with zinc and then restrain the ACE activity. In summary, in the interaction of ACE with ACKEP, the seven residues from the ACE active site (His383, His387, Glu384, Arg522, Asp358 Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.
Table 2 The interactions and interaction energies of ACKEP binding with ACE. Hydrogen bonds
Total interaction energy (KJ/mol)
Total Van der Waals interaction energy (KJ/mol)
Total electrostatic interaction energy (KJ/mol)
Distance (Å)
ALA356–O25 ARG522–O5 ARG522–O5 ASP358–H60 ASN70–H67
7.27 −253.68 −253.68 −168.83 −29.18
−2.38 −0.62 −0.62 −0.46 −0.63
9.65 −253.06 −253.06 −168.37 −28.55
7.1 5.1 5.1 5.7 6.5
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4. Conclusion The pistachio kernel exhibited anti-hypertension effects after digested by gastrointestinal enzymes, which indicate the pistachio kernel protein could be utilized for production of ACE inhibitory peptides. Peptide ACKEP was isolated and identified, that has same C-terminal construction as that of lisinopril and enalapril, which plays a key role in binding with ACE. The mechanism between ACKEP and ACE was explored by molecule docking to reveal that seven amino acids in the ACE active site and two atoms of ACKEP greatly contributed to the combinative stabilization. Acknowledgments This work was supported by “National Natural Science Foundation of China (No. 31301413)”, “National Major Science and Technology Projects of China (No. 2012ZX09304009)”, “the Fundamental Research Funds for the Central Universities”, P. R. China. The authors are highly thankful to Paramount Farms Ltd. Co. (San QiaoKun valley, CA, USA) for providing the pistachio nuts. References [1] Lopez AD, Murray CC. The global burden of disease, 1990–2020. Nat Med 1998;4:1241–3. [2] Kim SY, Je JY, Kim SK. Purification and characterization of antioxidant peptides from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. J Nutr Biochem 2007;18:31–9. [3] Matsui T, Yukiyoshi A, Doi S, Sugimoto H. Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. J Nutr Biochem 2002;13:80–6. [4] Alemán A, Giménez B, Pérez-Santin E, Gómez-Guillén MC, Montero P. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chem 2011;125:334–41. [5] Ferreira SH. A bradykinin-potentiating factor (BPF) present in the venom of Bothrops jararaca. Br J Pharmacol Chemoth 1965;24:163–9. [6] Tarek FTA, Graham AM. Angiotensin-converting enzyme-inhibitor in hypertension-potential problems. J Hypertens 1995;13:11–6. [7] Vermeirssen V, Van CJ, 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. [8] Gu RZ, Li CY, Liu WY, Yi WX, Cai MY. Angiotensin I-converting enzyme inhibitory activity of low-molecular-weight peptides from Atlantic salmon (Salmo salar L.) skin. Food Res Int 2011;44:1536–41. [9] Chung FM, Shieh TY, Yang YH, Chang DM, Shin SJ, Tsai JR, Chen TH, Tai TY, Lee YJ. The role of angiotensin-converting enzyme gene insertion/deletion polymorphism for blood pressure regulation in areca nut chewers. Transl Res 2007;150:58–65. [10] Ko SC, Kang N, Kim EA, Kang MC, Lee SH, Kang SM, Lee JB, Jeon BT, Kim SK, Park SJ, Park PJ, Jung WK, Kim D, Jeon YJ. A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats. Process Biochem 2012;47:2005–11. ˜ [11] Limón RI, Penas E, Martínez-Villaluenga C, Frias J. Role of elicitation on the health-promoting properties of kidney bean sprouts. LWT-Food Sci Technol 2014;56:328–34. [12] Khantaphanta S, Benjakula S, Kishimura H. Antioxidative and ACE inhibitory activities of protein hydrolysates from the muscle of brownstripe red snapper prepared using pyloric caeca and commercial proteases. Process Biochem 2011;46:318–27. [13] Kuba M, Tana C, Twata S, Yasuda M. Production of anigiotensin I-converting enzyme inhibitory peptides from soybean protein with Monuascus purpureus acid proteinase. Process Biochem 2005;40:2191–6. [14] Tsai JS, Chen JL, Pan BS. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria). Process Biochem 2008;43:743–7. [15] Suetsuna K, Maekawa K, Chen JR. Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. J Nutr Biochem 2003;15:267–72. [16] Vercruysse L, Van CJ, Morel N, Rouge P, Herregods G, Smagghe G. Ala–Val–Phe and Val–Phe: ACE inhibitory peptides derived from insect protein with antihypertensive activity in spontaneously hypertensive rats. Peptides 2010;31:482–8. [17] Boschina G, Scigliuolob GM, Restab D, Arnoldi A. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. Food Chem 2014;145:34–40.
