Research On Identification And Interaction Mechanisms Of Phenolic Compounds From Dandelion For Urease Inhibition

Helicobacter pylori（H.pylori）is one of the most common human pathogens,60-90%of gastric cancers are linked to H.pylori infection.Urease is considered one of the most important virulence factors,and urease inhibitors can be utilized to control H.pylori infections.Plants-derived compounds are rich sources for new urease inhibitors development.To date,several studies have reported the promising urease inhibition activity of plant extracts or single compounds,and their urease inhibition mechanism was widely discussed,such as inhibition types,interaction sites,inhibition kinetics and thermodynamic parameters.However,with the confusing synergistic,antagonistic,or additive effects in the activity deconvolution,the plant extract exerts sufficiently lower or higher anti-urease activity than corresponding concentration components isolated from the plant,which seriously restricts the efficient development of plant resources.Dandelion is one of the commonly used food and medicinal herbs and mainly used to treat H.pylori-related gastric diseases.Studies showed that the extracts and single components of dandelion showed urease inhibition activity,however,the main active components have not been elucidated.The inhibition rules of single phenolic components and the mechanism of the combination of phenolic compounds on the inhibition of urease need to be conducted in-depth.This paper took dandelion as the research subjects,screened and identified the urease components of different parts of dandelion,studied the inhibitory effects of single or combinations of four phenolic compounds on jack bean urease（JBU）and Helicobacter pylori urease（HPU）.The gastroprotective effect of phenolic combinations was verified by in vivo experiments,and the mechanism of the combination of phenolic compounds on urease was explored.The main findings and conclusions of this paper were as follows:（1）The inhibitory activity of H.pylori in vitro and in vivoThe urease activity of EE was higher than WE,the IC₅₀ values were 0.56±0.02 and1.53±0.15 mg/m L,respectively.The motility and adhesion assay showed that 1/4MIC extracts could effectively inhibit flagellar motility and adhesion to GES-1 cells,and then affected the in vitro colonization of H.pylori.14 days of daily administration of 50 or 200 mg/kg EE appeared efficient to eliminate H.pylori infection（>50%）,and reduce gastric mucosal bleeding and gastric mucosal tissue destruction.Meanwhile,the levels of Ig G,TNF-α,and IL-6 in the blood were reduced,which indicates that EE could alleviate gastric inflammation.（2）Screening and identification of urease inhibitors from dandelionHPLC-MS/MS was used to qualitatively and quantitatively analyze the types and contents of components in dandelion roots,leaves,and flowers fractions,and the urease inhibition rates of different fractions were measured,then urease inhibitors were screened by correlation analysis and molecular docking.The results showed that the petroleum ether fractions from dandelion roots,leaves and flowers had low enzyme inhibitory activity(IC₅₀>5 mg/m L).Ethyl acetate,n-butyl alcohol,and water fractions of roots,leaves,and flowers showed inhibition activity against JBU and HPU.Ethyl acetate fraction of flowers exhibited the strongest concentration-dependent inhibition activity against JBU and HPU with IC₅₀values of0.184±0.007 and 0.091±0.003μg/m L.The urease inhibition activity was positively correlated with phenolic compounds content,which were the main urease inhibitory components in dandelion.These above results together with the results of molecular docking,indicated that chlorogenic acid,caffeic acid,quercetin and luteolin were the major urease inhibitors.（3）The inhibitory rules of phenolic compounds and JBUUV spectrophotometry,fluorescence spectroscopy,isothermal titration calorimetry（ITC）and surface plasma resonance（SPR）were used to determine the inhibition type,interaction sites,Stern–Volmer quenching constants,thermodynamic and binding dynamics parameters of chlorogenic acid,caffeic acid,quercetin and luteolin and JBU.The results indicated that chlorogenic acid,caffeic acid,quercetin,and luteolin showed a non-competitive inhibition type,with IC₅₀values of 57.24±1.91,21.47±2.66,35.67±1.01,26.07±4.83μM.The interacted site may be the flap region of the urease.The quenching mechanism was proved to be static quenching,the number of binding sites was about 1,and the quenching constant