Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein

Peptides 33 (2012) 52–58

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Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein Jiwang Chen a,∗ , Yimei Wang a , Qixin Zhong b , Yongning Wu a,c , Wenshui Xia a,d,∗∗ a

College of Food Science and Engineering, Wuhan Polytechnic University, Wuhan 430023, China Department of Food Science and Technology, The University of Tennessee, Knoxville 37996-4591, USA c Institute of Nutrition and Food Safety, Chinese Centre for Disease Control and Prevention, Beijing 100021, China d School of Food Science and Technology, Jiangnan University, Wuxi 214122, China b

a r t i c l e

i n f o

Article history: Received 20 September 2011 Received in revised form 5 November 2011 Accepted 5 November 2011 Available online 11 November 2011 Keywords: Grass carp protein hydrolysate ACE-inhibitory peptide Purification Inhibition pattern Stability against gastrointestinal enzymes

a b s t r a c t Peptides inhibiting angiotensin-I converting enzyme (ACE, EC. 3.4.15.1) are possible cures of hypertension. Food-derived ACE-inhibitory peptides are particularly attractive because of reduced side effects. Previously, we reported ACE-inhibitory activity of grass carp protein hydrolysates. In this work, we report steps for purifying the ACE-inhibitory peptide from the hydrolysate and its biochemical properties. Following steps of ultrafiltration, macroporous adsorption resin, and two steps of reversed phase high performance liquid chromatography (RE-HPLC), a single Val-Ala-Pro (VAP) tripeptide was identified. The tripeptide with excellent ACE-inhibitory activity (IC50 value of 0.00534 mg/mL) was a competitive ACE inhibitor and stable against both ACE and gastrointestinal enzymes of pepsin and chymotrypsin. This is the first report of food-derived VAP. The identified unique biochemical properties of VAP may enable the application of grass carp protein hydrolysates as a functional food for treatments of hypertension. The developed purification conditions also allow the production of VAP for pharmaceutical applications. © 2011 Elsevier Inc. All rights reserved.

1. Introduction The population of adults affected by hypertension was estimated to be 972 million worldwide in 2000 and was predicted to reach 1.56 billion by 2025 [19]. This is a severe concern because hypertension is frequently linked with high risks of cardiovascular and renal diseases [6]. While there are many causes of hypertension, ACE is well recognized for its important physiological roles in the regulation of blood pressure [6]. Biochemically, ACE converts angiotensin-I, a decapeptide that is inactive, to angiotensin-II, an octapeptide that is a potent vasoconstrictor [29]. ACE also

Abbreviations: ACE, angiotensin-I converting enzyme; ACEIP, angiotensin-I converting enzyme inhibitory peptides; RE-HPLC, reversed phase high performance liquid chromatography; HPLC, high performance liquid chromatography; HHL, hippuryl-histidine-leucine; HA, hippuric acid; TFA, trifluoroacetic acid; DH, degree of hydrolysis; BV, bed volume; PTH, phenylthiohydantoin. ∗ Corresponding author. Tel.: +86 139 7130 9046; fax: +86 027 8392 4790. ∗∗ Corresponding author: Tel.: +86 136 0619 3362; fax: +86 0510 8532 9057. E-mail addresses: [email protected] (J. Chen), [email protected] (W. Xia). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.11.006

inactivates bradykinin, a vasodilator [29]. These established biochemical mechanisms have led to the synthesis of ACE inhibitors such as captopril, lisinopril, enalapril and fosinopril for treatment of hypertension [5,7]. Although effective, these synthetic ACE inhibitors cause side effects such as coughing, taste disturbances, skin rashes and angioneurotic edema, and their long term administration may cause aldosterone escape phenomenon and reduce the efficacy of ACE inhibitors [2,11,36]. Therefore, much interest has focused on ACEIP derived from food materials to reduce side effects while maintaining physiological functions like body weight reduction and immune regulation. Oshima [30] first reported ACEIP derived from food natural sources. Since then, a large number of ACEIP have been isolated and identified from proteins or their hydrolysates originating from milk [4], egg [27], rapseed [25], soybean [32,42], peanut [16], rice [22], corn [20], wheat [15], beef [14], porcine [18], chicken [12,33], among others. ACEIP derived from seafood proteins have also been reported. Kohama et al. [21] observed that an octapeptide in an acid extract from tuna muscle, with an amino acid sequence of GD at the amino terminus, showed strong anti-hypotensive activity. More recently, many ACEIP have been isolated from bonito [13,40],

