Inhibitory effects of adlay extract on melanin production and cellular oxygen stress in B16F10 melanoma cells

Abstract

The aim of this study was to determine the effects of adlay extract on melanin production and the antioxidant characteristics of the extract. The seeds were extracted by the supercritical fluid CO2 extraction (SFE) method. The effect of adlay extract on melanin production was evaluated using mushroom tyrosinase activity assay, intracellular tyrosinase activity, antioxidant properties and melanin content. Those assays were performed spectrophotometrically. In addition, the expression of melanogenesis-related proteins was determined by western blotting. The results revealed that the adlay extract suppressed intracellular tyrosinase activity and decreased the amount of melanin in B16F10 cells. The adlay extract decreased the expression of microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase related protein-1 (TRP-1) and tyrosinase related protein-2 (TRP-2). The extract also exhibited antioxidant characteristics such as free radical scavenging capacity and reducing power. It effectively decreased intracellular reactive oxygen species (ROS) levels in B16F10 cells. We concluded that the adlay extract inhibits melanin production by down-regulation of MITF, tyrosinase, TRP-1 and TRP-2. The antioxidant properties of the extract may also contribute to the inhibition of melanogenesis. The adlay extract can therefore be applied as an inhibitor of melanogenesis and could also act as a natural antioxidant in skin care products.

Figure 1

The inhibitory effects of adlay extract on melanogenesis. (A) The effects of adlay extract on mushroom tyrosinase activity; (B) The effects of adlay extract on melanin content in B16F10 cells; (C) The effects of adlay extract on tyrosinase activity in B16F10 cells. The results are presented as percentages of the control, and the data are presented as the mean ± S.D. of three separate experiments. Black bars and lines on each bar indicate the mean and standard deviation. The values are significantly different compared with the control. * p < 0.05, ** p < 0.01.

Figure 2

The effects of adlay extract on melanogenesis-related protein expression levels. (A) Western blotting of cellular proteins in B16F10 cells; (B) Total RNA from B16F10 cells treated with adlay extract (40 mg/mL) collected at the indicated time. microphthalmia-associated transcription factor (MITF) mRNA levels were examined by real time RT-PCR, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control. (* p < 0.05 vs. control).

Figure 3

The antioxidant characteristics of adlay extract. (A) DPPH scavenging capacity assay; (B) ABTS⁺ radical scavenging activity assay; (C) Determination of reducing capacity; (D) Measurement of total phenolic content; (E) ROS level assay. Different concentrations of the adlay extract and positive standards were used in the above assays. The results are expressed as percentages of the control. The data are presented as the mean ± S.D. * p < 0.05, ** p < 0.01.