Coinhibitory Effects of Resveratrol- and Protopanaxadiol-Enriched Rice Seed Extracts Against Melanogenic Activities in Melan-a Cells

Abstract

In the current study, we aimed to evaluate the combined antimelanogenic effects of resveratrol- and protopanaxadiol (PPD)-enriched rice seed extracts (DJ526 and DJ-PPD) in melan-a cells. The treatment antioxidant capacity was evaluated using the ABTS radical scavenging method. TR_3 (70% [wight (w)/w] of DJ526 and 30% [w/w] of DJ-PPD) markedly increased the antioxidant activity at a level similar to that of DJ526 and DJ-PPD alone. The antimelanogenic activities in melan-a cells were evaluated after co-culturing of treatments at the concentration of 100 μg/mL. The in vitro melan-a cell experiment showed that treatment with the DJ526 and DJ-PPD mixture significantly reduced the cellular tyrosinase activity and melanin content; suppressed the expression of melanogenesis-related genes and proteins; decreased the number and size of melanin-containing cells; upregulated phosphorylated extracellular signal-regulated kinase 1/2 and protein kinase B expression levels; and suppressed the expression of p-p38 MAPK. These results show that DJ-PPD does not interfere with the antioxidant and antimelanogeneic activities of DJ526 but enhances the antioxidant and antimelanogeneic activities of DJ526. These findings indicate the potential of resveratrol- and PPD-enriched rice seeds as novel agents for controlling hyperpigmentation.

Keywords: MAPK; NF-κB; antimelanogenic; antioxidant; hyperpigmentation; synergistic effect.

Figure 1

Antioxidant activity of DJ526 and DJ-PPD in combination. The data are presented as the mean ± standard deviation (n = 3). Lowercase letters represent the significant differences in ABTS radical scavenging activity between the treatments at the same concentration at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), and “c” represents significantly lower value than “b” (p < 0.05).

Figure 2

Effect of combined DJ526 and DJ-PPD on the viability of melan-a cells. The concentrations of DMSO and the treatments were 0.1% (v/v) and 100 µg/mL, respectively. The data are presented as the mean ± standard deviation (n = 3). The significant difference was determined at p < 0.05 compared to the medium group.

Figure 3

Effects of the combination of DJ526 and DJ-PPD on the melanin content and cellular tyrosinase activity in melan-a cells. (a) Melanin content (%) compared to the medium group, (b) representative of cell pellet collection, (c) cellular tyrosinase activity (%) compared to the medium group, and (d) representative of L-DOPA staining on melan-a cells. The concentrations of DMSO and the treatments were 0.1% (v/v) and 100 µg/mL, respectively. The data are presented as the mean ± standard deviation (n = 3). Significant differences among the treatments are indicated using the different letters (a, b, c, d, e, and f) at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), “c” represents significantly lower value than “b” (p < 0.05), “d” represents significantly lower value than “c” (p < 0.05), “e” represents significantly lower value than “d” (p < 0.05), “f” represents significantly lower value than “e” (p < 0.05), and the same letter represents no significant difference (p < 0.05).

Figure 4

Effects of the combination of DJ526 and DJ-PPD on melanogenesis-related gene expression levels. Effect on the (a) MITF expression level, (b) TRP-1 expression level, (c) TRP-2 expression level, and (d) tyrosinase expression level. The concentrations of DMSO and the treatments were 0.1% (v/v) and 100 µg/mL, respectively. The data are presented as the mean ± standard deviation (n = 3). Significant differences in gene expression levels among the treatments are indicated using the different letters (a, b, c, and d) at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), “c” represents significantly lower value than “b” (p < 0.05), “d” represents significantly lower value than “c” (p < 0.05), and the same letter represents no significant difference (p < 0.05).

Figure 5

Effect of DJ526 and DJ-PPD in combination on melanogenesis-related protein expression levels. The concentrations of DMSO and the treatments were 0.1% (v/v) and 100 µg/mL, respectively. The data are presented as the mean ± standard deviation. Significant differences in protein expression levels among the treatments are indicated using the different letters (a, b, c, and d) at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), “c” represents significantly lower value than “b” (p < 0.05), “d” represents significantly lower value than “c” (p < 0.05), and the same letter represents no significant difference (p < 0.05).

Figure 6

Effect of the combination of DJ526 and DJ-PPD on the morphology and differentiation of melan-a cells. Effect on (a) melanin-containing cells in the total of 1000 cells, (b) Fontana–Masson staining with scale bar at 50 μm (−), (c) melan-a cell differentiation score 1+, (d) melan-a cell differentiation score 2+, (e) melan-a cell differentiation score 3+, (f) melan-a cell differentiation score 4+. The concentrations of DMSO and the treatments were 0.1% (v/v) and 100 µg/mL, respectively. The data are presented as the mean ± standard deviation (n = 3). Significant differences in melanin-containing cells and melan-a cell differentiation score levels among the treatments are indicated using the different letters (a, b, c, d, and e) at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), “c” represents significantly lower value than “b” (p < 0.05), “d” represents significantly lower value than “c” (p < 0.05), “e” represents significantly lower value than “d” (p < 0.05), and the same letter represents no significant difference (p < 0.05).

Figure 7

Effect of the combination of DJ526 and DJ-PPD on the MAPK and PI3K/Akt signaling pathways. (a) Blot analysis, (b) p-ERK 1/2 expression, (c) p-p38 MAPK expression, and (d) p-Akt expression. The data are presented as the mean ± standard deviation. Significant differences in the protein expression levels among the treatments are indicated using the different letters (a, b, c, d, e, and f) at p < 0.05. The letter “a” represents the reference value, “b” represents significantly lower value than “a” (p < 0.05), “c” represents significantly lower value than “b” (p < 0.05), “d” represents significantly lower value than “c” (p < 0.05), “e” represents significantly lower value than “d” (p < 0.05), “f” represents significantly lower value than “e” (p < 0.05), and the same letter represents no significant difference (p < 0.05).