In the underlying study, the inhibitory effects of alpha-arbutin (4-hydroxyphenyl aa-glucopyranoside) on the production of melanin were investigated both in cultured human melanoma cells (HMV-II) and in a three-dimensional model of cultured human skin. The light-absorbing melanin is formed in the biochemical process of melanogenesis, which begins with the synthesis of the molecule tyrosinase. However, if there is an overproduction of melanin, hyperpigmentation of the skin can occur, which leads to characteristic dark spots and colorations on the skin.
Tyrosinase is involved in the first two steps of melanin synthesis, the hydroxylation of tyrosine to 3-(3,4-dihydroxyphenyl)-alanine (DOPA) and the subsequent oxidation of DOPA to dopaquinone. Tyrosinase is therefore also regarded as a key enzyme in this process. Various substances are used in cosmetics to inhibit the tyrosinase process and thus eliminate hyperpigmentation of the skin.
The application of alpha-arbutin caused a concentration-dependent inhibition of melanin synthesis in human melanoma cells (HMV-II). At concentrations lower than 1.0 mM, alpha-arbutin did not affect cell growth. At a concentration of 0.5 mM, the activity of tyrosinase within the cells was reduced to the level of 60% of untreated cells, whereas the expression of tyrosinase mRNA remained unaffected. The decrease in melanin synthesis in alpha-arbutin-treated cells correlated strongly with the inhibition of cellular tyrosinase activity by alpha-arbutin.
Therefore, it is likely that the inhibitory effects of alpha-arbutin on melanogenesis are caused by the direct inhibition of melanosomal tyrosinase activity and not by the suppression of cell growth or tyrosinase gene expression.
The effects of alpha-arbutin on the three-dimensional model of cultivated human skin were also investigated and evaluated. The inhibitory effect on melanin synthesis was confirmed by quantitative measurements of melanin and by macro- and microscopic examinations of tissue pigmentation.
To test the melanin synthesis of the HMV-II cells, they were coated with 10 ml medium in a 100 mm dish and then allowed to grow for 10 days. On days 1, 4 and 7 of the study, the medium was replaced with fresh medium containing different concentrations of alpha-arbutin.
The determination of melanin content was measured using the method of Hosoi and others, which was minimally modified. HMV-II cells grown in a 100 mm dish were washed with Mg21-, Ca21-free phosphate-buffered saline [PBS(2)]. After treatment with 0.25% trypsin, the cells were selected via centrifugation (100003 g for 10 min) and sonicated in 0.5 ml 1% Triton X-100/PBS(2) on ice. In contrast, the three-dimensional model of cultured human skin was selected and sonicated in 0.5 ml PBS(2). The lysate was centrifuged at 100003 g for 10 minutes and the pellet was then incubated with 0.04% proteinase K/PBS(2) at 45 °C for 18 hours. After centrifugation for 10 minutes at 100003 g, the pellet was sonicated in 0.5 ml 1% Triton X-100/PBS(2) on ice. For quantitative measurement of melanin content, acid-soluble materials were obtained by double extraction of the pellet using 10% trichloroacetic acid.
This precipitate was then washed once with 100% ethanol, dried and then dissolved with 1 ml of 1 N NaOH-10% dimethyl sulfoxide by incubation at 80 °C for two hours. In addition, a solution with synthetic melanin was prepared, which served as a standard solution. The absorbance of the solutions was measured at 470 nm and the melanin content was calculated as mg/106 cells.
Cells treated with alpha-arbutin and HMV-II cells (1.03106) determined for cell proliferation were allowed to grow for 6 days in 10 ml medium containing different concentrations of alpha-arbutin, with the medium being changed on day 3. The cells were treated with 0.25% trypsin and the number of cells was then determined using a hemocytometer.
The cellular activity of tyrosinase was tested using the method developed by Tomita et al. using L-DOPA as a substrate. A total of 2,03104 HMV-II cells in 100 ml medium containing different concentrations of alpha-arbutin were plated on a 96-well microtiter plate and incubated for 6 days. On day 1 and day 4, the medium was replaced with fresh medium. The alpha-arbutin treated cells were washed with PBS(2) and lysed with 100 ml 0.5% Triton-X/PBS(2) per well. The lysates were mixed by vibration and 50 ml of 10 mM L-DOPA was added to each well. After incubation at 37 °C for three hours, the absorbance was measured at 475 nm using a spectrophotometer.
The results of the study suggest that alpha-arbutin also influences melanogenesis in the three-dimensional model of cultivated human skin. No cell toxicity of this effect was detected. The inhibitory effects of hydroquinone on melanogenesis are attributed to the inhibition of tyrosinase in the melanocytes and its cell toxicity towards melanocytes. However, analysis of the HPLC showed that although 70% of the alpha-arbutin applied to the three-dimensional model of cultured human skin was retained in the medium, no hydroquinone could be detected after cultivation. These results suggest that the inhibitory effects of alpha-arbutin on melanin synthesis cannot be attributed to the effects of hydroquinone released from alpha-arbutin.
Conclusion
In summary, the results of the underlying studies support the usability of alpha-arbutin as a cosmetic skin lightening agent.
Underlying study:
Sugimoto, K. et al: Inhibitory Effects of aa-Arbutin on Melanin Synthesis in Cultured Human Melanoma Cells and a Three-Dimensional Human Skin Model; in: Biol. Pharm. Bull. 27(4) 510-514 (2004)