1 Characterization of Angiotensin-Converting Enzyme Inhibitory

Yhiya Amen,. ‡,§. Masashi Kusubata,. †. Kiyoko Ogawa-Goto,. †. Kuniyoshi Shimizu,. ‡ and Shunji Hattori. †. †. Nippi Research Institute o...
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Bioactive Constituents, Metabolites, and Functions

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-Gly-Type Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation Yuki Taga, Osamu Hayashida, Ahmed Ashour, Yhiya Amen, Masashi Kusubata, Kiyoko Ogawa-Goto, kuniyoshi shimizu, and Shunji Hattori J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03648 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 4, 2018

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Journal of Agricultural and Food Chemistry

Characterization of Angiotensin-Converting Enzyme Inhibitory Activity of X-Hyp-GlyType Tripeptides: Importance of Collagen-Specific Prolyl Hydroxylation

Yuki Taga,*,† Osamu Hayashida,† Ahmed Ashour,‡,§ Yhiya Amen,‡,§ Masashi Kusubata,† Kiyoko Ogawa-Goto,† Kuniyoshi Shimizu,‡ and Shunji Hattori†



Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan



Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581,

Japan §

Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516,

Egypt

To whom correspondence should be addressed: Yuki Taga Nippi Research Institute of Biomatrix, 520-11 Kuwabara, Toride, Ibaraki 302-0017, Japan Tel: +81-297-71-3046; Fax: +81-297-71-3041 E-mail: [email protected]

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ABSTRACT

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Hydroxyproline (Hyp) is a collagen-specific amino acid formed by post-translational

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hydroxylation of Pro residues. Various Hyp-containing oligopeptides are transported into the

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blood at high concentrations after oral ingestion of collagen hydrolysate. Here we investigated

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the angiotensin-converting enzyme (ACE) inhibitory activity of X-Hyp-Gly-type tripeptides. In

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an in vitro assay, ginger-degraded collagen hydrolysate enriched with X-Hyp-Gly-type

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tripeptides dose-dependently inhibited ACE, and various synthetic X-Hyp-Gly-type tripeptides

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showed ACE-inhibitory activity. In particular, strong inhibition was observed for Leu-Hyp-Gly,

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Ile-Hyp-Gly, and Val-Hyp-Gly with IC50 values of 5.5, 9.4, and 12.8 µM, respectively.

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Surprisingly, substitution of Hyp with Pro dramatically decreased inhibitory activity of X-Hyp-

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Gly, indicating that Hyp is important for ACE inhibition. This finding was supported by

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molecular docking experiments using Leu-Hyp-Gly/Leu-Pro-Gly. We further demonstrated that

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prolyl hydroxylation significantly enhanced resistance to enzymatic degradation by incubation

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with mouse plasma. The strong ACE-inhibitory activity and high stability of X-Hyp-Gly-type

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tripeptides highlight their potential for hypertension control.

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Keywords: collagen hydrolysate, hydroxyproline, X-Hyp-Gly, ginger, angiotensin-converting

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enzyme

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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Collagen is the most common protein in the body and is particularly abundant in connective

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tissues, such as skin, bone, and tendon. Features of the amino acid sequence of collagen are

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repeating Gly-Xaa-Yaa triplets and numerous post-translational modifications catalyzed by

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specific enzymes before triple helix formation in the endoplasmic reticulum.1 Most Pro residues

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lying at the Yaa position are hydroxylated to 4-hydroxyproline (4-Hyp) (~100 residues/1000

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amino acid residues in type I collagen), contributing to the stabilization of the collagen triple

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helix.2 Other collagen-specific post-translational modifications, including prolyl hydroxylation at

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the Xaa position forming 3-Hyp and lysyl hydroxylation/glycosylation at the Yaa position, are

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rare compared to 4-Hyp (hereinafter simply referred to as Hyp).3

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Oral ingestion of collagen hydrolysates prepared by enzymatic hydrolysis of gelatin has shown

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various beneficial effects, such as increasing bone density,4, 5 modulating lipid metabolism,6, 7

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lowering blood sugar levels,8,

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oligopeptides were identified in blood at markedly high concentrations after oral ingestion of

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collagen hydrolysate.14 The total concentration of Hyp-containing oligopeptides reaches 100 µM

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in the blood after the ingestion,15,

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conferred by the presence of Hyp within the peptide sequence.14, 17-19 Various Hyp-containing

