Importance of Terminal Amino Acid Residues to the Transport of

Aug 16, 2017 - Pentapeptides with N- or C-terminal Tyr also showed high permeability levels, with Papp values of about 10 × 10–6 cm/s. ..... of pep...
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Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides across Caco-2 Cell Monolayer Long Ding, Liying Wang, Zhipeng Yu, Sitong Ma, Zhiyang Du, Ting Zhang, and Jingbo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03450 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

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Importance of Terminal Amino Acid Residues to the Transport of Oligopeptides

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across Caco-2 Cell Monolayer

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Long Ding†, Liying Wang†, Zhipeng Yu‡, Sitong Ma†, Zhiyang Du†, Ting Zhang†,

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Jingbo Liu†*

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6

China

7



8

China

College of Food Science and Engineering, Jilin University, Changchun 130062, P.R.

College of Food Science and Engineering, Bohai University, Jinzhou 121013, P.R.

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Running title: Terminal Amino Acid Residues and Transport of Oligopeptides

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*Corresponding author: Tel.: +86 431 87836351. Fax: +86 431 87836391. E-mail:

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[email protected].

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ABSTRACT: The objective of this paper was to investigate the effects of terminal

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amino acids on the transport of oligopeptides across Caco-2 cell monolayer. Ala-based

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tetra- and penta-peptides were designed and the N-terminal or C-terminal amino acid

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residues were replaced by different amino acids. The results showed that the

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oligopeptides had a wide range of transport permeability across Caco-2 cell

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monolayer and could be divided into 4 categories: non/poor permeability, low

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permeability, intermediate permeability, and good permeability. Tetrapeptides with

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N-terminal Leu, Pro, Ile, Cys, Met, Val or C-terminal Val showed the highest

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permeability with apparent permeability coefficient (Papp) values over 10×10-6 cm/s (P

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< 0.05), suggesting that non-polar hydrophobic aliphatic amino acids or polar

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sulfur-containing amino acids were the best for the transport of tetrapeptides.

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Pentapeptides with an N-terminal or C-terminal Tyr also showed high permeability

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levels with Papp values about 10×10-6 cm/s. The amino acids Glu, Asn, Thr at the

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N-terminus or Lys, Asp, Arg at the C-terminus were also beneficial for the transport of

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tetra- and penta-peptides with Papp values ranging from 1×10-6 cm/s to 10×10-6 cm/s.

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In addition, peptides with amino acids replaced at the N-terminus generally showed

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higher permeability than those with amino acids replaced at the C-terminus (P < 0.05),

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suggesting that N-terminal amino acids were more important for the transport of

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oligopeptides across Caco-2 cell monolayer.

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KEYWORDS: oligopeptide, amino acid, transport permeability, Caco-2 cell

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monolayer, brush border membrane peptidase

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INTRODUCTION

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Bioactive peptides derived from enzymatic hydrolysis of food proteins have

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generated much attention for their potential human health-promoting functions and

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limited adverse effects. In the last few decades, a large number of bioactive peptides

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have been identified from food proteins, including animal and plant proteins, as well

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as marine proteins. These bioactive peptides have been shown to have different

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biological activities such as antihypertensive, antioxidant, anti-inflammatory,

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immunomodulatory, anticancer, antimicrobial, and calcium-binding activities.1

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However, it has been found that the in vitro bioactivities of peptides do not always

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correlate with in vivo activity.2 This is mainly because, most food-derived bioactive

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peptides have to overcome two important physiological barriers, extensive enzymatic

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degradation in the gastrointestinal tract, poor permeability through the intestinal

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epithelium, and be absorbed in an intact form into the circulation to exert their

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bioactive functions in vivo after oral administration.3 This implies the importance of

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resistance to various peptidases, including brush border membrane peptidases, and

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absorption through the intestine for the bioavailability of food-derived bioactive

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peptides.

