Inhibition Of Pentacyclic Triterpenoids On α-glucosidase And Protein Non-enzymative Glycation

Diabetes,characterized by high blood sugar,has become a global metabolic disease,whose incidence in China ranks first in the world.α-Glucosidase is a kind of hydrolase that catalyzes oligosaccharides into glucose,and increasedα-glucosidase activity could cause an increase in blood sugar.Long-term hyperglycemia accelerates non-enzymatic glycation in the human body,producing lots of advanced glycation end products（AGEs）,which could trigger a series of diabetic complications,such as cardiovascular and cerebrovascular diseases,kidney disease etc.At present,the use ofα-glucosidase and non-enzymatic glycation inhibitors is an important approach to treat diabetes and its complications.However,these clinical inhibitors for instance,acarbose,are synthetic drugs,which have high side effects,limiting their applications.Thus,seekingα-glucosidases and non-enzymatic glycation inhibitors with low side effects and high activities together with the research of their inhibitory mechanism have theoretical and practical significance to prevent and treat diabetes and its complications,which can also provide the experimental basis and scientific guidance for dietary nutrition.Natural plant active ingredients have good pharmacological activities and little toxic side effects,which have become an important resource for the research and development of drugs treating anti-diabetic and its complication.Due to its anti-diabete activity.pentacyclic triterpenoids gain great attentions In recent years.However,there are few studies on the inhibition and inhibitory mechanism ofα-glucosidase and non-enzymatic glycation.In this paper,three representative pentacyclic triterpenoids（oleanolic acid,ursolic acid and betulinic acid）were selected to investigate their inhibitory activities and kinetics onα-glucosidase.The binding interaction between pentacyclic triterpenoids andα-glucosidase,together with the conformational influences onα-glucosidase were analyzed to reveal the inhibitory mechanism.Besides,a non-enzymatic glycosylation model was established to study the inhibition of oleanolic acid,ursolic acid and betulinic acid on different stages of non-enzymatic glycation and protein oxidation.The free radical scavenging and protein structural protection abilities of pentacyclic triterpenoids were explored to reveal the possible inhibitory mechanism on non-enzymatic glycation.The main research contents and results are as follows:1.Oleanolic acid,ursolic acid and betulinic acid strongly inhibitedα-glucosidase activity.The inhibitory activities of oleanolic acid,ursolic acid and betulinic acid were better than that of the positive acarbose with their IC₅₀0 values of（6.35±0.02）×10^-6,（1.69±0.03）×10^-55 and（1.06±0.02）×10^-55 mol L^-1,respectively,which means that the order of their inhibitory capacities were that oleanolic acid（29）betulinic acid（29）ursolic acid.The inhibition kinetics research indicated that inhibition constant of oleanolic acid,ursolic acid and betulinic acid were(7.08×10^-6),(2.15×10^-5)and(8.53×10^-6)L mol^-1,respectively.Oleanolic acid and ursolic acid are non-competitive inhibitors while betulinic acid inhibitedα-glucosidase in a mixed manner.The inactivation kinetics research indicated that inactivations induced by three pentacyclic triterpenoids were monophasic processes in a first-order manner.2.Oleanolic acid,ursolic acid and betulinic acid could bind withα-glucosidase to form complex,quenching the endogenous fluorescence ofα-glucosidase.All of oleanolic acid,ursolic acid and betulinic acid have one binding site onα-glucosidase The binding constants betweenα-glucosidase and oleanolic acid/ursolic acid/betulinic acid were（2.04±0.02）×10³、（1.87±0.02）×10³、（2.11±0.02）×10³L mol^-11 at 25°C.The binding behaviours happened spontaneously under the drives of hydrophobic interactions and hydrogen bonds.The singular value decomposition algorithm showed that three significant reaction components existed in this complicated reaction system:BA,α-glucosidase and the BA-α-glucosidase complex.The pure spectra and concentration profiles of these three components were resolved from the overlapped UV-vis and fluorescence spectra by multivariate curve resolution with alternating least square（MCR-ALS）,which could be used to monitor the process of the reaction.3.Oleanolic acid,ursolic acid and betulinic acid bound withα-glucosidase to form a BA-α-glucosidase complex,resulting in the enhancement of the rayleigh scattering,which also altered the microenvironments of the Trp and Tyr residues.Besides,oleanolic acid,ursolic acid and betulinic acid induced a decline in the stability ofα-glucosidase with increasingα-helix content and decreasingβ-sheet content.The molecular simulation and chemical modification results showed that oleanolic acid and ursolic acid bound into allosteric cavity 2 and 4 ofα-glucosidase,which induced the allosteric regulation,perturbing the conformation ofα-glucosidase.As a result,the substrate bound with oleanolic acid/ursolic acid-α-glucosidase complex to form nonreactive oleanolic acid/ursolic acid-α-glucosidase-substrate complex,leading to the decline of theα-glucosidase catalytic activity.Betulinic acid bound into the active cavity ofα-glucosidase and mainly interaced with amino acids His,Tyr,Lys and Arg,which hindered the entrance of the substrate intoα-glucosidase,resulting in the decrease of enzyme activity.4.Oleanolic acid,ursolic acid and betulinic acid exhibited weak and moderate inhibitions on the formation of fructosamine and dicarbonyl compounds,the products of early and intermediate glycation stages,respectively,while they strongly inhibited the production of advanced glycation product（AGEs）.This phenomenon might due to the reduction of the precursors of the last reaction induced by the inhibition of early and intermediate glycation stages together with the inhibition on protein cross-linking and other reactions happened in the last stage.Oleanolic acid,ursolic acid and betulinic acid could also inhibit the formation of dityrosine,kynurenine and N’-formylkynurenine.Besides,oleanolic acid,ursolic acid and betulinic acid were found to scavenge hydroxyl radicals and superoxide anion radicals.They could protect the structure of protein from being further glycated.The free radicals scavenging ability and the protection of protein structure might be two inhibitory mechanisms of oleanolic acid,ursolic acid and betulinic acid on non-enzymatic glycation.