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peptides appear in the blood, including Pro-Hyp, Hyp-Gly, and X-Hyp-Gly-type tripeptides.14, 16-

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18, 20

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oligopeptides, such as growth stimulation of skin fibroblasts,17,

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differentiation,23, 24 and improvement of skin barrier dysfunction.25

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and lowering blood pressure.10-13 In 2005, Hyp-containing

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probably due to their high peptidase/protease resistance

Recent studies have demonstrated physiological activities of these collagen-specific 21, 22

promotion of osteoblast

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Angiotensin-converting enzyme (ACE) is a dipeptidyl carboxypeptidase that converts an

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inactive form of decapeptide, angiotensin I, into a potent vasoconstrictor, angiotensin II.26 This 3

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enzyme also catalyzes inactivation of a vasodilator, bradykinin. Therefore, inhibition of ACE is a

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major target for the prevention and treatment of hypertension. A wide variety of food proteins

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have been reported as a source of ACE-inhibitory peptides.26, 27 For example, well-established

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ACE-inhibitory peptides, Val-Pro-Pro and Ile-Pro-Pro, were isolated from sour milk and

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demonstrated to decrease blood pressure after oral administration.28, 29 To date, various types of

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enzymatic hydrolysates of collagen have been discovered to possess ACE-inhibitory activity and

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in vivo antihypertensive effects.10-13,

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peptides was determined in some studies, such as Gly-Pro-Leu,30, 31 Gly-Pro-Met,30 Gly-Pro-

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Val,31 Met-Gly-Pro,13 and Val-Gly-Pro-Val.32 In addition, Hyp-containing peptides possessing

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high ACE-inhibitory activity were identified in chicken breast muscle hydrolysate (Gly-Phe-

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Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe) and chicken leg collagen hydrolysate (Gly-Ala-Hyp-

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Gly-Leu-Hyp-Gly-Pro).11, 33

30, 31

The structure of collagen-derived ACE-inhibitory

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We recently developed a novel type of collagen hydrolysate using ginger protease.18 The

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unique substrate specificity of ginger protease recognizing Pro and Hyp at the P2 position enables

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efficient generation of X-Hyp-Gly-type tripeptides, which are almost undetectable in

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commercially available collagen hydrolysates. Although X-Hyp-Gly-type tripeptides appear in

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blood at high concentrations after oral ingestion regardless of the types of collagen hydrolysates,

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the ginger-degraded collagen hydrolysate (GDCH) shows more higher absorption of this type of

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tripeptide.18 Iwai et al. investigated the ACE-inhibitory activity of Hyp-containing peptides

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detected in human blood after oral intake of chicken collagen hydrolysate, such as Ala-Hyp (IC50

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= 0.177 mM) and Pro-Hyp (IC50 = 18.118 mM).34 The IC50 value against ACE was also

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measured for some X-Hyp-Gly-type tripeptides (0.601 mM for Ser-Hyp-Gly, 0.711 mM for Ala-

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Hyp-Gly, 1.129 mM for Pro-Hyp-Gly, and 82.330 mM for Glu-Hyp-Gly).34 The inhibitory 4

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activity of X-Hyp-Gly-type tripeptides was moderate in their study. However, in the current

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study, we found that X-Hyp-Gly-type tripeptides where X is branched-chain aliphatic amino

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acids strongly inhibit ACE. We also showed that Hyp is a contributing factor to the ACE-

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inhibitory activity of X-Hyp-Gly.

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MATERIALS AND METHODS

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Chemicals. ACE from porcine kidney and hippuric acid (Hip) were purchased from Sigma-

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Aldrich (St. Louis, MO, USA). Hippuryl-histidyl-leucine (HHL) was purchased from Peptide

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Institute (Osaka, Japan), and N-benzoyl-d5-glycine (d5-Hip) was purchased from CDN Isotopes

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(Quebec, Canada). Gly-Ala-Hyp (>99% purity), Ala-Pro-Gly (>99% purity), and Ile-Pro-Pro

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(>98% purity) were purchased from Bachem (Bubendorf, Switzerland), and other peptides

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(>95% purity) were custom synthesized by AnyGen (Kwangju, Korea).

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Ethics Statement. All animal studies were approved by the Experimental Ethical Committee of Nippi Research Institute of Biomatrix.

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Preparation of Collagen Hydrolysates. GDCH was prepared from bovine bone gelatin using

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ginger protease as reported previously,18 with a slight modification for industrial production.

