Stability and Transport of Spent Hen-Derived ACE-Inhibitory Peptides

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Bioactive Constituents, Metabolites, and Functions

Stability and transport of spent hen-derived ACE-inhibitory peptides IWHHT, IWH, and IW in human intestinal Caco-2 cell monolayers Hongbing Fan, Qingbiao Xu, Hui Hong, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03956 • Publication Date (Web): 03 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

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Journal of Agricultural and Food Chemistry

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Stability and transport of spent hen-derived ACE-inhibitory peptides IWHHT, IWH, and

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IW in human intestinal Caco-2 cell monolayers

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Hongbing Fan a, Qingbiao Xu a,b, Hui Hong a,c, Jianping Wu a,*

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a

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Alberta, Edmonton, Alberta T6G 2P5, Canada

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b

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430070, China

Department of Agricultural, Food and Nutritional Science, 4-10 Ag/For Building, University of

College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan

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c

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100083, China

College of Food Science and Nutritional Engineering, China Agricultural University, Beijing

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*Corresponding author: Tel.: +1 780 492 6885; fax: +1 780 492 4265.

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E-mail address: [email protected] (J. Wu)

1 ACS Paragon Plus Environment


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Abstract

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Ile-Trp-His-His-Thr (IWHHT), initially identified as an ACE inhibitory peptide, was shown

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to have antioxidant and anti-inflammatory activities in cells and blood pressure lowering activity

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in animals. IWHHT was degraded into IWH and IW during simulated gastrointestinal digestion.

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The purpose of this study was to investigate the stability, permeability, and transport pathways of

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IWHHT, IWH and IW, across intestinal epithelium using human intestinal Caco-2 cell

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monolayers. IWHHT, IWH, and IW were partly degraded by aminopeptidase N and/or

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dipeptidyl peptidase IV, but they were transported intact, with apparent permeability coefficients

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of (22.0 ± 1.42) ´ 10-8, (37.5 ± 1.11) ´ 10-8, and (19.6 ± 0.62) ´ 10-8 cm s-1, respectively. IWH

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was transported via both of PepT1 and paracellular route, while IW was via PepT1 and IWHHT

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was via paracellular route only. This study suggested that all three peptides could pass through

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the intestinal epithelium and that the degraded IWH and IW might also contribute to the

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antihypertensive activity of IWHHT.

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Keywords: transepithelial transport; spent hen; ACE inhibitory peptides; Caco-2 cells; brush

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border membrane peptidase.


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Introduction

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Hypertension, afflicting more than 20% of adults worldwide, is a known risk factor for

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cardiovascular diseases.1 Although the etiology of hypertension is complex and is not fully

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understood, renin-angiotensin system (RAS) is a key regulator of blood pressure.2 Activation of

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RAS by angiotensin-converting enzyme (ACE) converts angiotensin (Ang) I into Ang II, a

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potent vasoconstrictor that leads to hypertension. Pharmaceutical drugs targetting inhibition of

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ACE have been proved effective for treating hypertension but are associated with adverse effects

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such as dry cough and angioedema.3 Thus, food-derived alternatives especially ACE inhibitory

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(ACEi) peptides have gained increasing interests.2

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IWHHT (Ile-Trp-His-His-Thr) was an ACEi peptide identified from spent hen myofibrils

40

using in silico approach; its presence was then confirmed using the conventional activity-guided

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fractionation and identification.4,5 This peptide also showed anti-inflammatory and anti-oxidant

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activities in endothelial cells.4 Interestingly, the same peptide was also identified from bonito and

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showed blood pressure lowering activity in spontaneously hypertensive rats (SHRs),6 although

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IWHHT was not stable and digested into IWH and IW in simulated gastrointestinal digestion.4

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IWH and IW showed comparatively ACE-inhibitory, anti-inflammatory, and antioxidant

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activities as those of IWHHT.4,6 It is conceived that IWHHT, IWH, and IW could co-exist in the

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small intestine after oral administration.

