Study On The Interaction Between Polyphenols And Pepsin

With the improvement of people's material living standards,people pay more and more attention to healthy diet.Functional polyphenols are widely found in plants and have good antioxidant properties and reliable safety.They play an important role in preventing chronic diseases and ensuring human health.Natural polyphenol compounds are often added to food to improve its nutritional function.After the food is ingested by the human body,it will be digested and decomposed by digestive enzymes in the body and polyphenols will interact with digestive enzymes(pepsin),affecting pepsin activity and polyphenol bioactivity.Therefore,studying the mechanism of the interaction between polyphenols and pepsin and the effect on the inhibition of pepsin activity and the antioxidant properties of polyphenols will help to understand the utilization of polyphenols and also provide references for healthy diet and related functional food developments.In this paper,the mechanism of interaction between polyphenols and pepsin and its effect on their functional properties were investigated by fluorescence,ultraviolet and furrier infrared spectroscopy.The main conclusions obtained are as follows:1.The quenching mechanism of the interaction between quercetin,quercetin-3-O-glucoside,kaempferol,cinnamic acid,gallic acid and ferulic acid with pepsin found to be static quenching by fluorescence quenching spectroscopy and the quenching mechanism between rutin and kaempferol-3-O-rutinoside with pepsin is combined dynamic and static quenching.The addition of eight polyphenols quenched pepsin to varying degrees except quercetin-3-O-glucoside and the quenching rate of other polyphenols was less affected by temperature.The number of binding sites for the interaction of polyphenols and pepsin was almost unaffected by temperature and about one binding site was formed.The binding ability of quercetin,quercetin-3-O-glucoside,rutin,kaempferol,cinnamic acid and ferulic acid to interact with pepsin was increased by temperature.The stability of polyphenol-pepsin complex was increased and the binding strength of kaempferol-3-0-rutoside,gallic acid and pepsin was reduced.2.From the results of thermodynamic parameters,the interaction forces between pepsin and quercetin,quercetin-3-O-glucoside,rutin,kaempferol,cinnamic acid and ferulic acid are all hydrophobic forces(?H>0 and?S>0),the type of interaction force between kaempferol-3-O-rutin,gallic acid and pepsin is mainly electrostatic force(?H<0 and?S>0),and the presence of other forces can not be excluded.According to the theory of non-radiation transfer,pepsin and these eight polyphenols have undergone Forster's dipole-dipole non-radiative energy transfer and the energy transfer from pepsin to polyphenols may be one of the reasons for the quenching of pepsin fluorescence.The UV absorption spectrum reflects the static quenching of their interaction.At the same time,polyphenols changed the spatial structure of pepsin.3.Synchronous fluorescence spectroscopy and three-dimensional fluorescence results proved that the addition of polyphenols changed the microenvironment near the amino acid residues of pepsin.The binding sites of quercetin,kaempferol,kaempferol-3-O-rutinoside and quercetin-3-O-glucoside interacting with pepsin are closer to tyrosine residues,influences the microenvironment in which tyrosine residues are attached.The combination of cinnamic acid and gallic acid with pepsin enhances the polarity of the microenvironment near tyrosine residues and tryptophan residues,weakens the hydrophobicity and loosens the structure of pepsin.The interaction of ferulic acid with pepsin made the microenvironment near tyrosine an tryptophan residues of pepsin weakened and the hydrophobicity increased.The binding site of rutin interacting with pepsin is closer to tryptophan residues,which increases the polarity of the microenvironment near the tryptophan residues of pepsin,weakens the hydrophobicity and loosens the structure of pepsin.4.From the furrier infrared spectrum,it can be seen that before adding polyphenols,the content of?-helix in pepsin was 19.48%,however,after adding polyphenols,the content of each secondary structure of pepsin changed.With the increase of polyphenols concentration,the?-helix content decreased,and other secondary structures increased.The results showed that the addition of polyphenols changed the structure of pepsin,the peptide chain was stretched and the helical structure was loosened,which might affect the activity of pepsin.5.From the experiment of inhibiting the activity of pepsin by polyphenols,it was observed that the addition of polyphenols changed the structure of pepsin and the activity of pepsin was decreased and the inhibition was more significant with the increase of polyphenol concentration.The IC₅₀of quercetin,quercetin-3-O-glucoside,rutin,kaempferol,kaempferol-3-O-rutinoside,cinnamic acid,gallic acid and ferulic acid are respectively:17.99(?g/m L),22.68(?g/m L),98.88(?g/m L),18.69(?g/m L),17.99(?g/m L),12.63(?g/m L),17.24(?g/m L),17.85(?g/m L).The interaction between polyphenols and pepsin significantly decreased the reducing ability of polyphenols,scavenging ABTS·⁺free radicals and DPPH·free radicals,indicating that the addition of pepsin reduced the antioxidant properties of polyphenols.