Identification of New Anti-inflammatory Peptides from Zein Hydrolysate

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Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells Qiufang Liang, Meram Chalamaiah, Xiaofeng Ren, Haile Ma, and Jianping Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04562 • Publication Date (Web): 01 Dec 2017 Downloaded from http://pubs.acs.org on December 1, 2017

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Journal of Agricultural and Food Chemistry

Identification of New Anti-inflammatory Peptides from Zein Hydrolysate after Simulated Gastrointestinal Digestion and Transport in Caco-2 Cells Qiufang Lianga,b, Meram Chalamaiahb, Xiaofeng Rena,b, Haile Maa, Jianping Wub

a

School of Food and Biological Engineering, Jiangsu University, 301 Xuefu Road,

Zhenjiang, Jiangsu 212013, China b

Department of Agricultural, Food and Nutritional Science (AFNS), 4-10 Ag/For

Centre, University of Alberta, Edmonton, Alberta, Canada

Corresponding Author Dr. Jianping Wu E-mail: [email protected]; Fax: +1 780 492 4265; Tel: +1 780 492 6885

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ABSTRACT

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Chronic inflammation is an underlying contributor to various chronic diseases.

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The objectives of this study were to investigate the anti-inflammatory activity of zein

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hydrolysate after simulated gastrointestinal digestion and Caco-2 cell absorption, and

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to identify novel anti-inflammatory peptides after transport across Caco-2 cells. Three

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zein hydrolysates were prepared and further digested using gastrointestinal proteases;

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their transports were studied in Caco-2 cells. Anti-inflammatory activity was studied

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in endothelial EA.hy926 cells. Three zein hydrolysates and their digests significantly

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decreased

the

expression

of

tumor

necrosis

factor-α

(TNF-α)

induced

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pro-inflammatory vascular cell adhesion molecule-1 (VCAM-1) by 37.3-66.0%.

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Eleven novel peptides with 5-9 amino acid residues were sequenced; three peptides

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showed strong anti-inflammatory activity by inhibiting the VCAM-1 by 54-38.9% and

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intercellular cell adhesion molecule-1 (ICAM-1) by 36.5-28.6% at 0.2 mM. A new

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approach to identify novel anti-inflammatory peptides that could survive

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gastrointestinal digestion and absorption is developed.

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KEYWORDS: zein hydrolysate, bioactivity, simulated gastrointestinal digestion,

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peptide bioavailability

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INTRODUCTION

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Maize is the third most widely cultivated cereal in the world. Zein, a byproduct

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of corn starch processing, is the main storage protein accounting for ~ 50% of total

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endosperm protein.1 Zein is often used as a coating material, a drug carrier, or in

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tissue engineering;2 zein is also claimed as a good source of bioactive peptides. Zein

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derived bioactive peptides were shown to possess antioxidant,3 angiotensin converting

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enzyme (ACE) inhibitory,4 antihepatotoxic,5 anti-cancer, immunomodulatory6

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activities, and facilitating alcohol metabolism.7 The anti-inflammatory activity of a

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corn gluten hydrolysate was suggested in a rat model of experimental colitis,8

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although characterization of the responsible peptides has not been reported. Bioactive

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peptides released from food proteins have great potential as functional

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food/nutraceutical ingredients for improving human health.

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Endothelial cells, lining the inner layer of blood vessels, play a vital role in

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vascular biology, such as regulation of blood vessel tone, hemostasis, neutrophil

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recruitment, hormone trafficking, and fluid filtration.9 Vascular inflammation is a key

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factor that contributes to endothelial dysfunction and has been associated with a

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variety of disease states including atherosclerosis, diabetes, coronary artery disease,

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hypertension

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pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) phosphorylates nuclear

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factor κappa B (NF-kB), which leads to increased expression of intercellular adhesion

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molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the

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release of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells.11 These

and

hypercholesterolemia.10

Under

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conditions,

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adhesion molecules then recruit leukocytes (monocytes and macrophages) to the site

