Cellular Anti-Melanogenic Effects of a Euryale ferox Seed Extract Ethyl Acetate Fraction via the Lysosomal Degradation Machinery

Abstract

The aim of this study was to investigate the effect of ethyl acetate fraction of Euryale ferox seed extracts (Efse-EA) on melanogenesis in immortalized mouse melanocyte cell line, melan-a. Efse-EA showed strong dose-dependent mushroom tyrosinase inhibitory activity. Treatment of melan-a cells with 30 μg/mL Efse-EA produced strong inhibition of cellular tyrosinase and melanin synthesis. Efse-EA significantly reduced the levels of melanogenesis-related proteins, such as tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor. Because Efse-EA treatment reduced tyrosinase protein levels without changing its mRNA expression, we investigated whether this decrease was related to proteasomal or lysosomal degradation of tyrosinase. We found that chloroquine, a lysosomal proteolysis inhibitor, almost completely abolished both the down-regulation of tyrosinase and the inhibition of melanin synthesis induced by Efse-EA. These results suggested that Efse-EA may contribute to the inhibition of melanogenesis by altering lysosomal degradation of tyrosinase, and that this extract may provide a new cosmetic skin-whitening agent.

Figure 1

A kinetic curve of Efse-EA for antioxidant activity by oxygen radical absorbance capacity (ORAC) assay. The ORAC assay was carried out as described in Experimental Section. The final ORAC values were calculated using the regression equation for the Trolox concentration plotted against the net area under the fluorescence decay curve (AUC; area under the curve). Open triangle, vehicle; open diamond, Trolox 3.125 µg/mL; open circle, Trolox 6.25 µg/mL; closed circle, Trolox 12.5 µg/mL; closed diamond, Efse-EA 3.125 µg/mL closed triangle, Efse-EA 6.25 µg/mL.

Figure 2

Anti-tyrosinase and copper chelating effects of Efse-EA. Tyrosinase was preincubated with the test substances at 25 °C for 5 min prior to incubation with l-tyrosine for 30 min; absorbance was read at 490 nm. Each determination was made in triplicate and the data shown represent the mean ± SD. ** p < 0.01, Student’s t-test (A); copper chelating effects were measured using pyrocatechol violet (PV) as described in Experimental Section (B).

Figure 3

Effect of Efse-EA on melanogenesis in melan-a cells. Cells (1 × 10⁵ cells/mL) were seeded for overnight, and then the medium was replaced with fresh medium containing the indicated concentrations of Efse-EA or PTU (N-phenylthiourea). Cells were cultured for 72 h and further incubated for 24 h. After washing with PBS, the cells were lysed with 250 μL of 1 N NaOH and transferred to a 96 well plate. The melanin contents were estimated by measuring the absorbance at 405 nm. Each determination was made in triplicate and the data shown represent the mean ± SD. * p < 0.05, ** p < 0.01, Student’s t-test.

Figure 4

Effect of Efse-EA on intracellular tyrosinase activity. Melan-a cells (1 × 10⁵ cells/mL) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of Efse-EA or arbutin for 3 days. The cells were collected and lysed. After quantifying protein levels, tyrosinase activity was determined by l-DOPA zymography. Each determination was made in triplicate and the data shown represent the mean ± SD. ** p < 0.01, Student’s t-test.

Figure 5

Effect of Efse-EA on the levels of melanogenesis-related mRNA and proteins in melan-a cells. Cells (1 × 10⁵ cells/mL) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of Efse-EA or arbutin for three days. Total cell lysates were extracted and assayed by Western blotting using antibodies against tyrosinase, tyrosinase-related protein (TYRP)-1, TYRP-2, and microphthalmia-associated transcription factor (MITF). Equal amounts of protein loading were confirmed using β-actin (A); Relative intensity of melanogenesis-related protein expressions, the intensity of the protein expressions was compared to the control; The normalized data for each were plotted as bar graphs (B); Cells (1 × 10⁵ cells/mL) were cultured for 24 h; the medium was replaced with fresh medium containing the indicated concentrations of Efse-EA or arbutin for 1 day. The mRNA was extracted using TRIzol; mRNA expression was analyzed by reverse-transcription polymerase chain reaction (C).

Figure 6

Effect of Efse-EA on lysosomal tyrosinase degradation in melan-a cells. Cells (3 × 10⁵ cells/mL) were pretreated with 25 μg/mL cycloheximide for 1 h, as indicated. Cells were also pretreated with 10 μM MG132 or 50 μM chloroquine for 1 h, and then treated with Efse-EA for 6 h. Whole cell lysates were subjected to western blotting using an anti-tyrosinase antibody. Equal protein loading was confirmed using actin (A); β-Hexosaminidase assay was calculated as described in Experimental Section. In brief, melan-a cells (2 × 10⁵ cells/well) were seeded and sensitized with 1 μg/mL of dinitrophenyl (DNP)-immunoglobulin E (IgE) and stimulated with 20 μg DNP-bovine serum albumin (BSA). Following 1 h incubation, supernatant was transferred and the substrate for β-hexosaminidase (1 mM 4-nitrophenyl N-acetyl-β-d-glucosaminide (NAG; N9376, Sigma) was added. After adding stop solution, the sample was measured at 405 nm with a spectrophotometer (B). * p < 0.05, ** p < 0.01.