Transport of Egg White ACE-Inhibitory Peptide, Gln-Ile-Gly-Leu-Phe, in

Mar 26, 2014 - Gln-Ile-Gly-Leu-Phe (QIGLF) was an ACE-inhibitory peptide, with an IC50 value of 75 μM, identified from egg white ovalbumin. The resul...
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Transport of Egg White ACE-Inhibitory Peptide, Gln-Ile-Gly-Leu-Phe, in Human Intestinal Caco‑2 Cell Monolayers with Cytoprotective Effect Long Ding,† Yan Zhang,† Yiqun Jiang,‡ Liying Wang,† Boqun Liu,† and Jingbo Liu*,† †

Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, People’s Republic of China College of Life Science, Jilin University, Changchun 130021, People’s Republic of China

‡

ABSTRACT: The purpose of this study was to investigate the transepithelial transport and cytoprotective effect of Gln-Ile-GlyLeu-Phe (QIGLF), an ACE-inhibitory peptide derived from egg white ovalbumin, in human intestinal Caco-2 cell monolayers. The results showed that QIGLF could be absorbed intact through Caco-2 cell monolayers with a Papp value of (9.11 ± 0.19) × 10−7 cm/s (transport kinetic parameters: Km, 32.37 ± 12.59 mM; Vmax, 1.23 ± 0.49 μM/min cm2). The transport was not significantly decreased by sodium azide and Gly-Pro, an ATP synthesis inhibitor and a peptide transporter 1 (PepT1) substrate, respectively, suggesting that transport of QIGLF was not energy-dependent and carrier-mediated. In addition, wortmannin, a transcytosis inhibitor, had little effect on the transport, suggesting that endocytosis was not involved in the transport of QIGLF. However, the transport of QIGLF was increased significantly in the presence of cytochalasin D, a tight junction disruptor, suggesting that paracellular transport via tight junctions was the major transport mechanism for intact QIGLF across Caco-2 cell monolayers. Moreover, QIGLF was added to Caco-2 cells followed by addition of H2O2, and exhibited significant cytoprotective effect in Caco-2 cells against oxidative stress induced by H2O2. KEYWORDS: egg white, ACE-inhibitory peptide, Caco-2 cells, transport

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INTRODUCTION Angiotensin-converting enzyme (ACE) plays a critical role in regulating blood pressure in the rennin−angiotensin system and kallikrein−kinin system by converting inactive angiotensin I into the potent vasoconstrictor angiontensin II and catalyzing the degradation of bradykinin. In the past decades, various bioactive peptides have been found in protein hydrolysates and fermented products with ACE inhibitory activity in vivo and in vitro, providing an approach for the therapy of cardiovascular diseases caused by hypertension. In addition, the peptides derived from food proteins are considered to have no undesirable adverse effects as compared to synthetic drugs, that is, captopril, beyond supplying basic nutrients. As a good protein source for bioactive peptides, egg white accounts for 58% of the total egg weight and contains 10−12% proteins, which mainly comprise ovalbumin, ovotransferrin, ovomucoid, globulins, and lysozyme.1 Miguel et al. reported YAEERYPIL, FRADHPFL, and RADHPFL, which were all identified from ovalbumin, exhibited significant antihypertensive effects in spontaneously hypertensive rat.2,3 Majumder et al. reported three ACE-inhibitory peptides IQW, IRW, and LKP from ovotransferrin.4 Moreover, in the previous study, egg white protein was hydrolyzed by alcalase, and the hydrolysates were isolated with gel filtration. Next, 19 peptides were identified from the high activity fractions by LC tandem mass spectrometric 4000 Q Trap MS. Among these peptides, QIGLF, RVPSL, and TNGIIR showed good ACE inhibitory activities in vitro.5−8 To exert the ACE inhibitory activity in vivo, food-derived bioactive peptides have to overcome two important physiologic barriers, extensive enzymatic degradation in gastrointestinal © 2014 American Chemical Society