J. Chen et al. / Peptides 33 (2012) 52–58

cuttlefish [31], pipefish [37], sardine [26], squid [3], and yellowfin [17], and the “Katsuo-bushi oligopeptide” is now sold in Japan as a functional food product claiming anti-hypertension benefits [12,13]. We are interested in farm-raised freshwater fish as protein sources of ACEIP, particularly grass carp because its production ranks the first in China, about 3.7 million tons annually [24], and second in the world. Grass carp proteins are easy to extract and their functional and nutritional properties can be improved by methods such as enzymatic hydrolysis. Hydrolysates with unique properties add significant values to aquaculture. Previously, we hydrolyzed grass carp protein using alcalase and observed significant ACE-inhibitory activities in the derived peptides [8,9]. Because high-purity ACEIP are required for applications such as admission by hypertension patients, the major objective of this work was to purify ACEIP from grass carp protein hydrolysates using RP-HPLC. Our secondary objectives were to evaluate the ACE-inhibitory functions of the purified ACEIP and characterize its stability during simulated digestions by gastrointestinal enzymes. 2. Materials and methods 2.1. Materials Grass carp was purchased from the supermarket of Wushang Wholesale Chain Company (Wuhan, China). Alcalase, pepsin and chymotrypsin were purchased from Novozymes Biotechnology Company (Shanghai, China). ACE from rabbit lungs, HHL and HA were purchased from Sigma–Aldrich (St. Louis, MO, USA). Acetonitrile and TFA were HPLC grade, and other reagents were analytical grade, purchased from Sinopharm Chemical Reagent Company (Shanghai, China). 2.2. Preparation of grass carp protein hydrolysate Grass carp protein hydrolysates were prepared according to our previously described method [9]. The reactor (MUT-Tschamber, Roedermark, Germany) was equipped with a stirrer, a thermostatic water-circulator bath (Huayi, Gongyi, China) for temperature control, and a pH-adjustment system (Mettler Toledo, Shanghai, China) enabling a constant pH during hydrolysis. The solution with 5.5% (w/w) grass carp protein was homogenized at 0 ◦ C for 30 min and pre-incubated at 50 ◦ C for 5 min. The hydrolysis was performed using alcalase at a level of 48 AU per kg protein at a constant temperature of 50 ◦ C and pH 9. At a DH of 17.25%, the hydrolysis was terminated by adjusting pH to 4.5, followed by centrifugation at 10,000 × g for 10 min. The supernatant was transferred and lyophilized after adjusting pH to neutral. The final powder was collected and stored at −20 ◦ C until further experiments. 2.3. Measurement of ACE-inhibitory activity The ACE inhibitory activity was measured by the method of Wu et al. [38] with slight modifications. The 30 ␮L of a 2.5 mM HHL solution was mixed with 10 ␮L of an inhibitor solution, followed by incubation for 5 min at 37 ◦ C. Afterwards, 20 ␮L of a 0.1 U/mL ACE solution (prepared in a 50 mM borate buffer adjusted to pH 8.3, containing 0.3 M NaCl) was added, followed by incubation at 37 ◦ C for 60 min. The reaction was terminated by adding 70 ␮L of 1 M HCl before the following assays. A blank sample was prepared by replacing the inhibitor solution with the 50 mM borate buffer. Samples were filtered through a 0.45 ␮m nylon syringe filter and then separated by a C18 column (4.6 mm × 150 mm, 5 ␮m). HA and HHL were detected at 228 nm. The column was eluted at a flow rate of 0.8 mL/min with two solutions – (A) 0.05% TFA in water and (B) 0.05% TFA in acetonitrile – using a gradient of 10–60% B in 10 min

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and another 60–10% B in 2 min. Peak areas were used to quantify HA. The degree of ACE inhibition (in percentages) was calculated according to the following equation:



