The Inhibitory Effects Of Galloyl-based Polyphenols On The Enzymes For Starch Digestion

As the staple food of Chinese residents'daily life,the velocity of starch digestion affects the postprandial blood glucose level and is important for maintaining the glucose metabolism and absorption in body.Both?-amylase and?-glucosidase are the key enzyme for starch digestion and catalyze the hydrolysis of starch to produce glucose.Inhibiting the activity of enzymes has been proved to be one of the important ways to regulate the blood glucose.The polyphenols in plants have a wide range of sources and many types of structures,which have been shown to inhibit enzyme through non-covalent forces.The functional groups in polyphenols have great influence on the enzyme inhibition.Galloyl moiety(GM)is an important structural constituent of dietary polyphenols and enhances the physiological activity of polyphenols.Based on the importance of GM to the structural composition and enzyme inhibition of polyphenols,the interaction between polyphenols and two digestive enzymes was further explored.The structure-activity relationship between molecular structure and enzyme inhibition can be recognized through comparing the inhibitory results of polyphenols on enzymes from the perspective of structure.The present study provided novel theoretical basis for interaction between galloyl-based polyphenols and enzymes and could guide the development and utilization of plant foods rich in galloyl-based polyphenols.The main findings of this paper are as follows:(1)Nine galloyl-based polyphenols mainly composed of GM(Gallic acid,Hamamelitannin,Ellagic acid,1,3,6-Tri-O-galloyl-?-D-glucose,Corilagin,Punicalin,1,2,3,4,6-O-pentagalloylglucose,Punicalagin,Tannic acid)were selected,including different GM numbers and connection methods.Among them,GM that connected to the sugar core only through ester bond was defined as free GM,while GM that had additional chemical bonds was defined as unfree GM.The results showed that the polyphenols with free GMs exhibited much higher PPA inhibition than that with unfree GMs.The inhibition was in a competitive mode competing with starch for the active site of PPA and increased with an increase in the number of free GMs.Interestingly,all the constants that indicate binding affinity of polyphenols to PPA,including reciprocal of competitive inhibition constant(1/K_ic),fluorescence quenching constant(K_FQ)and binding energy(E_b)increased with an increase in the number of free GMs.Besides,the respective orders of 1/K_ic,K_FQand E_b for the galloyl-based polyphenols was contrary to that of IC₅₀,indicating that the polyphenol-PPA binding interactions resulted in enzyme inhibition.Additionally,the binding interactions were suggested to result from hydrogen bonding and?-?conjugation forces between the free GMs and amino acid residues at the active sites of PPA,quenching the fluorescence intensity of PPA in a static mode.Polyphenols with unfree GM may not interact with the essential amino acid residues of PPA in a flexible posture due to their chemical bonds with other groups,weakened the PPA inhibition.Furthermore,the hypoglycemic effects of two polyphenols with 5 free GMs indicated that GM may be considered as functional fragment to inhibit enzymes and reduce the digestibility of starch.(2)The?-glucosidase inhibition by galloyl-based polyphenols with free GM increased greatly with an increase in the number of free GMs.For the polyphenols with unfree GMs,the?-glucosidase inhibition was depended on their structure.The chemical bond weakened the inhibitory effects,but ellagic acid and hexahydroxydiphenoyl group(HHDP)contributed to the enzyme inhibition.Free GMs could bind not only with the active site of?-glucosidase(competitive inhibition),but also with the non-active sites(uncompetitive inhibition);however,the former binding interaction was stronger than the latter one.Complex polyphenols due to the connection between unfree GM were bound to the non-active site of the enzyme,showing uncompetitive inhibition.The number of amino acid residues involved in polyphenol-enzyme binding interactions(hydrogen bonding and?-conjugations)increased with the inhibitory activity increasing.The above results indicated that the number of GM and the structural flexibility(caused by the intramolecular bonds)had significant influence on the enzyme inhibition by galloyl-based polyphenols.As characteristic substituent,GM interacted with amino acid residues at the active/non-active site of enzymes through non-covalent forces,quenching the fluorescence intensity and preventing the binding of the substrate and enzyme,thereby reducing digestibility of substrate.This study provided the structure-activity relationship between galloyl-based polyphenols and enzyme inhibition.GM can be used as characteristic group to screen potential enzyme inhibitors in natural plants,or used for the development of functional factors that regulate starch digestion.