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Only when peptides pass through the intestinal epithelium and remain as active forms, their

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bioactivities can be exerted; however, transport of IWHHT, IWH, and IW remain unknown. The

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intestinal epithelium is composed of a vast array of well-differentiated and polarized epithelial

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cells.7 Human colon carcinoma cell line (Caco-2) is a widely-used model to mimic the transport

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of drugs and nutrients. After differentiation, Caco-2 cells form monolayers with tight junctions



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(TJs) and function like mature enterocytes, with stable expression of transporters and brush

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border membrane (BBM) peptidases.8 Thus, the stability of peptides against BBM peptidases is

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the pre-requisite for them to be absorbed. Many antihypertensive peptides, such as IRW, IQW,

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AAATP, AAPLAP, and KPVAAP, have been either partially or completely degraded after

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transport.9-11

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Transport of many food-derived bioactive peptides including from egg, milk, and meat has

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been investigated.9-22 Peptide transport pathways are determined by peptide length, sequence,

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hydrophobicity, and amino acid compositions.12,13 At least three pathways are involved in

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peptide transport. H+-coupled peptide transporter 1 (PepT1) participates in transporting small

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peptides (di-/tri-peptides),14 while, transcytosis route is preferred for proteins or larger peptides

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or certain peptides, which are easily internalized on the apical surface.11,15 Paracellular diffusion

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route exists in transporting peptides with various length, such as WQ,16 RWQ,16 VPP,17 IRW,9

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AHLL,15 VGPV,18 GPRGF,18 RVPSL,19 QIGLF,20 KVLPVP,21 and GAXGLXGP.22 Although

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the molecular mechanism of paracellular diffusion is not clear, it appears to be preferred by small

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hydrophilic peptides.2 Additionally, peptides might be transported via either one or multiple

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routes. Given IWHHT, IWH, and IW share a similar N-terminal structure but possess different

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charges, peptide length, and hydrophobicity, it is interesting to see how they perform differently

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in transport study. The aim of this study was to investigate the stability, permeability, and

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transport pathways of IWHHT, IWH, and IW across the intestinal epithelium using human

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intestinal Caco-2 cell monolayers.

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Materials and Methods Materials. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), 0.25%

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(w/v) trypsin-0.53 mM EDTA, Hanks balanced salt solution (HBSS with Ca and Mg), 4-(2-68


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hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), nonessential amino acids (NEAA), and

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penicillin-streptomycin were obtained from Gibco Invitrogen (Burlington, ON, Canada). HPLC-

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grade water, acetonitrile (ACN), and trifluoroacetic acid (TFA) were purchased from Fisher

79

Scientific (Ottawa, ON, Canada). Dimethyl sulfoxide (DMSO), sodium azide, wortmannin,

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cytochalasin

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Tris(hydroxymethyl)aminomethane (Tris), and aminopeptidase N (164599) were obtained from

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Sigma (Oakville, ON, Canada). AlamarBlue was purchased from Thermo Scientific (Burlington,

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ON, Canada). Peptides (IWHHT, IWH, IW, and Gly-Pro) were synthesized in Genscript Corp

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(purity: > 97%; Piscataway, NJ, USA). Diprotin A (Ile-Pro-Ile, ab145599) was purchased from

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Abcam (Cambridge, UK). Caco-2 cells (HTB-37) were obtained at passage 18 from American

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Type Culture Collection (Manassas, VA, USA). Transwell plate (12-well, 0.4 µm polyester

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membranes) was purchased from Corning (Corning, NY, USA).

D,

2-(N-Morpholino)ethanesulfonic

acid

(MES),

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Cell culture. Caco-2 cells at passages 22 to 30 were seeded onto the semipermeable

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polyester insert of a 12-well transwell at a density of 1.0 × 105 cells/cm2, with 10% DMEM

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containing 10% FBS, 1% NEAA, and 1% penicillin-streptomycin (cell culture medium). Culture

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medium at both apical (0.5 mL) and basolateral chamber (1.5 mL) were changed every 2 days

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until day 21. After one week seeding, transepithelial electrical resistance (TEER) was monitored

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every 2 days using an ohmmeter (World Precision Instruments, Sarasota, FL, USA) and, on day

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21, only wells with TEER values higher than 400 Ω/cm2 were used for transport experiments.

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Caco-2 cells were incubated at 37 °C in a 100% humidified atmosphere with 5% CO2 for all

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transport processes.

