Inhibitory Effect Of Paeonol Structural Analogues And Chemical Modifications On Tyrosinase

Tyrosinase (EC1.14.18.1), known as polyphenol oxidase, is a copper-containingmetalloenzyme widely distributed in the human body, animals, plants, andmicroorganisms, which is the key enzyme in the synthesis of melanin. Paeonol,known as2-hydroxy-4-methoxy acetophenone, is the primary bioactive componentsof peony, with a very wide range of pharmacological activity. Paeonol can inhibit theactivity of tyrosinase, reduce the production of melanin and make skin whitening, butthe inhibition mechanism of its analogues and chemical modifications on tyrosinase,is rarely reported in the previous literature.In this paper, paeonol (a) being as a lead compound, enzyme kinetics and UVspectrometry were used to study the inhibition mechanism of paeonol and itsstructural analogs (a)-(g) on the diphenolase of mushroom tyrosinase. It is necessaryfor us to obtain mother structures with excellent anti-tyrosinase activity, and sum upthe relevant structure-activity relationship. Enzyme kinetics showed that compounds(a)-(g) have been found having remarkable inhibition on mushroom tyrosinase andthe inhibition was displayed as reversible. Among them, compound (a) had the mostpotent inhibition activity, IC50=0.207mM. Replacement of o-hydroxy and p-methoxymoieties of acetophenone is more favorable for the activity enhancement. Theinhibition type was induced by the kind of substitutions at ortho or para position. Inthe copper ions chelation assay of compounds (a)-(g), the maximum absorptionwavelengths of hydroxy substituted acetophenone showed different significant blueshifts while that of methoxy substituted acetophenone didn’t make any change.On this basis, paeonol and its structural analogs a-e were selected as the initialstructure to synthesize10paeonol thiosemicarbazone analogues a1-e1and a2-e2.enzyme kinetics was used to study the inhibitory effect of compounds a1-e1and a2-e2on the diphenolase of mushroom tyrosinase. The results showed that paeonolthiosemicarbazone analogues have been found exhibiting more remarkable inhibitionthan their indexcompounds on mushroom tyrosinase. Among them, compound d1hadthe most potent inhibition activity, IC50=0.006mM, which was about208times stronger than that of its indexcompound d. phenyl substituents a1-e1had more potentactivity than methyl substituents a2-e2. No matter the hydrogen was replaced by phenylmoiety or methyl moiety, the inhibitory ability of o-or p-substituted acetophenonethiosemicarbazones was: di-substituted acetophenone thiosemicarbazones> mono-substituted acetophenone thiosemicarbazones> non-substituted acetophenonethiosemicarbazones.Enzyme kinetics, UV spectrometry and fluorescence spectrometry were used toinvestigate the inhibition mechanism of compounds d1on the diphenolase ofmushroom tyrosinase and reveal the specific association between structure andactivity. Enzyme kinetics indicated that compounds d1was a reversible andcompetitive tyrosinase inhibitor. Copper ions chelation assay ratio indicated a1.5:1binding ratio of compound d1with copper ions. In the fluorescence quenching study,compound d1behaved stronger fluorescence quenching on tyrosinase towardsd1-Cu2+complex.This paper not only introduced a series of new, efficient, high-quality tyrosinaseinhibitors, but also summed up the underlying structure-activity relationship,providing the theoretical basis for further search of novel, efficient and safetyrosinase inhibitors. Research on inhibition mechanism and structure-activityrelationship of inhibitors, provide a more scientific theory for the application of thecompounds in whitening cosmetics.