Study On The Active Components With The Inhibitory Effect Of Sugar Metabolism Enzyme In Euonymus Fortunei

Diabetes is a metabolic disease characterized by high blood sugar caused by insulin insufficiency or insulin dysfunction and has become the third largest non-communicable disease after cardiovascular disease and tumor.Inhibition of glucose metabolism enzyme activity is one of the effective ways to control postprandial blood glucose in diabetic patients.In the study of screening medicinal plant resources withα-glucosidase and glycogen synthase kinase-3β（GSK-3β）inhibition activities,we found that Euonymus fortunei（Euonymus fortunei（turcz.）hand.-Mazz.）had inhibitory effects on both of them.In this study,a combination of chemical components isolation and identification and activity evaluation was used to search for chemical constituents withα-glucosidase and GSK-3βinhibition activities from Euonymus fortunei,aiming to find the double target lead compounds which can inhibit both of them.Modern separation techniques,such as silica gel column chromatography,octadecyl silane column chromatography,preparative high performance liquid chromatography,and identification methods such as ¹H-NMR,¹³C-NMR,MS were used to systematically study the chemical constituents of Euonymus fortunei.A total of28 compounds were isolated from the crude extract of Euonymus fortunei,including13 triterpenoids:fortunenone A（1）,fortunenone B（2）,sasanquylacetate（4）,lupeol acetate（5）,friedelin（6）,epifriedelanol（7）,D:B-friedoolean-5-en-3β-ol（8）,baroul（9）,echinochlorin D（10）,β-amyrenone（11）,lupenone（12）,wilforlide A（13）,wilforlide B（14）;3 sterides:β-sitosterol（15）,β-sitostenone（16）,daucosterol（17）;1 flavone:kaempferol-3-O-β-D-glucopyranyl（6→1）-β-D-glucopyranoside（18）;4 aliphatic hydrocarbons:ethyl linoleate（19）,peplusol（20）,palmitic acid（21）,isophytol（22）;7 other compounds:3’,4’,4’’-trihydroxy-α-truxillic acid（3）,ferruginol（23）,α-tocopherol（24）,methoxytocopherol（25）,protocatechuic acid（26）,syringaldehyde（27）,ehydrololiolide（28）.Among them,1,2 and 3 were new compounds,and 4,10,20,22-25 and 28 were compounds isolated from the Euonymus for the first time.Theα-glucosidase activity screening model was used to evaluate the activity and enzymatic kinetics of the compounds isolated from Euonymus fortunei.The results showed that wilforlide A,lupenone,echinochloride D and wilforlide B had significant inhibitory activities againstα-glucosidase with IC₅₀ values of 207.2±1.9μM、246.6±3.6μM、312.7±0.9μM、388.3±2.7μM,respectively.Enzyme kinetics analysis by Lineweaver-Burk plots revealed that the inhibition types of four active compounds were all mixed inhibition with K_i values of 3.36×10^-4 mol/L,5.75×10^-4 mol/L,5.38×10^-4 mol/L,6.01×10^-4 mol/L,respectively.Molecular docking was used to further explore the interactions between active compounds andα-glucosidase.The results revealed that hydrogen bonds and hydrophobic interactions played an important role in performingα-glucosidase inhibitory activity.Molecular docking virtual screening combined with GSK-3βactivity verification were used to evaluate the activity of the isolated compounds from Euonymus fortunei.The results showed that wilforlide A,α-tocopherol,3’,4’,4’’-trihydroxy-α-truxillic acid showed remarkable activities against GSK-3βwith IC₅₀values of 4.54±0.2μM,11.37±2.3μM and 59.78±8.6μM,respectively.Molecular docking was used to further explore the binding mode of the active compounds with GSK-3β.The results showed that all three compounds could bind to the characteristic amino acids of the active pocket through hydrogen bonds of different strengths and hydrophobic interactions.This is an important reason why the compounds bind to GSK-3βand produce inhibitory effects.In addition,the binding energy of wilforlide A andα-tocopherol to the substrate binding pocket was significantly higher than that of the ATP binding pocket,indicating that wilforlide A andα-tocopherol were selective to the GSK-3βpockets,and were more inclined to bind to ATP binding pocket to compete with ATP.The binding energies of 3’,4’,4’’-trihydroxy-α-truxillic acid with two binding pockets of GSK-3βwere similar,indicating that the compound had a weak selectivity to the two binding pockets,and could bind to both ATP binding pocket and substrate binding pocket.In this study,28 compounds were isolated and identified from Euonymus fortunei including 3 new compounds and 9 compounds isolated from Euonymus for the first time.Fourα-glucosidase inhibitors and three GSK-3βinhibitors were screened out from the 28compounds by usingα-glucosidase and GSK-3βactivity screening models in vitro,which providing reference for the development and application of Euonymus fortunei resources in the treatment of diabetes.Among them,triterpenoid wilforlide A was found to inhibit bothα-glucosidase and GSK-3βfor the first time,which laid a foundation for the development of glucose metabolism enzyme inhibitors as a lead compound.