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Cytotoxicity assay. Cell viability was detected by alamarBlue fluorescence assay followed

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the protocol provided by Thermo Fisher Scientific (Burlington, ON, Canada). Cells were seeded



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on a 96-well plate at a concentration of 1.0 × 104 cells/well. After 24 h incubation with 10 mM

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peptides, 200 µL of 10% alamarBlue (dissolved in culture medium) were added to the wells and

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incubated for another 4 h (protected from direct light). The control was without any peptide

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treatment. The fluorescence signal was detected at 590 nm; the excitation wavelength was at 570

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nm.

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Transepithelial transport study. To better mimic the acidic microenvironment at the apical

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surface, HBSS was adjusted to pH 6.0 (by 25 mM MES and Tris) for the apical side and to pH

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7.4 (by 25 mM HEPES and Tris) for the basolateral side.11 All stability and transport tests

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incorporated 0.5 mL apical HBSS (pH 6.0) and 1.5 mL basolateral HBSS (pH 7.4) in each well,

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respectively. IWHHT, IWH, and IW were dissolved in the apical HBSS and re-adjusted to pH

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6.0 (using MES and Tris) to avoid pH shift before adding to the apical chambers. All wells were

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tested the TEER (> 400 Ω/cm2) values before and after transport study.

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1) Stability test and apical-to-basolateral transport. Caco-2 cells were gently washed 3 times

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with pre-warmed (37 °C) HBSS (pH 6.0 or 7.4), followed by pre-incubating for another 30 min.

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Peptide samples (5 mM) were then added to the apical chambers. An aliquot of 200 µL sample

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from the basolateral chamber was collected at selected time intervals (0, 30, 60, 90, and 120 min).

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After each sampling, 200 µL fresh HBSS (pH 7.4) was added to the basolateral chamber. The

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apparent permeability coefficients (Papp, cm s-1) and accumulated concentrations of peptides

117

were calculated based on formulas reported previously.2,23 Treatment with only HBSS was used

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as a control. Ultra-performance liquid chromatography (UPLC) and liquid chromatography-mass

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spectrometry/mass spectrometry (LC-MS/MS) were used to determine peptide concentrations

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and sequences, respectively.


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2) Peptidase inhibitor. Diprotin A, an inhibitor of dipeptidyl peptidase IV (DPP IV, EC

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3.4.14.5), was used to study the role of DPP IV in hydrolyzing peptides.24 Caco-2 monolayers

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were pre-incubated with 1 mM diprotin A for 30 min and followed by co-incubation with 5 mM

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peptides. After 60 min incubation, samples from the basolateral chambers were collected and

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analyzed by UPLC.

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3) Transport pathway study. PepT1 competitor (Gly-Pro, 10 mM), ATP synthesis inhibitor

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(sodium azide, 10 mM), TJs disruptor (cytochalasin D, 1 µg/mL), and transcytosis inhibitor

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(wortmannin, 1 µM) were added to the apical chambers.11,20 Gly-Pro was prepared in HBSS (pH

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6.0), while sodium azide, cytochalasin D, and wortmannin were dissolved in DMSO and then

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diluted to the above final concentrations (with 0.05% DMSO in HBSS) for transport study. The

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control was set with only HBSS (with 0.05% DMSO). To better understand the effects of these

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compounds on transport, they were pre-incubated 30 min with cells, followed by co-incubation

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of peptides (5 mM) for another 60 min. Samples from the basolateral chambers were collected

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and analyzed using UPLC.

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Stability of peptide against aminopeptidase N (APN). One-half milliliter of peptide sample

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(3 mM) and 20 µL of APN solution (0.3 U) were co-incubated at 37 °C for 60 min in a 2-mL

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polypropylene centrifuge tube. The reaction was performed using an Eppendorf Thermomixer R

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(Brinkmann Instruments, NY, USA) with continuous agitation at 450 rpm. Then, the mixture

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was heated in boiling water for 10 min to terminate the reaction, and analyzed by UPLC. Both

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peptide and APN solutions were prepared and reacted in HBSS (pH 6.0).

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UPLC Analysis. The peptides were quantified using a Waters Acquity UPLC system

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(Waters, Milford, MA, USA), equipped with an Acquity PDA eλ detector and an Acquity UPLC

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BEH C18 column (1.7 µm, 2.1 × 100 mm). Chromatographic separation was performed using a



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gradient elution of chromatographic grade water (0.1% TFA, solvent A) and ACN (0.1% TFA,

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solvent B) as follows: 1% B (0-3 min) and 1%-23% B (3-28 min), with a flow rate of 0.3

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mL/min. Injection volume was 10 µL; peaks were detected at 220 nm. Peptides were quantified

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based on their standard curves and that of Trp is shown in Figure S1).