ACE inhibition (%) = 1 −

A

inhibitor

Ablank



× 100

(1)

where Ainhibitor and Ablank are peak areas corresponding to HA for an inhibitor sample and the blank sample, respectively. The IC50 value was defined as the concentration of inhibitor required to reduce the HA peak area by 50% (indicating 50% inhibition of ACE). 2.4. Purification of ACE inhibitory peptides from hydrolysates The lyophilized hydrolysate was dissolved at 50 mg/mL in deionized water. The solution was filtered using two ultrafiltration membranes with 3 and 10 kDa molecular-weight-cut-off to obtain three fractions corresponding to MW below 3 kDa, between 3 and 10 kDa, and above 10 kDa. Each fraction was assayed for ACEinhibitory activity. The fraction with the highest ACE-inhibitory activity was lyophilized, and the powder obtained was prepared at 10 mg/mL and loaded to a DA 201-C macroporous resin column at a flow rate of 1 BV/h. After loading, the column was washed with deionized water at a flow rate of 1 BV/h until the eluent had the same conductivity as deionized water. Then, four fractions were obtained from step-elution using 25%, 50%, 75% and 90% aqueous ethanol solution at a flow rate of 3 BV/h. The fraction with the highest ACE-inhibitory activity was lyophilized and further purified in two RP-HPLC (model 1525-2998, Waters Corp., Torrance, USA) steps. A Jupiter C18 column (5 ␮m, 10 mm × 250 mm, Phenomenex Co., Torrance, CA, USA) was adopted in the first step. After equilibrating the column with 5% acetonitrile, 2 mL sample, prepared with the lyophilized powder at 10 mg/mL, was injected. A linear gradient of 5–60% acetonitrile (in deionized water, containing 0.1% TFA) was carried out in 60 min at a flow rate of 2 mL/min, with the absorbance of eluent monitored at 220 nm. Fractions were collected according to the elution peaks and lyophilized immediately. The lyophilized fraction with the highest ACE-inhibitory activity was dissolved at 10 mg/mL and separated for the second step RP-HPLC using a Luna C18 column (5 ␮m, 4.6 mm × 250 mm, Phenomenex Co., Torrance, CA, USA). After injecting 250 ␮L sample, the elution was conducted at a flow rate of 0.8 mL/min using an isocratic step with 10% acetonitrile for 10 min and a subsequent linear gradient of 10–25% acetonitrile in 45 min. The detection of eluent was also based on the absorbance at 220 nm. Similar to the first step, fractions were collected and lyophilized immediately for further use. Another RP-HPLC analysis was performed for the fraction showing the highest ACE-inhibitory activity from the second step to evaluate the purity of peptide. The separation was achieved with a Jupiter C18 column (5 ␮m, 4.6 mm × 250 mm, Phenomenex Co., Torrance, CA, USA) and a sample injection volume of 20 ␮L. To elude the adsorbed peptide, an isocratic step with 10% acetonitrile for 5 min and a subsequent linear gradient of 10–100% acetonitrile in 25 min were used at a flow rate of 0.8 mL/min. The absorbance at 220 nm was monitored during elution. 2.5. Amino acid sequencing of the purified peptide The purified peptide was dissolved in 1% TFA (in deionized water) solution and then transferred onto a polyvinylidene fluoride membrane. After fixing by polybrene, the amino acid sequence of the peptide was characterized using an automated protein/peptide sequencer (model PPSQ-33A, Shimadzu, Tokyo, Japan) equipped with an online detection system for PTH-amino acids.

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Fig. 1. Reversed phase-HPLC chromatograms demonstrating two steps used to purify the ACE-inhibitory peptide from the grass carp protein hydrolysate after ultrafiltration and separation using a DA 201-C macroporous resin column. In the first step (a), the mixture was separated using a Jupiter C18 column. A total of 8 fractions was collected, with the fraction 6 (marked with an arrow) showing the highest ACE-inhibitory activity. In the second step (b), the fraction 6 from the first step was separated using a Luna C18 column. A total of 6 fractions were collected, and the fraction 6-1 (marked with an arrow) showed the highest ACE-inhibitory activity. The chromatogram in (c) shows the purity of the fraction 6-1 from the second step, also separated using a Jupiter C18 column and reversed phase-HPLC.

2.6. Determination of ACE-inhibition pattern of the purified peptide

the intercept of the Lineweaver–Burk lines. All experiments were conducted in triplicate.