K values were1.0⁵×10⁷,6.76×10⁷,1.62×10⁷,and 1.29×10⁷ L/mol,indicating a high inhibitory activity.ITC test showed that the binding constants Ka of the phenolic compounds were 4.15×10⁴,7.20×10⁴,4.52×10⁴,and 1.64×10⁴L/mol,respectively.The Gibbs free energy（ΔG）was found to be negative,indicating that their binding was a spontaneous process.The binding of caffeic acid/quercetin and urease was driven by both favorable entropy and favorable enthalpy,whereas the binding of chlorogenic acid/luteolin and urease were enthalpy-driven.SPR test results showed that the binding equilibrium constant K_A of phenolic compounds ranged from 10³ to10⁴,among which chlorogenic acid bound to urease rapidly and quickly entered the active center.The combination of phenolic compounds showed an additive effect on the urease inhibition activity.（4）The inhibitory rules of phenolic compounds and HPUThe results indicated that chlorogenic acid,caffeic acid,quercetin,and luteolin showed the mixed-type inhibition type,with IC₅₀values of 36.37±3.34,16.63±0.83,38.94±0.86 and18.75±0.91μM.The interacted site may be the flap region and Ni²⁺of urease.The quenching mechanism was proved to be static quenching,the number of binding sites was about 1,and the quenching constant K values were 2.19×10⁷,4.47×10⁷,1.82×10⁷,and 3.89×10⁷L/mol,indicating a high inhibitory activity.ITC test showed that the binding constants Ka of the phenolic compounds were 1.26×10⁴,1.82×10⁴,1.48×10⁴,and 9.90×10⁴ L/mol,respectively.The Gibbs free energy（ΔG）was found to be negative,indicating that their binding was a spontaneous process.The binding of phenolic acid and urease was drivn by both favorable entropy and favorable enthalpy.SPR test showed that the binding mode of the target phenolic compounds with urease was fast binding and fast dissociation mode,and the binding equilibrium constant K_Avalue was 10⁴.The combination of phenolic compounds showed that chlorogenic acid-luteolin and caffeic acid-luteolin were synergistic effects on the urease inhibition activity,chlorogenic acid-quercetin and caffeic acid-quercetin showed synergistic or additive effect on the urease inhibition activity,and clorogenic acid-caffeic acid and quercetin-luteolin was additive effect on the urease inhibition activity.（5）Gastroprotection of combination of phenolic compounds in H.pylori infectionAll phenolic compounds had inhibitory activity against H.pylori,with MIC values of 64,128,128 and 256μg/m L,respectively,and caffeic acid and luteolin combination was selected to evaluate the gastroprotective effects by RUT test,visual and histopathological analyses,and evaluation inflammatory factors and Ig G expression.In caffeic acid-luteolin combination groups,the eradication rate was similar to that of the positive drugs group,gastric mucosal bleeding was reduced and gastric mucosal tissue integrity was better after 14 days of intervention.The level of Ig G antibody and inflammatory factors were decreased.The combination of phenolic compounds had a significant protective effect against gastritis,with no significant difference of the eradication rate in comparison to positive drugs.The levels of Ig G antibody decreased by 50.19%,32.04%,48.95%and 45.33%,and the levels of TNF-αand IL-6 decreased by 23.19%,32.34%,33.88 and 35.20%and 23.3%,27.60%,27.80%and27.91%,respectively.（6）The mechanism of the combination of phenolic compounds on the inhibition of urease.Single and multi-molecule docking and molecular dynamics simulation were used to analyze the characteristics of urease binding pocket,pose properties of phenolic compounds,and parameters of binding stability,binding energy and the hydrogen bond number.The results showed that the structure of JBU and HPU were highly consistent,but the binding pocket of JBU was larger than that of HPU.The binding of phenolic compounds led to structural changes in the urease flap region and volume contraction of the binding site.The number of hydrogen bonds with JBU was 2-4 and the binding energy was between 150 and 350 KJ/mol,while the number of hydrogen bonds with HPU was 1-5 and the binding energy was between 150 and350 KJ/mol.Multi-molecule docking showed that the binding pocket of JBU contained only one molecule,and the other one bound to different structural regions of urease,showing additive effects of inhibitory activity.The HPU binding pocket is small,and the two phenolic compounds entered and bound stably in the catalytic region with minimal steric hindrance,showing a synergistic effect of inhibitory activity.