Antihypertensive effects, molecular docking study and isothermal

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Antihypertensive effects, molecular docking study and isothermal titration calorimetry assay of the angiotensin Iconverting enzyme inhibitory peptides from Chlorella vulgaris Jingli Xie, Xujun Chen, Junjie Wu, Yanyan Zhang, Yan Zhou, Lujia Zhang, Yajie Tang, and Dong-Zhi Wei J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04294 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

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Journal of Agricultural and Food Chemistry

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Antihypertensive effects, molecular docking study and isothermal titration

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calorimetry assay of the angiotensin I-converting enzyme inhibitory peptides

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from Chlorella vulgaris

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Jingli Xie†, ¶, *, Xujun Chen†, Junjie Wu†, Yanyan Zhang♣, Yan Zhou†, Lujia Zhang†, Yajie Tang‡,

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Dongzhi Wei†, ¶, *

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†

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School of Biotechnology, East China University of Science and Technology, Shanghai 200237,

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People’s Republic of China

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‡

State Key Laboratory of Bioreactor Engineering, Department of Food Science and Technology,

Key Laboratory of Fermentation Engineering (Ministry of Education), Hubei Provincial

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Cooperative Innovation Center of Industrial Fermentation, Hubei Key Laboratory of Industrial

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Microbiology, Hubei University of Technology, Wuhan 430068, People’s Republic of China

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♣

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Republic of China

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¶

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People’s Republic of China

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* Corresponding author. Mail: P.O. Box 283, East China University of Science and Technology, 130

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Meilong Road, Shanghai 200237, People’s Republic of China. Tel: +86-21-64251803; Fax:

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+86-21-64251803; E-mail address: [email protected] (J. Xie); [email protected] (D. Wei)

Department of Food Science, Shanghai Business School, Shanghai 200235, China People’s

Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), Shanghai 200237,

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ACS Paragon Plus Environment 1


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Abstract The aim of this work is to explore angiotensin I-converting enzyme (ACE) inhibitory peptides

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from Chlorella vulgaris (C. vulgaris) and discover the inhibitory mechanism of the peptides. After C.

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vulgaris proteins were gastrointestinal digested in silico, several ACE inhibitory peptides with

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C-terminal tryptophan were screened. Among them, two novel non-competitive ACE inhibitors,

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Thr-Thr-Trp (TTW) and Val-His-Trp (VHW), exhibited the highest inhibitory activity indicated by

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IC50 values, 0.61 ± 0.12 and 0.91 ± 0.31 µM respectively. Both the peptides were demonstrated

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stable against gastrointestinal digestion and ACE hydrolysis. The peptides were administrated to

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spontaneously hypertensive rats (SHRs) in the dose 5 mg/Kg body weight, and VHW could decrease

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50 mmHg systolic blood pressure of SHRs (p < 0.05). Molecular docking displayed that both TTW

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and VHW formed six hydrogen bonds with active site pockets of ACE. Besides, isothermal titration

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calorimetry assay discovered that VHW could form more stable complex with ACE than TTW.

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Therefore, VHW was an excellent ACE inhibitor.

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Keywords: Chlorella vulgaris; ACE inhibitory peptides; antihypertensive activity; molecular

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docking; isothermal titration calorimetry

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INTRODUCTION

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Microalgae species, Spirulina, Chlorella, Dunaliella, Haematococcus, and Schizochytrium, are

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recognized as GRAS (Generally Regarded as Safe) food sources according to the GRAS category

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issued by the U.S. FDA (Food and Drug Administration).1 Among these microalgae, the most

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commonly used ones are Chlorella and Spirulina. Chlorella vulgaris, a kind of green microalgae

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which have a relative long history as food, is famous for its prominent nutritional value, such as 55–

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67% protein, 9–18% dietary fiber, 1–4% chlorophyll and plenty of n-3 and n-6 fatty acids, minerals

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and vitamins.2-5 In the nutraceutical market of U.S., China, Japan and other countries, Chlorella is a

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widely accepted health food or health supplement.5 Notably, the Chlorella’s protein is composed of

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all the essential amino acids for humans and animals,2-4 which indicates that Chlorella protein is with

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good nutritional quality. Bioactivities associated with Chlorella proteins, protein hydrolysate and

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peptides, according to the review of Samarakoon and Jeon, include anti-oxidative activity in vitro,

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angiotensin I-converting enzyme (ACE, EC 3.4.15.1) inhibitory activity in vitro, antiproliferation

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activity in vivo, and immune-stimulant activity in vivo.6 Several ACE inhibitory peptides were

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identified from C. vulgaris or its industrial by-products. Their molecular size ranged from 3 amino

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acid residues to hendeca-peptide, and ACE inhibitory IC50 value ranged from 11.4 to 315 µM.7, 8

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These reports suggested C. vulgaris might be a fine resource of ACE inhibitory peptides.

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Unfortunately, the information of antihypertensive activity in vivo of such reported peptides is absent,

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which may hinder the application of C. vulgaris in hypertension.

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ACE is a ubiquitous enzyme in mammalian tissues involved in the renin-angiotensin system and

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kinin nitric oxide system, and considered as a useful therapy target for treating hypertension9-11. A

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large number of drugs such as captopril, lisinopril, enalapril, ramipril were developed as orally drugs



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to treat hypertension with potent and highly selective ACE inhibitory activity. At same time,

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food-derived biologically active peptides with the same function as drugs were also focused, because

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of their natural origin and safety. Studies have shown that bioactive peptides as natural active

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ingredients may offer therapeutic value for either prevention of disease or reducing symptoms.12 The

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cardioprotective bioactive peptides from foods have already been reported with lots of potential

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health benefits, among which, the effects on control and regulation of blood pressure of such

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peptides are most prominent.13 Therefore, ACE inhibitory peptides used in the treatment of

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antihypertension have been the most intensively studied over the last few decades.14

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In more recent, due to the developments in the fields of computational biology and structural

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biology, as well as the elucidation of ACE structure, the molecular docking becomes a frequently

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used tool for the mechanism illustration of ACE inhibitory peptides.15-19 It is an efficient and

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convenient tool to illustrate the structure details of the ligand-receptor complex, including the

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conformation and interactions. However, docking procedure is a molecular simulation method. To

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get more accurate binding information between the inhibitor and ACE, experimental means is

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necessary. Isothermal titration calorimetry (ITC), a method for full thermodynamic characterization

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of an interaction, can be used under unrestricted molecular size, shape or chemical compostion.

