Anti-Melanogenic Effects of Ethanol Extracts of the Leaves and Roots of Patrinia villosa (Thunb.) Juss through Their Inhibition of CREB and Induction of ERK and Autophagy

Abstract

Patrinia villosa (Thunb.) Juss is a traditional herb commonly used in East Asia including Korea, Japan, and China. It has been administered to reduce and treat inflammation in Donguibogam, Korea. The mechanism for its anti-inflammatory effects has already been reported. In this study, we confirmed the efficacy of Patrinia villosa (Thunb.) Juss ethanol extract (Pv-EE) for inducing autophagy and investigate its anti-melanogenic properties. Melanin secretion and content were investigated using cells from the melanoma cell line B16F10. Pv-EE inhibited melanin in melanogenesis induced by α-melanocyte-stimulating hormone (α-MSH). The mechanism of inhibition of Pv-EE was confirmed by suppressing the mRNA of microphthalmia-associated transcription factor (MITF), decreasing the phosphorylation level of CREB, and increasing the phosphorylation of ERK. Finally, it was confirmed that Pv-EE induces autophagy through the autophagy markers LC3B and p62, and that the anti-melanogenic effect of Pv-EE is inhibited by the autophagy inhibitor 3-methyl adenine (3-MA). These results suggest that Pv-EE may be used as a skin protectant due to its anti-melanin properties including autophagy.

Keywords: CREB; ERK; Patrinia villosa (Thunb.) Juss; autophagy; melanogenesis.

Figure 1

Anti-melanogenesis effects of ethanol extracts of Patrinia villosa prepared fron leaf (lPv-EE) and root (rPv-EE) in α-melanocyte stimulating hormone (α-MSH)-treated B16F10 Cells (A–D) The levels of melanin secretion and contents in B16F10 cells treated with -MSH (100 nM) in the presence or absence of lPv-EE (400 and 800 μg/mL), rPv-EE (100 to 800 μg/mL), or arbutin (1 mM) for 48 h. (E,F) Viability, determined using the MTT assay after 24 h, of B16F10 cells treated with various concentrations (400 and 800 μg/mL) of lPv-EE or (100 to 400 μg/mL) of rPv-EE. ** p < 0.01 compared to the control group.

Figure 2

Effect of lPv-EE and rPv-EE on the MITF mRNA expression condition in α-MSH-treated B16F10 cells. (A,B) The effect of lPv-EE (400 to 1600 μg/mL), rPv-EE (100 and 200 μg/mL), or kojic acid (300 μM) on mushroom tyrosinase activity was determined by quantifying the activity of purified tyrosinase. (C–F) The mRNA levels, as determined by RT-PCR, of B16F10 cells treated with 100 nM α-MSH and lPv-EE (400 or 800 μg/mL) or rPv-EE (100 or 200 μg/mL) or 1 mM of arbutin for 24 h.

Figure 2

Effect of lPv-EE and rPv-EE on the MITF mRNA expression condition in α-MSH-treated B16F10 cells. (A,B) The effect of lPv-EE (400 to 1600 μg/mL), rPv-EE (100 and 200 μg/mL), or kojic acid (300 μM) on mushroom tyrosinase activity was determined by quantifying the activity of purified tyrosinase. (C–F) The mRNA levels, as determined by RT-PCR, of B16F10 cells treated with 100 nM α-MSH and lPv-EE (400 or 800 μg/mL) or rPv-EE (100 or 200 μg/mL) or 1 mM of arbutin for 24 h.

Figure 3

Anti-melanogenic mechanism of lPv-EE and rPv-EE in α-MSH-treated B16F10 cells. (A,B) The promoter binding activity of the transcription factor CREB was analyzed using a reporter gene assay. B16F10 cells were transfected with plasmids driving the expression of CREB-Luc (1 μg/mL) and β-gal (as a transfection control). After 24 h, some cells were treated with 100 nM α-MSH and lPv-EE (400 or 800 μg/mL) or rPv-EE (100 or 200 μg/mL) for 24 h. Luciferase activity was measured using a luminometer. (C–F) Levels of phosphorylated and total CREB, MITF, ERK, p38, JNK, and β-actin proteins were determined in B16F10 cells using phospho-specific or total antibodies for each protein. ** p < 0.01 compared to the normal group and * p < 0.05 compared to the control group.

Figure 4

Effect of ethanol extract of Patrinia villosa (Pv-EE) on autophagy. (A–C) Levels of total LC3B, p62, and β-actin proteins were determined in B16F10 cells using the total antibodies for each protein. (D,E) The promoter binding activity of the transcription factor CREB was analyzed using a reporter gene assay. B16F10 cells were transfected with plasmids driving the expression of CREB-Luc (1 μg/mL) and β-gal (as a transfection control). After 24 h, some of the cells were treated with 100 nM α-MSH and lPv-EE (800 μg/mL) or rPv-EE (200 μg/mL) or 3-MA (10 mM) for 48 h. Luciferase activity was measured using a luminometer. (F–H) The levels of melanin secretion and contents in B16F10 cells treated with α-MSH (100 nM) in the presence or absence of lPv-EE (800 μg/mL) or 3-MA (10 mM) or chloroquine (20 μM) or Wortmannin (2 μM) for 48 h were then determined. (I) The phytochemical profile of rPv-EE was analyzed by HPLC using standard compounds (quercetin, luteolin, and kaempferol). ** p < 0.01 compared to the normal group and * p < 0.05 compared to the control group.

Figure 4

Effect of ethanol extract of Patrinia villosa (Pv-EE) on autophagy. (A–C) Levels of total LC3B, p62, and β-actin proteins were determined in B16F10 cells using the total antibodies for each protein. (D,E) The promoter binding activity of the transcription factor CREB was analyzed using a reporter gene assay. B16F10 cells were transfected with plasmids driving the expression of CREB-Luc (1 μg/mL) and β-gal (as a transfection control). After 24 h, some of the cells were treated with 100 nM α-MSH and lPv-EE (800 μg/mL) or rPv-EE (200 μg/mL) or 3-MA (10 mM) for 48 h. Luciferase activity was measured using a luminometer. (F–H) The levels of melanin secretion and contents in B16F10 cells treated with α-MSH (100 nM) in the presence or absence of lPv-EE (800 μg/mL) or 3-MA (10 mM) or chloroquine (20 μM) or Wortmannin (2 μM) for 48 h were then determined. (I) The phytochemical profile of rPv-EE was analyzed by HPLC using standard compounds (quercetin, luteolin, and kaempferol). ** p < 0.01 compared to the normal group and * p < 0.05 compared to the control group.

Figure 5

Putative inhibitory pathway of lPv-EE and rPv-EE.