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GDCH was heated at 81 °C and pH 5.5 for 6 h to prepare heat-treated GDCH (H-GDCH). The

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collagen hydrolysates were dissolved in 0.1% formic acid for liquid chromatography–mass

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spectrometry (LC–MS) analysis of oligopeptides.

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ACE Inhibition Assay. ACE-inhibitory activity of collagen hydrolysates and synthetic

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peptides was measured as reported previously with some modifications.35, 36 Peptide samples at

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varied concentrations were pre-incubated with ACE in 75 mM Tris-HCl (pH 8.3) at 37 °C for 10

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min, and the reaction was started by adding HHL as a substrate. The final concentrations of ACE

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and HHL in the reaction mixture were 1 mU/mL and 0.1 mM, respectively. Aliquots were taken

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at 5, 10, 20, and 30 min during incubation at 37 °C, and an equal volume of 1% formic acid

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containing d5-Hip was added to stop the reaction. An external calibration curve was prepared

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using a standard of Hip after mixing with an equal volume of 1% formic acid containing d5-Hip.

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Hip was quantified by LC–MS in multiple reaction monitoring (MRM) mode using a hybrid

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triple quadrupole/linear ion trap 3200 QTRAP mass spectrometer (AB Sciex, Foster City, CA)

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coupled to an Agilent 1200 Series HPLC system (Agilent Technologies, Palo Alto, CA).

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Samples were loaded onto an Ascentis Express C18 HPLC column (5 µm particle size, L × I.D.

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150 mm × 2.1 mm; Supelco, Bellefonte, PA) at a flow rate of 500 µL/min and separated by a

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binary gradient as follows: 100% solvent A (0.1% formic acid) for 2 min, linear gradient of 0–

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70% solvent B (100% acetonitrile) for 4 min, 90% solvent B for 2 min, and 100% solvent A for

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2 min. The MRM transitions of Hip and d5-Hip were m/z 180.2→105.2 and m/z 185.2→110.2,

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respectively, with collision energy of 17 V. Concentrations of liberated Hip in test samples were

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estimated using the external calibration curve with correction using d5-Hip.

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Enzymatic activity of ACE was calculated from the time course of the liberated Hip

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concentration (nmol/min), and inhibitory activity was calculated from the ACE activity of the

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sample and blank control as follows: inhibitory activity (%) = 100 × (blank − sample)/blank. The

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IC50 value was determined by plotting the logarithm of ACE-inhibitory activity against peptide

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sample concentration.

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The mode of ACE inhibition and Ki value were determined by Dixon plot37 under the same

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reaction conditions with two substrate concentrations of 0.1 and 1.0 mM. Three peptide

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concentrations were used for the reaction (0, 1.9, and 4.7 µM for Leu-Hyp-Gly and 0, 50.1, and

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125.2 µM for Leu-Pro-Gly).

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Peptide Degradation Using Mouse Plasma. The experiment was performed as reported

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previously.18 In brief, a synthetic tripeptide mixture (Ala-Hyp-Gly, Leu-Hyp-Gly, Ala-Pro-Gly,

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Leu-Pro-Gly, and Ile-Pro-Pro) was incubated with fresh plasma prepared from male ICR mice

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(15 weeks of age; Japan SLC, Shizuoka, Japan) at a concentration of 20 µg/mL each at 37 °C.

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Time-course samples were collected at 0, 15, 30, 60, and 90 min during the incubation. The

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reactants were deproteinized by adding three volumes of ethanol, and the ethanol-soluble

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fractions were diluted with 0.1% formic acid for LC–MS analysis.

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LC–MS Analysis of Oligopeptides. We quantified oligopeptides in collagen hydrolysates and

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peptide degradation samples by LC–MS in MRM mode. Sample separation was performed using

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an Ascentis Express F5 HPLC column (5 µm particle size, L × I.D. 250 mm × 4.6 mm; Supelco)

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at a flow rate of 400 µL/min with a binary gradient as follows: 100% solvent A (0.1% formic

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acid) for 7.5 min, linear gradient of 0–90% solvent B (100% acetonitrile) for 12.5 min, 90%

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solvent B for 5 min, and 100% solvent A for 5 min. The MRM transitions of the oligopeptides

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are shown in Table S1.

