Structure And Transport Of ACE-inhibitory Peptides Derived From Egg White

The intact absorption of peptide means that some peptides can be absorbed intoblood circulation in intact form and without degradation by enzymes ingastrointestinal tract and intestinal epithelium after oral administration. Since up tonow, the intact absorption of many dipeptides and tripeptides have been widelyaccepted. But this issue is still obscure for oligopeptides with more than three aminoacids. It is a critical problem that can limit the development of study and applicationof food derived bioactive peptides today because most of the bioactive peptidespeople have found are of length more than three amino acids.The purpose of this study was to investigate the intact transepithelial transport ofegg white derived ACE-inhibitory peptides and relationship between the structuresand transport of peptides across Caco-2cell monolayer. Firstly, a Caco-2cellmonolayer model was established. Then, the Papp and possible transport pathway ofegg white derived ACE-inhibitory peptides across Caco-2cell monolayer weredetermined. At last, the relationship between the structures and transport was studiedby analyzing the Papp of a series of peptides, which were reformed and synthesizedbased on the sequence of RVPSL. The whole study could be divided into three partsas follow:1. The building and integrity evaluation of Caco-2cell monolayer.The Caco-2cells were seeded on the12-well transwell insert plates at a densityof1×105cell/insert. After continuous incubation for21to23day, the integrity ofCaco-2cell monolayer was evaluated by determining the TEER value, ALP activityand observing the cell morphous using confocal laser scanning microscopy. Resultsshowed that the TEER value of Caco-2cell monolayer became stable at about400cm2from19to23day, suggesting that the tight junctions had been formed. The ALP activity of AP surface was13.66folds of BL surface at21day, indicating that Caco-2cells had been differentiated completely with high cell polarity. To visualize thedistribution and formation of tight junctions, Caco-2cell layers were double-stainedwith FITC-labeled phalloidin and DAPI. After21days of incubation, the Caco-2cellsdisplayed thin monolayer with high tight junctions. In all, the Caco-2cell monolayerin this study had been well built, and could be used to simulate the intestinalepithelium in vitro and experiment the transport of egg white derived ACE-inhibitorypeptides.2. The study of intact transport of egg white derived ACE-inhibitory peptidesacross Caco-2cell monolayer, including QIGLF (IC50:75μM) and RVPSL (IC50:20μM).(1) The transport study of QIGLF, an egg white derived ACE-inhibitory peptidewhich was stable to gastrointestinal tract.It was found that more than96%of the initial QIGLF were recovered afterincubation for2h with Caco-2cell monolayer. The transport rate of QIGLF wasincreased with the increase of transport time and initial concentration. The kineticparameters had been determined to Km:32.37±12.59mM, Vmax:1.23±0.49uM/mincm2. When the initial peptide concentration of1mM and the transport time of2hwere used, the transport of QIGLF exhibited the highest rate with Papp value of(9.11±0.19)×10-7cm/s. Moreover, the result of the study for transport mechanismsuggested that paracellular diffusion might be the main pathway for the intacttransport of QIGLF across Caco-2cell monolayer.(2) The transport study of RVPSL, an egg white derived ACE-inhibitory peptidewhich was susceptive to gastrointestinal tract.It was found that about36.31%of the initial RVPSL were degraded by the brushborder membrane enzymes, which were distributed on the AP surface of Caco-2cellmonolayer, after incubation for2h with Caco-2cell monolayer. However, diprotin A,an inhibitor of DPPIV, could decrease this degradation to23.49%. The results of the bidirectional transport of RVPSL across Caco-2cell monolayer showed that the Papp(AP-BL) value of (6.97±1.11)×10-6cm/s was significantly higher than the Papp(BL-AP) value of (2.52±0.08)×10-6cm/s. Diprotin A could also increase the Papp(AP-BL) value to (6.53±0.08)×10-6cm/s, which was not differed from the Papp(AP-BL) value of (7.29±0.81)×10-6cm/s, which was determined in the presence ofdiprotin A. All these results indicated that the bidirectional transport of RVPSL acrossCaco-2cell monolayer was passive and non-polar. In addition, the paracellulardiffusion was experimented might to be the main pathway for the intact transport ofRVPSL across Caco-2cell monolayer.3. Study on the relationship between the structures and transport of peptidesacross Caco-2cell monolayer.The Papp of a series of peptides, which were reformed and synthesized based onthe sequence of RVPSL, were determined across Caco-2cell monolayer. Resultsshowed that N-terminal amino acid residue replacement by Pro of RVPSL but notC-terminal amino acid residue, the Papp of these peptides were arranged in the orderof PPP-X-X> RPP-X-X> PVP-X-X> RVP-X-X (X represented Pro, Leu or Ser),suggesting that N-terminal Pro residue might be more beneficial to the transportacross Caco-2cell monolayer than Val and Arg residues. Furthermore, the tetrapeptideRVPS had a higher Papp than RVPSL, but VPSL showed the lowest Papp value.Dipeptide RV and SL, tripeptide RVP and PSL were all more easily transported thanRVPSL. It might be due to that dipeptide and tripeptide could be transported by PepT1mediated transport pathway, which had higher transport efficiency than paracellularpathway.