The Study Of The Interaction Between Cinnamic Acid Derivatives And Pancreatic Lipase And Its Effect On Lipid Digestion

Pancreatic lipase,a major esterase enzyme synthesized and secreted by the pancreas,is responsible for the hydrolysis of 50%-70%of total dietary fat and plays a key role in the digestion of triglycerides.Clinically,pancreatic lipase inhibitors headed by orlistat have very strong effects in inhibiting lipase activity and reducing body fat content,but there are certain side effects and potential recurrence risks,making its application limited to a certain extent.Therefore,the search for healthy,safe and efficient pancreatic lipase inhibitors has become one of the hotspots in recent years.Cinnamic acid derivatives are a class of natural active small molecules that widely exist in fruits and vegetables.They are favored by people because of their low toxic and side effects and strong pharmacological activity.In this study,porcine pancreatic lipase（PPL）was used as the research object,focusing on the interaction mechanism of cinnamic acid（CA）,caffeic acid（CaA）,ferulic acid（FA）and chlorogenic acid（Ch A）with PPL and their effects on lipid digestion in vitro.In addition,sodium hyaluronate（HA-Na）and dextran 20（DEX 20）were used to construct a crowded environment to study the effects of cinnamic acid derivatives on the structure and function of PPL in intestinal-like environment,and to compare the binding affinity and similarities and differences between cinnamic acid derivatives and PPL in dilute solution and polysaccharide medium.（1）Research on the interaction mechanism between cinnamic acid derivatives and PPL.Spectral experiments show that CA,CaA,FA and Ch A can effectively quench the endogenous fluorescence of PPL,in which CA is static quenching,and the quenching type of CaA,FA and Ch A to PPL is a mixed quenching dominated by static quenching,indicating the formation of complexes between the four phenolic acids and PPL.Through kinetic experiments,it is found that the interaction between CA,CaA,FA,Ch A and PPL is a single exponential reaction,that is,phenolic acid directly acts on PPL with no intermediate state,and the apparent rate constant increases with the increase of phenolic acid concentration.In the presence of CA,CaA,FA and Ch A,the peptide chain of PPL stretched,the content ofα-helix decreased,the conformation was loose and the active site was exposed.The results of fluorescence quenching constant,binding constant and apparent rate constant of rapid kinetics show that there is a law of Ch A>CaA≈FA>CA at the same concentration.（2）Effects of cinnamic acid derivatives on PPL enzyme activity and lipid digestion.CA,CaA,FA and Ch A can effectively reduce the activity of PPL,and the type of inhibition is reversible mixed competitive inhibition,which can bind to either free enzyme or enzyme-substrate complex through non-covalent interaction with PPL.Because the K_i values of CA,CaA,FA and Ch A on PPL inhibition are all less than K_is,it shows that the binding power of these four phenolic acids to free enzyme is stronger than that to enzyme-substrate complex.In addition,it was found that CA,CaA,FA and Ch A could reduce the rate and degree of lipid digestion when simulating lipid digestion in vitro.The results of IC₅₀,apparent Michaelis constant K_m^app and inhibition rate of lipid digestion showed that at the same concentration,the result was Ch A>CaA≈FA>CA.（3）Research on the interaction between cinnamic acid derivatives and PPL in polysaccharide medium.The human intestinal crowded environment was constructed by using HA-Na and DEX 20as polysaccharide media,and the interaction between CA,CaA,FA,Ch A and PPL in the crowded environment was studied.The results showed that when the inhibitor was not added in HA-Na solution,the fluorescence intensity of PPL and the content ofα-helix increased with the increase of the solution concentration,indicating that the stability of PPL was improved,which led to the increase of its activity and the hydrolysis rate of lipid digestion.After the addition of CA,CaA,FA and Ch A,with the increase of crowded medium concentration,K_svand K_a decreased,α-helix content decreased and PPL activity decreased,resulting in a decrease in lipid digestion,which may be related to the network structure formed in the high concentration of HA-Na solution.When no inhibitor was added in DEX 20 solution,the fluorescence intensity of PPL and the content ofα-helix decreased with the increase of solution concentration,indicating that the activity of PPL decreased due to the extension of peptide chain and the loose conformation of protein,which eventually led to the decrease of lipid digestion rate and final hydrolysis rate.With the addition of CA,CaA,FA and Ch A,K_sv and K_a increased with the increase of crowded medium concentration,theα-helix content in the low concentration of DEX 20 solution decreased,while theα-helix andβ-fold content decreased in the high concentration of DEX 20 solution,which may be due to the decrease of effective volume in the high concentration of DEX 20 solution,the interaction intensity between phenolic acid and PPL increased,and the domain of two active sites of PPL were destroyed.As a result,the activity of PPL and the degree of lipid digestion decreased significantly.In different polysaccharide solutions,the fluorescence quenching constant,binding constant,the value of IC₅₀ and lipid digestion inhibition rate all have the rule of Ch A>CaA≈FA>CA.To sum up,different concentrations and different kinds of polysaccharides were selected to construct a crowded environment,and the interaction mechanism between cinnamic acid derivatives and PPL in dilute solution and polysaccharide medium and their effects on PPL enzyme activity and lipid digestion were studied systematically.Finally,it is found that both in dilute solution and polysaccharide environment,the affinity of cinnamic acid derivatives with PPL has the rule of Ch A>CaA≈FA>CA,indicating that the substitution of hydroxyl at position 3 and 4 of cinnamic acid can enhance its binding affinity to PPL,while the substitution of methoxy group at position 3 has no obvious effect on the binding affinity to PPL,while esterification greatly enhances the binding affinity to PPL.On the basis of previous studies about cinnamic acid derivatives in the treatment of obesity symptoms,this paper provided a further illustration about the effects of cinnamic acid derivatives on the structure and function of pancreatic lipase,and explored the relationship between the structure of cinnamic acid derivatives and their binding affinity,so as to provide a theoretical basis for the interaction between phenolic acids and digestive enzymes.