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LC-MS/MS. Transported peptides collected at the basolateral chambers were analyzed using

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a nanoAcquity UPLC system, connected with a Micromass Quadrupole Time-of-Flight (Q-TOF)

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premier mass spectrometer and an Atlantis dC18 (75 µm × 150 mm, 3 µm) UPLC column

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(Waters, Milford, MA, USA). The mobile phases were LC/MS grade water (0.1% formic acid,

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solvent A) and ACN (0.1% formic acid, solvent B). Samples were pre-desalted, dissolved in

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solvent A, injected (5 µL), and separated at a flow rate of 0.35 mL/min using the following

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gradient: 1% B (0-2 min), to 60% B (2-40 min), and to 95% B (40-55 min). Samples were

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ionized using electrospray ionization technique (ESI) in a positive ion mode (a capillary voltage

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of 3.4 kV and a source temperature of 100 °C). Peptide mass was detected using a Q-TOF

157

analyzer and the spectra were acquired with m/z ranges of 200-1200 in MS mode and 50-1000 in

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MS/MS mode, respectively. Data acquisition and interpretation were carried out by Mass Lynx

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software version 4.1 (Waters), coupled with manual de novo sequencing.

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2.10 Statistical analysis

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All analyses were run in triplicate (except for cytotoxicity replicated 6 times). Data were

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expressed as mean values with standard deviations of means. The apical stability of peptides,

163

effect of diprotin A on peptide transport, and effect of APN on IW were analyzed by t-test; all

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other results were performed by one-way analysis of variance (ANOVA) followed by Dunnett’s

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multiple test (except for apical-to-basolateral transport analyzed by two-way ANOVA by


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Tukey’s test) using GraphPad Prism version 6 (San Diego, CA, USA). The significant level was

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set at 5%.

168

Results

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Cytotoxicity of IWHHT, IWH, and IW. Peptides might exert cytotoxicity on cells after

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prolonged incubation.11 Therefore, prior to transport experiments, effects of peptides on viability

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of Caco-2 cells were performed (Figure S2). The results indicated that cell viability was not

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affected (P > 0.05) by incubating peptides at 10 mM for 24 h, which were higher than the peptide

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dose (5 mM) and much longer than the duration (2 h) in later transport studies. Thus, IWHHT,

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IWH, and IW were considered not cytotoxic against Caco-2 cells.

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Stability of IWHHT, IWH, and IW in Caco-2 monolayers. Peptides (5 mM) were added

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to the apical chambers to test the stability against BBM peptidases. After 120 min, samples from

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both apical and basolateral chambers were collected and analyzed. UPLC chromatograms

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showed the stability of IWHHT, IWH, and IW (Figure 1A); IW kept the highest residual

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concentration (86.8%) at the apical side, followed by IWHHT (81.3%) and IWH (77.4%).

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Although slightly degraded, all three peptides were found transported intact and were the major

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components at the basolateral side, especially IWHHT (Figure 1B and Figure 2).

182

To further understand the composition of major degraded peptide fragments, the basolateral

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effluxes were analyzed by LC-MS/MS (Figure 2). Comparing with the chromatograms of IWH

184

and IW (Figure 1B), WH was found in the basolateral sample of IWH; W was detected in both

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samples of IWH and IW. IWHHT was degraded in a more complicated way, being cleaved to at

186

least three major fragments (WHHT, HHT, and W) during transport (combined with the

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chromatograms in Figure 1). The MS/MS spectra of the three peptides after transport are

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presented in Figure 2.



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Apical-to-basolateral transport of IWHHT, IWH, and IW. Papp is a widely used

190

parameter to measure the permeability of compounds across the intestinal epithelium. Time-

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dependent transports of the three peptides (5 mM) from apical to basolateral surface up to 120

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min were studied (Figure 3A), showing a linear uptake trend. Papp values of all three peptides

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increased significantly (P < 0.05) with time. After 120 min, Papp value of IWHHT was (22.0 ±

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1.42) ´ 10-8 cm s-1, comparable to (19.6 ± 0.62) ´ 10-8 cm s-1 for IW (P > 0.05). However, both

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of them were significantly (P < 0.05) lower than that of IWH, (37.5 ± 1.11) ´ 10-8 cm s-1.