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Molecular docking. A molecular binding experiment was designed to predict the binding

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mode of Leu-Hyp-Gly and Leu-Pro-Gly at the active sites of ACE. The crystal structure of ACE

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was downloaded from the Protein Data Bank (www.rcsb.org; access code 4CA5)38 and then

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imported into the work place of the software AutoDock Vina implemented in PyRx.39 The ligand

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with the most favorable binding energy (increasingly negative value) was considered a potential

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inhibitor.

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RESULTS

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ACE-Inhibitory Activity of Collagen Hydrolysates Prepared Using Ginger Protease. We

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used two types of collagen hydrolysates for the ACE inhibition assay. GDCH was prepared by

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enzymatic hydrolysis of bovine gelatin using ginger protease, and GDCH was further subjected

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to heat treatment at 81 °C for 6 h to prepare H-GDCH. The contents of X-Hyp-Gly- and X-Pro-

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Gly-type-tripeptides in these collagen hydrolysates are shown in Table 1. Consistent with

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previous observations,18, 40, 41 we detected substantial amounts of various X-Hyp-Gly in GDCH.

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Pro-Hyp-Gly was only slightly generated because ginger protease cannot cleave the Gly-Pro

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bond.18 We also detected small amounts of X-Pro-Gly originating from partial hydroxylation of

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Pro residues. In contrast, all X-Hyp-Gly-type tripeptides were significantly diminished in H-

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GDCH due to efficient cyclization into cyclo(X-Hyp) through heating as reported recently.40

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Similar heat-induced decreases were also observed for X-Pro-Gly, probably due to conversion

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into cyclo(X-Pro). Gly-Pro-Y-type tripeptides, which are another major type of oligopeptides in

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GDCH, were only slightly decreased by the heat treatment (data not shown) as reported

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previously.41

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We used LC–MS to detect Hip liberated from HHL by porcine kidney ACE for measurement of

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ACE-inhibitory activity of the collagen hydrolysates. Figure 1 shows ACE inhibition curves of

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GDCH and H-GDCH. Both collagen hydrolysates dose-dependently inhibited ACE activity, and

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the IC50 value of GDCH (0.210 mg/mL) was markedly lower than that of H-GDCH (0.336

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mg/mL). Since the most striking difference between GDCH and H-GDCH was the X-Hyp-Gly

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content (Table 1), it was suggested that this type of Hyp-containing tripeptide has high ACE-

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inhibitory activity.

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ACE-Inhibitory Activity of X-Hyp-Gly-Type Tripeptides. We investigated ACE-inhibitory

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activity of X-Hyp-Gly using a series of synthetic peptides (Table 1). The IC50 value of Ile-Pro-

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Pro used as a positive control was 0.9 µM. This was lower than a previously reported value (5

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µM) estimated by a conventional method with UV detection of Hip,28 probably due to the low

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substrate concentration achieved by sensitive LC–MS detection. Among the X-Hyp-Gly-type

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tripeptides, Leu-Hyp-Gly most strongly inhibited ACE (IC50 = 5.5 µM). This ACE-inhibitory

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activity was lower than, but not far from, that of the known ACE-inhibitory peptide, Ile-Pro-Pro.

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In addition, higher ACE-inhibitory effects were also detected for Ile-Hyp-Gly and Val-Hyp-Gly

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with IC50 values of 9.4 µM and 12.8 µM, respectively. Other X-Hyp-Gly-type tripeptides,

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including Ala-Hyp-Gly, Glu-Hyp-Gly, Phe-Hyp-Gly, Pro-Hyp-Gly, and Ser-Hyp-Gly, displayed

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moderate inhibition (IC50 = 130.6–404.5 µM). ACE-inhibitory activity of Leu-Pro-Gly was

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extremely low (IC50 = 373.8 µM) compared to that of Leu-Hyp-Gly. Other X-Pro-Gly-type

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tripeptides (X = Ala, Pro, Ser, and Val) also showed 5- to 10-fold decreases in ACE-inhibitory

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activity compared to corresponding X-Hyp-Gly-type tripeptides. In contrast, ACE-inhibitory

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activity of another type of Hyp-containing tripeptide, Gly-X-Hyp, was lower than that of

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corresponding Gly-X-Pro-type tripeptides, showing 5- and 16-fold increases in IC50 values for

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Gly-Ala-Hyp and Gly-Leu-Hyp, respectively (Table S2). A pronounced change was observed by

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removing the C-terminal Gly residue from Leu-Hyp-Gly (IC50 = 3462.1 µM for Leu-Hyp).