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Furthermore, ITC data underpin understanding the correlation between thermodynamic parameters

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and molecular structural specifics correlated with a change from one state to another.20 Therefore,

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ITC experiment was once carried out for the admeasurement of the inhibition mode of

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Thr-Pro-Thr-Gln-Gln-Ser, an ACE inhibitory peptide purified from yeast.21 Thus, ITC may be a

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useful method to describe the inhibitory mechanism of ACE inhibitory peptide combined with

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molecular docking.


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In present study, several ACE inhibitory peptides were in silico screened from the products

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generated by virtual gastrointestinal digestion of the proteins from C. vulgaris. Then, the stability of

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such peptides against gastrointestinal digestion and ACE hydrolysis were assessed. The

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antihypertensive activity of the peptides were in vivo tested on spontaneously hypertensive rats

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(SHRs). Finally, the mechanism of ACE inhibition was studied from the angles of binding structure

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and thermodynamic nature through molecular docking and ITC assay.

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MATERIALS AND METHODS Chemicals. The enzyme ACE (10 U/mg protein) from rabbit lung, and its substrate

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hippuryl-histidyl-leucine (HHL); the enzymes for the hydrolysis, pepsin (EC 3.4.23.1, 503 U/mg

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solid) and porcine pancreatin (8 × USP) were supplied by Sigma Chemical Co. (St. Louis, MO,

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USA). Acetonitrile as mobile phase used in high performance liquid chromatography (HPLC) were

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from Fisher Scientific (Pittsburgh, PA, USA). Lisinopril (95% purity) was purchased from Sangon

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Biotech Co., Ltd. (Shanghai, China). All other chemicals used in this study were analytical grade.

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Virtually Gastrointestinal Digestion of C. vulgaris Proteins. Forty three piece of C. vulgaris

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proteins were selected from the UniProt database (http://www.uniprot.org/) for this study. The

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information of such proteins, such as entry name, length and molecular weight obtained from

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UniProt were shown in Table 1.

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Above proteins were virtually gastrointestinal digested by pepsin (pH < 2), trypsin (EC 3.4.21.4), and chymotrypsin (EC 3.4.21.1) with BIOPEP

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(http://www.uwm.edu.pl/biochemia/index.php/pl/biopep). Then di- and tri-peptides were picked out

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to compose a peptide library. ChemBioDraw Ultra 11.0 was used to generate structures of such di-



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and tri-peptides, and energy was minimized with the consistent force field (CFF, force fields

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adapting to organic compounds).

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Screening of ACE Inhibitory Peptides. LibDock, a usually used module of Discovery Studio 3.5

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software (DS 3.5, Accelrys, San Diego, CA, USA), was used as molecular docking tool. In the

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docking experiment, the crystal structure of human tACE (PDB ID: 1O8A,

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http://www.rcsb.org/pdb/explore/explore.do?structureId=1O8A) in the presence of cofactors (zinc

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and chloride ions) was as the binding receptor for the di- and tri-peptides. Rigid residues were

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residues within the sphere with 10Å radius and zinc as the center. The site sphere was generated by

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the selected rigid residues. The docking parameters are listed in Table 2. After the LibDock scores

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were obtained, the ACE inhibitory IC50 were predicted according to the relationship between

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Libdock score and IC50 value previously proposed by Wu et al.22

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Peptides Synthesis. Peptides synthesis were carried out by Synpeptide Co., Ltd. (Shanghai,

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China). The purity of the peptides was ≥ 95%, which was verified by HPLC (LC-20A, Shimadzu Co.

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Ltd., Kyoto, Japan) with a column (Inertsil ODS-SP: 4.6 mm × 250 mm × 5µm, Shimadzu Co. Ltd.,

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Kyoto, Japan). Molecular weight of the peptides was determined by electrospray ion trap mass

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spectrometry (LCQ Deca XP MAX, ThermoFinnigan, St. Clara, CA, USA).

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Measurement of ACE Inhibition. ACE inhibition was assayed according to Cushman and

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Cheung.23. ACE was dissolved in 100 mM sodium borate buffer (pH 8.3) to 310 mU/mL for the

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assay. Synthetic peptides were dissolved in distilled water to seven concentration levels. A certain

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concentration of peptide (25 µL) was mixed with ACE solution (10 µL). The mixture was

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pre-incubated at 37 °C for 5 min, then the addition of 50 µL of 5 mM HHL (100 mM sodium borate

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buffer, pH 8.3) started the reaction. The reaction was maintained at 37 °C for 1 h, then 500 µL 1 M


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HCl was added to stop the reaction. Ten microliters of the reaction solution was injected into a

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RP-HPLC (LC1200, Agilent Technologies Inc, St. Clara, CA, USA) fixed with a Thermo BDS-C18

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column (3.0 mm × 250 mm × 5 µm, Thermo Scientific Co. Ltd., Waltham, MA, USA). The mobile

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phase (acetonitrile : water = 2:8 (v/v), with 0.1% trifluoroacetic acid) flew in the rate of 0.8 mL/min.

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The absorbance was detected at 228 nm. All determinations were triplicate. The activity of ACE

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inhibition was calculated as following. ACE inhibitory activity (%) =

− × 100%

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Where AInhibitor is the relative area of the hippuric acid (HA) peak obtained from the reaction of

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ACE and HHL with inhibitor. And AControl is the relative area of the hippuric acid (HA) peak obtained

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from the reaction of ACE and HHL without inhibitor. IC50 value is defined as the concentration of

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peptide that can inhibit half of the ACE activity.