196

Effect of diprotin A on transport. Caco-2 cell monolayers were pre-incubated with diprotin

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A (1 mM) for 30 min, followed by co-incubation with peptides (5 mM) for up to 60 min. In the

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presence of diprotin A, the Papp values of IWHHT, IWH, and IW increased by 66.1% (P < 0.01),

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46.3% (P < 0.01), and 14.9% (P > 0.05), respectively (Figure 3B), which suggested that DPP IV

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played a profound role in cleaving the IWHHT and IWH during transport.

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Effect of aminopeptidase N (APN) on peptide stability. Results from transepithelial

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stability tests indicated a possible cleavage site between I and W, reflecting the formation of

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WHHT, WH, and W from IWHHT, IWH, and IW, respectively. Therefore, APN and

204

IW/IWH/IWHHT were used to investigate whether APN was involved in these cleavages (Table

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1). The results confirmed that APN played an important role in cleaving Ile residues from the

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three peptides. Additionally, incubation of IWH/IWHHT with APN indicated that APN also

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participated in cleaving Trp but with less efficiency than that of Ile, reflecting more cleavage of

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WH/WHHT than that of IW (Table 1).

209

Transport pathways of IWHHT, IWH, and IW. To identify which pathways were

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involved in the transport of three peptides, effects of different inhibitors were investigated

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(Figure 4). As indicated, the Papp values of IWH and IW decreased by 35.81% (P < 0.05) and


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57.25% (P < 0.01), respectively, in the presence of Gly-Pro, suggesting that their transports were

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mediated by PepT1. In comparison, transport of IWHHT was not via this route since its Papp

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value was not significantly affected (P > 0.05) by the addition of Gly-Pro. These results were

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further supported by the sodium azide study, similar to the trend of Gly-Pro, indicating that

216

transports of IWH and IW, but not IWHHT, were energy-dependent. While the Papp values of

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IWHHT and IWH were enhanced by 43.06% (P < 0.05) and 66.70% (P < 0.01) in the presence

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of cytochalasin D, respectively, but not for that of IW (P > 0.05). These results indicated that

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IWHHT and IWH, but not IW, were transported via paracellular diffusion. No significant

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changes (P > 0.05) were found with the addition of wortmannin (P > 0.05), suggesting that no

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peptides were transported across Caco-2 cell monolayers via transcytosis.

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Discussion

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Peptides must overcome an array of biochemical and physical barriers in the gastrointestinal

224

tract before reaching the site of action. Therefore, understanding the stability of peptides against

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these barriers is essential for their in vivo efficacy and mechanisms.25 Simulated gastrointestinal

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digestion and Caco-2 cell monolayers are two widely-used inexpensive models to study the

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stability and transport of bioactive peptides. Our previous study showed that a spent hen-derived

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antihypertensive peptide IWHHT could be gastrointestinal digested to IWH and IW which

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retained its biological activities.4 In this study, we further investigated the transport of IWHHT,

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IWH, and IW using Caco-2 cell model.

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Incubation of the three peptides on the apical surface indicated their resistance to digestion of

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BBM peptidases, resulting from the degradation varying from 13.2% to 22.6% after 2 h (Figure

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2A). They showed better stability than RVPSL, YFCLT, and GLLLPH, which were degraded

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36%-75% after 2 h of incubation,19,26 and many others such as AAATP, AAPLAP, KPVAAP,



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and YAEERYPIL that were degraded completely.10,27 Numerous BBM peptidases are secreted

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by the well differentiated and polarized Caco-2 cells and participate in peptide hydrolysis, such

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as aminopeptidases (e.g. APN and DPP IV), carboxypeptidases (e.g. ACE), and endopeptidases

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(e.g. neprilysin).7,28 Of them, aminopeptidases are the most abundant ones, in which APN and

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DPP IV might strongly affect peptide stability during transport.19,26,29 APN prefers N-terminal

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neutral aliphatic residues, while DPP IV cleaves two N-terminal residues with preference for

241

peptide with a Pro at the P1 position.28 In our study, transports of IW, IWH, and IWHHT

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produced mainly W, WH, and WHHT, respectively (Figure 1 & 2), indicating a major cleavage

243

site located between I and W. It suggested that hydrolysis of the peptides was initiated mainly

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from the N-terminus by aminopeptidases, possibly APN, which was confirmed by a co-

245

incubation study of APN with the three peptides (Table 1). APN has been speculated responsible

246

for cleaving Gly from Gly-Pro-Hyp, a collagen-derived bioactive peptide.29 However, our study

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showed that APN was also involved in degrading antihypertensive peptides during transport.