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We estimated the mode of ACE inhibition and Ki value for Leu-Hyp-Gly and Leu-Pro-Gly by

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Dixon plot (Fig. 2). Both peptides were determined to be competitive inhibitors, and the Ki value

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significantly differed between the peptides (5.5 µM for Leu-Hyp-Gly and 270.6 µM for Leu-Pro-

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Gly). These values were close to their IC50 values against ACE.

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Molecular docking 9

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Molecular docking is a computational procedure that is useful for predicting the optimal pose

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and binding affinity of a ligand (tripeptide) when it interacts with a target protein (ACE) to form

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a stable complex. Guided by the docking scores, it is noted that Leu-Hyp-Gly showed excellent

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binding activity with a binding energy value of −7.7 kcal/mol compared to that of Leu-Pro-Gly

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(−7.1 kcal/mol). Careful investigation of the interactions of Leu-Hyp-Gly with the target enzyme

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indicated that the hydroxyl group showed strong binding interactions with the Tyr-523 residue of

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the amino acid sequence of the binding site of ACE (Fig. S1), while the Tyr residue did not

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interact with Leu-Pro-Gly (Fig. S2). These results could at least in part explain the superior

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ACE-inhibitory activity of Leu-Hyp-Gly over that of Leu-Pro-Gly.

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Stability of X-Hyp-Gly-Type Tripeptides in Mouse Plasma. We previously showed that Ala-

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Hyp-Gly was highly stable as well as Pro-Hyp in mouse plasma, while Gly-Pro-Ala and Gly-

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Ala-Hyp were rapidly degraded.18 The data suggested that X-Hyp-Gly is resistant to enzymatic

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digestion due to the presence of Hyp located in the middle position. In the present study, we

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similarly studied the stability of Ala-Hyp-Gly and Leu-Hyp-Gly by incubation with mouse

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plasma using comparisons among Ala-Pro-Gly, Leu-Pro-Gly, and Ile-Pro-Pro. As shown in Fig.

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3, Ala-Pro-Gly and Leu-Pro-Gly were completely degraded after incubation for 60 min, but

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stability significantly increased for corresponding X-Hyp-Gly. Ala-Hyp-Gly remained

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unchanged during the 90 min incubation, consistent with the previous observation.18 In addition,

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Leu-Hyp-Gly exhibited only modest reduction comparable to Ile-Pro-Pro, which was reported to

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have high resistance to enzymatic degradation.42 The high stability of X-Hyp-Gly-type

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tripeptides would help exert their high ACE-inhibitory activity in vivo.

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DISCUSSION 10

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In the present study, we demonstrated that X-Hyp-Gly-type tripeptides, especially of which X

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is branched-chain aliphatic amino acids, have high ACE-inhibitory activity. It was clearly shown

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that prolyl hydroxylation is critical for ACE inhibition by X-Hyp-Gly. Saiga et al. previously

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identified Hyp-containing ACE-inhibitory peptides, including Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-

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Leu-Hyp-Gly-Phe (IC50 = 42.4 µM) and Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro (IC50 = 29.4 µM),

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derived from chicken collagen.11,

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antihypertensive effects against spontaneously hypertensive rats.43,

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evidence that these peptides are transported into the blood in an intact form. We speculate that X-

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Hyp-Gly-type tripeptides are generated by partial hydrolysis in the gastrointestinal tract and

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blood and are main contributors to the antihypertensive effects. In fact, Leu-Hyp-Gly showing

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the highest ACE-inhibitory activity is present both within the previously established Hyp-

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containing antihypertensive peptides.

33

The two kinds of Hyp-containing peptides showed 44

However, there is no

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Surprisingly, substitution of Hyp with Pro dramatically decreased the ACE-inhibitory activity

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of X-Hyp-Gly regardless of the amino acid residue at the N-terminal. Alemán et al. investigated

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the effect of Hyp on ACE-inhibitory activity using Gly-Pro-X-Gly-X-X-Gly-Phe-X-Gly-Pro-X-

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Gly-X-Ser where X was either Leu or Hyp.45 While higher inhibition of ACE was observed when

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all X were Leu, a corresponding peptide with X being Hyp did not show any inhibitory activity.