tract and poor permeability through intestinal epithelium, and to be absorbed in intact form into blood circulation and target sites after orally administration.9 Studies in Caco-2 cell monolayer systems, a good intestinal model for the absorption of drugs first described systematically by Hidalgo,10 have shown that the size and lipophilicity of peptides are two critical roles in determining their permeability through intestinal epithelium.11−13 In addition, a good correlation between the permeability in Caco-2 cell monolayers and absorption in human and rat intestines was observed.14 In general, passive transport and carrier-mediated transport coexist in biological membrane permeation to various extents.15 In terms of bioactive peptides, di- and tripeptide are mainly H+-dependent PepT1 mediated transported through intestinal epithelium.16,17 Oligopeptides are possibly absorbed intact via paracellular passive diffusion and transcytosis.18 Paracellular pathway is regulated by tight junctions, which comprise several proteins, such as zonula occludens proteins, occludins, and claudins. Tight junctions play an important role in maintaining the integrity of intestinal epithelial cells by creating a rate-limiting barrier to diffusion of solutes, for instance, water-soluble lowmolecular substances and minerals.19 However, a large number of food-derived substances including bioactive peptides are found to transepithelial transport across intestine via paracellular pathway, which was modulated by tight junction. Furthermore, in another point of view, it is possible that these Received: Revised: Accepted: Published: 3177

December March 14, March 26, March 26,

16, 2013 2014 2014 2014

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peptides interact with tight junctions or epithelial cells.20−23 Unfortunately, the interaction between food-derived substance and epithelium is still obscure. Gln-Ile-Gly-Leu-Phe (QIGLF) was an ACE-inhibitory peptide, with an IC50 value of 75 μM, identified from egg white ovalbumin. The result of simulated gastrointestinal digestion by pepsin and trypsin showed that QIGLF had a strong resistance to gastrointestinal proteases, providing a possibility for oral administration.8 However, the bioavailability and permeability through intestinal epithelium have not been studied. Therefore, the objective of the present study was to investigate the transport of QIGLF in human intestinal Caco-2 cell monolayers. Furthermore, the effect of QIGLF on the viability of Caco-2 cells in oxidative stress induced by H2O2 was evaluated.

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Transport Experiments. The transepithelial transport of ACE inhibitory peptide QIGLF was investigated as previously described by Hubatsch with minor modifications.25 The Caco-2 cell monolayers were rinsed with HBSS twice and then incubated with HBSS for 30 min at 37 °C in 5% CO2 before the transport experiment. We then aspirated the HBSS from both apical side and basolateral side of all wells. The peptide of 1−10 mM QIGLF in HBSS buffer (pH 7.4) was added in apical side, and fresh HBSS (pH 7.4) was added in basolateral side. All of the plates were incubated at 37 °C in 5% CO2 for 2 h. At the appointed time points of 0, 30, 60, 90, and 120 min, an aliquot of 50 μL of sample was collected from the basolateral side. After each sampling, an equivalent volume of HBSS buffer with 25 mM HEPES was added in basolateral side to maintain a constant volume. The QIGLF concentration in each sample was determined by HPLC. To identify which transport routes were involved in the transepithelial absorption of QIGLF, the Caco-2 cell monolayers were preincubated with transport inhibitor cytochalasin D (a tight junction disruptor, 0.5 μg/mL), wortmannin (a transcytosis inhibitor, 500 nM), sodium azide (an ATP synthesis inhibitor, 10 mM), or Gly-Pro (a peptide transport PepT 1 substrate, 10 mM) for 30 min followed by addition of 1 mM QIGLF to apical side. The cytochalasin D, wortmannin, and sodium azide were dissolved in DMSO (final concentration in HBSS 0.044%), and Gly-Pro was dissolved in HBSS. As a control, DMSO was used. All of the experiments were conducted in triplicate. Induction of Oxidative Stress. The induction of in vitro oxidative stress was conducted as previously described by Katayama with modifications.26,27 Caco-2 cells were seeded in 24-well culture plates (Costar, Corning, NY) at a density of 2 × 105 cells/mL and incubated at 37 °C in 5% CO2. The oxidative stress was induced until the confluent Caco-2 cell monolayers formed by addition of H2O2. The Caco-2 cell monolayers were rinsed with HBSS twice, and then incubated with 1 and 5 mM QIGLF (dissolved in HBSS, pH 7.4) for 2 h followed by addition of various concentrations of H2O2 (250, 500, and 1000 μM) for 6 h. MTS was added in the plates (20 μL of MTS per 100 μL of culture medium was used) and incubated at 37 °C in 5% CO2 for another 2 h followed by detection of the absorbance at 595 nm using a microplate reader (BioTek Instruments, U.S.).28 RP-HPLC. The peptide was quantified using a LC-2010HT HPLC system (SHIMADZU, JP) equipped with an autosampler, a quaternary pump, an online degasser, a UV detector, and a LC-2010HT workstation. The analytical column was a C18 reverse-phase column (ZORBAX SB-C18 column, 250 × 4.6 mm, particle size 5 μm, Agilent, U.S.). The mobile phases were solvent A (0.1% TFA in water) and solvent B (0.1% TFA in ACN). Samples were eluted with a gradient of solvent B from 25% to 50% in 25 min, from 50% to 100% in 10 min, and 100% for 5 min at a flow rate of 1.0 mL/min. The injection volume was 10 μL, and the absorbance detector wavelength was set at 220 nm. The peptide QIGLF concentration was calculated by a linear regression analysis of the peak area as described previously.29 Statistical Analysis. All data were expressed as the mean ± SD (n = 3). The difference was carried out by one-way analysis of variance (ANOVA) with the significance level at P < 0.05 by SPSS 19.0 software. The apparent permeability coefficient (Papp, cm/s) was calculated as follows:

MATERIALS AND METHODS

Materials. Caco-2 cell lines were purchased from American Type Culture Collection. The ACE-inhibitory peptide QIGLF with a purity of 95% was synthesized by GL Biochem Ltd. (Shanghai, China) by the solid-phase procedure peptide using Fmoc protected amino acids synthesis methods. Dulbeco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, penicillin-streptomycin, Hank’s balanced salt solution (HBSS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Gibco BRL Life Technology. Nonessential amino acid, trypsin, L-glutamine, dimethyl sulfoxide (DMSO), ethylene diamine tetraacetic acid (EDTA), cytochalasin D, wortmannin, sodium azide, glycyl-proline (Gly-Pro), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT), acetonitrile, trifluoroacetic acid (TFA), and methanol were all purchased from Sigma-Adrich. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega Biotechnology Co. Ltd. All other reagents were analytical grade. Cell Culture. The Caco-2 cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 1% nonessential amino acid solution, 100 units/mL penicillin, 100 μg/mL streptomycin, and 4 mM L-glutamine at 37 °C in an atmosphere of 5% CO2 and 90% relative humidity. Stock cultures were grown in 75 cm2 tissue culture flasks and were split at 80−90% confluency using 0.25% trypsin and 0.02% EDTA solution. The cells from passage numbers of 10−20 were used and seeded on the permeable polycarbonate inserts (0.4 μm pore size, 12 mm diameter, 1.12 cm2 grown surface, Costar, Corning, NY) in 12 transwell plates at a density of 1 × 105 cells/insert. The culture medium (0.5 mL in the apical side and 1.5 mL in the basolateral side) was replaced every 2 days for the first week and then daily until they were used for experiments 21 days after the seeding. The integrity of the cell monolayer was evaluated by measuring transepithelial electrical resistance (TEER) values with an EVOM epithelial volt/ohmmeter (Millicell-ERS, Millipore, U.S.). Only the cell monolayers with TEER values higher than 250 Ω cm2 were used for the transport studies. Cytotoxicity Assay. MTT was used to determine the cytotoxicity of QIGLF in Caco-2 cells as described previously.24 Caco-2 cells were grown in 96-well culture plates and incubated with 1−10 mM QIGLF, which dissolved in DMEM, at 37 °C in 5% CO2 for 24 h followed by addition of 0.05 mg of MTT and incubation for another 4 h. The culture medium then was replaced with 150 μL of DMSO to dissolve the MTT crystals, and the absorbance at 595 nm was measured using a multimode microplate reader (BioTek Instruments, U.S.). Stability Assay. The stability of QIGLF in the apical surface of Caco-2 cells was studied. The Caco-2 cell monolayers were rinsed with HBSS twice and then incubated with HBSS for 30 min at 37 °C in 5% CO2. The medium in the surface apical was replaced with 1 mM QIGLF (dissolved in HBSS, pH 7.4) and incubated at 37 °C in 5% CO2. At the indicated time points, an aliquot of 50 μL from the apical surface was collected and analyzed by HPLC.