The purified peptide was mixed with ACE at an enzyme/substrate ratio of 6 mU/mg peptide and incubated at 37 ◦ C for 3 h. After boiling for 15 min to inactivate the enzyme, the mixture was neutralized for acidity and centrifuged at 12,000 × g for 25 min. The supernatant was transferred and assayed for ACE inhibitory activity. The supernatant was also analyzed for possible complexation with ACE using the RP-HPLC conditions described above. To characterize the ACE inhibition pattern of the purified peptide, the kinetics of ACE inhibition was established at different reaction durations, and the data was analyzed using Lineweaver–Burk plots, where the reciprocal of HHL concentration is used as an independent variable (X-axis) and the reciprocal of production rate of HA as a dependent variable (Y-axis). After linear regression, the inhibitory constant (Ki ) was determined as

2.7. Stability of the purified peptide during in vitro digestion by gastrointestinal enzymes The 1% (w/w) pepsin solution was prepared in a 0.1 mM KCl–HCl buffer adjusted to pH 2.0, while the 1% (w/w) chymotrypsin solution in a 0.1 mM KCl–NaOH buffer was adjusted to pH 7.0. The purified peptide was dissolved at 0.02 mg/mL in the pepsin or chymotrypsin solution and reacted at 37 ◦ C for 4 h. In a separate experiment, the purified peptide was subsequently treated by the pepsin and chymotrypsin at 37 ◦ C for 4 h in each step. Reactions were terminated by boiling for 15 min. After centrifugation at 12,000 × g for 25 min, the supernatant was transferred, neutralized for acidity, and assayed for ACE-inhibitory activity. The supernatant was also analyzed using the above RP-HPLC conditions to evaluate the changes of peptide after treatment by digestive enzymes.

J. Chen et al. / Peptides 33 (2012) 52–58 Table 1 Summary for IC50 values after different steps of purifying an ACE-inhibitory peptide from grass carp protein hydrolysate. Purification step Protein hydrolysate Fraction with MW < 3 kDa after ultrafiltration 50% ethanol elution fraction from DA201-C resin column Fraction 6 after the first step RP-HPLC Fraction 6-1 after the second step RP-HPLC

IC50 values (mg/mL)a

Purification fold

0.872 ± 0.003 0.308 ± 0.005

0.00 2.83

0.130 ± 0.002

6.71

0.0553 ± 0.0004 0.00534 ± 0.00003

15.77

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Table 2 IC50 values and chromatography peak areas of the Val-Ala-Pro (VAP) tripeptide before and after treatment by ACE. Treatment

IC50 valuesa (mg/mL)

Peak area (mAU/s)

Without ACE With ACE

0.00538 ± 0.00003 0.00542 ± 0.00005

1,462,414 1,434,764

a

Results are means and S.D. of triplicate measurements (p < 0.05).

3.3. Determination of ACE inhibition pattern of the purified tripeptide

163.30

a

IC50 value was defined as the concentration of inhibitor required to inhibit 50% of the ACE activity. All data are means and S.D. of triplicate measurements (p < 0.05).

Control treatments were prepared by mixing inactivated pepsin and/or chymotrypsin (by boiling for 15 min) with the purified peptide. After incubation, the mixture was processed and analyzed as above. 2.8. Statistical analysis

There are two possible causes of the reduced HA production during the assays. One is that VAP serves as an inhibitor by competing the binding sites on ACE and the other by reducing the ability of HHL binding to ACE. The VAP before and after interacting with ACE at 37 ◦ C for 3 h had similar IC50 values (Table 2) and similar retention time and peak area on chromatograms (Fig. 2a vs. b). The results indicate no degradation of VAP by ACE (that would reduce the IC50 value). Therefore, VAP is an inhibitor of ACE. To further illustrate the mode of inhibition, a Lineweaver–Burk plot (Fig. 2c) was constructed using three concentrations of VAP.