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Neway and Smith first found that some dipeptides, such as glycyl-glycine, could be

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absorbed through the intestine without hydrolysis.4,5 Two important H+-coupled

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transporters for dipeptides, peptide transporter 1 (PepT1) and peptide transporter 2

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(PepT2), were successfully cloned and identified in the 1990s, providing evidence for

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the intact absorption of dipeptides at the molecular level.6,7 Moreover, many 3

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tripeptides or peptidomimetic drugs were also demonstrated to be transported by

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PepT1 and PepT2.8 However, the exact transport pathways for oligopeptides,

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especially peptides containing 4 to 10 amino acid residues, remain unknown.

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Compared with di- or tri-peptides, oligopeptides are generally more easily hydrolyzed

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by brush border membrane peptidases and have a lower permeability through the

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intestine. It is speculated that some oligopeptides might be transported through the

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intestinal epithelium by transcytosis and the tight junction-mediated paracellular

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pathway,9 although convincing empirical evidence is lacking. In addition, Ananth and

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colleagues10,11, and Chothe and colleagues12,13 reported two novel Na+-coupled

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oligopeptide transporters SOPT1 and SOPT2, that can transport peptides consisting of

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five or more amino acids. These two new peptide transporters differ from PepT1 and

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PepT2, as the transport of oligopeptides through SOPT1 and SOPT2 can be

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stimulated or inhibited by some di- or tri-peptides. Nevertheless, the genes encoding

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SOPT1 and SOPT2 and their protein structures have not yet been fully identified.

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Actually, regardless of the oligopeptide transport pathway, it is important to

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understand the relationship between transport and structural features of oligopeptides

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or whether there are structural determinants. In our previous study, the relationship

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between the structure of RVPSL and transport across Caco-2 cell monolayer was

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investigated. A series of pentapeptides derived from RVPSL was obtained by amino

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acid replacement with proline. It was found that N-terminal Pro was more beneficial

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for the transport of pentapeptides across Caco-2 cell monolayer than Arg and Val

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residues, while no similar results were observed at the C-terminus.14 To further verify 4

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the effects of terminal amino acid residues on the transport of oligopeptides, up to 78

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Ala-based tetra- and penta-peptides with different N-terminal or C-terminal amino

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acids were designed and their transport across Caco-2 cell monolayer was

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investigated in the present study. In addition, the enzymatic degradation of peptides

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by brush border membrane peptidases of Caco-2 cells was studied.

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MATERIALS AND METHODS

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Chemicals. Caco-2 cell lines were purchased from American Type Culture

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Collection. All peptides were synthesized by solid-phase procedures using

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Fmoc-protected amino acid synthetic methods with purity over 95% by ChinaPeptide

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Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal

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bovine serum, penicillin-streptomycin, nonessential amino acid solution, and Hanks’

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balanced salt solution (HBSS) were purchased from Gibco BRL Life Technology

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(Carlsbad, CA, USA). Trypsin, L-glutamine, dimethyl sulfoxide (DMSO),

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ethylenediaminetetraacetic acid (EDTA), and collagen type I were all purchased from

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Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN), trifluoroacetic acid (TFA),

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and methanol were purchased from Fisher Scientific (Waltham, MA, USA). All other

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reagents were analytical grade.

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Cell culture. Caco-2 cells were grown in DMEM containing 10% fetal bovine

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serum, 1% nonessential amino acid solution, 100 units/mL penicillin, 100 µg/mL

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streptomycin, and 4 mM L-glutamine at 37 °C in an atmosphere of 5% CO2 and 90%

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relative humidity. Stock cultures were grown in 75 cm2 tissue culture flasks and were

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split at 80% to 90% confluency using 0.25% trypsin and 0.02% EDTA solution. The 5

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cells from passage numbers of 30−40 were used and seeded on collagen type I-coated

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permeable polycarbonate inserts (12 mm diameter, 0.4 µm pore size, 1.12 cm2 grown

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surface area; Costar, Corning, NY, USA) in 12 transwell plates at a density of 1 × 105

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cells/insert. The culture medium (0.5 mL on the apical side (AP) and 1.5 mL on the

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basolateral side (BL)) was replaced every 2 days for the first week and then daily until

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the cells were used for experiments 21 days after seeding. The integrity of the cell

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monolayer was evaluated by measuring transepithelial electrical resistance (TEER)

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values with an EVOM epithelial volt/ohmmeter (Millicell-ERS, Millipore, Billerica,

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MA, USA). Only Caco-2 cell monolayers with TEER values higher than 300 Ω·cm2

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were used for transport studies.