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of inflammation and enhance the plaque formation between endothelial cells and

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vascular smooth muscle cells that eventually contributes to increased blood pressure

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and initiation of atherosclerosis.10, 12 Therefore, targeting inflammation of endothelial

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cells is a strategy to reduce endothelial cells’ inflammation thus improve endothelial

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function. A number of food bioactive components were reported to exert

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anti-inflammatory activity. Recently, milk-derived hydrolysates have been reported to

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exhibit anti-inflammatory activity in endothelial cells by down regulation of

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expression of VCAM-1, ICAM-1 and E-selectin.13 Furthermore, IRW, IQW, GWNI

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and GW peptides isolated from egg ovotransferrin, and VPP peptide derived from

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casein showed anti-inflammatory effects in endothelial cells.14-16

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Bioactive peptides composed of amino acids are susceptible to proteases that

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present in the gastrointestinal tract; some peptides render inactive while more potent

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peptides may also be released after the proteolytic digestion.17 Therefore it is vital to

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study the stability of bioactive peptides in the gastrointestinal tract. As the ethical

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regulation on animal studies have become more stringent, simulated gastrointestinal

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digestion, which is considered to be simple, rapid, and inexpensive, has been widely

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used for assessment of the stability of a wide range of food derived bioactive peptides

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such as ham peptides,18 loach protein hydrolysates,19 etc. As the absorption of

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peptides mainly occurs through the epithelium of the small intestine after

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gastrointestinal digestion, another key factor affecting the bioactivity of bioactive

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peptides is their transport through the gut epithelium.20 Caco-2 cell line, a human 4

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intestinal epithelial cell model derived from a colon carcinoma, is a well-established

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model of intestinal absorption in vitro.21 Recently, Caco-2 monolayers have been

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successfully used to study the transport of some purified peptides and protein

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hydrolysates.22-23 Since studies related to anti-inflammatory effect of zein derived

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peptides are scanty, the objectives of this study were to investigate the

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anti-inflammatory activity of zein hydrolysate after simulated gastrointestinal

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digestion and Caco-2 cell absorption, and to identify novel anti-inflammatory peptides

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after transport across Caco-2 cells.

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MATERIALS AND METHODS

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Chemicals

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Neutral protease (from bacteria Bacillus subtilis) was obtained from BIA-CAT

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(Troy, NY, USA). Zein, Alcalase® 2.4L (from Bacillus licheniformis, Subtilisin A),

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thermolysin (from Geobacillus stearothermophilus) were bought from Sigma

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Chemical Co. (St.Louis, MO, USA). Recombinant human TNF-α was obtained from

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R&D System (Minneapolis, MN, USA). Fetal bovine serum (FBS) and Dulbecco’s

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modified eagle medium (DMEM) were bought from Gibco/Invitrogen (Carlsbad, CA,

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U.S.A.). The antibodies of VCAM-1 and ICAM-1 were purchased from Santa Cruz

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Biotechnologies (Santa Cruz, CA, USA). α-Tubulin antibody (rabbit polyclonal

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antibody) was purchased from Abcam (Cambridge, MA, USA). Goat anti-rabbit and

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donkey anti-mouse fluorochrome-conjugated secondary antibodies were obtained

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from Licor (Licor Biosciences, Lincoln, NE, USA). All other chemicals and reagents

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used were analytical grade. 5

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Enzymatic hydrolysis of zein

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Zein powder was dissolved in distilled water to obtain 50 mg/mL protein

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concentration. The pH and the temperature of enzymes (Alcalase, neutral protease,

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and thermolysin) were adjusted according to the manufacturer’s recommendations

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(Table 1). The enzymes were added at a ratio of 50:1 (w/w, zein to protease) to start

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the hydrolysis. The hydrolysis was performed in a jacketed beaker connected to a

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circulating water bath for maintaining constant temperature and Titrando (Metrohm,

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Herisan, Switzerland) for maintaining constant pH with 0.5 M NaOH. After 3 h of

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hydrolysis, the mixture was kept in boiling water for 10 min and centrifuged at

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10,000g for 15 min. The supernatant was concentrated and freeze-dried for further

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use.