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In contrast, Saiga et al. suggested the importance of Hyp for ACE inhibition by replacing all Hyp

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within Gly-Phe-Hyp-Gly-Thr-Hyp-Gly-Leu-Hyp-Gly-Phe with Pro, which decreased the

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inhibitory activity 10-fold.33 However, in a subsequent paper, they concluded that Phe at the C-

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terminal most critically affects the ACE-inhibitory activity of the peptide.43 These previous data

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were obtained for the relatively long peptides. In contrast, our findings are based on observations

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of tripeptides, which would more directly reflect the effect of Hyp on ACE-inhibitory activity. In 11

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addition to X-Hyp-Gly-type tripeptides, we analyzed Gly-Ala-Hyp and Gly-Leu-Hyp with

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comparison to Gly-Ala-Pro and Gly-Leu-Pro, which were previously reported to strongly inhibit

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ACE.46, 47 The ACE-inhibitory activity was dramatically decreased by prolyl hydroxylation in the

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case of this type of Hyp-containing tripeptide, indicating that the position of Hyp is also

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important for the enhancement of ACE inhibition. Molecular docking experiments comparing

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Leu-Hyp-Gly with Leu-Pro-Gly suggested that the hydroxyl group of Hyp contributes to the

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ACE-inhibitory activity of X-Hyp-Gly through its interaction with the enzyme. However, further

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studies using various types and lengths of Hyp-containing peptides are needed to clarify the

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essential requirements for enhancement of ACE inhibition by prolyl hydroxylation.

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Although a relationship between the peptide primary structure and ACE-inhibitory activity has

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not been completely elucidated, common features can be found among ACE-inhibitory peptides;

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for example, peptides having hydrophobic amino acids at terminal positions tend to show high

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ACE inhibition.26, 48, 49 Among X-Hyp-Gly-type tripeptides, Leu-Hyp-Gly, Ile-Hyp-Gly, and Val-

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Hyp-Gly exhibited especially strong ACE-inhibitory activity in this study. All of these peptides

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have branched-chain aliphatic amino acids at the Xaa position. This is consistent with previous

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observations that branched-chain aliphatic amino acids at the N-terminal are suitable for binding

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to ACE.47-49 A dipeptide, Leu-Hyp, displayed extremely weak ACE-inhibitory activity that was

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600-fold lower than that of Leu-Hyp-Gly, indicating that C-terminal Gly is also important for

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ACE inhibition by X-Hyp-Gly-type tripeptides. Taken together, all constituents of X-Hyp-Gly

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are indispensable to exert high ACE-inhibitory activity.

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One of the advantages of collagen-derived Hyp-containing peptides is their high

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bioavailability. While other food-derived ACE-inhibitory peptides, including Ile-Pro-Pro from

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milk casein and Val-Tyr from sardine muscle, are detected in blood at nM levels after oral 12

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ingestion,50, 51 X-Hyp-Gly-type tripeptides show remarkably higher blood concentrations at µM

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levels.15, 16 We consider that two factors contribute to the high bioavailability of X-Hyp-Gly. One

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is that -X-Pro-Gly- sequences are frequently observed in collagen (Fig. S3). Since most Pro

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residues at the Yaa position are hydroxylated, large amounts of X-Hyp-Gly are potentially

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generated during production of collagen hydrolysate and in the gastrointestinal tract after oral

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ingestion. In contrast, only one -Ile-Pro-Pro- sequence is present in bovine β- and κ-casein,

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respectively, and bovine αs1- and αs2-casein do not have the sequence. Although Ile-Hyp-Gly and

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Val-Hyp-Gly displayed higher ACE-inhibitory activity next to Leu-Hyp-Gly, their contents in

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GDCH were small due to the low occurrence of -Ile-Pro-Gly- and -Val-Pro-Gly- sequences in

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bovine type I collagen (Fig. S3). The use of other animal sources of collagen may increase the

256

yield of these peptides.

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Another factor responsible for the high bioavailability of X-Hyp-Gly-type tripeptides is their

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high resistance to peptidase/protease. A previous study reported that Val-Pro-Pro and Ile-Pro-Pro

259

were not very susceptible to digestive enzymes and Caco-2 cells expressing various brush-border

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peptidases, suggesting that orally administered these antihypertensive tripeptides are absorbed

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intact into the blood.42 We showed that Ile-Pro-Pro was stable but slightly decreased (~30%)

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during incubation for 90 min with mouse plasma. Although Ala-Pro-Gly and Leu-Pro-Gly were

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rapidly degraded, the stability of corresponding X-Hyp-Gly-type tripeptides was higher (Ala-

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Hyp-Gly) or comparable (Leu-Hyp-Gly) to that of Ile-Pro-Pro. The high bioavailability of the

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collagen-derived Hyp-containing peptides possessing high ACE-inhibitory activity highlights

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their potential for hypertension control. Further in vivo experiments are warranted to evaluate the

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antihypertensive effects of X-Hyp-Gly-type tripeptides and GDCH enriched with the peptides.