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Isolation and characterization of angiotensin I-converting enzyme inhibitory peptides from wheat gliadin hydrolysate. Mol Nutr Food Res 2003;47:354–8. [38] Ruiz-Giménez P, Marcos JF, Torregrosa G, Lahoz A. Novel antihypertensive hexa- and heptapeptides with ACE-inhibiting properties: from the in vitro ACE assay to the spontaneously hypertensive rat. Peptides 2011;32:1431–8. [39] Murray BA, FitzGerald RJ. Angiotensin converting enzyme inhibitory peptides derived from food proteins: biochemistry, bioactivity and production. Curr Pharm Des 2007;13:773–91. [40] Terashima M, Oe M, Ogura K, Matsumura S. Inhibition strength of short peptides derived from an ACE inhibitory peptide. J Agric Food Chem 2011;59: 11234–7. [41] Douglas RG, Sharma RK, Masuyer G, Lubbe L, Zamora I, Acharya KR, Chibale K, Sturrock ED. Fragment-based design for the development of N-domain-selective angiotensin-1-converting enzyme inhibitors. Clin Sci 2014;126:305–13. 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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
In vitro and in vivo ACE inhibitory of pistachio hydrolysates and in silico mechanism of identified peptide binding with ACE Peng Li a , Jia Jia b , Ming Fang a , Lujia Zhang a , Mingrong Guo a , Jingli Xie a,∗ , Yuelan Xia a , Li Zhou a , Dongzhi Wei a a State Key Laboratory of Bioreactor Engineering, Department of Food Science and Engineering, East China University of Science and Technology, Shanghai 200237, PR China b Shandong Silk Textile Vocational College, Zibo 255300, Shandong, PR China
a r t i c l e
i n f o
Article history: Received 8 October 2013 Received in revised form 12 February 2014 Accepted 13 February 2014 Available online 22 February 2014 Keywords: ACE inhibitory peptide Molecular docking Pistacia vera L. Purification Spontaneously hypertensive rats (SHRs)
a b s t r a c t The ACE inhibitory activity of pistachio (Pistacia vera L.) kernel’s hydrolysates by gastrointestinal enzymes was studied. Results indicated that hydrolysate successively hydrolyzed by pepsin and trypsin, Pe–Tr–H, presented in vitro ACE inhibitory activity as IC50 0.87 ± 0.04 mg/ml. The Pe–Tr–H can in vivo decrease around 22 mmHg in systolic blood pressure (SBP) and 16 mmHg in the diastolic blood pressure (DBP) at 4 h after the oral administration, however the pistachio kernel powder can slightly lower SBP and DBP. The Pe–Tr–H with the highest activity was then separated by ultrafiltration membrane of 3 kDa, size exclusion chromatography on Sephadex G-15 and G-10 columns and reversed phase high-performance liquid chromatography (RP-HPLC) consecutively. A novel ACE inhibitory peptide, ACKEP, with the IC50 value of 126 M, was identified by MALDI–TOF/TOF system. ACKEP has the same C-terminal residue as Lisinopril and Enalapril, which plays a key role in binding with ACE. The binding mechanism was explored at a molecular basis by docking experiments, which revealed that seven residues from ACE active site (His383, His387, Glu384, Arg522, Asp358, Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Hypertension is a considerable public health problem worldwide and poses a major risk factor for cardiovascular diseases, the number one cause of death in the world. The WHO estimated that by 2020, heart disease and stroke will have surpassed infectious diseases to become the leading cause of death [1]. On the other hand, health food and diet could be an aid to prevent the cardiovascular diseases, for instance, the antioxidant, vitamins, minerals and the bio-functional peptide from foods. The angiotensin Iconverting enzyme (ACE) inhibitory peptide, which can be released by gastrointestinal enzyme hydrolysis and regulate blood pressure through binding to ACE [2,3]. ACE is a membrane anchored dipeptidyl peptidase that hydrolyses angiotensin I to the potent vasoconstrictor angiotensin II and by abrogating a potent vasodilator bradykinin to its inactive fragments, which play a key role in the blood pressure homeostasis. Thus, inhibition of ACE is considered as the first line of therapy for treating hypertension [4]. The
∗ Corresponding author. Tel.: +86 21 64251803; fax: +86 21 64252563. E-mail address: [email protected] (J. Xie). http://dx.doi.org/10.1016/j.procbio.2014.02.007 1359-5113/© 2014 Elsevier Ltd. All rights reserved.
