Study On The Interaction Between Oenothein B And Protein In The Digestive Tract

Oenothein B(OeB)is a dimeric macrocyclic tannin with a variety of biological activities.It has been shown that OeB can exert various physiological activities after oral administration into the body.However,OeB will pass through oral,pharyngeal,esophageal,stomach and small intestine gastrointestinal organs before oral administration into the body's blood.It contains a series of human digestive enzymes and food-derived proteins,which will have a certain impact on the activity of OeB,and OeB also It affects the digestion and absorption of the human body by affecting people's digestive enzyme activity.Since OeB is a macrocyclic dimer tannin,its structural characteristics are very different from those of small-molecule polyphenols,and the process of its interaction with proteins is not completely consistent with that of small-molecule polyphenols.At present,there is a lack of systematic and in-depth research on the interaction between OeB and other oligomers tannins with foreign proteins in the digestive tract.Therefore,by means of spectroscopy,microtitration and molecular modeling,this paper systematically studied the interaction of OeB with digestive enzymes and food-derived proteins,and revealed the molecular mechanism of the interaction of OeB with digestive tract and foreign proteins after oral administration into the digestive system.For the same kind of macromolecule polyphenols.The study of protein interaction provides a reference.The results of this study are as follows:(1)The stability of OeB in simulated digestive tract in vitro.Through the stability experiment of OeB in different pH and simulated gastrointestinal fluid environment,it was found that OeB is stable under acidic conditions,and the retention rate is more than 96%at pH 4 for 4 h,and it will not be catalyzed by pepsin at the same time.Influencing and producing new metabolites,it is speculated that the loss of OeB in simulated gastric fluid is only related to the complexation with pepsin;the retention rate of OeB 30 min before weak alkaline conditions is above 90%,but the long-term stability is poor After 4 h at pH 7.8,the loss rate reached 42.69%.The peak time of OeB metabolites in simulated intestinal fluid was consistent with that under weak base conditions.The production rate of metabolites was 2%and was less than 4%under weak base conditions,indicating that OeB is not affected by pancreas.The catalytic action of proteases and the interaction between OeB and trypsin reduce the degradation of OeB by weak bases and protect the structural stability of OeB.In general,the main factor affecting free OeB status in a short period of time in an in vitro simulated gastrointestinal environment is its interaction with pepsin and trypsin.(2)Effect of OeB on pepsin and trypsin.By measuring the inhibitory rate and inhibition kinetics of OeB on pepsin and trypsin,the effects of OeB on the activities of two digestive enzymes were studied.The results showed that OeB inhibited the activities of the two enzymes,the highest inhibition rate was above 90%,and the semi-inhibitory concentrations of pepsin and trypsin were 2.66×10~-44 mol/L and 1.54×10~-44 mol/L,respectively.L,indicating that it has a stronger inhibitory effect on trypsin.The inhibition of pepsin by OeB is a combination of competitive inhibition and non-competitive inhibition,indicating that OeB binds to both active sites of pepsin and inactive sites on the surface of protein substrates or enzymes,increasing OeB The inaccessibility to the substrate prevents the enzymatic hydrolysis of the substrate by pepsin in two aspects.The inhibition type of trypsin is mainly non-competitive inhibition,which increases the inaccessibility of OeB to the substrate,thereby preventing trypsin from enzymatic hydrolysis of the substrate,resulting in reduced enzyme activity.(3)Effect of internal and external proteins on OeB activity.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of OeB were determined by 96-well plates and Oxford Cup antibacterial experiments.The effect of three internal and external proteins on the size of the OeB zone was also studied.The results showed that OeB exhibited strong antibacterial activity against three strains of tetracycline-resistant Staphylococcus aureus(SA).The MICs for SA 85 and SA 103 were both 250?g/mL and the MBC was 1000?g/mL.mL and 500?g/mL.The MIC for strain 86was 500?g/mL and the MBC was greater than 4000?g/mL.The results of the Oxford Cup antibacterial experiment showed that the antibacterial activity of foreign proteins in the digestive tract had a certain inhibitory effect on OeB.With the increase of the concentration of exogenous proteins in the digestive tract,the inhibition zone diameter of OeB appeared to varying degrees.At the concentration of 1000?g/mL of pepsin,trypsin,and lactoferrin,the diameter of the zone of inhibition decreased by 4.68 mm,10.16 mm,and 7.40 mm,respectively,indicating that the inhibitory effect of different proteins on the antibacterial activity of OeB is trypsin>Lactoferrin>Pepsin.