Papp =

dQ 1 1 × × dt A C0

(1)

where dQ/dt was the permeability rate (mmol/s); A was the area of membrane (cm2); and C0 was the initial concentration of QIGLF in the donor chamber (mM). After each sampling, the QIGLF concentration on the receiving side was diluted, so a correction equation was used as follows: n−1

Q = CnV +

∑ CiVi i=0

(2)

where Q was the accumulation of QIGLF on the receiving side (mmol); Cn and Ci were the concentrations of QIGLF in sample n and 3178

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sample i, respectively; and V and Vi were the volumes of receiving chamber and sample i, respectively. To evaluate the kinetics in the transepithelial transport of QIGLF, Km and Vmax were calculated using the Michaelis−Menten equation, and the transport rate (V, μM/min cm2) was calculated as follows: V=

dC 1 × dt A

(3)

where dC/dt was the change of QIGLF concentration in basolateral side (μM/min).

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RESULTS AND DISCUSSION Validation of QIGLF. The primary structure and basic properties of QIGLF (the 204−208 residues of egg white ovalbumin, molecular weight 576.69 g/mol) were evaluated as described.30 It was a neutral pentapeptide at pH 7.4 with a log D value of −2.287. The isoelectric point was 6.00, and the pKa value was 3.76. Moreover, QIGLF had two intramolecular hydrogen bonds. The volume was 383.47 Å3, and the total surface area was 603.88 Å2 with polar surface area of 222.80 Å2. In the present study, the retention time for QIGLF (dissolved in HBSS, pH 7.4) was 17.59 min, and the total run time was 30 min in the chromatogram of RP-HPLC analysis. Cytotoxicity of QIGLF in Caco-2 Cells. As shown in Table 1, the result of MTT assay demonstrated that QIGLF did

Figure 1. Stability of QIGLF (1 mM in HBSS, pH 7.4) in the AP side of Caco-2 cell monolayers within 2 h. One mM QIGLF (dissolved in HBSS, pH 7.4) was added to AP side, and samples were collected from AP side for HPLC analysis at 30, 60, 90, and 120 min. The recovery analysis revealed that the degradation of QIGLF was not significant within 2 h (P > 0.05). All values were the means ± SD (n = 3).

a strong resistance to these enzymes. Iwan et al. reported that treating Caco-2 cell monolayers with diprotin A, a dipeptidyl peptidase IV inhibitor, resulted in lower degradation of βcasomorphin-5 and -7 in the donor chamber and higher transport rate.33 Cakir-Kiefer et al. also found that the presence of bile salts appeared to modulate the peptidase activities of the Caco-2 cells leading to higher degradation of α-casozepine but lower degradation of αs1-casein-(f91−97).34 Transport of QIGLF across Caco-2 Cell Monolayers. The time-dependent uptake of QIGLF across Caco-2 cell monolayers was as shown in Figure 2. The accumulated

Table 1. Cytotoxicity of QIGLF in Caco-2 Cells As Evaluated by MTT Assay QIGLF concentrations (mM) 0 (HBSS, control) 1 2 4 8 10

absorbance at 595 nma,b 0.4993 0.4932 0.4875 0.4814 0.4806 0.4796

± ± ± ± ± ±

0.03 0.04 0.04 0.02 0.02 0.02

a

No significant difference as compared to control group existed. bAll values were the means ± SD (n = 3).

not have any cytotoxic effect in Caco-2 cells at the concentrations of 1−10 mM (dissolved in HBSS, pH 7.4), which were used in the transport experiments. Moreover, the transport experiments were performed within 2 h, which was much shorter than the time used for MTT assay. Therefore, the QIGLF (1−10 mM) showed no cytotoxicity in Caco-2 cell monolayers in our studies. Stability of QIGLF on Caco-2 Cell Monolayers. The peptide of QIGLF at 1 mM (dissolved in HBSS, pH 7.4) was added to the apical surface Caco-2 cell monolayers and incubated 2 h. The recovery of QIGLF within 2 h was as shown in Figure 1. QIGLF was hardly degraded within 1 h, and had a good stability to Caco-2 cell monolayers with a recovery of 96.33% after 2 h incubation time. This result was associated with the strong resistance of QIGLF to gastrointestinal proteases described previously,8 and comparable to the stability of VLPVP31 and VYIHPF30 to Caco-2 cell monolayers. In general, food-derived protein and peptide are degraded by proteases and peptidases in the gastrointestinal tract prior to being absorbed into the systemic blood circulation and target sites. The extensive enzyme system in gastrointestinal lumen, epithelial cells, and mucosal membrane constituted an important enzymatic barrier, and the brush border membrane enzymes might mainly cleave the peptide entered into Caco-2 cells to free amino acids or dipeptides.32 However, QIGLF had

