RESUMO
Biotransformation of the soy isoflavone genistein by sequential 3'-hydroxylation using recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium and then methylation using another recombinant E. coli expressing O-methyltransferase from Streptomyces peucetius was conducted. The results showed that two metabolites were produced from the biotransformation, identified as 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone, respectively, based on their mass and nuclear magnetic resonance spectral data. 5,7,4'-Trihydroxy-3'-methoxyisoflavone showed potent antiproliferative activity toward mouse B16 melanoma cells with an IC50 value of 68.8 µM. In contrast, the compound did not show any cytotoxicity toward mouse normal fibroblast cells, even at 350 µM concentration. The results of the present study offer insight on the production of both 5,7,4'-trihydroxy-3'-methoxyisoflavone and 5,7,3'-trihydroxy-4'-methoxyisoflavone by two recombinant E. coli strains and the potential anti-melanoma applications of 5,7,4'-trihydroxy-3'-methoxyisoflavone.
Assuntos
Antineoplásicos Fitogênicos/biossíntese , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Isoflavonas/biossíntese , Metiltransferases/metabolismo , Monofenol Mono-Oxigenase/metabolismo , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Bacillus megaterium/química , Bacillus megaterium/enzimologia , Proteínas de Bactérias/genética , Biotransformação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/genética , Fibroblastos/efeitos dos fármacos , Genisteína/metabolismo , Hidroxilação , Concentração Inibidora 50 , Isoflavonas/isolamento & purificação , Isoflavonas/farmacologia , Melanoma Experimental/patologia , Engenharia Metabólica/métodos , Metilação , Metiltransferases/genética , Camundongos , Monofenol Mono-Oxigenase/genética , Especificidade de Órgãos , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismo , Streptomyces/química , Streptomyces/enzimologia , TransgenesRESUMO
Biotransformation of 8-hydroxydaidzein by recombinant Escherichia coli expressing O-methyltransferase (OMT) SpOMT2884 from Streptomyces peucetius was investigated. Two metabolites were isolated and identified as 7,4'-dihydroxy-8-methoxy-isoflavone (1) and 8,4'-dihydroxy-7-methoxy-isoflavone (2), based on mass, 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectrophotometric analysis. The maximum production yields of compound (1) and (2) in a 5-L fermenter were 9.3 mg/L and 6.0 mg/L, respectively. The two methoxy-isoflavones showed dose-dependent inhibitory effects on melanogenesis in cultured B16 melanoma cells under non-toxic conditions. Among the effects, compound (1) decreased melanogenesis to 63.5% of the control at 25 µM. This is the first report on the 8-O-methylation activity of OMT toward isoflavones. In addition, the present study also first identified compound (1) with potent melanogenesis inhibitory activity.
Assuntos
Biotransformação , Escherichia coli/genética , Escherichia coli/metabolismo , Isoflavonas/biossíntese , Metiltransferases/genética , Metiltransferases/metabolismo , Streptomyces/genética , Animais , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fermentação , Expressão Gênica , Isoflavonas/química , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Melanoma Experimental , Camundongos , Streptomyces/enzimologiaRESUMO
The inhibitory effect of a water extract from Pemphis acidula on melanogenesis in mouse B16 melanoma cells was investigated. The results showed that the P. acidula extract (PAE) inhibited melanogenesis in 3-isobutyl-1-methylxanthin (IBMX)-stimulated B16 cells in a dose-dependent manner, with an IC50 value of 33.5 µg/ml. In addition, PAE also inhibited cellular tyrosinase activity. Moreover, western blot and real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses respectively confirmed that PAE down-regulated levels of tyrosinase protein and its mRNA in IBMX-stimulated B16 cells. These results demonstrated that PAE inhibits melanogenesis of B16 cells by reducing tyrosinase gene expression. From the present study, PAE is proven to be a good candidate as a skin-whitening agent for treatment of skin hyperpigmentation.