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APN preferably cleaves N-terminal neutral aliphatic amino acid residues, which might restrain

249

small ACEi peptides to be absorbed as bioactive forms, since potent ACEi di-/tri-peptides

250

usually contain a N-terminal aliphatic residue, such as IRW, IQW, IPP, and VPP,9,11,17,30 as well

251

as IWH and IW in this study. In addition, W was another fragment of IWH and IWHHT after

252

transport (Figure 1), suggesting a second cleavage site located between W and H. Further

253

incubation of IWH/IWHHT with APN implied that APN indeed hydrolyzed Trp following Ile

254

cleavage (Table 1). Both IWH and IWHHT were substantially hydrolyzed (> 85%) after 15 min,

255

when Ile cleavage (WH/WHHT accumulation) was much higher than that of Trp (W

256

accumulation). Therefore, it was conceived that Ile cleavage of IWH/IWHHT by APN was

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higher than that of Trp under the transport conditions (with both degradations < 25% after 2 h in


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the apical side). In addition, DPP IV was another aminopeptidase cleaving the W-H peptide bond

259

of IWH and IWHHT (Figure 3B). Interestingly, transport of IW was slightly increased after

260

adding diprotin A (Figure 3B), although it was deemed not a substrate of DPP IV. The N-

261

terminal Ile of diprotin A (Ile-Pro-Ile) could be cleaved by APN to a certain extent (Figure S3),

262

albeit with a less affinity (higher Km) than that of IW due to a penultimate Pro residue.28

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Cleavage of the three peptides is illustrated in Figure 5A.

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Larger peptides are generally more susceptible to proteases due to an increased likelihood of

265

cleavage sites.11 A unexpectedly higher degradation of IWH (22.6%) than IWHHT (18.7%) was

266

possible due to limited action of other peptidases, other than APN and DPP IV, on IWHHT. For

267

example, a dipeptide carboxypeptidase, ACE, could cleave HT from IWHHT and liberated IWH

268

(Figure 2A), but its expression is very low compared with other BBM peptidases.6,7,28 Moreover,

269

IWH, transported partially by PepT1 (Figure 4B), was more easily degraded by BBM peptidases

270

which are mainly expressed on the apical membrane.28

271

IWH and IW were both good substrates for PepT1. Besides, the greater inhibition of Gly-Pro

272

on IW (by 57.25%) than that of IWH (by 35.81%) was possibly due to (i) a higher degradation

273

that IWH underwent than IW during transport, and (ii) the presence of a positively charged His

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residue, weakening the binding between IWH and PepT1 pocket.14 The effect of sodium azide on

275

transports of IWH and IW further supported these conclusions. Paracellular diffusion was

276

another pathway for transports of IWHHT and IWH. This pathways is involved in transporting

277

many bioactive peptides varying in length,11,16-19 and it is mediated by TJs which is overall

278

negatively charged and contains numerous pores ranging from 5.8 to 10.4 Å.31,32 Therefore,

279

small hydrophilic peptides, especially with positive charges, were preferentially transported by

280

this route.16,32 In our study, IWH and IWHHT had radii approximately of 5 Å and 6 Å,



281

respectively, sharing positive charges and were thus capable to be transported via paracellular

282

diffusion. A higher degree of increase in transport of IWH than that of IWHHT, after adding

283

cytochalasin D, was similar to the result that WQ was more preferred by paracellular route than

284

RWQ.16 These comparisons suggested an important role of peptide size in paracellular

285

diffusion.16 However, the size factor might be contradicted by comparing the effects of

286

cytochalasin D on IWH and IW. Given IW is smaller than IWH, it was supposedly easier to be

287

transported via this route. Interestingly, however, transport of IW was slightly impacted (P >

288

0.05) after adding cytochalasin D. Therefore, we proposed that TJs did not support transporting