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Determination of Inhibitory Pattern. Peptide solutions in 1 mg/mL and 0.5 mg/mL, and HHL in

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4 mg/mL, 2 mg/mL and 1 mg/mL were used. Twenty five microliters of peptide solution was mixed

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with ACE as referred in above section. The ACE inhibition was assayed under different level of HHL.

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Lineweaver-Burk plots was used to confirm ACE inhibitory pattern of the peptides. The inhibitory

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constant (Ki) was the intercept of X-axis of the plot of which the Y-axis displayed the slopes of

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Lineweaver-Burk line and the X-axis indicated peptide concentrations.23

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Gastrointestinal Stability of Peptides. Gastrointestinal stability of peptides was evaluated in

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vitro.24, 25 Peptides was dissolved in 0.1 M KCl-HCl buffer (pH 2.0) to the concentration 0.1 mg/mL

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and 0.5 mg/mL, respectively. Pepsin (503 U/mg) was added to peptide solution to the final

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concentration of 0.4 mg/mL, then the mixture was digested at 37 °C for 4 h. After the reaction was

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terminated by boiling for 15 min, pH of the reaction solution was adjusted to 7.0 with 1 M NaOH.



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The reaction solution was centrifuged at 12,000 rpm for 5 min. One milliliter of the supernatant was

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taken for the determination of ACE inhibition. The rest supernatant further reacted with pancreatin

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(10 mg/mL, 8 × USP) at 37 °C for another 4 h. The reaction was also stopped by boiling for 15 min

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and then the reaction solution was centrifuged at 12,000 rpm for 5 min. This supernatant was also

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used to detect ACE inhibition. Stability of Peptides against ACE. The stability of peptides against ACE was assessed in vitro.26,

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borate buffer, pH 8.3) at 37 °C for 24 h, respectively. The enzyme was inactivated by boiling for 10

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min. Then the ACE inhibitory activity was measured.

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Each peptides (30 µL, 0.1 mg/mL) was incubated with 100 µL ACE (310 mU/mL with 100 mM

Antihypertensive Effect on SHRs. SHR/NCrlVr (male,10-week-old, 250-320 g body weight,

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specific pathogen-free) were obtained from Vital River Laboratory Animal Co., Ltd. (Beijing, China).

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SHRs were randomly separated into a control group, a positive group, and two experiment groups.

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There were six SHRs in each group. The rats were housed under 12 h day/night cycle at 22 ± 2 °C,

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and accessed regular laboratory diet (SLAC Laboratory Animal Co. Ltd., Shanghai, China. according

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to “Laboratory Animal-Nutrients for Formula Feeds, GB14924.3-2010” standard issued by National

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Technical Committee for Standardization of Laboratory Animals, China) and tap water ad libitum.

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After the tail systolic blood pressure (SBP) of animal was above 180 mmHg, the gavage

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administration could start. The animal experiment was carried out following the ethics guidelines

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approved by the Shanghai Laboratory Animal Administration Office by Laboratorial Animal Center

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of Shanghai University of Traditional Chinese Medicine.

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Normal saline was used as both solvent of synthesized peptides and the control reagent for gavage

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administration. The dosage of peptides was 5 mg/kg body weight for the two experiment groups, and


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same dose of Lisinopril for the positive group and the same volume of normal saline for the control

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group. Tail-cuff method was performed for SBP and diastolic blood pressure (DBP) measurement

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with an ALC-NIBP-TY2928 BP system (Alcott Biotech Co., Ltd., Shanghai, China). The measuring

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points are just before oral gavage (0 h), and 1, 2, 4, 6 and 8 h after the administration. Before each

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measurement, SHRs were always warmed up in a 37 °C thermostat chamber for 10 min.

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Molecular Docking of Peptide with Human ACE. The docking condition was similar as above

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illustration. The best pose of each peptide binding with crystal structure of human tACE (PDB ID:

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1O8A) was established according to LibDock score and binding energy. Interactions between the

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peptide and ACE, including van der Waals force, coordination interaction and secondary forces such

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as hydrogen bonds, electrostatic, hydrophobic, hydrophilic forces, were identified.

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Isothermal Titration Calorimetry Binding Assays. Assays were performed using an iTC200

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microcalorimeter (General Electric, Fairfield, CT, USA) and according to the guidelines in the

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iTC200 microcalorimeter user manual. tACE (2 U/mg, 50 mM borate buffer with 300 mM NaCl, pH

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8.3) was heterologous expressed by sf 9 cells and purified by affinity chromatography. The peptides

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were dissolved in the same buffer as that of tACE. The reaction cell was maintained at 25 °C, with

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the peptide concentration in the syringe as 800 to 1400 nM, and the ACE concentration in the cell as

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80 to 140 nM. Then heat of the reaction was measured. Thermodynamic parameters (∆G, ∆H, and

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∆S) and dissociation constant Kd of the reaction were calculated using Origin 7.0 with the iTC200

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MicroCal Software Addon.

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Statistical analysis. All results were displayed as mean value ± standard deviation (SD) of

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triplicate. ANOVA one-way analysis with Minitab v17 (Minitab Ltd., UK) was used for statistical

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treatment of data. A Tukey pairwise comparison was used to compare the means and identify the



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diﬀerences. Significant difference was taken into account when p value was below 0.05.