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Transport experiments. The transepithelial transport of peptides across Caco-2

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cell monolayer was conducted according to the method described by Hubatsch.15 The

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Caco-2 cell monolayers were rinsed with HBSS twice and then incubated with HBSS

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for 30 min at 37 ℃ in 5% CO2 prior to transport experiments. The HBSS was

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removed and replaced with 0.5 mL of 3.0 mM peptides (dissolved in HBSS, pH 7.4)

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on the AP and 1.5 mL fresh HBSS on the BL (pH 7.4). All plates were incubated at

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37 ℃ in 5% CO2 for 2 h. Peptide concentrations on both the AP and BL were

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determined by RP-HPLC analysis.

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The apparent permeability coefficient (Papp, cm/s) was calculated as follows:  =

 1 1 × × ×  

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where dC/dt was the change of peptide concentration on the BL (mM/s); A was the

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area of the membrane (cm2); C0 was the initial peptide concentration on the AP (mM); 6

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V was the volume of the BL (mL).

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During the process of absorption, peptides were partly degraded by various brush

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border membrane enzymes into smaller peptides and amino acids and this might have

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decreased the concentrations of peptides on the AP and affected the permeability of

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peptides. For many peptides, the more they were degraded by brush border membrane

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enzymes, the less well absorbed they were. Thus, we introduced a new permeability

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coefficient (Papp’) here to account for the final peptide concentrations on the AP in the

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present study.

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The Papp’ (cm/s) was calculated as follows:  ′ =

 1 1 × × ×  

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where dC/dt was the change of peptide concentration on the BL (mM/s); A was the

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area of the membrane (cm2); Ct was the peptide concentration on the AP after 2 h

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(mM); V was the volume of the BL (mL).

134 135

Degradation of peptides by various brush border membrane enzymes (cytosolic peptidases were not included) was calculated as follows: Degradation % = 1 −

 × +  × " × 100%  ×

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where C0 and Ct were the initial and final peptide concentrations on the AP (mM),

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respectively, and C was the peptide concentration on the BL (mM); V0 and V were the

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volumes of the AP and BL (mL), respectively.

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RP-HPLC. Peptides were quantified using a Shimadzu HPLC system equipped

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with LC solution software, an LC-20AD binary gradient pump, an SIL-20AC

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autosampler, a CTO-20AC column oven and SPD-20AV UV detector (Shimadzu, 7

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Kyoto, Japan). The analytical column was a C18 reverse-phase column (Symmetry C18

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column, 250×4.6 mm, particle size 5 mm, Waters, Milford, MA, USA). The mobile

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phases were 0.1% TFA in water (solvent A) and 0.1% TFA in ACN (solvent B).

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Samples were eluted with a gradient of solvent B from 15% to 55% over 15 min and

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held at 55% for 2 min at a flow rate of 0.5 mL/min. The injection volume was 20 µL,

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and the absorbance detector wavelength was set at 220 nm. Peptide concentrations

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were calculated by a linear regression analysis of known concentrations ranging from

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1 µM to 3000 µM of peptide standard and corresponding peak areas. The calibration

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curves of peak area vs concentration and the coefficients were then analyzed.

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Statistical analysis. All data were expressed as the mean ± SD (n = 3). Differences

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between two means were detected by unpaired Student’s t-tests. When there were

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more than two means, the differences were analyzed by one-way analysis of variance

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(ANOVA) followed by the least significant difference (LSD) test with the significance

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level at P < 0.05 using SPSS 19.0 software.