Analysis of variance was performed with SPSS 13.0 software (Armonk, NY, USA). Linear regression was performed using the Microsoft Excel-2003 (Microsoft Corp., Seattle, WA, USA). 3. Results 3.1. Purification of ACE-inhibitory peptide from grass carp protein hydrolysates Ultrafiltration was firstly applied to separate grass carp protein hydrolysates to three fractions with MW distributions of below 3 kDa, between 3 and 10 kDa and above 10 kDa. The fraction corresponding to MW < 3 kDa had the highest ACE-inhibitory activity, with an IC50 value of 0.308 ± 0.005 mg/mL, while the > 10 kDa fraction showed the lowest activity. The <3 kDa fraction was applied to the DA 201-C macroporous resin column. The fraction corresponding to elution conditions of 50% ethanol had the highest ACE-inhibitory activity, with an IC50 value of 0.130 ± 0.002 mg/mL, and was further separated by two steps of RP-HPLC, with chromatograms presented in Fig. 1. The fraction 6 collected in the first step RP-HPLC (Fig. 1a) had the highest ACE-inhibitory activity among the eight fractions collected, corresponding to an IC50 value of 0.0553 ± 0.0004 mg/mL. The 6th fraction from the first step was applied in the second step RP-HPLC where the fraction 6-1 (Fig. 1b) was observed to have the highest ACE-inhibitory activity, with an IC50 value of 0.00534 ± 0.00003 mg/mL or 18.6 ␮M. After separation using the Jupiter C18 column for purity, the chromatogram showed a major peak (Fig. 1c) suggesting the fraction 6-1 had a purity satisfactory for amino acid sequencing. Purification performances are summarized in Table 1. The IC50 value decreased by 163 folds after the four purification steps, indicating 163 times improvement in purity. The second RP-HPLC step had the highest purification performance, with a decrease of IC50 value by 10 times. 3.2. Amino acid sequence and IC50 value of the purified peptide The amino acid sequence of the fraction 6-1 was determined to be Val-Ala-Pro (VAP). This is the first time that the tripeptide VAP has been isolated from food proteins. Compared to a synthesized VAP and captopril that has an IC50 value of 2.0 ␮M [34] and 0.022 ␮M [13], respectively, the VAP in this study had a higher IC50 value estimated at 18.6 ␮M.

Fig. 2. Chromatograms for the Val-Ala-Pro (VAP) tripeptide before (a) and after (b) ACE treatment. (c) Lineweaver–Burk plot for three (0, 0.002, 0.004 ␮g/mL) VAP concentrations.

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Fig. 3. Chromatograms for the Val-Ala-Pro (VAP) tripeptide after treatments by gastrointestinal enzymes. The VAP was treated by pepsin (b) or chymotrypsin (d) alone or in combination (f). For comparison, the VAP was also treated by inactivated pepsin (a), inactivated chymotrypsin (c) or both (e).

The lines corresponding to two VAP concentrations showed the same intercept as the one without VAP, indicating that VAP is a competitive inhibitor. The Ki value of VAP was determined to be 3.34 nM. The Lineweaver–Burk plot indicates that VAP competes with HHL for the binding sites of ACE.

The IC50 values are summarized in Table 3, showing no impacts of digestive enzymes on ACE-inhibitory activity of VAP. This was confirmed by the same elution time and areas (Fig. 3 and Table 3) of the peaks corresponding to VAP. Our results suggest that VAP is stable against gastrointestinal enzymes of pepsin and chymotrypsin.

3.4. Stability of the purified peptide against gastrointestinal enzymes

4. Discussion

In addition to inhibitory activity, peptides have to survive possible hydrolysis by gastrointestinal proteases after oral administration [4,12,23]. In this work, VAP was subjected to in vitro separate and combined digestions by pepsin and chymotrypsin.

Food-derived ACE-inhibitory peptides are particularly attractive because of reduced side effects. It is common to obtain ACE-inhibitory peptides by enzymatic hydrolysis. In this work, the hydrolysates with ACE inhibitory activity were obtained by hydrolyzing grass carp protein with alcalase. Matsui et al. [26]

J. Chen et al. / Peptides 33 (2012) 52–58 Table 3 IC50 values and chromatography peak areas of the Val-Ala-Pro (VAP) tripeptide after treatment by gastrointestinal enzymes with comparison to controls with inactivated enzymes. Treatment

IC50 valuesa (mg/mL)

Inactivated pepsin Pepsin Inactivated chymotrypsin Chymotrypsin Sequential steps of inactivated pepsin and chymotrypsin Sequential steps of pepsin and chymotrypsin