289

very hydrophobic peptides (with only hydrophobic residues). Instead, IW might be transported

290

via transcellular diffusion due to a high hydrophobicity, besides PepT1-mediated transport.33

291

None of the three peptides was absorbed via transcytosis. Proteins or large peptides might pass

292

through the intestinal epithelium through transcytosis via internalization, such as β-CN (193−209)

293

(TQEPVLGPVRGPFPIIV),34 BCM-5 (YPFPG), bradykinin (RPPGFSPFR),35 and gliadin-

294

derived peptides.36 It appeared that large peptides rich in Pro residue are more likely to be

295

transported via transcytosis; antihypertensive peptides are usually small peptides and less

296

preferred by this route. Transepithelial transport pathways of the three peptides are illustrated in

297

Figure 5B.

298

Although susceptible to peptidase degradation, the three peptides were transported intact to

299

the basolateral surface. They possessed Papp values of approximately 10-7 cm s-1 (transport rate of

300

0.32%-0.60%), according with those of other antihypertensive peptides ranging in magnitude

301

from 10-8 to 10-6 cm s-1.11,16,20,21,30 Transport of IWH was higher than that of IWHHT and IW,

302

due to a combination of PepT1-mediated transport and paracellular diffusion. Bioavailability of

303

orally-ingested proteins or peptides is less than 1-2%.37 However, the in vivo absorption is more


Page 14 of 30

Page 15 of 30


304

efficient than that of the Caco-2 cell model, since (i) in vivo ileum possess higher expression of

305

PepT1 and lower TJs than Caco-2 cell monolayers,38 and (ii) the active transcellular transport of

306

nutrients after a meal further opens TJs and promotes paracellular diffusion via osmotic force.39

307

Moreover, ACEi peptides could exert in vivo efficacy even with a lower bioavailability. For

308

instance, three ACEi peptides IRW, IQW, and LKP were transport 0.3-0.4% in Caco-2 cells

309

while reduced blood pressure of SHRs significantly (p < 0.05);9,11,40,41 others such as IPP, LPP,

310

and VPP exerted antihypertensive efficacy with a bioavailability of approximately 0.1%.42 Oral

311

administrating IWHHT, IWH, and IW with 60 mg/kg BW to SHRs (equivalent to ~10 mg/kg

312

BW to human) produced a short-term blood pressure reduction;6,43 however, a long-term study of

313

these peptides are warranted in the near future.

314

In conclusion, the present study demonstrated that IWHHT and its degraded fragments IWH

315

and IW could be transported intact across Caco-2 cell monolayers. In addition to being

316

susceptible to DPP IV degradation, our study indicated an important role of aminopeptidase N in

317

affecting stability of ACEi peptides during transport. Besides, IWHHT and IW were transported

318

via paracellular diffusion and PepT1, respectively, while IWH was transported via both of them.

319

The results supported that small hydrophobic peptides (constituted of only highly hydrophobic

320

amino acid residues) might be unfavorable for paracellular diffusion, which could facilitate

321

understanding the effects of peptide structural features on their preferences for transport

322

pathways. However, it should be noted that the digestive profile of peptide in human is harder to

323

mimic using in vitro gastrointestinal digestion/absorption models due to a greater complexity

324

which includes hormonal/nervous control, peristaltic movements, interaction of food matrices,

325

and many other factors;44 therefore, in vivo study on the bioavailability and efficacy of these

326

peptides are warranted.



327

Abbreviation Used

328

ACE, angiotensin-converting enzyme; ACEi; ACE inhibitory; ACN, acetonitrile; Ang I/II,

329

angiotensin I/II; APN, aminopeptidase N; BBM, brush border membrane; BW, body weight;

330

DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethyl sulfoxide; DPP IV, dipeptidyl

331

peptidase IV; FBS, fetal bovine serum; HBSS, Hanks balanced salt solution; HEPES, 4-(2-68

332

hydroxyethyl)-1-piperazineethanesulfonic acid; IWHHT, Ile-Trp-His-His-Thr; IWH, Ile-Trp-His;

333

IW, Ile-Trp; LC-MS/MS, liquid chromatography-mass spectrometry/mass spectrometry; MES,

334

2-(N-Morpholino) ethanesulfonic acid; NEAA, nonessential amino acids; RAS, renin-

