Discovery And Structure-activity Relationship Of Flavonoids Inhibitors Against Human Pancreatic Lipase

Objective:Pancreatic lipase（PL）is secreted by pancreas and then released into gastrointestinal system,which cooperates with bile acid salts secreted by liver to decompose fat into fatty acids and glycerol.PL is responsible for the hydrolysis of about 50%～70%of dietary fat and a key enzyme to regulate the absorption of lipid substances in mammals.In the past few decades,porcine pancreatic lipase（p PL）is widely used as the enzyme source for screening PL inhibitors,which generates a wide range of p PL inhibitors.By contrast,the efficacious inhibitors against human pancreatic lipase（h PL）are rarely reported.Therefore,this study aims to set a high-throughput screening and evaluate method of inhibitors with h PL as enzyme source.Following screening of the inhibition potentials of more than 100 herbal medicines（HMs）against h PL,discovering the naturally occurring h PL inhibitors from HMs and to characterize the inhibitory mechanisms of the newly identified h PL inhibitors.Then,we further explained the weight-loss and lipid-lowering effects of HMs and provided candidate drugs for the treatment of clinical obesity and related metabolic diseases.In addition,this study measured the inhibitory effects of 64 natural and synthetic flavonoids against h PL,and discussed the structural inhibition relationship of flavonoids as h PL inhibitors,which provided valuable research basis for pharmaceutical chemists to improve flavonoids derivatives as h PL inhibitors.Methods:（1）A fluorescence-based high-throughput assay for screening h PL inhibitors was established by us,in which h PL was used as the enzyme source and a near-infrared fluorogenic substrate（DDAO-ol）was used as probe substrate.With the help of this newly developed h PL inhibition assay,the inhibitory activity of more than 100 HMs and 64 natural and synthetic flavonoids were assayed;（2）After then,performing LC-TOF-MS/MS to identify bioactive compounds in HMs;（3）The IC₅₀,inhibition kinetics analysis and inhibition constant（K_i）of the newly identified potent inhibitors were measured.（4）Through docking simulation and molecular dynamics of inhibitor and human pancreatic lipase protein crystal structure,the interaction site and intensity of inhibitor and human pancreatic lipase were further verified.Result:（1）The screening and evaluation system of h PL inhibitors was constructed with h PL specific near-infrared probe DDAO-ol.With the help of high-throughput screening method,it was found that Ampelopsis grossedentata extract（AGE）had anti-h PL bioactivity,and a panel of techniques were used to identify effective h PL inhibitors.The research results showed that almost all flavonoids in AGE were naturally occurring h PL inhibitors,among which two flavonoids（dihydromyricetin and iso-dihydromyricetin）were moderate h PL inhibitors,while myricetin and quercetin were identified as potent h PL inhibitors,with IC₅₀value of about 1.5μM.The inhibition kinetics analysis showed that myricetin and quercetin inhibited h PL in a non-competitive manner,with the K_ivalues of less than 2.5μM.Molecular dynamics simulation showed that myricetin and quercetin could bind h PL tightly at allosteric sites.（2）By studying the inhibitory activity of 64 flavonoids against h PL,the results showed that the number and position of phenolic hydroxyl groups on the flavonoid skeleton were crucial to the inhibitory effect against h PL.The catechol or pyrogallol group at C-5 position could enhance h PL inhibitory capacity,while hydroxyl group at C-6,C-7 position became O-methylation that could lead to the loss of h PL inhibition.Glycosylation at C-3,C-6,C-7 and C-8 positions could reduce the inhibition of h PL.Introduction of a hydrophilic group at C-8 position could lead to loss of h PL inhibition.Subsequently,the introduction of hydroxyl or amino group into C-4’of B ring promoted the inhibition of h PL.The catechol group at C-4’on ring B could enhance h PL inhibitory capacity.Finally,C-2and C-3 position form unsaturated double bonds that can enhance h PL inhibitory effects.Conclusion:In this study,more than 100 HMs were screened,and it was found that Ampelopsis grossedentata（AGE）had significant inhibitory activity against h PL.Then,the study clearly demonstrated that various flavonoids in AGE were naturally occurring inhibitors against h PL.Finally,we collected 64 flavonoids and further discussed the structure-activity relationship of flavonoids against h PL.These findings provide direct evidence for the anti-obesity effect of AGE,and these newly identified h PL inhibitors could be used as lead compounds for developing new drugs.