0.00540 0.00538 0.00549 0.00553 0.00567

± ± ± ± ±

0.00003 0.00002 0.00002 0.00003 0.00004

0.00571 ± 0.00003a

Peak areas (mAU/s) 749,760 751,242 689,804 690,335 677,955 668,328

a Results are means and S.D. of triplicate measurements (p < 0.05) on the IC50 values.

reported that the ACE inhibitory activity of alkaline protease hydrolysate derived from sardine muscle increased markedly with an increase in DH. Previously, we reported high ACE-inhibitory activity of grass carp protein hydrolysates using alcalase, with a high DH due to its enzymatic endo-actions and specific hydrolysis for carboxyl-terminal hydrophobic amino acids [8,9]. The ultrafiltration, macroporous adsorption resin, and two steps of RE-HPLC were utilized to sequentially purify the ACE-inhibitory peptide from the hydrolysates, and a single VAP tripeptide was identified. Jung et al. [17] fractionated yellowfin frame protein hydrolysates using two ultrafiltration steps, and the fraction with the lowest MW (below 5 kDa) showed the most potent ACEinhibitory activity. The non-polar macroporous resin DA201-C was used by Zhang et al. [41] to efficiently separate peptides in hydrolysates of grass carp fish scale. In the literature, the tripeptides of LKP from dried bonito, VKP from freshwater clams, VPP from sour milk, RIY from rapeseed, and VYP from cheese were reported, with IC50 values of 0.32, 3.7, 9, 28 and 288.4 ␮M, respectively [1,13,25,28,35]. The ACE-inhibitory activity of VAP in this work (with an IC50 value of 18.6 ␮M) is higher than that of RIY and VYP but lower than those of LKP, VKP and VPP. In addition, a pentapeptide FFVAP isolated from the hydrolysate of casein, with an IC50 value of 6.0 ␮M [34], showed a stronger ACEinhibitory activity than the VAP tripeptide in this study. Further, we used quantitative structure–activity relationships (QSAR) to model ACE inhibitory peptides and predict the most potent peptide. The amino acid at the C-terminus had the most significant effect on the ACE-inhibitory activity of tripeptides. The tripeptides with an aromatic residue at the C-terminal and a hydrophobic amino acid residue at the N-terminal had strong ACEinhibitory activity (data not shown), consistent with the reports of Cheung et al. [10] and Wu et al. [39]. The impact of the center amino acid residue on the ACE-inhibitory activity of tripeptides differs in the literature and our study. Wu et al. [39] reported that tripeptides have stronger ACE-inhibitory activity when the center amino acid residue is a positively charged, e.g., lysine and arginine. Our results showed that tripeptides consisting of a center amino acid residue with low electronic properties and hydrophilicity and bulky side amino acid residues such as alanine, valine, proline and methionine have strong ACE-inhibitory activities. Based on the inhibition mechanism, ACE-inhibitory peptides derived from food proteins can be classified into 3 groups [12,13]. The inhibitor-type peptides, i.e., competitive ACE inhibitors, have IC50 values that are not affected after treatment by ACE or gastrointestinal proteases. The substrate type peptides are hydrolyzed by ACE to products with weaker activity. The prodrug-type peptides are converted to true inhibitors by ACE or gastrointestinal proteases. The VAP in this work is a competitive ACE inhibitor because the inhibitory activity of the peptide is not affected by ACE, pepsin and chymotrypsin, and the Lineweaver–Burk plot