first peptide inhibitors of ACE were reported from snake venom of Bothrops jararaca [5]. The drugs, captopril, lisinopril, enalapril for medical intervention are based on the snake venom peptide scaffold. Although these drugs show dramatically activity, however, along with various side effects such as coughing, skin rashes, and angioedema are concerned [6,7]. In recent years, ACE inhibitory peptides from food sources are promising natural bio-functional alternatives to the synthetic drugs and are currently the best known class of bioactive peptides. These specific sequences of bioactive peptides can be released through proteolysis either by food processing or by gastrointestinal digestion. Various ACE inhibitory peptides from enzymatic hydrolysates derived from different sources have been reported, such as the Atlantic salmon [8], areca nut [9], Chlorella ellipsoidea [10], kidney bean [11], brownstripe red snapper [12], soy bean [13], hard clam [14], wakame [15], insect protein [16], lupin [17], egg [18], peas [19], and so on. In recent years, computational biology was used to predict the interaction between protein and small molecular such as bioactive peptides, thus, computational (in silico) methods can be used for inhibitory mechanism study as an assistive tool and the design of novel enzyme inhibitors [20–23]. An increasing number of
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researches are booming for the quantitative structure–activity relationship of ACE inhibitory peptide and the mechanism of peptide binding with ACE by using the molecular simulation on computer, and the common software are Discovery Studio, Autodock and Dock [24–26]. Pistachio (Pistacia vera L.) is a nutritional food, which is rich in protein, vitamins, minerals and oil, consuming around the world [27]. However, little study has been reported on the pistachio kernel’s anti-hypertension functionality related to ACE inhibition. An attempt was made in this study to discuss the ACE inhibitory activity of gastrointestinal enzymatic hydrolysates from the pistachio kernel in vitro and in vivo. Then a novel ACE inhibitory peptide was isolated and identified from the pepsin–trypsin hydrolysate (Pe–Tr–H) with highest ACE inhibitory activity. Extend study was conducted to explore the binding mechanism including hydrogen bond, hydrophobic interaction, electrostatic interactions, Van der Waals interaction force and total energy between peptide and ACE by docking experiments. 2. Materials and methods 2.1. Materials The pistachio (P. vera L.) was obtained from Paramount Farms Ltd. Co. (San QiaoKun valley, CA, USA). Pepsin, trypsin, ACE (EC 3.4.15.1) from rabbit lung and hippuryl–histidyl–leucine (HHL) as a substrate peptide of ACE were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile and methanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). All other reagents used in this study were analytical grade chemicals. 2.2. Enzymatic hydrolysis Pistachios were shelled to obtain the kernel, and then were milled into powder in a food processor (JYL-B031, Joyoung, Jinan, China) to obtain pistachio kernel powder (PKP). PKP was mixed with distilled water (10%, w/v) and digested with pepsin (5000 U/g powder, pH 1.8) at 37 ◦ C for 4 h. The hydrolysis reaction was terminated by heating in a boiling water bath for 10 min, and then the pH was adjusted to 7.8. The mixture was further digested with trypsin (5000 U/g powder) at the same condition for another 6 h. After heating in a boiling water bath for 10 min, the hydrolyzed solution was then centrifuged at 9000g at 4 ◦ C for 25 min. The supernatant was collected and lyophilized as the pepsin–trypsin hydrolysate (Pe–Tr–H). The pepsin hydrolysate (Pe–H) and trypsin hydrolysate (Tr–H) were obtained at the same condition. 2.3. ACE inhibition measurement The ACE inhibitory activity was measured according to the method of Cushman and Cheung [28] with slight modifications. Ten milligram sample was dissolved in 1 ml distilled water and then diluted to seven different concentrations for ACE inhibitory measurements. Fifteen microliter of sample solution in certain concentration added with 15 l substrate HHL (8.3 mM Hip–His–Leu in 50 mM sodium borate buffer containing 0.5 M NaCl at pH 8.3) was pre-incubated at 37 ◦ C for 5 min, and the reaction was initiated by adding 5 l of ACE solution (310 mU/ml) and incubated for 60 min at the same temperature. The reaction was terminated by the addition of 1.0 M HCl (200 l). Ten microliters of the reaction solution were injected directly onto a Thermo BDS-C18 column (3.0 mm × 250 mm, 5 m, Thermo Scientific Co. Ltd., USA). The mobile phase was 10% acetonitrile and 90% water with 0.1% trifluoroacetic acid (TFA). The flow rate was 0.7 ml/min and monitored at 228 nm to evaluate the ACE inhibition activity of hydrolysates. All