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Degree of hydrolysis (DH) and yield

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DH of zein was measured by 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)

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method.24-25 Zein was completely hydrolyzed using 6 M HCl at 115 °C for 24 h to

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determine the total amino group. The nitrogen content was determined according to

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the modified Kjeldahl method using LECO Truspec total CN analyser (LECO, USA).

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The yield was calculated by the equation:  % =

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      ℎ ℎ   × 100

      ℎ     

Simulated gastrointestinal digestion

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Zein and zein derived hydrolysates were subjected to simulated gastric and

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intestinal digestion based on the previous method.26 Simulated gastric and intestinal

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fluids were prepared according to the U.S. Pharmacopeia.27 Briefly, zein and three 6

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zein hydrolysates were digested with gastric fluid at 1:20 (w/v) for 4 h in a shaking

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incubator (at 150 rpm) at 37 °C. Then pH was adjusted to 6.8 and pancreatin was

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added to form the intestinal fluid. The mixture was incubated for a further 6 h to

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mimic the intestinal digestion. The digestion was terminated by keeping the digests in

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boiling water for 10 min. The digests were allowed to cool down and centrifuged at

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10,000 g for 10 min to collect the supernatant. The control (using water instead of

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samples) was performed under the same conditions and used to correct interference

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from the simulated digestive juices.

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Cell culture

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The endothelial cell line, EA.hy926 (CRL-2922TM), and human colon

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adenocarcinoma cell line, Caco-2 (HTB-37TM), were purchased from American-type

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culture collection (ATCC, Manassas, VA, USA). DMEM supplemented with 10%

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FBS, 2.5% HEPES, 1% antibiotics and 1% non-essential amino acids was used as cell

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growth medium. The medium was replaced each other day, and the cells were

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subcultured using 0.25% trypsin-EDTA treatment. The cells were incubated in a

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humidified atmosphere with 5% CO2 at 37 °C.

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Measurement of cytotoxicity

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The cell cytotoxic properties were monitored using an Alamar Blue assay

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described by Huang et al.28 Caco-2 cells were seeded in 96-well plates at a density of

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1×104 cell/well for 24 h. Then the cells were treated with various concentrations

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(10-50 mg/mL) of zein hydrolysate for another 24 h in a fresh medium. After 24 h

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treatment, the media was discarded, and the fresh medium with 10% Alamar Blue 7

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reagent was added and incubated for 4 h at 37 °C. The fluorescence intensity of the

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wells was measured at an emission wavelength of 590 nm and an excitation

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wavelength of 560 nm. The viability of the treated cell was expressed as the

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percentage as compared to untreated cells.

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Transport studies

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The transport experiments were performed according to the modified method

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of Bejjani et al.29 Caco-2 cells were grown in 12-well Transwell® plates (0.4 µm pore

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size, 12 mm diameter, 1.12 cm2 grown surface, Corning Costar Corporation,

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MA, USA) at a concentration of 1×105 cells/cm2. The Caco-2 monolayers that showed

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transepithelial electrical resistance values > 300 Ω/cm2 could be used for the

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experiment. Before initiation of transport experiments, the culture medium of the

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Caco-2 cells was replaced by HBSS buffer and the Caco-2 cells were pre-incubated

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for 0.5 h at 37 °C. Transport of zein hydrolysate digests was performed by adding the

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digests at 20 mg/mL (dissolved in HBSS buffer) to the apical (AP) surface. AP surface

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samples at 0 h and basal surface (BL) samples at 0.5, 1, 2, 4 h were collected for

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further analysis.

