Study On Interaction Of Four Dietary Polyphenols With Pepsin And Trypsin

Polyphenols are natural active ingredients widely existing in plants,with good antioxidant activity.They play a variety of biological activities and pharmacological effects in organisms,such as preventing cardiovascular disease,preventing cancer,anti-aging and improving immunity.Studies have shown that some polyphenols will interact with digestive enzymes in the digestive tract after being ingested by organisms,causing changes in enzyme activity and affecting the digestion and absorption of nutrients in the body.Polyphenols are also affected by digestive enzymes,so their biological activities cannot be fully exerted.Therefore,the study on the mechanism of the interaction between polyphenols and enzymes will be beneficial to take effective measures to reduce the influence of the interaction between polyphenols and digestive enzymes on their biological activities.In this paper,the interaction characteristics of four polyphenols(Resveratrol,Gallic acid,Aloe-emodin and Hesperitin)with pepsin and trypsin were studied through spectroscopic experiments,molecular docking simulation,antioxidant and enzyme activity experiments,and the interaction mechanism was elucided,which would provide theoretical basis for the effective utilization of four dietary polyphenols in vivo.The research results were as follows:1.Four dietary polyphenols such as resveratrol,gallic acid,aloe-emodin,hesperitin interacted respectively with pepsin through one action site to form complex spontaneously(?G < 0).The four polyphenols had quenching effect on endogenous fluorescence of pepsin and the quenching mechanism was static quenching.The main binding force of resveratrol,gallic acid and aloe-emodin to pepsin was hydrogen bond and van der Waals force,while the main binding force of hesperitin to pepsin was hydrophobic.Four dietary polyphenols interacted respectively with trypsin through one action site to form complex spontaneously(?G < 0).The four polyphenols had quenching effect on endogenous fluorescence of trypsin and the quenching mechanism was static quenching.The main binding forces of aloe-emodin and trypsin were hydrogen bond and van der Waals force,while the main binding forces of resveratrol,gallic acid and hesperitin were hydrophobic.2.After the interaction of four polyphenols with pepsin and trypsin,the microenvironment around the tryptophan and tyrosine groups of the enzyme were affected,resulting in changes in the secondary structure of pepsin and trypsin.The combination of resveratrol,gallic acid and pepsin led to the enhancement of polarity and the decrease of hydrophobicity of microenvironment of tryptophan residues and tyrosine residues.The combination of aloe-emodin and pepsin increased the polarity of the microenvironment of tryptophan residues and had little effect on the microenvironment of tyrosine residues.The combination of hesperitin and pepsin led to the decrease of the polarity and the increase of hydrophobicity of the microenvironment of tryptophan residues and tyrosine residues.After four polyphenols were combined with pepsin respectively,the ?-helix content of pepsin was decreased,the random coil content was increased,and changes of ?-sheet and ?-turn content were different.The combination of resveratrol,gallic acid and trypsin led to the increase of the microenvironment polarity of tryptophan residues and tyrosine residues,and the decrease of hydrophobicity.The combination of aloe-emodin,hesperitin and trypsin led to the decreased polarity and enhanced hydrophobicity of tryptophan residues.After the combination of four polyphenols with trypsin,the secondary structure of trypsin showed a decreasing trend of ?-helix content and an increasing trend of random coil content.3.Molecular docking results visually show the specific binding sites of four polyphenols interacting with pepsin and trypsin,phenolic hydroxyl groups and amino acid residues involved in the interaction,and maintain the main force of polyphenol-enzyme complex.The binding sites of resveratrol and pepsin were located in the catalytic activity centers of hydrophobic grooves(Asp32 and Asp215),while the binding sites of gallic acid,aloe-emodin and hesperitin to pepsin were located in the substrate binding cavity of hydrophobic grooves.The main binding forces of resveratrol,gallic acid,aloe-emodin and pepsin were hydrogen bond and van der Waals force,while the main binding forces of hesperitin and pepsin were hydrophobic.The binding site of resveratrol,gallic acid,aloe-emodin and trypsin was located in the hydrophobic cavity of the S1 binding pocket(the main substrate binding site)of trypsin,while the binding site of hesperetin and trypsin was located in the catalysis center(a catalytic triad consisting of His57,Asp102 and Ser195),partially the hydrophobic cavity of the S1 binding pocket.The main interaction of resveratrol,gallic acid,hesperetin and trypsin was hydrophobic,while the main forces of aloe-emodin and trypsin binding were hydrogen bonding and van der Waals forces.The results of molecular docking experiments and spectroscopy experiments verified each other,revealing the mechanism of interaction between four polyphenols and pepsin and trypsin.4.The results of enzyme activity and antioxidant experiments showed that the enzyme activities of pepsin and trypsin were significantly inhibited after the interaction of resveratrol,gallic acid,aloe-emodin and hesperitin with pepsin and trypsin,respectively(P<0.05),and the ability of four polyphenols to scavenge superoxide anions and DPPH free radicals were significantly decreased(P<0.05).