Mechanism Of Corn Peptides On Hepatocyte Apoptosis Induced By Ethanol,metabolism And Structure-activity Relationship Of Qllpf

With the development of scientific research,the various bioactivities of corn peptides have been developed.Our research group has conducted relatively systematical study on the facilitating alcohol metabolism and hepatoprotective activity of corn peptides.However,there is little research focused on the primary structure of the corn peptides which has facilitating alcohol metabolism and hepatoprotective activities.The molecular mechanism and effective form of the corn peptides are remained to be solved.QLLPF(Gln-Leu-Leu-Pro-Phe),a pentapeptide which was previously identified from the corn peptides in our research group,was synthesized by solid phase peptide synthesis in the study.The acitivity of facilitating alcohol metabolism,and the stability of corn peptides on temperature,pH value were investigated.The molecular mechanism of the pentapeptide and mixed corn peptides on the inhibition of hepatocyte apoptosis was also conducted.Based on the structure of the pentapeptide,the metabolites in vitro and in vivo and the structure-activity relationship of the facilitating alcohol metabolism were clarified.The main results are as follows:1 The facilitating alcohol metabolism activity and stability of corn pentapeptide QLLPFThe pentapeptide QLLPF had a significant effect on eliminating alcohol in vivo.Compared with the mixed peptides(200 mg/kg bw)which had an alcohol eliminating rate of 27.40%,the synthetic pentapeptide with a 1/20 dose(10 mg/kg bw)of that of mixed peptides had a higher alcohol eliminating rate of 35.00%.The results of the stability test of the pentapeptide in vitro showed that the residual contents of pentapeptide remained more than 95%after treatment of pH 4-10 and the temperature below 70? which indicated that the pentapeptide was stable to temperature and acid-base.We found that the residual contents of pentapeptide were about 90%after treated with pepsin and chymotrypsin for 2,respectively.The results indicated that the pentapeptide was also stable to pepsin and chymotrypsin.2 The metabolism of corn peptides attenuating hepatocyte apoptosis induced by ethanolFrom the perspective of hepatocyte apoptosis,the hepatoprotective effect of the mixed corn peptides and the corn-derived peptide QLLPF were investigated in an acute and subacute model.The corn peptides can reverse the change of the biochemical indices AST,ALT,MDA and GSH.Whats more,in the subacute model,it showed in dose-dependent manner.Generally,the effect of the mixed corn peptides(50 mg/kg bw and 200 mg/kg bw)is better than that of pentapeptide(10 mg/kg bw and 30 mg/kg bw).The peptides treatment can up-regulate the ADH and ALDH levels which could accelerate the clearance of toxic substances.And the protein expression of MnSOD was also detected,and the up-regulation of MnSOD indicated the attenuation of the oxidative stress.Besides,the results from the corn peptides inhibiting hepatocyte apoptosis showed that corn peptides can inhibit the hepatocyte apoptosis through regulating Bcl-2 family proteins,inhibiting the release of cyt C into the cytosol,regulating the cell surface receptor Fas,its ligand FasL and the important nuclear factor NF-?B,and preventing the activation of caspase-3.Specifically,the corn peptides may suppress the hepatocyte apoptosis by participating in both the intrinsic and extrinsic pathways,which is likely to be an important mechanism for preventing alcoholic liver injury.3 Metabolic characteristic of the pentapeptide QLLPFThe main metabolites in phase I of pentapeptide QLLPF in rat blood,liver and kidney were investigated in vitro and in vivo.The precipitant used for the termination reaction in the process of sample preparation was firstly screened.The result showed that the residual content of pentapeptide was about 95%after 24 h in methanol solvent(adding 0.1%of formic acid),so methanol solvent was selected as the precipitant.The detection result of the incubation metabolites of the pentapeptide in rat blood,liver and kidney in vitro showed that the pentapeptide had the fastest degradation in kidney,second fastest degradation in liver,and the slowest degradation in blood.A minimum of metabolites were found in blood,and 5 kinds of peptides:Leu-Pro,Pro-Phe,Leu-Pro-Phe,Gln-Leu-Leu-Pro,Leu-Leu-Pro-Phe were detected.Compared with the samples in blood,another kind of peptide Leu-Leu-Pro was detected in liver and two other kinds of peptides Leu-Leu-Pro and Gln-Leu-Leu were detected in kidney.We analyzed the main metabolites of pentapeptide QLLPF in rat blood,liver and kidney of SD rats in vivo intragastrically(400 mg/kg bw).We found the pentapeptide Gln-Leu-Leu-Pro-Phe in blood.The result indicated that the pentapeptide can directly be transported into the blood.We also found 3 kinds of peptides Leu-Leu-Pro-Phe,Leu-Pro-Phe and Pro-Phe in blood.One kind of metabolite(Pro-Phe)and 2 kinds of metabolites(Leu-Pro and Leu-Leu)were detected in liver and kidney,respectively.Among the detected metabolites,the Leu-Leu was not found in all of the in vitro samples and the in vivo samples of blood and liver.Our results indicated that liver and kidney were the main places of metabolism of the pentapeptide QLLPF,the half-life of the pentapeptide is very short and the dipeptide Leu-Leu might be the vital structure unit for biological activity of the pentapeptide,and the Pro-Phe structure was more resistant to the effect of protease in vivo.4 Purification and identification of corn peptides facilitating alcohol metabolism and the structure-activity relationship on the basis of QLLPFIn order to establishing a clear structure-activity relationship between corn peptides and facilitating alcohol metabolism,the corn peptides with good activity of facilitating alcohol metabolism were fractionated through ultrafiltration technology,purified by semi-preparative RP-HPLC,and a total of 16 peptides sequences were identified from fractions which had significant activity of facilitating alcohol metabolism by NanoLC-ESI-MS/MS.We found that two strong hydrophobic fractions F3-H4 and F3-H5 displayed higher activity of eliminating alcohol in vivo.The fractions of F3-H4 and F3-H5 were found to contain large amounts of hydrophobic amino acids including Ala,Leu,Pro and Phe.Thus,the 4 kinds of amino acids may contribute to the facilitating alcohol metabolism activity of corn peptides.Furthermore,we synthetized a series of structure related short peptides by the molecular design on the basis structure of Gln-Leu-Leu-Pro-Phe.The activities of facilitating alcohol metabolism for synthetic short peptides were measured by acute ingesting model evaluation.The results showed that the deletion of Leu-Leu(the tripeptide Gln-Pro-Phe)or Pro-Phe(the tripeptide Gln-Leu-Leu)greatly decreased or forfeited the activity of facilitating alcohol metabolism,compared with the parent peptide Gln-Leu-Leu-Pro-Phe.Our results indicated that the Leu-Leu and Pro-Phe were the key structure units in Gln-Leu-Leu-Pro-Phe for its facilitating alcohol metabolism activity.