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determination was carried out at least in triplicate. The inhibition activity was calculated using the following equation:
ACE inhibition (%) = 1 Ainhibitor /Acontrol
× 100
where Ainhibitor and Acontrol are, respectively, the relative areas of the hippuric acid (HA) peak of the assay with inhibitor and of the control sample without inhibitor. The IC50 value was defined as the concentration of inhibitor that could inhibit 50% of the ACE activity. 2.4. SHRs and measurement of blood pressure In vivo testing was conducted by the methods of Matsui [3] with some modifications. The Spontaneously hypertensive rats (SHRs, 10-week-old, male, specific pathogen-free, 250–320 g body weight) with tail systolic blood pressure over 180 mmHg were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). SHRs were then housed individually in steel cages (6 SHRs per group) in a room kept at 22 ± 2 ◦ C with a 12 h light–dark cycle, and fed a standard laboratory diet. Tap water was freely available. The SHRs experiment was conducted by Shanghai Second Military Medical University (Shanghai, China). The study was approved by the Shanghai Second Military Medical University Animal Care and Use Ethics Committee, and the animals were cared for in accordance with the institutional ethical guidelines. PKP and gastrointestinal enzymatic hydrolysate powders were mixed in saline and were orally injected into SHR at a dose of 1 g per kg body weight, Control rats were administrated with the same volume of saline solution. After oral administration, SHRs were warming up in a chamber maintained at 37 ◦ C for 10 min. The systolic blood pressure (SBP) and the diastolic blood pressure (DBP) were measured by tail-cuff method with a Softron BP system (Softron BP-98A, Tokyo, Japan) and all the results were measured three times. 2.5. Purification and identification of ACE inhibitory peptide The Pe–Tr–H powder was first separated by UF membrane with 3 kDa molecular weight cut-off (Millipore Co., Beverly, USA). Two fractions with molecular weights of <3 kDa, and >3 kDa were collected to assay the ACE inhibition in vitro and then lyophilized for further use. The lyophilized powder was dissolved in distilled water and was applied to a Sephadex G-15 followed by G-10 column (1.0 cm × 80 cm; Pharmacia, Sweden), eluted with ultrapure water at a flow rate of 48 ml/h and were monitored at 215 nm by way of fast protein liquid chromatography (FPLC) (AKTA P-900; GE, USA). Fractions were collected automatically and the highest active fraction obtained was pooled and further purified using RP-HPLC on an Ultimate 3000 C18 semi-prep column (10 mm × 150 mm; DIONEX, USA). The mobile phase was 0.1% TFA in water (v/v) as eluent A and acetonitrile containing 0.1% TFA as eluent B. A linear gradient was conducted by the eluent B (5–50% in 60 min) at a flow rate of 1.5 ml/min and monitored at 215 nm. Fractions were collected based on the profile of the eluted peptides, and then lyophilized. Molecular weight of purified peptide from pistachio kernel’s hydrolysates was determined by electrospray ion trap mass spectrometry (LCQ Deca Model 5890, HP, USA), and the amino acid sequence was determined in positive ion mode by de novo sequencing method of MALDI–TOF/TOF system. The sample was dissolved in the water and acetonitrile (1:1), and Cyano-4-hydroxycinnamic acid (CHCA) was used as substrate. MALDI–TOF MS spectra were acquired with an AB Sciex 4800plus, MALDI–TOF/TOF MS equipped with a Nd:YAG laser (emitting at 355 nm, operated at 200 Hz). The spectra were recorded in the reflectron positive ion mode and externally calibrated with “TOF/TOF calibration” standard solution.