Figure 2. Effects of incubation time on the transport of QIGLF across Caco-2 cell monolayers. One mM QIGLF (dissolved in HBSS, pH 7.4) was added to AP side, and samples were collected from BL side for HPLC analysis at 30, 60, 90, and 120 min. All values were the means ± SD (n = 3).

amount of QIGLF in the basolateral side increased linearly up to 2 h, and the corresponding Papp values increased from 2.43 × 10−7 cm/s at 30 min to 9.11 × 10−7 cm/s at 120 min. Figure 3 showed the effect of QIGLF concentration in apical side on the transport. The transport kinetic parameters were also examined (Km, 32.37 ± 12.59 mM; Vmax, 1.23 ± 0.49 μM/min cm2). The Papp value of QIGLF at 1 mM from the apical to basolateral side for 2 h was (9.11 ± 0.19) × 10−7 cm/s, suggesting that less than 2% QIGLF was transepithelial transported intact across Caco-2 cell monolayers. This was higher than that of KVLPVP,35 HLPLP,36 Gly-Ala-Hyp-Gly-Leu-Hyp-Gly-Pro,37 VYIHPF,30 3179

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significantly alter the Papp value of QIGLF (P > 0.05, Figure 4), suggesting that transcytosis was not the main mechanism for the transport of QIGLF. In general, beside the transporter-mediated transport for basic molecules and the transcytosis for macromolecules, there are two other pathways for the intestinal transport of nutrients, transcellular passive diffusion for hydrophobic substances, and paracellular transport for water-soluble low-molecular substances. The transcellular passive diffusion depends on the liposolubility of substance to the lipid bilayers. However, the paracellular permeability is regulated by tight junctions, which could be evaluated by measuring the TEER. In the present study, the effect of paracellular pathway on QIGLF transport was also examined. Cytochalasin D, a tight junction disruptor, was added to Caco-2 cell monolayers and preincubated for 30 min to reduce the tight junction. The result exhibited that the TEER value decreased 10% (data not show), and the Papp value of QIGLF increased significantly (P < 0.05, Figure 4), suggesting that the QIGLF was transepithelial transported through Caco-2 monolayers possible mainly via paracellular pathway. This result resembled the transport route of oligopeptides VLPVL,31 KVLPVP,35 HLPLP,36 Gly-Ala-HypGly-Leu-Hyp-Gly-Pro,37 GGYR,40 and tripeptide VPP,41 which were all described previously. Besides cytochalasin D, bile salts34 and sodium deoxycholate35 were also often used to increase peptides paracellular permeability across Caco-2 cell monolayers by regulating the tight junction of Caco-2 cells and influencing the hydrolysis of peptides on the microvilli. What should be noted was that Caco-2 cell monolayers had a higher tight junction than human intestinal epithelial cells, indicating that the paracellular permeability of QIGLF across Caco-2 cell monolayers was lower than in vivo human intestine.42 Cytoprotective Effects of QIGLF on Caco-2 Cells Exposed to H2O2. To determine the effects of QIGLF on the viability of Caco-2 cells exposed to H2O2, Caco-2 cells were cultured with varying concentrations of QIGLF (1 and 5 mM) for 2 h followed by exposure to varying concentrations of H2O2 (250, 500, and 1000 μM) for another 6 h, and the cell viability was evaluated by MTS assay. As shown in Figure 5 A, the viability of Caco-2 cells decreased significantly when exposed to H2O2 with a dose−response effect (P < 0.05). When the concentration of H2O2 was up to 500 μM, 90.52% viability was maintained through the test period (P < 0.05); however, 1000 μM H2O2 led to only 86.90% viability remained (P < 0.01), which would bring excessive damage to Caco-2 cells. Therefore, 500 μM H2O2 was selected in the present study. Interestingly, treating with QIGLF could significantly improve the Caco-2 cells viability by protecting from oxidative damage induced by H2O2 (P < 0.05, Figure 5B). The QIGLF at high concentration of 5 mM offered a strong protection by enhancing H2O2decreased cell viability up to 99.48%, with a slightly greater effect than that shown by 1 mM (95.53%), and those concentrations were all lower than the toxic concentration. Human intestinal epithelium composes a nature barrier that selectively regulates the absorption of nutrients. However, the oxidative damage of intestinal epithelium might result in the loss of barrier function and chronic diseases.43 It is well-known that H2O2 is not only an important member of the reactive oxygen species (ROS) family, but also can penetrate the cell membrane and react with intracellular metal ions to form hydroxyl radicals, both of which can cause severe damage to cultured cells. In addition, H2O2 and ROS are involved in the modulation of nuclear factor erythroid 2-related factor (Nrf2),