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RESULTS AND DISCUSSION Pool of Di- and Tri-Peptide. Total 4,334 pieces of peptides (including all repetitive and

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non-repetitive peptides) were produced when the selected C. vulgaris proteins were in silico digested

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by gastrointestinal enzymes with BIOPEP (Figure 1). Among these peptides, 468 pieces of

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non-repetitive di- and tri-peptide were chosen for a peptide library for the screening of ACE

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inhibitory peptides in silico. ACE inhibitory peptides with higher activity usually have the C-terminal

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residue such as hydrophobic, large steric, aromatic amino acids with a polar functional group.28

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According to the sequence analysis of the 468 pieces of peptides, the statistical results of C-terminus

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were 31.3% Phe, 17.8% Tyr, 13.9% Trp, 10.4% Leu and 8.7% Ala, respectively. Thus, the possibility

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to harvest potent ACE inhibitory peptides from this peptide library is predictable.

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Screening of the ACE Inhibitory Peptides. LibDock was used in the ACE inhibitory peptides

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screening. About 48.8% of the peptides had LibDock scores greater than 100 (data not shown),

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which indicated that these peptides were more likely to have lower IC50 according to the relation

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between Libdock score and IC50, Libdock score = 10.063 lg (1/IC50) + 68.08.22 The other principle

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for the screening was the type of C-terminal amino acid. Aromatic or cyclic amino acids like Trp, Tyr

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and Pro were the most common C-terminal residues found in the structure of highly active ACE

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inhibitors.29, 30 For instance, a piece of designed peptide, Val-Lys-Trp, was reported with IC50 of

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0.020 µM.17 Ile-Gln-Try and Ile-Arg-Try from enzymatic digestion of egg ovotransferrin, had the

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IC50 values as 0.59 and 2.8 µM, respectively.31 Therefore, 10 pieces of peptides with C-terminal Trp

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and higher LibDock scores (ranging from 154 to 179) were picked out for further study (Table 3).


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The ACE inhibitory IC50 values of these tripeptides ranged from 0.61 to 590 µM. The two pieces of

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peptides, Thr-Thr-Trp (TTW) and Val-His-Trp (VHW), respectively presented IC50 values as 0.61 ±

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0.12 and 0.91 ± 0.31 µM, which indicated they are strong inhibitors to ACE inhibition.

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The Inhibitory Pattern of the Peptides on ACE. ACE inhibitory peptides were reported to play

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in all types, such as competitive, non-competitive, and mixed-competitive inhibition of ACE.32 TTW

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and VHW were all confirmed as non-competitive inhibitors for ACE by Lineweaver-Burk plots

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(Figure 2). There are also some reported peptides from amounts of food stuffs working in

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non-competitive inhibition, for example Ile-Phe-Leu from fermented soybean food,33 and

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Tyr-Phe-Pro from yellowfin sole.34

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Table 4 shows the kinetic parameters of the two peptides binding to ACE. Combining with the

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results shown in Figure 2, Km value is constant whatever inhibitors and their concentrations, which is

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one of important properties of non-competitive inhibition. The chance is equal for inhibitor (peptide)

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or substrate (HHL) to bind with ACE in non-competitive pattern. Therefore, the apparent affinity of

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substrate binding to ACE is constant. However, the reactions against variety of competitive inhibitors

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displayed diverse Km values,23 because the apparent affinity of substrate to its binding site in ACE

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was decreased when the inhibitor present.

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The Vmax of uninhibited ACE was 6.81 mg/mL⋅min, however, this velocity decreased

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dose-dependently with the peptides. The peptide binds with ACE at another site than substrate to

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form ACE-HHL-peptide and ACE-peptide complexes. The formation of these complexes depress the

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apparent catalytic efficiency of ACE and reduce the enzymatic reaction rate. The Ki values of TTW

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and VHW are 0.36 and 0.45 mM, which directly correlate with their activity of ACE inhibition,

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indicating their high binding affinity to ACE.



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Stability of the ACE Inhibitory Peptides. TTW and VHW are tri-peptides that can cross the

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intestinal wall and sequentially enter the blood circulation. However, the peptides should resist

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gastrointestinal digestion and keep their integrity before above step, otherwise their biological

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activity may be activated or inactivated.35 The fate of peptides under oral administration could be

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imitated by the simulated gastrointestinal digestion in vitro.25 As Figure 3 shows, in vitro incubation

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with gastric juice and intestinal liquid containing the corresponding digestion protease doesn’t cause

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any obvious change on the ACE inhibitory activity of peptides, despite the peptides concentration.

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This result suggests that the peptides are gastrointestinal stable and can maintain their significant

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activity when they get into the blood.

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ACE is a carboxypeptidase with very broad substrate specificity.36 Hence, some ACE inhibitory

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peptides may be the substrate of ACE and hydrolyzed to smaller fragments. As a result, the ACE

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inhibitory activity changes during the reaction. For instance, heptapeptide

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Phe-Lys-Gly-Arg-Tyr-Tyr-Pro from chicken muscle could be hydrolyzed by ACE to produce three

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shorter peptides Phe-Lys-Gly, Arg-Tyr and Tyr-Pro, meanwhile IC50 value increased from 0.55 µM to

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34 µM.36 ACE Prodrug-type or substrate type inhibitors, such as

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Ile-Val-Gly-Ala-Pro-Ala-His-Gln-Gly from both chicken muscle and dried bonito, could be

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hydrolyzed into His-Gln-Gly and Ile-Val-Gly-Ala-Pro-Ala by ACE. Then His-Gln-Gly obtained

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significant ACE inhibitory activity and antihypertensive activity.36 Accordingly, the peptides stability

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upon ACE need to be taken into account. Figure 4 shows IC50 values of TTW and VHW are stable

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within the incubation of peptides with ACE for 24 h. The result proves that TTW and VHW are

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stable ACE inhibitors but not substrate.

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Antihypertensive Activity on SHRs. With an ACE inhibitor type drug, Lisinopril, as positive


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control, the antihypertensive activity in vivo of the two peptides was assessed on SHRs (Figure 5).