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RESULTS

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Transport of peptides. An Ala-based tetrapeptide (Ala-Ala-Ala-Ala) and

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pentapeptide (Ala-Ala-Ala-Ala-Ala) were firstly designed and their N-terminal or

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C-terminal amino acid residues were then replaced with other amino acids. In total, 78

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tetra- and penta-peptides with different N-terminal or C-terminal amino acid residues

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were obtained and synthesized. The permeability coefficients of Papp and Papp’ of these

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oligopeptides were determined in Caco-2 cell monolayer.

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N-terminal amino acids had a large influence on the transport of tetrapeptides 8

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(Figure 1A and B). It was found that tetrapeptides with N-terminal amino acid

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residues of Leu, Ile, Pro, Cys, Met or Val had the highest transport permeability with

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Papp and Papp’ values over 20×10-6 cm/s and 30×10-6 cm/s, respectively. Tetrapeptides

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with N-terminal Tyr, Glu, Asn, Thr, Asp, Gly or His all had relatively low

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permeability with Papp and Papp’ values ranging from 1×10-6 cm/s to 10×10-6 cm/s,

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respectively. Tetrapeptides with N-terminal Phe, Gln, Ser, Lys, Ala, Arg and Trp were

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either poorly transported or not transported at all. Compared with the N-terminal

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amino acids, tetrapeptides with altered C-terminal amino acid residues generally

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showed lower permeability (Figure 1C and D). Only the tetrapeptide with the Val at

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C-terminus had a Papp value greater than 10×10-6 cm/s, followed by that with Cys, Glu,

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Lys, Asp, Thr and Arg, ranging from 1×10-6 cm/s to 10×10-6 cm/s. However, the two

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tetrapeptides with C-terminal Val and Cys residues both exhibited Papp’ values greater

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than 40×10-6 cm/s. The amino acids Tyr, Trp, Ala, Ile, Asn, Gln, Ser, His, and Gly at

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the C-terminus all caused tetrapeptides to be either poorly transported or not

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transported at all across Caco-2 cell monolayer.

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Pentapeptides with the N-terminal amino acids Asp, Gly and Tyr had Papp values

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greater than 10×10-6 cm/s, followed by Asn, Arg, Glu, Gln, Thr, Lys, His and Cys that

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ranged from 1×10-6 cm/s to 10×10-6 cm/s (Figure 2A). Pentapeptides with N-terminal

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Ser, Ala, Pro, Leu, Phe, Trp, and Val residues had poor permeability or were not

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transported at all. However, what should be noted was that the pentapeptide with an

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N-terminal Tyr residue had the highest Papp’ value of approximately 70×10-6 cm/s and

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it was significantly higher than pentapeptides with N-terminal Asp, Gly and Asn 9

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residues (Figure 2B). Interestingly, the pentapeptide with C-terminal Tyr also showed

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the highest Papp value compared with other C-terminal amino acids, although the Papp

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value was only approximately 7×10-6 cm/s. Pentapeptides with the C-terminal amino

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acids Pro, Lys, Asp, Cys, Gln, Arg and Val had lower Papp values ranging from 1×10-6

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cm/s to 5×10-6 cm/s. Pentapeptides with Ala, Phe, Met, Ile, Trp, Leu, Glu, and Thr at

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the C-terminus were non-permeable or had poor transport permeability (Figure 2C).

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Notably, pentapeptides with different C-terminal amino acids showed lower Papp’

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values compared with N-terminal replaced pentapeptides. Only the pentapeptides with

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C-terminal Cys and Tyr had Papp’ values greater than 10×10-6 cm/s. However, it was

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found that the Papp’ value of the pentapeptide with C-terminal Cys was higher than

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that with Tyr. Pentapeptides with C-terminal Asp, Lys, Pro, Val, Gln, Arg, Gly, Thr,

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Asn and Leu also showed intermediate permeability with Papp’ values ranging from

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1×10-6 cm/s to 10×10-6 cm/s (Figure 2D).