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indicates that VAP competes with HHL for the binding sites of ACE (Fig. 2c). Although competitive ACE inhibitors have been frequently reported, there are several reports of other ACE-inhibitory peptides, e.g., MAW and VYAP from cuttlefish and MIFPGlAGGPEL from yellowfin sole [17,31]. Finally, although captopril and other peptides have stronger ACE inhibition activity than the VAP in this work, VAP is a novel peptide derived from readily available food protein. The purified VAP may be used for therapy of hypertension and admitted orally. The hydrolysate can be used as nutraceutics alone or together with other compounds as functional foods to prevent and/or treat hypertension. Acknowledgements This work was supported by the National Advanced Science and Technology (863) Key Program (2010AA023003), 111 Project-B07029, PCSIRT0627, the earmarked fund for Modern Agroindustry Technology Research System (NYCYTX-49) and Hubei Provincial Ministry of Education (C2010040). References [1] Abubakar A, Saito T, Kitazawa H, Kawai Y, Itoh T. Structural analysis of new antihypertensive peptides derived from cheese whey protein by proteinase K digestion. J Dairy Sci 1998;81(12):3131–8. [2] Alderman CP. Adverse effects of the angiotensin-converting enzyme inhibitors. Ann Pharmacother 1996;30(1):55–61. [3] Alemán A, Giménez B, Pérez SE, 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(2):334–41. [4] Ana Q, María del Mar C, Mercedes R, Lourdes A, Isidra R. Stability to gastrointestinal enzymes and structure–activity relationship of ␤-casein-peptides with antihypertensive properties. Peptides 2009;30(10):1848–53. [5] Atkinson AB, Morton JJ, Brown JJ, Davies DL, Fraser R, Kelly P, et al. Captopril in clinical hypertension. Changes in components of renin–angiotensin system and in body composition in relation to fall in blood pressure with a note on measurement of angiotensin II during converting enzyme inhibition. Br Heart J 1980;44(3):290–6. [6] Bonesi M, Loizzo MR, Satti GA, Michel S, Tillequin F, Menichini F. The synthesis and angiotensin converting enzyme (ACE) inhibitory activity of chalcones and their pyrazole derivatives. Bioorg Med Chem Lett 2010;20(6):1990–3. [7] Brown NJ, Vaughan DE. Angiotensin-converting enzyme inhibitors. Circulation 1998;97:1411–20. [8] Chen JW, Sun Q, Xia WS. Macroporous adsorption resin isolation and stability of angiotensin-converting enzyme (ACE) inhibitory activity of antihypertensive peptides derived from grass carp protein. Food Sci 2009;30(18):25–8. [9] Chen JW, Xia WS, Huang AN, Wang FA. Preparation and physical and chemical characterization of antihypertensive peptides from fish protein by enzymatic hydrolysis. J Fish China 2010;31(4):512–7. [10] Cheung HS, Wang FL, Ondetti MA, Sabo EF, Cushman DW. Binding of peptide substrate and inhibition of angiotensin converting enzyme importance of the COOH-terminal dipeptides sequence. J Biol Chem 1980;255(2):401–7. [11] Cicoira M, Zanolla L, Rossi A, Golia G, Franceschini L, Cabrini G, et al. Failure of aldosterone suppression despite angiotensin-converting enzyme (ACE) inhibitor administration in chronic heart failure is associated with ACE DD genotype. J Am Coll Cardiol 2001;37(7):1808–12. [12] Fujita H, Yokoyama K, Yoshikawa M. Classification and antihypertensive activity of angiotensin I-converting enzyme inhibitory peptides derived from food proteins. J Food Sci 2000;65(4):564–9. [13] Fujita H, Yoshikawa M. LKPNM: a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 1999;44(1–2):123–7. [14] Jang A, Lee M. Purification and identification of angiotensin converting enzyme inhibitory peptides from beef hydrolysates. Meat Sci 2005;69(4):653–61. [15] Jia JQ, Ma HL, Zhao WR, Wang ZB, Tian WM, Luo L, et al. The use of ultrasound for enzymatic preparation of ACE-inhibitory peptides from wheat germ protein. Food Chem 2010;119(1):336–42. [16] Jimsheena VK, Gowda LR. Arachin derived peptides as selective angiotensin Iconverting enzyme (ACE) inhibitors: structure–activity relationship. Peptides 2010;31(6):1165–76. [17] Jung WK, Mendis E, Je JY, Park PJ, Son BW, Kim HC, et al. Angiotensin Iconverting enzyme inhibitory peptide from yellowfin sole (Limanda aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. Food Chem 2006;94(1):26–32. [18] Katayama K, Jamhari, Mori T, Kawahara S, Miake K, Kodama Y, et al. Angiotensin-I converting enzyme inhibitory peptide derived from porcine skeletal muscle myosin and its antihypertensive activity in spontaneously hypertensive rats. J Food Sci 2007;72(9):702–6.

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