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The 4000 series Explorer and DATA Explorer (AB Sciex, America) were used for data acquisition and processing. The acceleration voltage was set to 20 kV and the extraction delay time used was 450 ns.
2.8. Statistical analysis for the results of experiments All results were presented as means ± standard deviation (S.D.). Significance of differences between two samples was determined by the Student t-test. A p value of less than 0.05 was taken as significant.
2.6. Synthesis of purified peptide The identified peptide was synthesized by the GL Biochem Co. Ltd. (Shanghai, China), so as to confirm its ACE inhibitory activity.
2.7. Molecular docking of peptide with human ACE The 3D structure of human ACE (1O8A) was derived from the Protein Data Bank. The energy minimized conformation of peptide was obtained by means of ChemOffice 2008. Molecular docking study borrowed ideas from Pan and Cao [29] with some modification. The docking was implemented using the Flexible Docking tool of DS 3.5 software with the cofactors (zinc and chloride ions) and the default pH setting of Discovery Studio 3.5 in docking was 7.0. The docking runs, centered at zinc, were carried out with a radius ˚ of 15 A.
Fig. 2. Elution profile with ACE inhibitory activity of Pe–Tr–H I on Sephadex G-15 and Sephadex G-10.
Fig. 1. Effects of signal oral administration on SHR’s SBP (a) and DBP (b). Saline was used as a control. Single oral administration was performed with a dose of 1 g/kg body weight. And SBP and DBP were measured 0, 1, 2, 4, 6 and 8 h after oral administration. PKP for pistachio kernel powder; Pe–H for pepsin hydrolysate; Tr–H for trypsin hydrolysate; Pe–Tr–H for pepsin–trypsin hydrolysate. * p < 0.05, ** p < 0.01 compared with control.
Fig. 3. Elution profile with ACE inhibitory activity of F-3 on RP-HPLC. (a) F-3 was separated into four fractions; (b) IC50 of four fractions.
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3. Results and discussion 3.1. Effect of signal oral administration on SHRs The antihypertensive effect of PKP, Pe–H, Tr–H and Pe–Tr–H were evaluated by measuring the changes of SBP (Fig. 1a) and DBP (Fig. 1b) in SHR at 0, 1, 2, 4, 6, and 8 h after the oral administration of 1 g/kg body weight. The control group was injected with the same volume of saline. The Results indicated that PKP could slightly low SBP and DBP at first 6 h and keep stable from 6 h to 8 h
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after the oral signal administration. The Pe–H showed a smoothly decreasing in SBP for around 10 mmHg at 8 h and 9 mmHg in DBP at 6 h. The Tr–H and Pe–Tr–H exhibited a similar progress. The SBPs and DBPs were dramatically decreased by both hydrolsyates at 1 h after oral administration, and then continuously dropped around 22 mmHg and 16 mmHg till 4 h, respectively, and such situations were maintained to 8 h (Fig. 1). Oral administration of gastrointestinal enzymatic hydrolysates displayed a higher in vivo antihypertensive activity, suggesting that the intake of hydrolysates could achieve more efficient blood
Fig. 4. The peptide obtained from hydrolysate was identified by MALDI–TOF/TOF mass spectrometer.