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The effects of the replacement of different amino acids at the N-terminus and

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C-terminus on the transport permeability of Ala-based tetra- and penta-peptides across

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the Caco-2 cell monolayer were compared. As shown in Figure 3, the replacement of

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N-terminal amino acids generally improved the transport permeability of Papp (P
10

> 20

Leu, Ile, Pro, Cys, Met and Val at N-terminus; Val and Cys at C-terminus

Category

Tetrapeptides

Pentapeptides

Lys, Ser, Gln and Phe at N-terminus; Trp and Tyr at C-terminus His and Trp at N-terminus; Pro, His, Gly, Ser and Gln at C-terminus

Ser at N-terminus; Trp, Met, Ile, Phe and Ala at C-terminus

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Met, Val, Trp, Phe and Ile at N-terminus; Gly, His, Asn, Ser, Thr, Glu and Leu at C-terminus Asn, Arg, Glu, Gln, Thr, Lys and Cys at N-terminus; Tyr, Pro, Lys, Asp, Cys, Gln, Arg and Val at C-terminus Tyr, Asp and Gly at N-terminus

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Figure legends Figure 1. Effects of N-terminal (A, B) and C-terminal (C, D) amino acid residues on the permeability coefficients Papp (A, C) and Papp’ (B, D) for tetrapeptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 2. Effects of N-terminal (A, B) and C-terminal (C, D) amino acid residues on the permeability coefficients Papp (A, C) and Papp’ (B, D) for pentapeptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 3. Effects of different amino acids at the N-terminus and C-terminus on the transport permeability of Papp (A) and Papp’ (B) for Ala-based tetra- and penta-peptides across Caco-2 cell monolayer. All values are means ± SD (n = 3). Figure 4. Correlation between the transport permeability of Papp and Papp’ of a number of Ala-based tetra- and penta-peptides with different N-terminal or C-terminal amino acids across Caco-2 cell monolayer. The solid line represents the fitted equation for all plots: y=2.5460x+0.6116; R2=0.8420, n=78; The dotted line represents the fitted equation that did not include the plots of AAAC, AAAV and YAAAA: y=2.3538x-0.0817; R2=0.8626, n=75. Figure 5. Effects of N-terminal amino acid residues on the degradation of tetrapeptides (A) and pentapeptides (B) by brush border membrane enzymes of Caco-2 cells. All values are means ± SD (n = 3). Figure 6. Effects of C-terminal amino acid residues on the degradation of tetrapeptides (A) and pentapeptides (B) by brush border membrane enzymes of Caco-2 cells. All values are means ± SD (n = 3). 25

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Papp (1×10 , cm/s) 8 -6

15

10

Papp' (1×10 , cm/s)

-6

-6

Papp (1×10 , cm/s) 20

10

6

4

FA A Q A AA SA A A KA A A RA A A AA A W AA AA HA A A DA A A G A AA NA A A EA A A TA A A YA A A VA A A PA A A M A AA A IA A CA A A LA A AA

16

Papp' (1×10 , cm/s)

FA A Q A AA SA A A KA A A AA A A RA A W AA AA HA A A G A AA DA A A TA A A NA A A EA A A YA A A VA A A M A AA CA A A IA A A PA A A LA A AA

35

AA AA AY A AA W A AA N A AA A A AA Q A AA S AH AA AA AI A AA G A AA P A AA M A AA F A AA L A AA D A AA T A AA R A AA E A AA K A AA C AV

AA AA AY A AA W A AA A AA AI A AA N A AA Q A AA S A AA F A AA G A AA M A AA H A AA L A AA P A AA R A AA T AD AA A AA K A AA E A AA C AV

Journal of Agricultural and Food Chemistry

(A)

Papp 90

30

5

0

(C) 80

2 10

0 0

(B)

Papp'

80

25 70

60

50

40

30

20

10 0

14

(D)

12 70

60

50

40

30

20

Figure 1

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Papp (1×10 , cm/s) 4 -6

5

3

2

Papp' (1×10 , cm/s)