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Lisinopril resulted in maximal antihypertensive effect on SHRs. Both TTW and VHW could

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decrease the SBP of SHRs. Furthermore, VHW could significantly decrease the SBP at 1 and 2 h (p
0.05) as well. Both TTW and VHW could also play influences on

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DBP. Although VHW affected SBP more significant, TTW led to a significant reduction of the DBP

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from 180 to 140 mmHg at 2 h (p < 0.05), while VHW decreased the DBP from 174 to 153 mmHg at

353

1 h (p > 0.05) at most. The results implied that VHW and TTW could be used to treat different

354

hypertensive phenotypes or cooperate together because of their significant influence respectively on

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SBP or DBP.

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When a non-competitive ACE inhibitory peptide from the muscle of cuttlefish,

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Val-Glu-Leu-Tyr-Pro, with IC50 value of 5.22 µM, was gavage administrated to SHRs in 10 mg/kg

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bodyweight, maximum decline of SBP as 20 mmHg was realized at 6 h.37 Val-Trp from sardine

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muscle, displaying competitive inhibitory pattern, has a maximum SBP decrease (18 mmHg) at 1 h

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after the orally administration to SHRs in the dosage of 100 mg/kg body weight.38 Non-competitive

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inhibitor of ACE from hydrolysate of oyster proteins, Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, with

362

IC50 value of 66 µM, showed the maximal drop of SBP as 10 mmHg at 6 h when the peptide was

363

gavaged to SHRs in dosage of 20 mg/kg body weight.39 Compared with these reported ACE

364

inhibitory peptides, TTW and VHW achieved more blood pressure decrease in relatively low dosage,



365

suggested that TTW and VHW had a considerable potential as functional food for blood pressure

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control.

367

Molecular Docking. VHW and TTW were docked into the binding pockets of ACE through

368

LibDock to discover the ligand-receptor interaction mechanism. The pose with the lowest binding

369

energy was recognized as the most stable conformation for further structural analysis. The 3-D and

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2-D structures of the peptide-ACE complexes are displayed in Figure 6.

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There are three main active site pockets in molecule of ACE. S1 pocket has three residues, Ala354,

372

Glu384 and Tyr523. S2 pocket comprises Gln281, His353, Lys511, His513 and Tyr520. S1’ pocket

373

only includes residue Glu162.40, 41 Both TTW and VHW locate in deep active channel of ACE, and

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they are surrounded by the electron cloud of hydrophobic interactions which form a hydrophobic

375

pocket (Figure 6a and c). The peptides and the residues of ACE are linked through the main

376

interaction forces involving van der Waals force and some secondary interactions such as hydrogen

377

bonds, hydrophobic, and electrostatic forces (Figure 6b and d). The hydrogen bond is the main

378

interaction force to form a stable peptide-ACE complex. There are six hydrogen bonds formed

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between VHW and the residues His353, Ala354, Glu384, Glu411 and Tyr520 of ACE (Figure 6b,

380

Table 4). Namely, VHW constructs three hydrogen bonds with S1 pocket (Ala354 and Glu384) and

381

two more with S2 pocket (His 353 and Tyr 520). Among the residues connecting with VHW, Glu384

382

is the one formed coordination effect with Zn2+.19 Thereafter, VHW may also interfere ACE from

383

combining with Zn2+ that may contribute to the ACE inhibitory activity.

384

TTW also forms 6 hydrogen bonds with ACE. The involved residues are His353, Ala354, His513

385

and Pro407 (Figure 6d, Table 4). Ala354 belonging to S1 pocket links with TTW through 3 hydrogen

386

bonds. His353 and His513 are part of S2 pocket, generating 2 hydrogen bonds with TTW. His353,


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Ala354 and His513 were reported as important residues interacting with Lisinopril which formed 6

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hydrogen bonds with ACE when H2O molecule absent.19, 40, 42 According to the docking results,

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VHW and TTW achieve strong ACE inhibitory activity mainly through the hydrogen bonds with the

390

active site pockets of ACE.

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ITC Assays. ITC has been reported as an important method to explain the binding mechanism of

392

Lisinopril, Enalaprilat and Captopril.43 A typical ITC profile of the peptides reacting with

393

recombinant human tACE is shown in Figure 7. According to the forward titration curve (Figure 7a),

394

the binding rate of Lisinopril is quick so that the reaction equilibrium is achieved at about 14 min.

395

The fitting curve of molar reaction heat against molar ratio (inhibitor concentration over ACE

396

concentration) is like a typical “S” curve, and reaction reaches saturation when the molar ratio is

397

approximately 1.3. Both of the titration curve and the fitting curve of VHW are looked similar as

398

those of Lisinopril (Figure 7b). However, the reaction equilibrium of TTW binding to ACE is

399

achieved at approximate 16 min and the fitting curve is far from a typical “S” curve (Fig. 7c).

400

Table 6 shows the thermodynamic parameters of the reactions resulted by ITC. The ∆G values of

401

Lisinopril and VHW binding to ACE are approximately same, but that of TTW binding to ACE is

402

lower. Moreover, according to The Second Law of Thermodynamics, the ∆G of the three reactions are

403

mainly contributed by -T∆S, which indicated the three reactions are all entropically-driven. The

404

value of -T∆S indicates the degree of conformational changes of ACE before and after the reaction,

405

which results in sufficient interactions between ACE and peptides. The comparison of the -T∆S

406

values of VHW and TTW suggests that VHW achieves more stable interaction with ACE. The

407

dissociation constant Kd of the ACE-peptide complex can reflect the binding affinity as well. The Kd

408

value of VHW is close to that of Lisinopril, but that of TTW is much higher. This result indicates that



409

VHW and Lisinopril can form more stable complex with ACE than that TTW can. Although TTW

410

and VHW displayed approximate in vitro ACE inhibitory activity, VHW was thermodynamically

411

verified to form more stable complex with ACE by ITC, which might partly interpret the difference

412

of antihypertensive activity between TTW and VHW. Therefore, ITC is an efficient method for the

413

binding mechanism illustration of ACE inhibitory peptide through the thermodynamic analysis.