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pressure drop than the intake of whole kernel powder. The PKP could be digested by the gastrointestinal enzymes in vivo to form the ACE inhibitory peptides. However the effect of hydrolysis was not as optimal as those of the gastrointestinal enzymatic hydrolysis in vitro, which resulted in the comparative lower antihypertensive effects of PKP than those of the gastrointestinal enzymatic hydrolysates. The results were in coincidence with the works of Kim [2] and Matsui [3]. Moreover, the hydrolysates were continuously hydrolyzed in digestive tract, which could generate more active peptides although some previous peptides would be degraded. Accordingly, the gastrointestinal enzymatic hydrolysates released from pistachio kernel exhibited anti-hypertension effects in vivo, which indicate the pistachio kernel protein could be utilized for production of ACE inhibitor. The Pe–Tr–H displayed a higher anti-hypertension activity among the hydrolysates, thus, it was used for further investigation. 3.2. Separation of ACE inhibitory peptide from pepsin–trypsin hydrolysate of pistachio kernel The ACE inhibitory activity of Pe–Tr–H was measured as 0.87 ± 0.05 mg/ml IC50 . Then Pe–Tr–H was separated into two molecular weight (MW) groups, Pe–Tr–H I (MW < 3 kDa) and Pe–Tr–H II (MW > 3 kDa) by UF membrane cut-off of 3 kDa. Pe–Tr–H I exhibited higher ACE inhibitory activity with IC50 value of 0.58 ± 0.02 mg/ml than that of the Pe–Tr–H II with IC50 value of 1.15 ± 0.06 mg/ml. The activity of Pe–Tr–H I was stronger than that of the hydrolysate (<3 kDa) we obtained from a marine animal, Phascolosoma esculenta [30] and weaker than walnut’s hydrolysate (<3 kDa) reported by Liu [31]. The fraction Pe–Tr–H I with lower molecular weight, yet exhibited higher ACE inhibitory activity, might because that peptides with lower molecular weight were thought to be much easier to reach the active site of ACE to realize the inhibitory activity [4,30–36]. Then, Pe–Tr–H I was selected for the further purification of the ACE inhibitory peptide. As shown in Fig. 2, the Pe–Tr–H I was separated into four fractions by tandem size exclusion chromatography Sephadex G-15 and Sephadex G10 columns, among which the fraction F-3 showed a higher ACE inhibitory activity with IC50 of 0.26 ± 0.009 mg/ml. The fraction F-3 was eluted into four portions through RP-HPLC (Fig. 3a), the fraction P-2 exhibited the potent ACE inhibitory activity with IC50 value of 0.074 ± 0.007 mg/ml (Fig. 3b). The ACE inhibitory activity of Pe–Tr–H was improved more than 11 times after three steps of purification (Table 1). 3.3. Identification of the ACE inhibitory peptide and synthesis The fraction P-2 was then analyzed by ESI–MS for molecular weight (MW) determination and MALDI–TOF/TOF for the characterization of peptide (Fig. 4). A pentapeptide ACKEP with molecular weight 547.2 Da was identified. The peptide was synthesized to confirm the ACE inhibitory activity, and the IC50 value of synthesized peptide was 126 M. Some previous studies have been discussed, that biological activities of protein hydrolysates are related to amino acid composition, sequence and configuration of peptides [2,15,33,37,38]. ACE was implied to prefer inhibitors containing hydrophobic amino acid residues at the C-terminal pos-
Fig. 5. The structural comparison of lisinopril, enalapril and ACKEP. The same construction of the proline residue at the C-terminal was marked with red box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