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UPLC analysis

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The samples collected from the AP and BL surfaces were analyzed using an

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Acquity Ultra-Performance Liquid Chromatograph (UPLC) system. 15 µL sample

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was loaded on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d., 1.7 µm,

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Waters, Milford, MA, USA). Mobile phases were solvent A (0.1% trifluoroacetic acid

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in Milli-Q water) and solvent B (0.1% trifluoroacetic acid in acetonitrile). The 8

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peptides were eluted with a gradient of solvent A (100-75% in 25 min, 75-50% in

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25-35 min) at 0.3 mL/min. The detection wavelength was 220 nm. Transport percent

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was expressed as the percentage of total peak area calculated at different time points

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in the BL surface as compared to 0 h in the AP surface.

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EA. hy926 cells experiment

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The EA. hy926 cells with passage number < 12 were grown in 48-well plates.

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The cells reaching 80−90% confluence were treated with various concentrations (2.5

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mg/mL of hydrolysates and digests, 0.2 mM and 3.0 mM of synthetic peptides) of the

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samples for 18 h. Then the cells were stimulated with TNF-α at 10 ng/mL and

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incubated for an additional 6 h in order to induce inflammation.

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Measurement of ICAM-1 and VCAM-1

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The level of ICAM-1 and VCAM-1 was determined by Western blot analysis

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according to our previous study.14 After the treatment period, the culture medium of

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the EA. hy926 cells was discarded and the boiling Laemmle buffer containing 0.2%

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TritonX-100 and 50 µM dithiothreitol was added to lysate the cells. The cell lysates

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were then run on 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The

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gels were transferred onto nitrocellulose membranes, and immunoblotted with

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anti-ICAM-1/anti-VCAM-1 antibodies. The concentration of antibody to α-tubulin

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used was 0.4 µg/mL, while all other antibodies were 0.1 µg/mL. The protein bands

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were scanned using Licor Odyssey BioImager (Licor Biosciences, Lincoln, NB, USA)

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and quantified by densitometry using Image Studio Lite 5.2. The expression of

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ICAM-1 and VCAM-1 were normalized using the control α-tubulin. All the data were 9

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expressed as the percentage change of the corresponding positive control (cells treated

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with TNF-α alone).

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Identification of anti-inflammatory peptides

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The peptides were subjected to liquid chromatography−electrospray ionization

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tandem mass spectrometry (LC−ESI-MS/MS) analysis using Waters ACQUITY

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UPLC coupled to Waters Micromass Q-TOF MS Premier Instrument. The sample (5

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µL) was desalted by loaded onto a peptides trap column (180 µm×20 mm, Symmetry

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C18 nanoAcquity column, Waters) and desalted at 10 µL/min for 5 min using 1%

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acetonitrile in water (containing 0.1% formic acid). Then the desalted sample was

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loaded onto a nano analytical column (150 mm×75 µm, Atlantis d C18 nanoAcquity

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column, Waters) and separated by a gradient of acetonitrile with 0.1% formic acid

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(1-6% in 0-2 min, 6-25% in 2-25 min, 25-45% in 25-40 min, 45-75% in 40-45 min,

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75-95% in 45-50 min, keeping at 95% in 50-55 min) at 350 µL/min. The mass

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spectrometer was operated in a positive mode with capillary voltage 3.6 kV, source

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temperature 100 °C, scanning m/z range 200-1000 in MS mode and 50-1990 in

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MS/MS mode. The MS/MS data were processed by Peaks Viewer 4.5 (Bioinformatics

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Solutions Inc., Waterloo, ON, Canada) in combination with manual de novo

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sequencing. Identified peptide sequences were synthesized, validated using

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LC-MS/MS and its purity (>98%) was further validated using HPLC by Genscript

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Corp (Piscataway, NJ, USA). All synthetic peptides were further used for

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anti-inflammatory assays.

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Statistical analysis 10

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The data were presented as the mean ± standard deviation of 4-6 determinations.

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Statistical analyses were performed using one-way analysis of variance and

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differences with P values