414

In a summary, two novel ACE inhibitory peptides, TTW and VHW, screened from the peptide

415

library generated form C. vulgaris protein through gastrointestinal digestion in silico, were firstly

416

reported. Both the peptides were stable against in vitro simulated gastrointestinal digestion, as well

417

as the incubation with ACE. The animal procedures showed both the peptides could significant cut

418

down the blood pressure of the SHRs in relatively low dosage. Together with the results of molecular

419

docking and ITC assay, the two peptides were verified as excellent ACE inhibitors and could be used

420

in the treatment of different hypertensive phenotypes. Compared with the reported ACE inhibitory

421

peptides from C. vulgaris, the two peptides display the highest in vitro inhibitory activity and in vivo

422

anti-hypertensive activity. The Trp content of C. vulgaris protein is about 2.2%, suggesting that the

423

generation of peptides with Trp as C-terminus by enzymatic hydrolysis are with palpable possibility,

424

and C. vulgaris may be a good resource for ACE inhibitory peptides. Therefore, the coming work

425

will focus on the enrichment of ACE inhibitory peptides with C-terminal Trp by enzymatic

426

hydrolysis of C. vulgaris protein. Besides, the effects of C-terminal Trp on the ACE inhibition will

427

be discovered by the crystal structural study of VHW-ACE complex. The inhibitory mechanism

428

insights by the combination of molecular docking and ITC assay in present work indicates a novel

429

way for the in vitro inhibitory mechanism study according to both binding model and actual

430

thermodynamic features.


Page 16 of 39

Page 17 of 39


431 432 433

FUNDINF SOURCES This work was supported by the National Natural Science Foundation of China (No. 31301413,

434

31772007), Open Funding Project of Key Laboratory of Fermentation Engineering (Ministry of

435

Education) of China and the National High Technology Research and Development Program of

436

China (No. 2013AA102109).

437

Notes

438

The authors declare no competing financial interest.



439

REFERENCES

440

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441

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442

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446

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447

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pupa protein and molecular insight into inhibition mechanism. J. Agric. Food Chem. 2017, 65,

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I-converting enzyme inhibitory peptides. Food Res. Int. 2002, 35, 367-375.

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nutraceutically potential ACE-Inhibitory peptides from leatherjacket (Meuchenia sp.) protein

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hydrolysates. LWT-Food Sci. Technol. 2017, 80, 430-436.

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protein. Immunopharmacol. 1999, 44, 123-127.

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peptides during gastrointestinal proteolysis: a bioinformatic, in silico approach. Eur. Food Res.

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Technol. 2005, 221, 712-716.

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2004, 134, 980-988.

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protein digests. Biochimie 2004, 86, 231-239.

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(31) Majumder, K.; Wu, J. A new approach for identification of novel antihypertensive peptides from

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egg proteins by QSAR and bioinformatics. Food Res. Int. 2010, 43, 1371-1378.

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(32) Maeno, M.; Yamamoto, N.; Takano, T. Identification of an antihypertensive peptide from casein

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hydrolysate produced by a proteinase from lactobacillus helveticus, cp790. J. Dairy Sci. 1996, 79,

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1316-1321.

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(33) Kuba, M.; Tanaka, K.; Tawata, S.; Takeda, Y.; Yasuda, M. Angiotensin I converting enzyme

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inhibitory peptides isolated from fermented soybean food. Biosci. Biotechnol. Biochem. 2003, 67,

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1278-1283.

520

(34) Jung, W. K.; Mendis, E., Je; J. Y., Park; P. J., Son; B. W.; Kim, H. C. Angiotensin i-converting

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enzyme inhibitory peptide from yellowfin sole (limanda aspera) frame protein and its

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antihypertensive effect in spontaneously hypertensive rats. Food Chem. 2006, 94, 26-32.

523

(35) Escudero, E.; Mora, L.; Toldrá, F. Stability of ACE inhibitory ham peptides against heat

524

treatment and in vitro digestion. Food Chem. 2014, 161, 305-311.

525

(36) Fujita, H.; Yokoyama, K.; Yoshikawa, M. Classification and antihypertensive activity of

526

angiotensin I-converting enzyme inhibitory peptides derived from food proteins. J. Food Sci. 2000,



527

65, 564-569.

528

(37) Cao, W.; Zhang, C.; Hong, P.; Ji, H.; Hao, J. Purification and identification of an ace inhibitory

529

peptide from the peptic hydrolysate of acetes chinensis and its antihypertensive effects in

530

spontaneously hypertensive rats. Int. J. Food Sci. Technol. 2010, 45, 959-965.

531

(38) Matsui, T.; Hayashi, A.; Tamaya, K.; Matsumoto, K.; Kawasaki, T.; Murakami, K. Depressor

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suppression of human circulating renin-angiotensin system. Clin. Exp. Pharmacol. P. 2003, 30,

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262-275.

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(39) Wang, J.; Hu, J.; Cui, J.; Bai, X.; Du, Y.; Miyaguchi, Y. Purification and identification of a ACE

536

inhibitory peptide from oyster proteins hydrolysate and the antihypertensive effect of hydrolysate in

537

spontaneously hypertensive rats. Food Chem. 2008, 111, 302-308.

538

(40) Wu, Q.; Jia, J.; Yan, H.; Du, J.; Gui, Z. A novel angiotensin-I converting enzyme (ACE)

539

inhibitory peptide from gastrointestinal protease hydrolysate of silkworm pupa (Bombyx mori)

540

protein: Biochemical characterization and molecular docking study. Peptides 2014, 68, 17-24.

541

(41) Rohit, A. C.; Sathisha, K.; Aparna, H. S. A variant peptide of buffalo colostrum

542

beta-lactoglobulin inhibits angiotensin I-converting enzyme activity. Eur. J. Med. Chem. 2012, 53,

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211-219.