(4)Determination of the interaction between OeB and foreign proteins in the digestive tract.In this section,the interactions between OeB and foreign proteins in the digestive tract were studied using fluorescence(FL),circular dichroism(CD),isothermal titration calorimetry(ITC)and molecular docking.The fluorescence quenching of OeB on exogenous proteins in the digestive tract was found to be a combination of static and dynamic quenching through FL.FL data calculated the affinity of OeB for the three proteins:lactoferrin>pepsin>trypsin After the addition of OeB,the FL profiles of the three proteins showed a red shift,indicating that the addition of OeB decreased the hydrophobicity of the protein.Using ITC to further characterize the thermodynamic properties of OeB binding to the three proteins,the data shows that the binding energy of the three proteins decreases with increasing temperature and is a typical hydrogen bonding method,indicating that the OeB and the foreign proteins in the digestive tract are The energy released during the binding process mainly comes from the hydrogen bond.In the process of binding with lactoferrin,?H<0,?S>0,it is proved that the interaction between OeB and lactoferrin has electrostatic and hydrophobic forces.participate.CD spectroscopy showed that the degree of expansion and relaxation of three proteins caused by OeB was different.Among them,OeB had little effect on pepsin and lactoferrin.The proportion of different structures changed within 5%,while the effect of trypsin was greater.About 30%of the?-sheet structure in the protease structure is converted into an?-helical structure.Simulations of molecular docking showed that hydrogen bonding and hydrophobic interactions exist between OeB and the three proteins,and the binding point to pepsin lies in the gap between its domains,masking some of its active sites;docking with trypsin The site is on the surface of the protein and hydrogen bonds with the amino acid of its active site,but does not mask the active site;the binding site with lactoferrin is in the hydrophobic cavity of lactoferrin.(5)The interaction mechanism of OeB with digestive tract proteins.The above results show that non-covalent bonds are the driving forces in the interaction between the three proteins and OeB.They enter the hydrophobic cavities of different proteins through hydrophobic interactions,and bind to protein amino acid residues through hydrogen bonds,further enhancing OeB and protein.interaction.The positively charged alkaline region of lactoferrin can also interact with the negatively charged free OeB in the solution by positive and negative ion exchange and adsorb to the surface of the protein molecule,indicating that the electrostatic interaction also participates in the interaction between OeB and some positively charged proteins.in.The order of the affinity of OeB to different proteins in the digestive tract was lactoferrin>pepsin>trypsin,indicating that with the increase of the molecular weight and hydrophobic amino acid content of the protein,the affinity of the pro-protein and OeB also increased.The effect of OeB on the secondary structure of different proteins was trypsin>>lactoferrin>pepsin.As the concentration of OeB increased,the proportion of antiparallel structures in?-sheets of three proteins decreased uniformly,indicating that OeB was added.The stability of the anti-parallel structure in the protein has some influence,but there is no absolute link between the affinity of OeB protein and its degree of influence on the protein structure.Based on the data of structural changes,molecular docking,and enzyme activity changes,we hypothesized that OeB binds to specific sites of digestive enzymes through hydrogen bonds and hydrophobic interactions to form relatively stable complexes that change the conformation of the digestive enzymes.Affects the conformation of the active center of the enzyme,thereby affecting the biological function of digestive enzymes.The inhibitory effect of the enzyme activity is related to the degree of influence of OeB on the structure of the enzyme.The greater the structural change of the enzyme,the greater the degree of influence on the enzyme activity.The type of inhibition of the enzyme activity is related to the OeB binding site.If the binding site is in the same hydrophobic cavity as the active site of the enzyme,OeB will mask the active site of the enzyme,resulting in competitive inhibition;if the binding site No overlap in the active site of the enzyme with the enzyme does not result in competitive inhibition.In general,the structural characteristics of different digestive enzymes have largely affected the inhibitory effect and mechanism of OeB on different enzymes.