335

angiotensin system; Papp, apparent permeability coefficients; PepT1, peptide transporter 1; SHRs,

336

spontaneously hypertensive rats; TEER, transepithelial electrical resistance; TFA, trifluoroacetic

337

acid; TJs, tight junctions; Tris, Tris(hydroxymethyl)aminomethane; UPLC, ultra-performance

338

liquid chromatography;

339

Acknowledgments

340

This work was supported by funding from Natural Sciences and Engineering Research

341

Council of Canada, Egg Farmers of Canada and Burnbrae Farms Ltd. H. F. is the receipt of

342

Scholarships from China Scholarship Council and Alberta Innovates Technology Features -

343

Graduate Student Scholarships.

344

Supplementary Materials Standard curve of tryptophan (Figure S1); Cell cytotoxicity (Figure S2); Hydrolysis of

345 346

diprotin A by APN (Figure S3).

347

Notes Authors declare no competing financial interest.

348


Page 16 of 30

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474

Figure captions

475

Figure 1. Stability and chromatograms of IWHHT, IWH, and IW in the apical (A) and

476

basolateral (B) chamber after 2 h transport. The initial peptide concentration was 5 mM. The

477

inserts of (A) indicate the recovery (remaining percentage) of peptides in the apical chamber

478

after 2 h incubation. IWHHT, IWH, IW, and W were identified based on the retention time of the

479

corresponded standards.

480

Figure 2. LC-MS/MS of basolateral sample of Caco-2 cell monolayer after 2 h incubation with

481

IWHHT, IWH, and IW. Results were presented with MS of each basolateral sample and MS/MS

482

of the parent peptide. For MS/MS interpretation, a water (-18) or ammonium (-17) loss of the

483

b/y-ions was noted as (b/y-H2O/NH3); (b-28) was noted for the a-ions.

484

Figure 3. Transport of IWHHT, IWH, and IW (5 mM) across Caco-2 cell monolayers. (A)

485

Transport of IWHHT, IWH, and IW cross Caco-2 cell monolayer for 120 min; (B) Effect of

486

diprotin A on the transports of IWHHT, IWH, and IW across Caco-2 cell monolayers for 60 min;

487

**, P < 0.01; ns, not significant.

488

Figure 4. Effects of Gly-Pro (a PepT1 competitor), sodium azide (a ATP synthesis inhibitor),

489

wortmannin (a transcytosis disruptor), and cytochalasin D (a tight junction disruptor) on the

490

transports of IWHHT (A), IWH (B), and IW (C) across Caco-2 cell monolayers for 60 min. *, **,

491

and *** indicate P < 0.05, 0.01, and 0.001, respectively, compared to the control (without

492

treatment).

493

Figure 5. Schematic cleavage (A) and transport pathways (B) of IWHHT, IWH, and IW across

494

Caco-2 cell monolayers. APN, DPP IV, and ACE, indicate aminopeptidase N, dipeptidyl

495

peptidase IV, and angiotensin converting enzyme, respectively; PepT1, peptide transporter 1.



Page 24 of 30

Table 1 Effect of aminopeptidase N on the formation of cleaved fragments from their respective peptides IW, IWH, and IWHHT Time (min)

Peptides (µM) Precursor

Fragments

IW

0

15

30

60

3000

361 ± 5.40a 2620 ± 25.0c 18.1 ± 1.40c

6.96 ± 0.800b 2890 ± 20.6a 101 ± 2.70b

2540 ± 50.1 419.5 ± 21.6 7.90 ± 1.30b 2770 ± 32.4b 227 ± 5.10a

W IWH

3000 WH W

426.0 ± 16.0a 44.9 ± 4.64b 12.8 ± 4.40c a a WHHT 2060 ± 11.8 2040 ± 10.2 1890.1 ± 8.37b W 510 ± 4.15c 917.0 ± 14.9b 1100 ± 12.2a Results are presented as means ± standard deviations (n = 3). Same superscript lowercase letters within a row indicate no significance (P > 0.05).

IWHHT

3000


Page 25 of 30


Figure 1



Figure 2


Page 26 of 30

Page 27 of 30


Figure 3



Figure 4


Page 28 of 30

Page 29 of 30


Figure 5



TOC Graphic


Page 30 of 30

Stability and Transport of Spent Hen-Derived ACE-Inhibitory Peptides

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