544

(42) Jimsheena, V. K.; Gowda, L. R. Arachin derived peptides as selective angiotensin I-converting

545

enzyme (ACE) inhibitors: structure-activity relationship. Peptides 2010, 31, 1165-1176.

546

(43) Andújar-Sánchez, M.; Cámara-Artigas, A.; Jara-Pérez, V. A calorimetric study of the binding of

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lisinopril, enalaprilat and captopril to angiotensin-converting enzyme. Biophys. Chem. 2004, 111,

548

183-189.

549 ACS Paragon Plus Environment 22

Page 22 of 39

Page 23 of 39


550

Figure 1. Length distribution of peptides after in silico gastrointestinal digestion of proteins of C.

551

vulgaris with BIOPEP.

552

Figure 2. Lineweaver–Burk plots of ACE inhibited by the peptides. 1/V and 1/S represents the

553

reciprocal of reaction velocity and substrate concentration, respectively. (a) TTW; (b) VHW.

554

Figure 3. The stability of VHW and TTW under simulated gastrointestinal digestion. The final

555

concentration of peptides were 0.1 and 0.5 mg/mL, respectively for (a) and (b). GC, peptide sample

556

and simulated gastric fluid without pepsin; G, peptide sample with gastric fluid with pepsin; G + IC,

557

peptide sample and simulated gastrointestinal fluid without pepsin and pancreatin; G + I, peptide

558

sample with gastrointestinal fluid with pepsin and pancreatin. Values represent mean ± SD (n = 3).

559

Figure 4. The stability of VHW and TTW against ACE. The concentration of peptides were 0.1

560

mg/mL, values represent mean ± SD (n = 3).

561

Figure 5. Changes of SBP and DBP after oral administration of Lisinopril, VHW and TTW. Normal

562

saline was used as control. Single oral administration was performed in dose of 5 mg/kg body weight.

563

Blood pressures were measured before and 1, 2, 4, 6 and 8 h after oral administration of Lisinopril or

564

the peptides. The differences with a value of p < 0.05 were considered to be significant. (a) SBP

565

changes; (b) DBP changes.

566

Figure 6. Molecular docking of ACE and the peptides. (a) 3-D details of ACE and VHW (green)

567

interaction; (b) 2-D interaction details of VHW and ACE; (c) 3-D details of ACE and TTW (green)

568

interaction; (d) 2-D interaction details of TTW and ACE. Green dotted lines represent hydrogen bond

569

that supply electrons by the main chain, and blue dotted lines represent hydrogen bond that supply

570

electrons by the side chain. Atoms in green mean van der Waals interaction force, and pink atoms

571

mean electrostatic interaction force and gray atom means zinc. Difference density map (blue clouds)



572

is the electron cloud of hydrophobic interactions (For interpretation of the references to color in this

573

figure legend, the reader is referred to the web version of this article).

574

Figure 7. ITC profile of Lisinopril, VHW and TTW binding to ACE. (a) Lisinopril; (b) VHW; (c)

575

TTW.


Page 24 of 39

Page 25 of 39


Table 1. Proteins of C. vulgaris in UniProt. Length of amino

MW

Length of amino

MW

acid sequence

(kDa)

acid sequence

(kDa)

A9XHY8 (Antifreeze protein)

104

10.8

P56325 (Photosystem II reaction center protein M)

36

4.01

C6ZE77 (Actin)

157

17.6

P56327 (Photosystem II reaction center protein T)

31

3.60

G4WUD7 (Antifreeze protein)

178

18.7

P56338 (Photosystem II reaction center protein J)

42

4.25

G4WUD8 (Antifreeze protein)

178

18.7

P56339 (Photosystem II reaction center protein L)

38

4.39

G4WUE0 (Antifreeze protein)

178

18.7

41

4.43

M4HPI0 (Heat shock protein 90)

703

80.7

751

83.2

138

16.5

P56340 (Photosystem I reaction center subunit IX) P56341 (Photosystem I P700 chlorophyll a apoprotein A1) P56342 (Photosystem I P700 chlorophyll a apoprotein A2)

734

81.8

133

14.6

P56348 (Photosystem II reaction center protein K)

42

4.68

81

8.82

P56349 (Chloroplast envelope membrane protein)

266

31.1

P56305 (Cytochrome b6-f complex subunit 5)

37

4.01

819

94.7


31

3.44

36

3.95

P56307 (Photosystem II CP47 reaction center protein)

508

56.1

P56370 (Uncharacterized membrane protein ycf78) P58214 (Photosystem I reaction center subunit VIII) Q7M1S5 (Cytochrome c6)

88

9.29

P56308 (Photosystem II CP43 reaction center protein)

473

52.1

Q8H0E4 (Actin)

238

26.5

P56313 (Photosystem II reaction center protein Z)

62

6.76

Q8H0E5 (Actin)

238

26.5

P56314 (Photosystem I reaction center subunit XII)

31

3.31

Q96405 (Actin)

280

31.5

P56316 (Cytochrome f)

315

34.2

Q96406 (Actin)

205

23.2

P56318 (Photosystem II protein D1)

353

39.0

Q9LRG1 (Urf59 protein)

69

7.89

P56319 (Photosystem II D2 protein)

352

39.4


177

19.4

P56321 (Cytochrome b6)

215

24.1


126

13.7


160

17.4

Q9LRG7 (Alpha-tubulin)

149

16.6

P56323 (Photosystem II reaction center protein H)

80

8.54

Q9SAR8 (Actin)

212

24.0

P56324 (Photosystem II reaction center protein I)

38

4.38

Name

O20118 (Uncharacterized 16.5 kDa protein in psaC-atpA intergenic region) O20120 (Uncharacterized 14.6 kDa protein in psaC-atpA intergenic region) P56301 (Photosystem I iron-sulfur center)

25

Name

ACS Paragon Plus Environment


Page 26 of 39

Table 2. Parameters for molecular docking experiments performed with the LibDock of DS 3.5. Input site sphere

