Screening, Isolation And Identification For Tyrosinase Inhibitors From Extracts Of Entomogenous Fungi

In this study, tyrosinase inhibitory activity was used as screening index to screen extracts from50entomogenous fungi strains for strains with high tyrosinase inhibitory activity. Bioactive compounds in the extracts of the high active strains were isolated and identifiedResults of the screening on the50methanol extracts from solid cultured mycelia of the50entomogenous fungi strains showed that tyrosinase inhibitory activity of the tested extracts was very different, and all of the extracts from strains of Paecilomyces gunnii exhibited relative stronger tyrosinase inhibitory activity. Comparison of tyrosinase inhibitory and radical scavenging activity between methanol extracts from solid cultivated mycelia or liquid cultivated fermentation broth of7strains revealed that solid cultivated mycelia extract of RCEF0199had the strongest tyrosinase inhibitory and radical scavenging activity. Therefore, the mycelia of RCEF0199were used for further studies.Results of tyrosinase inhibitory and radical scavenging activity comparison between different solvent extracts from mycelia of RCEF0199showed that the methanol extract had the strongest bioactivity. The half inhibition concentration (IC50) of the extracts against the diphenols enzyme was0.048mg/mL. Inhibition mechanism studies revealed that the inhibition of the extract against tyrosinase diphenol enzyme was reversible and the type of inhibition was competitive inhibition. The Michaelis constant (Km) increased with the increasing of the concentration of the extract. This indicated that the affinity of the enzyme on the substrate was reduced while the extract concentration increased. The extract hindered the binding of the enzyme and substrate and made the catalytic activity of the enzyme reduce.when there was no extract in the reaction system, the obtained Michaelis constant (Km) was1.02mmol/L. The maximum velocity (Vmax) was186.44U/min according to the slope of the straight line and Km values. And the inhibition constants (KI) of the extract on free enzyme was555.56μg/mL.The lag time of the reaction and the catalytic efficiency about the sample was determined by tracking the progress of the reaction. The result showed that the effect of methanol extract on monophenol enzyme activity was prolonging the reaction lag time, and there was little effect on the steady-state activity of the catalytic reaction. The results of DPPH radical scavenging activity tests of the methanol extract showed that radical scavenging activity of the extrat increased gradually with increasing of the concentration of the extract. When the extract concentration was1.0mg/ml, the clearance rate reached94.66%.Different components of the extract were separated with semi-preparative column and tested with tyrosinase inhibitory activity and free radical scavenging activity assay model. Results showed that components p-1,p-2andp-3had tyrosinase inhibitory activity and radical scavenging activity. By two times separation using high-speed countercurrent chromatographythe three bioactive components were successfully prepared. The solvent system of the first separation was n-hexane: ethyl acetate: methanol: acetic acid: water=3.5:5:3.5:0.15:5(V/V/V//V/V) and the solvent system of the second separation was n-hexane: ethyl acetate: methanol: acetic acid: water=2.5:5:3.5:0.15:5(V/V/V//V/V). The purity of p-1,p-2andp-3were91.7%,94.5%and96.8%respectively. The color of component p-1was yellow-green, and component p-2and p-3showed yellow.Results of LC-TOF-MS and NMR showed that the molecular formula of compound p-3was C15H12O6, and the structure was6,7,8,9-tetrahydroxy-3-methoxy-4-methyl-1-phenalenone. The molecular formula of compound p-1was C15H13NO5, and the structure was6,7,8-trihydroxy-9-amino-3-methoxy-4-methyl-1-phenalenone. It was structural analogues of p-3with9-OH substituted by NH2. The molecular formula of compound p-2was C15H10O6, and the structure was probably1,6-dihydroxy--3-methoxy-4-methyl-7,8,9-trihyphenalenone. It was also probably structural analogues of p-3. Natural products database query result showed that three compounds were novel compounds belonging to phenalenone. They all had tyrosinase inhibitory activity and radical scavenging activity. The three compounds were found for the first time existed abundantly.