itions [39], and the existence of the proline residue at the C-terminal most likely strengthens the inhibitory activity of peptides to ACE [40]. As shown in Fig. 5, the peptide ACKEP has same C-terminal proline residue as lisinopril and enalapril, which plays a key role in binding with ACE and strengthening the inhibitory activity of these peptides. In the deeper speculate, maybe the imidazole ring of proline residue is easy to combine with the amino acid residues which in the active center of ACE [40]. 3.4. In silico simulation In order to explore the structural–functional mechanism between ACKEP and ACE, the computation of molecular docking was made. The most stabilized pose of the ACKEP bond with ACE was obtained, and their 3D and 2D structures were exhibited in Fig. 6a and b, respectively. The inhibitor combined with the residues of ACE through the main interaction forces of hydrogen bonds, hydrophobic, Van der Waals and electrostatic force. The hydrogen bonds interaction force played a main role. The molecular docking of ACKEP on the ACE binding site revealed that ACKEP was encompassed by a hydrophobic pocket which formed by the electron cloud of hydrophobic interactions (Fig. 6c), what was in accordance with some recent reports [41,42]. The ACKEP
Table 1 Purification of ACE inhibitory peptide from Pe–Tr–H. Purification steps
Sample
IC50 (mg/ml)
Enhanced fold of activity
Pepsin and Trypsin hydrolysate UF Sephadex G-15 and G-10 C18 semi-prep column
Pe–Tr–H Pe–Tr–H I F-3 P-2
0.87 ± 0.05 0.58 ± 0.02 0.26 ± 0.009 0.074 ± 0.007
1.0 1.5 3.4 11.8
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Fig. 6. 3D and 2D structures of ACKEP binding with ACE. (a) General overview of theoretical docking poses (gray) at the ACE catalytic site. (b) ACKEP interaction with ACE residues in 2D, atoms in green means Van der Waals interaction force, and pink atoms means electrostatic interaction force and gray atom means zinc. (c) Details of ACE (gray) and ACKEP (green) interaction after automated docking. Dotted line in black stand for hydrogen bonds, and difference density map (blue clouds) was the electron cloud of hydrophobic interactions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
makes contact via Van der Waals interactions with His353, His513, Val351, Val518, Phe391, Phe512, Glu384, Glu403, Ala354, Trp357, Tyr360, Asn406, Pro407, Gly404 of ACE, as well the electrostatic interactions with Ser355, Lys368, His383, Ala356, Asn70, His387, Tyr523, His410, Arg522, Asp358, Tyr394, Arg402 of ACE (Fig. 6b). The interaction energy of ACKEP binding with ACE was summarized in Table 2. The O25 and H67 of the ACKEP formed hydrogen bonds with Ala356 (7.27 kJ/mol) and Asn70 (−29.18 kJ/mol) of ACE, respectively. According to the total energy, those two hydrogen bonds between ACE and ACKEP were not stable, thus they were not significant for the ACE inhibitory activity. On the other hand, the O5 of ACKEP formed two hydrogen bonds with Arg522
(−507.37 kJ/mol), also the H60 formed hydrogen bonds with Asp358 (−168.83 kJ/mol), which contributed significantly to stability especially. ACE contains the zinc binding motifs, His383, E411, His387, and a downstream Glu384 residue [43,44]. Molecular docking showed that the ACKEP makes contact via Van der Waals interactions with Glu384 as well as the electrostatic interactions with His383 and His387, resulting ACKEP may prevent ACE from binding with zinc and then restrain the ACE activity. In summary, in the interaction of ACE with ACKEP, the seven residues from the ACE active site (His383, His387, Glu384, Arg522, Asp358 Ala356 and Asn70) and two atoms of ACKEP (O5, H60) greatly contributed to the combinative stabilization.