-6

12 60

10 50

6

4

Papp' (1×10 , cm/s)

-6

Papp (1×10 , cm/s) 8

8 20

SA A AA AA A LA AA A IA AA W AAA AA FA AA A VA AA A M AA AA HA AA A PA AA A KA AA A CA AA A TA AA A Q AA AA EA AA A RA AA A NA AA A G AA AA DA AA A YA AA AA A

SA A AA AA A PA AA A LA AA AA IA A A FA AA W AAA AA VA AA A M AA AA CA AA A HA AA A KA AA A TA AA A Q AA AA EA AA A RA AA A NA AA A YA AA A G AA AA DA AA AA A

14

AA A AA AA A AA AF AA AA M AA AA A I AA AW A AA AE A AA AH A AA AS A AA AL A AA AN A AA AT A AA AG A AA AR A AA AQ A AA AV A AA AP A AA AK A AA AD A AA AY AA C

AA A AA AA A AA AF AA AA M AA AA AA I AA W A AA AL A AA AE A AA AT A AA AS A AA AN A AA AH A AA AG A AA AV A AA AR A AA AQ A AA AC A AA AD A AA AK A AA AP AA Y

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(A)

Papp 70

2 10

0 0

(C)

7 18

1

0

(B)

Papp'

40

30

20

(D)

6 16

14

12

10

8

6

4

2

0

Figure 2

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N-terminus C-terminus

30

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Papp (1×10 cm/s)

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25 20 15 10 5 0 0 90

5

10

15

20

25

30

35

40

35

40

number of peptides

(B)

N-terminus C-terminus

80

60 50

-6

Papp' (1×10 cm/s)

70

40 30 20 10 0

0

5

10

15

20

25

number of peptides

30

Figure 3

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y=2.5460x+0.6116, 2 R =0.8420, n=78

80

YAAAA 70

AAAV

-6

Papp' (1×10 cm/s)

60 50

AAAC

y=2.3538x-0.0817, 2 R =0.9626, n=75

40 30 20 10 0 0

5

10

15

20

25

30

-6

Papp (1×10 cm/s) Figure 4

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H A R AA A A G AA A A D AA AA A TA AA EAAA A KAAA A A IA A A Q AA A A W AA A A C AA AA A N A A A VAAA A FAAA A M AA AA A LA A A YAAA A AAAA A PAAA A SAAA AAA A

Degradation (%) PA LAAA VAAA H AA AA IA A C AA A YAAA M AA A D AA A EAAA N AA A SAAA TAAA G AA A W AA AA R A A KAAA Q AA A FAAA AAAA AA

Degradation (%)

Journal of Agricultural and Food Chemistry

90

80

100

90

(A)

70

60

50

40

30

20

10

0

(B)

80

70

60

50

40

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20

10

0

Figure 5

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AA AAAAH AAAA Y AAAA P AAAA S AAAA K AA AA E AAAA G AAAA D AA AA R AAAA Q AAAA N AA AA V AAAAC AAAAT AA AA A L AA AM A A AF AA AA A AI AA AW AA A

Degradation (%) AA AA AE AAAD AAAH AA AT AA AS AAAN AA AP AAAQ AA AK AAAG AA AV AAAC AAAR AAAA AAAY A AA L AA AI AA AM A AA W AF

Degradation (%)

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100 90

100

90

(A)

80

70

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10

0

(B)

80

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Figure 6

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XAAA/XAAAA AAAX/AAAAX Caco-2 cell monolayers

Good permeability 35 30

-6

Papp (1×10 , cm/s)

25

Intermediate permeability

20

Non/Poor permeability 15 10

Low permeability

5

AA AA S W SAAA A A AA AA W AW A AAAA AA AS W A AA G AA AA AA A H AA AA S A AA A AA H A G AAAA R TAAA AAR A A AAAA A D AAAD TAAK N AA AAAA A A EAAK EA A A N AA AA A YA A A G AA AA A D A AA A AAAA VAAV M AA A C AA AA IA A PAAA LAAA AA

0

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