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ABBREVIATIONS USED

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Hyp, hydroxyproline; ACE, angiotensin-converting enzyme; GDCH, ginger-degraded collagen

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hydrolysate; Hip, hippuric acid; HHL, hippuryl-histidyl-leucine; H-GDCH, heat-treated ginger-

272

degraded collagen hydrolysate; LC–MS, liquid chromatography–mass spectrometry; MRM,

273

multiple reaction monitoring

274 275

Supporting Information. Figure S1: Molecular docking between Leu-Hyp-Gly and ACE.

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Figure S2: Molecular docking between Leu-Pro-Gly and ACE. Figure S3: Amino acid

277

sequences of the triple helical region of bovine type I collagen. Table S1: MRM transitions of

278

dipeptides and tripeptides. Table S2: Effect of prolyl hydroxylation on ACE-inhibitory activity

279

of Gly-X-Pro-type tripeptides. This material is available free of charge via the Internet at

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http://pubs.acs.org.

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Figure captions

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Figure 1. ACE inhibition rate of (A) GDCH and (B) H-GDCH. The data represent the mean ±

437

SD of three separate measurements.

438 439

Figure 2. Dixon plot of (A) Leu-Hyp-Gly and (B) Leu-Pro-Gly. The kinetic assays were

440

performed at substrate concentrations of (▲) 0.1 and (■) 1.0 mM. The data represent the mean ±

441

SD of three separate measurements.

442 443

Figure 3. Residual ratio of tripeptides after incubation with mouse plasma. Ala-Hyp-Gly, Leu-

444

Hyp-Gly, Ala-Pro-Gly, Leu-Pro-Gly, and Ile-Pro-Pro were incubated with freshly prepared

445

mouse plasma at 37 °C for 0, 15, 30, 60, and 90 min. The residual ratio was calculated using the

446

peak area of the respective peptides in LC–MS analysis. The data represent the mean ± SD (n =

447

3).

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Tables Table 1. ACE-inhibitory Activity of Collagen-derived Oligopeptides and Their Contents in GDCHs

a

Ala-Hyp-Gly Ala-Pro-Gly

IC50 (µM)a,c 151.3 ± 6.8 1293.3 ± 286.6

Content in GDCH (mg/g)b,c 2.205 ± 0.087 0.204 ± 0.008

Content in H-GDCH (mg/g)b,c 0.197 ± 0.017 0.056 ± 0.004

Glu-Hyp-Gly

404.5 ± 51.4

1.204 ± 0.057

0.310 ± 0.009

Ile-Hyp-Gly

9.4 ± 2.2

0.076 ± 0.002

0.018 ± 0.002

Leu-Hyp-Gly Leu-Pro-Gly Leu-Hyp

5.5 ± 1.5 373.8 ± 77.2 3462.1 ± 1012.2

1.857 ± 0.254 0.008 ± 0.001 ND

0.131 ± 0.018 0.002 ± 0.001 ND

Phe-Hyp-Gly

210.9 ± 37.1

0.380 ± 0.022

0.013 ± 0.001

Pro-Hyp-Gly Pro-Pro-Gly

257.8 ± 41.9 1372.6 ± 297.8

0.017 ± 0.001 ND

0.010 ± 0.004 ND

Ser-Hyp-Gly Ser-Pro-Gly

130.6 ± 4.5 1029.5 ± 163.0

0.183 ± 0.021 0.071 ± 0.003

ND ND

Val-Hyp-Gly Val-Pro-Gly

12.8 ± 0.7 125.9 ± 4.5

0.018 ± 0.001 0.093 ± 0.008

0.007 ± 0.001 0.037 ± 0.001

Ile-Pro-Pro 0.9 ± 0.2 The data were obtained by ACE inhibition assay using synthetic peptides. bThe data were

obtained by LC–MS analysis of the collagen hydrolysates. ND, not detected. cThe data represent the mean ± SD of three separate measurements.

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