Table 2 The interactions and interaction energies of ACKEP binding with ACE. Hydrogen bonds
Total interaction energy (KJ/mol)
Total Van der Waals interaction energy (KJ/mol)
Total electrostatic interaction energy (KJ/mol)
Distance (Å)
ALA356–O25 ARG522–O5 ARG522–O5 ASP358–H60 ASN70–H67
7.27 −253.68 −253.68 −168.83 −29.18
−2.38 −0.62 −0.62 −0.46 −0.63
9.65 −253.06 −253.06 −168.37 −28.55
7.1 5.1 5.1 5.7 6.5
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4. Conclusion The pistachio kernel exhibited anti-hypertension effects after digested by gastrointestinal enzymes, which indicate the pistachio kernel protein could be utilized for production of ACE inhibitory peptides. Peptide ACKEP was isolated and identified, that has same C-terminal construction as that of lisinopril and enalapril, which plays a key role in binding with ACE. The mechanism between ACKEP and ACE was explored by molecule docking to reveal that seven amino acids in the ACE active site and two atoms of ACKEP greatly contributed to the combinative stabilization. Acknowledgments This work was supported by “National Natural Science Foundation of China (No. 31301413)”, “National Major Science and Technology Projects of China (No. 2012ZX09304009)”, “the Fundamental Research Funds for the Central Universities”, P. R. China. The authors are highly thankful to Paramount Farms Ltd. Co. (San QiaoKun valley, CA, USA) for providing the pistachio nuts. References [1] Lopez AD, Murray CC. The global burden of disease, 1990–2020. Nat Med 1998;4:1241–3. [2] Kim SY, Je JY, Kim SK. Purification and characterization of antioxidant peptides from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. J Nutr Biochem 2007;18:31–9. [3] Matsui T, Yukiyoshi A, Doi S, Sugimoto H. Gastrointestinal enzyme production of bioactive peptides from royal jelly protein and their antihypertensive ability in SHR. J Nutr Biochem 2002;13:80–6. [4] Alemán A, Giménez B, Pérez-Santin E, Gómez-Guillén MC, Montero P. Contribution of Leu and Hyp residues to antioxidant and ACE-inhibitory activities of peptide sequences isolated from squid gelatin hydrolysate. Food Chem 2011;125:334–41. [5] Ferreira SH. A bradykinin-potentiating factor (BPF) present in the venom of Bothrops jararaca. Br J Pharmacol Chemoth 1965;24:163–9. [6] Tarek FTA, Graham AM. Angiotensin-converting enzyme-inhibitor in hypertension-potential problems. J Hypertens 1995;13:11–6. [7] Vermeirssen V, Van CJ, 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. [8] Gu RZ, Li CY, Liu WY, Yi WX, Cai MY. Angiotensin I-converting enzyme inhibitory activity of low-molecular-weight peptides from Atlantic salmon (Salmo salar L.) skin. Food Res Int 2011;44:1536–41. [9] Chung FM, Shieh TY, Yang YH, Chang DM, Shin SJ, Tsai JR, Chen TH, Tai TY, Lee YJ. The role of angiotensin-converting enzyme gene insertion/deletion polymorphism for blood pressure regulation in areca nut chewers. Transl Res 2007;150:58–65. [10] Ko SC, Kang N, Kim EA, Kang MC, Lee SH, Kang SM, Lee JB, Jeon BT, Kim SK, Park SJ, Park PJ, Jung WK, Kim D, Jeon YJ. A novel angiotensin I-converting enzyme (ACE) inhibitory peptide from a marine Chlorella ellipsoidea and its antihypertensive effect in spontaneously hypertensive rats. Process Biochem 2012;47:2005–11. ˜ [11] Limón RI, Penas E, Martínez-Villaluenga C, Frias J. Role of elicitation on the health-promoting properties of kidney bean sprouts. LWT-Food Sci Technol 2014;56:328–34. [12] Khantaphanta S, Benjakula S, Kishimura H. Antioxidative and ACE inhibitory activities of protein hydrolysates from the muscle of brownstripe red snapper prepared using pyloric caeca and commercial proteases. Process Biochem 2011;46:318–27. [13] Kuba M, Tana C, Twata S, Yasuda M. Production of anigiotensin I-converting enzyme inhibitory peptides from soybean protein with Monuascus purpureus acid proteinase. Process Biochem 2005;40:2191–6. [14] Tsai JS, Chen JL, Pan BS. ACE-inhibitory peptides identified from the muscle protein hydrolysate of hard clam (Meretrix lusoria). Process Biochem 2008;43:743–7. [15] Suetsuna K, Maekawa K, Chen JR. Antihypertensive effects of Undaria pinnatifida (wakame) peptide on blood pressure in spontaneously hypertensive rats. J Nutr Biochem 2003;15:267–72. [16] Vercruysse L, Van CJ, Morel N, Rouge P, Herregods G, Smagghe G. Ala–Val–Phe and Val–Phe: ACE inhibitory peptides derived from insect protein with antihypertensive activity in spontaneously hypertensive rats. Peptides 2010;31:482–8. [17] Boschina G, Scigliuolob GM, Restab D, Arnoldi A. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. Food Chem 2014;145:34–40.
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