Fermented Aronia melanocarpa Inhibits Melanogenesis through Dual Mechanisms of the PI3K/AKT/GSK-3β and PKA/CREB Pathways

Abstract

UV light causes excessive oxidative stress and abnormal melanin synthesis, which results in skin hyperpigmentation disorders such as freckles, sunspots, and age spots. Much research has been carried out to discover natural plants for ameliorating these disorders. Aronia melanocarpa contains various polyphenolic compounds with antioxidative activities, but its effects on melanogenesis have not been fully elucidated. In this study, we investigated the inhibitory effect of fermented Aronia melanocarpa (FA) fermented with Monascus purpureus on melanogenesis and its underlying mechanism in the B16F10 melanoma cell line. Our results indicate that FA inhibited tyrosinase activity and melanogenesis in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. FA significantly downregulated the PKA/CREB pathway, resulting in decreased protein levels of tyrosinase, TRP-1, and MITF. FA also inhibited the transcription of MITF by increasing the phosphorylation levels of both GSK3β and AKT. Interestingly, we demonstrated that these results were owing to the significant increase in gallic acid, a phenolic compound of Aronia melanocarpa produced after the fermentation of Monascus purpureus. Taken together, our research suggests that Aronia melanocarpa fermented with Monascus purpureus acts as a melanin inhibitor and can be used as a potential cosmetic or therapeutic for improving hyperpigmentation disorders.

Keywords: Aronia melanocarpa; Monascus purpureus; anti-melanogenesis; fermentation; gallic acid; melanin.

Figure 1

Cell viability (%) of B16F10 cells after 48 h of treatment with UA (A) and FA (B). Cell viability was not significantly altered by UA and FA concentrations of up to 800 μg/mL. Each value is presented as the mean ± SD.

Figure 2

Melanin content (%) of B16F10 cells and cell pellets after 48 h of treatment with UA and FA. FA significantly inhibited melanin synthesis compared to UA. Each value is presented as the mean ± SD. Significant difference versus control: ### p < 0.001. Significant difference versus α-MSH-treated group: *** p < 0.001. Positive control: 1 mM of arbutin.

Figure 3

(A) Protein levels of tyrosinase, TRP-1, TRP-2, and MITF were examined in UA-and FA-treated B16F10 cells with or without α-MSH. FA significantly inhibited melanin synthesis compared to UA. Each value is presented as the mean ± SD. Significant difference versus control: ### p < 0.001. Significant difference versus α-MSH-treated group: * p < 0.05, ** p < 0.01, *** p < 0.001. Positive control: 1 mM of arbutin. (B–E) Relative expression levels of proteins. Each value was calculated from the ratio of the signal intensity for the indicated protein to that of GAPDH. Data are shown as mean ± standard deviation values from two separate experiments. Each value is presented as the mean ± SD. Significant difference versus control: ### p < 0.001. Significant difference versus α-MSH-treated group: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

(A–C) Effect of UA and FA on α-MSH-induced protein expression of p-GSK3β and p-AKT in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p < 0.001; in comparison with the α-MSH-treated group, as *p < 0.05 ** p < 0.01 and *** p < 0.001. ns, not significant.

Figure 5

(A–C) Effect of UA and FA on α-MSH -induced protein expression of p-PKA and p-CREB in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001; in comparison with the α-MSH-treated group, as * p < 0.05 and *** p < 0.001. ns, not significant.

Figure 6

HPLC chromatograms of gallic acid determined with a 10 μL injection of UA and FA. HPLC chromatograms are recorded at 270 nm. The red line was a baseline. (A) UA—unfermented Aronia; (B) FA—fermented Aronia; (C) GA—gallic acid.

Figure 7

(A–E) Effect of gallic acid (100uM and 150uM) on α-MSH-induced protein expression of tyrosinase, TRP-1, TRP-2, and MITF in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001. ns, not significant; in comparison with the α-MSH-treated group, as ** p < 0.01 and *** p < 0.001. (F–H) Effect of gallic acid on α-MSH-induced protein expression of p-GSK3β and p-AKT in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001. ns, not significant; in comparison with the α-MSH-treated group, as ** p < 0.01 and *** p < 0.001. (I–K) Effect of gallic acid on α-MSH-induced protein expression of p-PKA and p-CREB in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001; in comparison with the α-MSH-treated group, as *** p < 0.001.

Figure 7

(A–E) Effect of gallic acid (100uM and 150uM) on α-MSH-induced protein expression of tyrosinase, TRP-1, TRP-2, and MITF in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001. ns, not significant; in comparison with the α-MSH-treated group, as ** p < 0.01 and *** p < 0.001. (F–H) Effect of gallic acid on α-MSH-induced protein expression of p-GSK3β and p-AKT in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001. ns, not significant; in comparison with the α-MSH-treated group, as ** p < 0.01 and *** p < 0.001. (I–K) Effect of gallic acid on α-MSH-induced protein expression of p-PKA and p-CREB in B16F10 cells. Each value is presented as the mean ± SD: in comparison with the control group, as ### p <0.001; in comparison with the α-MSH-treated group, as *** p < 0.001.

Figure 8

(A) Effects of FA and LY 294002 (PI3K inhibitor) on melanin content (%) in α-MSH-treated B16F10 cells after 48 h. Each value is presented as the mean ± SD. ### p < 0.001 (significant difference versus control); *** p < 0.001 (significant difference versus α-MSH-treated group). (B) Effects of FA and LY 294002 (PI3K inhibitor) on protein expression of MITF in α-MSH-treated B16F10 cells after 48 h. Each value is presented as the mean ± SD. *** p < 0.001 (significant difference versus α-MSH-treated group).

Figure 9

(A) Effect of FA and H-89 (PKA/CREB inhibitor) on melanin content (%) in α-MSH-treated B16F10 cells after 48 h. Each value is presented as the mean ± SD. ### p < 0.001 (significant difference versus control); *** p < 0.001 (significant difference versus α-MSH-treated group). (B) Effects of FA and H-89 (PKA/CREB inhibitor) on protein expression of MITF in α-MSH-treated B16F10 cells after 48 h. Each value is presented as the mean ± SD. ### p < 0.001 (significant difference versus control); *** p < 0.001 (significant difference versus α-MSH-treated group).