Inhibitory Mechanism Of Guanidine Compounds On Tyrosinase And Their Biological Effects

Tyrosinase(TYR)(EC 1.14.18.1),a copper-containing enzyme in the biological events,is usually found in human,animals,plants and microorganisms.TYR can catalyze the synthesis of melanin.For human and animals,in response to the UV light,the organism produces more melanin pigment that darkens the skin,but abnormal accumulations of skin pigmentation may cause serious pigmentation disorders.TYR is also the key enzyme in fruits and vegetables enzymatic browning and the growth and development of insects.Taken these together,the regulation of melanin generation and accumulation by the inhibition of TYR is becoming a hot spot in current research.Guanidine compounds(GCs)play an important role in fields of chemical synthesis,medicine,antibacterial and ecology.In this study,the inhibitory effects on mushroom tyrosinase(MT)of four GCs,including guanylthiourea(GT),guanidine thiocyanate(GTC),phenylguanidine(PG),and phenethyldiguanide(PTDG)were investigated.From the results,four GCs reduced monophenolase and diphenolase activities of MT.The IC50 values of GT,GTC,PG,and PTDG were 0.27,3.53,6.68,and 5.18 mmol/L for monophenolase,respectively,and were 0.23,3.24,3.97 and 2.50 mmol/L for diphenolase,respectively.The inhibitions of diphenolase activity were reversible for the four GCs.The inhibition types of GT and PTDG were competitive,while the inhibition types of GTC and PG were mixed.We further studied the mechanism of four GCs on MT by UV spectroscopy,fluorescence quenching analysis,Native-PAGE electrophoresis and molecular docking simulation as well.The results implied that four GCs could interact with the amino acid residues near the active center of TYR,GT and PTDG could form metal interactions with the copper ions of TYRBased on the inhibitory mechanism of GCs on MT,furthermore,we demonstrated the melanogenesis regulatory effect of GT and GTC on B16F10 cells and zebrafish,the results showed that the two compounds had significant anti-melanogenic activity by down-regulation of the expression of microphtalmia-associated transcription factor(MITF),tyrosinase(TYR)and dopachrome-tautomerase(DCT).The antioxidant activities of four GCs were determined by FRAP,ABTS,DPPH and DNA damage protection assays.Results revealed that GT possessed potent antioxidant activity.Based on this result,the effect of four GCs on the fresh-cut iron stick yam preservation was also presented.The results suggested that it had great potential for GCs applied to enhance freshness and quality in fruits and vegetables,and the treatment with GT had preferable storage effects.Therefore,our study provided a new idea for the research and development of GCs,the results suggested that these GCs are potential candidate of novel small molecule TYR inhibitors and GT could have broader application prospects.