Inhibitory Effect Of Substituted Benzaldehyde Thiosemicarbazone And Flavonoids From Plantaginis On Xanthine Oxidase

Hypoxanthine is catalyzed by Xanthine oxidase（XO）,then it is convert to xanthine,and XO catalyzes the direct conversion of xanthine to uric acid.Therefore,xanthine oxidase is the key enzyme that produces uric acid in the human body.Gout、hyperuricemia and other illnesses are caused by the accumulation and decomposition of uric acid in the body.Therefore,the search for xanthine oxidase inhibitors has important implications for the clinical treatment of these diseases.Xanthine oxidase inhibitors have been reported in recent years.However,there are few studies on the interactions between inhibitors and enzymes.Therefore,artificially synthesized compounds and natural flavonoids in plantagogra were selected as inhibitors.Inhibitory effect of target compound on xanthine oxidase was studied and the relationship between the structure of compounds and the inhibition of enzyme activity was explored by using a variety of modern analytical techniques such as inhibition kinetics、fluorescence spectroscopy combined with molecular modeling techniques and so on to provide a theoretical basis for the search for chemical precursors for the treatment of gout drugs.A series of substituted benzaldehyde thiosemicarbazide compounds（1-7）as xanthine oxidase（XO）target screening inhibitors were synthesized.The interactions between substituted benzaldehyde thiosemicarbazide compounds（1-7）and XO were studied by UV,fluorescence spectroscopy,and molecular docking in this article.The fluorescence results showed that hydrogen bond and hydrophobicity consisted of the main interactions between substituted benzaldehyde thiosemicarbazide compounds and XO,and introducing-OH at para-position could increased modifier’s inhibitory activity(Compound 4,IC₅₀=19.32μM).However,Molecular docking showed that the interaction between the amino-terminated benzene ring and aromatic amino acid residues such as phenylalanine residues interacts with the hydrophobic cavity of XO byπ-πstacking force,which greatly increased the modifier’s inhibitory activity.(Compound 7,IC₅₀=0.04μM).We concluded that introducing Ph-group at the amino terminal of compound 4 and-OH group at para-position of benzene ring was a good route obtaining novel XO inhibitors,which deserved further studies.In addition,the interactions and combinations of flavonoids and xanthine oxidase in plantain were also explored.The activity test showed that all five flavonoids had good enzyme inhibition.Among them,the strongest inhibitory effect on XO was Eupatilin(IC₅₀=0.72μM).The ANS probe experiment demonstrated that the compounds had a competitive relationship with the ANS probe,and the degree of decrease in the area of the fluorescence peak was corresponded to the strength of the inhibition effect,the hydrophobicity of the specific site group of the compound was an important reference for measuring and predicting the inhibitory effect was suggest in this part.The strong inhibitory effect of Eupatilin on the enzyme was manifested by hydrogen bonds,vander Waals forces,π-sigma bonds,and hydrophobic forces.This phenomenon was demonstrated by nuclear magnetic titration and molecular docking.Therefore,we could know that the subtle differences in the structure of flavonoids often had different inhibitory effects due to the strength of hydrophobic and other interactions.At the same time,the research in this paper was also beneficial to the development of efficient and safe xanthine oxidase inhibitors to relieve hyperuricemia and gout.