Inhibitory Effect of Arctigenin from Fructus Arctii Extract on Melanin Synthesis via Repression of Tyrosinase Expression

Abstract

To identify the active compound arctigenin in Fructus Arctii (dried seed of medicinal plant Arctium lappa) and to elucidate the inhibitory mechanism in melanogenesis, we analyzed melanin content and tyrosinase activity on B16BL6 murine melanoma and melan-A cell cultures. Water extracts of Fructus Arctii were shown to inhibit tyrosinase activity in vitro and melanin content in α -melanocyte stimulating hormone-stimulated cells to similar levels as the well-known kojic acid and arbutin, respectively. The active compound arctigenin of Fructus Arctii displayed little or no cytotoxicity at all concentrations examined and decreased the relative melanin content and tyrosinase activity in a dose-dependent manner. Melanogenic inhibitory activity was also identified in vivo with zebrafish embryo. To determine the mechanism of inhibition, the effects of arctigenin on tyrosinase gene expression and tyrosinase promoter activity were examined. Also in addition, in the signaling cascade, arctigenin dose dependently decreased the cAMP level and promoted the phosphorylation of extracellular signal-regulated kinase. This result suggests that arctigenin downregulates cAMP and the tyrosinase enzyme through its gene promoter and subsequently upregulates extracellular signal-regulated kinase activity by increasing phosphorylation in the melanogenesis signaling pathway, which leads to a lower melanin content.

Figure 1

The inhibitory effect of FAE on tyrosinase activity in vitro (a) and melanin content in murine B16BL6 cells stimulated with 100 nM α-MSH (b). Cells were treated with KA (60 μg/mL) or arbutin (100 μg/mL) and FAE at different doses (1, 100, 500 μg/mL). In vitro tyrosinase activities were measured based on the absorbance at 475 nm, and melanin contents are depicted as a percentage (%) of control. Each value was expressed with the mean ± SD of three independent experiments.

Figure 2

Chemical structure (a) and cytotoxicity of ATG at different treatment doses (b). Values of cytotoxicity were represented as a percentage (%) of control, with mean ± SD of three independent experiments.

Figure 3

Effect of ATG (1, 10, and 50 μM) on α-MSH (100 nM) stimulated murine B16BL6 melanoma cells ((a) and (b)) and melan-A cells ((c) and (d)). Data are represented as relative melanin contents and tyrosinase activity compared with arbutin (100 μg/mL). ATG concentration without α-MSH was 10 μM. All values were represented from three independent experiments with mean ± SD values.

Figure 4

ATG treatment suppressed pigment deposition without affecting cell migration. Moderate concentration of ATG (10 μM) treated embryos showed reduced embryonic pigmentation without causing any discernible pigment cells migration defect (red arrowhead). Treatment of high dose ATG (100 μM) caused severe embryonic developmental retardation and growth was stalled at 18–20 somite stage (data not shown). All live embryos were treated with ATG for different dosages at 15 hpf (12–14 somite stage) followed by taken picture at 40 hpf. Pictures were shown by overview (a) and lateral view (b).

Figure 5

Inhibitory effect of ATG (10 μM) on tyrosinase gene expression was analyzed by real-time qPCR (a) and western blot (b) in B16BL6 cells stimulated with α-MSH. The effect of ATG (1, 10, 20, and 50 μM) on tyrosinase promoter activity was determined using the luciferase reporter system (c). Dose-dependent inhibitory effect of ATG (1, 10, and 50 μM) was observed in the CRE-luciferase assay (d), and the level of P-ERK increased in a time-dependent manner (e).