Figure 3. Effects of concentration of QIGLF on the transport across Caco-2 cell monolayers. Different concentrations of QIGLF (1, 2, 4, 8, and 10 mM; dissolved in HBSS, pH 7.4) were added to AP side, and samples were collected from BL side for HPLC analysis at 120 min. All values were the means ± SD (n = 3).

YPFPG, and SRYPSY,38 but lower than that of VLPVP,31 all of which had been identified with significant ACE inhibitory activities. The high absorptivity of QIGLF might be due to its hydrophobic uncharged residues such as Ile, Leu, and Phe. Effects of Various Compounds on QIGLF Transport. To determine which transport pathways were involved in the transepithelial absorption of QIGLF, some inhibitors were added to Caco-2 cell monolayers to preincubate for 30 min before the transport experiment. As shown in Figure 4, Gly-Pro,

Figure 4. Effects of various compounds on the transport of QIGLF across Caco-2 cell monolayers. One mM QIGLF (dissolved in HBSS, pH 7.4) was added to AP side following the preincubation with transport inhibitors (wortmannin, sodium azide, and Gly-Pro and cytochalasin D) for 30 min, and samples were collected from BL side for HPLC analysis at 120 min. #P < 0.05 as compared to the control group. All values were the means ± SD (n = 3).

a good substrate for peptide transport PepT 1, had little effect on the transport (P > 0.05), suggesting that transport of QIGLF across the Caco-2 cell monolayers was not mediated by PepT1. The finding was unchanged when adjusted for the use of sodium azide, an ATP synthesis inhibitor, suggesting that QIGLF transport was energy-independent. It is known that PepT1 was mainly responsible for the transepithlelial transport of many di- and tripeptides, such as IRW and YPI,29,39 bur not for the QIGLF in our present study. Furthermore, the effect of transcytosis initiated by endocytosis at the apical cell membrane on QIGLF transepithelial transport was examined by a 30 min preincubation for Caco-2 cell monolayers with wortmannin, a transcytosis inhibitor. However, the presence of wortmannin did not 3180

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Figure 5. Cytoprotective effects of QIGLF on Caco-2 cells. (A) The cytotoxicity of H2O2 on viability in normal Caco-2 cells. Caco-2 cells were exposed to H2O2 at indicated concentrations of 250, 500, and 1000 μM for 6 h at 37 °C in 5% CO2. (B) The cytoprotective effect of QIGLF on oxidative damage induced by H2O2 in Caco-2 cells. Caco-2 cells were incubated with couple concentrations of QIGLF (1 and 5 mM, dissolved in HBSS, pH 7.4) for 2 h at 37 °C in 5% CO2 followed by the addition of 500 μM H2O2 for another 6 h incubation. The cell viability was evaluated by MTS assay. All values were the means ± SD (n = 3).

an emerging regulator of cellular resistance to oxidative stress.44 In the present study, we observed that the treatment with 5 mM QIGLF could improve Caco-2 cells viability by protecting from oxidative damage induced by H2O2. However, QIGLF showed very low DPPH radical-scavenging activity in vitro in our previous work.6 Thus, in synthesis, the cytoprotective effect might be involved in the Keap1/Nrf2/ARE signaling pathway, thereby upregulating the gene expression of antioxidant enzyme.

