Study Of Tyrosinase Inhibitors From The Gomphrena Globosa And The Inhibitory Mechanisms

Tyrosinase is a rate-limiting oxidoreductase and widely exists in biosystem. It plays asignificant role in physiological functions. The excessive expression of tyrosinase may causehyperpigmentation diseases in body and unfavorable browning in food products. In thepresent study, the tyrosinase inhibitors were extracted from Gomphrena globosa (GG) and themechanism of tyrosinase inhibition was further discussed, which could reveal thedehyperpigmentation function of GG. In addition, the obtained results would provide newevidence to elucidate the unique molecular mechanism of GG and provide basic informationand theoretical basis for the exploration of tyrosinase inhibitors from natural plants. Asfollows, it is most content in this paper:The extraction and separation of tyrosinase inhibitors from natural plantsBased on the results of single factor experiment, the extraction conditions was optimizedby the response surface methodology (RSM). The best extraction condition was80℃,withdraws for3hours, ratio of liquid to material60:1(v/m), as well as the concentration ofethanol50%. Under the above conditions, the inhibition rate of tyrosinase by the crudeextract of GG was (53.86±2.11)%.Following the biological effects to the tyrosinase, seven kinds of constitutes wereobtained by means of solvent extraction, column chromatography and preparativechromatography. The chemical structures were identified to be VA (VA), tricin,p-hydroxy-cinnamic acid (HCA), and p-methoxycinnamic acid (MCA) by modern analysistechniques such as nuclear magnetic resonance, mass spectrometry and X-ray single crystaldiffraction. The IC50values of VA, tricin, HCA and MCA were510.01μg/mL,454.86μg/mL,15.82μg/mL, and20.37μg/mL, respectively.The inhibitory mechanisms of inhibitors from natural plants on tyrosinaseThe molecular mechanisms of inhibitors from Gomphrena globosa (VA, HCA, tricin,and MCA) on tyrosinase were elucidated by means of enzyme kinetics and spectroscopytechniques, and molecular docking was used for interaction model establishment betweeninhibitors and tyrosianse.VA could interact with residues in the entrance to the dicopper center. VA could form a more stable complex with tyrosinase than the complexes for substrates. Moreover, VA couldprolong the lag time of tyrosinase for oxidation of L-tyrosine and decrease the initial reactionvelocity of monophenolase and diphenolase of tyrosinase by a mixed type of inhibitorymechanism.Quenching mechanism of tyrosinase by tricin was static mechanism. And tricin couldform complexes with tyrosinase by forming hydrogen bonds with residues near the catalyticcore domain and hydrophobic associations with residues in the hydrophobic pocket oftyrosinase. And these interactions extended the lag time of tyrosinase for oxidation ofL-tyrosine and decreased the initial reaction velocity of monophenolase and diphenolase oftyrosinase. Tricin behaved as a mixed-type inhibitory mechanism, in which thenon-competitive mechanism played the dominant roles.HCA and MCA could occupy the binding sites of substrates in the active center oftyrosinase by forming hydrogen bonds and hydrophobic force with residues of tyrosinaseactive center. Moreover,-OH of HCA could coordinate to Cu²⁺and form hydrogen bondswith His-57, whereas-OCH₃of MAC couldn't. As a result, HCA could inhibit bothmonophenolase and diphenolase activity of tyrosinase, and MCA could only function asmonophenolase inhibitor of tyrosinase. In addition, HCA could be oxidized by tyrosinase tocorresponding quinone slowly.