Parameter value

Input site sphere

Parameter value

x

48.65

Keep hydrogrns

FALSE

y

82.55

Max conformation hits

30

z

54.04

Max start conformations

1000

Number of hotpot

100

Steric fraction

0.1

Docking tolerance

0.25

Final cluster radius

0.5

Docking preference

User Specified

Apolar SASA cutoff

15

Max hits to save

10

Polar SASA cutoff

5

Max number of hits

100

Surface grid steps

18

Minimum LibDock score

100

Conformation method

Best

Final score cutoff

0.5

Minimization algorithm

Do not minimize

Max BFGS steps

50

Parallel processing

FALSE

Rigid optimization

FALSE


Page 27 of 39


Table 3. Summary of the LibDock score and ACE inhibitory IC50 of the selected peptides. IC50 values are mean ± confidence interval. Means for a variable not sharing a common symbol (a, d, c, d, e, f, g, h) are significantly different (p < 0.05). Peptide

MW

LibDock score

IC50 (µM)

Precursor protein b

Val-Asp-Trp

418

179

21.5 ± 6.9

Val-His-Trp

441

177

0.91 ± 0.31a

His-Asn-Trp

455

168

126 ± 20d e

Alpha-tubulin (Q9LRG7) Photosystem II D2 protein (P56319)

Asp-Thr-Trp

420

165

260 ± 26

Photosystem II CP43 reaction center protein (P56308)

Thr-Thr-Trp

406

162

0.61 ± 0.12a

Photosystem I P700 chlorophyll a apoprotein A1 (P56341)

Thr-Asn-Trp

419

161

517 ± 52g

Photosystem I P700 chlorophyll a apoprotein A1(P56341); Actin (Q96405; Q9SAR8)

Val-Val-Trp Pro-Ile-Trp Val-Pro-Trp Thr-Asn-Trp

402 414 400 420

160 158

c

76.7 ± 10.1

Photosystem II protein D1 (P56318); Heat shock protein 90 (M4HPI0)

f

Uncharacterized membrane protein ycf78 (P56370)

590 ± 20

154

383 ± 18

154

c

61 ± 8.9

Photosystem I P700 chlorophyll a apoprotein A1 (P56341)

g

Actin (Q96405; Q9SAR8)

27

ACS Paragon Plus Environment


Page 28 of 39

Table 4. Kinetics parameters of ACE-catalyzed reactions in different peptide concentrations. kinetics parameters

Control

Km (mM) Vmax (mg/mL⋅min) Ki (mM)

TTW 1 mg/mL

VHW 0.5 mg/mL

4.00 ± 0.12 6.81 ± 1.02

1.56 ± 0.14

1.89 ± 0.21

0.36 ± 0.08


1 mg/mL

0.5 mg/mL 4.00 ± 0.12

1.88 ± 0.13

2.24 ± 0.19

0.45 ± 0.11

Page 29 of 39


Table 5. The information of hydrogen bonds of TTW and VHW binding with ACE. Peptide

TTW

VHW

Distance

Hydrogen bond

(Å)

ACE active pockets

H30-His353

2.17

S2

H31-Ala354

2.08

S1

H35-His513

2.48

S2

H39-Ala354

2.02

S1

H42-Ala354

2.46

S1

H53-Pro407

1.89

-

H34-Glu411

2.08

-

H48-Ala354

2.41

S1

H51-Ala354

1.96

S1

H54-His353

2.45

S2

H58-Tyr520

2.23

S2

H63-Glu384

1.93

S1



Page 30 of 39

Table 6. Thermodynamic parameters for the inhibitors binding to ACE. ∆S

-T∆S

∆H

∆G

Kd

(Kcal/mol)

(Kcal/mol)

(Kcal/mol)

(Kcal/mol)

(nM)

VHW

24.2

-6.73

-4.30

-10.5

20.0

TTW

22.4

-6.23

-2.02

-8.75

377

Lisinopril

25.9

-7.21

-3.21

-10.4

20.0

Peptides


Page 31 of 39


Figure 1.

Amout of peptides

1200 None-repeated peptides

1000

Repeated peptides

800 600 400 200 0

Xie et al.



Page 32 of 39

Figure 2.

1000

1 mg/mL

800

1/V (mg-1⋅mL⋅min)

0.5 mg/mL Control

600 400 200 0

-3

-2

-1

0 -200

1 1/S (mM-1)

2

1 1/S (mM-1)

1.5

(a)

1000

1 mg/mL 1/V (mg-1 ⋅mL⋅ min)

0.5 mg/mL Control

800 600 400 200 0

-2

-1.5

-1

-0.5

0

0.5

-200 (b) Xie et al.


Page 33 of 39


Figure 3. 100

GC

ACE inhibitory activity (%)

G 80

G+IC G+I

60 40 20 0

TTW

VHW (a)

ACE inhibitory activity (%)

100

GC G

80

G+IC G+I

60 40 20 0 TTW

VHW (b)

Xie et al.



Page 34 of 39

Figure 4. ACE inhibitory activity (%)

100 VHW

TTW

90 80 70 60 50 0

4

8

12

16

Time (h)

Xie et al.


20

24

Page 35 of 39


Systolic blood pressure (mmHg)

Figure 5.

260 240 220 200 ∗

*

180

* **

160 0

2

4 Time (h)

Control

Lisinopril

6 VHW

8 TTW

Diastolic blood pressure (mmHg)

(a)

190

170

150

*

* * * 130 0

2 Control

4 Time (h) Lisinopril

6 VHW

(b) Xie et al.


8 TTW


Page 36 of 39

Figure 6.

(a)

(b)

(c)

(d)

Xie et al.


Page 37 of 39


Figure 7.

(a)

(b)



(c) Xie et al.


Page 38 of 39

Page 39 of 39


TOC Graphic


Antihypertensive effects, molecular docking study and isothermal

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