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(2) Miguel, M.; Recio, I.; Gomez-Ruiz, J. A.; Ramos, M.; LopezFandino, R. Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis. J. Food Prot. 2004, 67, 1914−1920. (3) Miguel, M.; Lopez-Fandino, R.; Ramos, M.; Aleixandre, A. Longterm intake of egg white hydrolysate attenuates the development of hypertension in spontaneously hypertensive rats. Life Sci. 2006, 78, 2960−2966. (4) Majumder, K.; Wu, J. Purification and characterisation of angiotensin I converting enzyme (ACE) inhibitory peptides derived from enzymatic hydrolysate of ovotransferrin. Food Chem. 2011, 126, 1614−1619. (5) Liu, J.; Yu, Z.; Zhao, W.; Lin, S.; Wang, E.; Zhang, Y.; Hao, H.; Wang, Z.; Chen, F. Isolation and identification of angiotensinconverting enzyme inhibitory peptides from egg white protein hydrolysates. Food Chem. 2010, 122, 1159−1163. (6) Yu, Z.; Liu, B.; Zhao, W.; Yin, Y.; Liu, J.; Chen, F. Primary and secondary structure of novel ACE-inhibitory peptides from egg white protein. Food Chem. 2012, 133, 315−322. (7) Yu, Z.; Yin, Y.; Zhao, W.; Wang, F.; Yu, Y.; Liu, B.; Liu, J.; Chen, F. Characterization of ACE-inhibitory peptide associated with antioxidant and anticoagulation properties. J. Food Sci. 2011, 76, C1149−C1155. (8) Yu, Z.; Zhao, W.; Liu, J.; Lu, J.; Chen, F. QIGLF, a novel angiotensin I-converting enzyme-inhibitory peptide from egg white protein. J. Sci. Food Agric. 2011, 91, 921−926. (9) Li, P.; Nielsen, H. M.; Mullertz, A. Oral delivery of peptides and proteins using lipid-based drug delivery systems. Expert Opin. Drug Delivery 2012, 9, 1289−1304. (10) Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 1989, 96, 736−749. (11) Linnankoski, J.; Makela, J.; Palmgren, J.; Mauriala, T.; Vedin, C.; Ungell, A.-L.; Lazorova, L.; Artursson, P.; Urtti, A.; Yliperttula, M. Paracellular porosity and pore size of the human intestinal epithelium in tissue and cell culture models. J. Pharm. Sci. 2010, 99, 2166−2175. (12) Shimizu, K.; Sato, M.; Zhang, Y.; Kouguchi, T.; Takahata, Y.; Morimatsu, F.; Shimizu, M. Molecular size of collagen peptide reverses the permeability of Caco-2 cells. Biosci. Biotechnol. Biochem. 2010, 74, 1123−1125. (13) Artursson, P.; Palm, K.; Luthman, K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Delivery Rev. 2001, 46, 27−43. (14) Yee, S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man–fact or myth. Pharm. Res. 1997, 14, 763−766.

AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 431 87836351. Fax: +86 431 87836391. E-mail: [email protected]. Funding

This work was funded by the National Natural Science Foundation of China (no. 31271907), Specialized Research Fund for the Doctoral Program of Higher Education (no. 20130061110088), and Project of National Key Technology Research and Development Program (no. 2012BAD33B03). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED QIGLF, Gln-Ile-Gly-Leu-Phe; ACE, angiotensin-converting enzyme; PepT1, peptide transporter 1; DMEM, Dulbeco’s Modified Eagle’s Medium; HBSS, Hank’s balanced salt solution; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetra-zolium bromide; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium; TFA, trifluoroacetic acid; TEER, transepithelial electrical resistance; Nrf2, nuclear factor erythroid 2-related factor 2

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REFERENCES

(1) Yu, Z.; Yin, Y.; Zhao, W.; Yu, Y.; Liu, B.; Liu, J.; Chen, F. Novel peptides derived from egg white protein inhibiting alpha-glucosidase. Food Chem. 2011, 129, 1376−1392. 3181

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Journal of Agricultural and Food Chemistry

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dx.doi.org/10.1021/jf405639w | J. Agric. Food Chem. 2014, 62, 3177−3182