Biotechnology Letters

, 30:743

Inhibitory effects of arbutin-β-glycosides synthesized from enzymatic transglycosylation for melanogenesis

Authors

  • So-Young Jun
    • Department of Biological Sciences, College of Natural SciencesPusan National University
  • Kyung-Min Park
    • Department of Biological Sciences, College of Natural SciencesPusan National University
  • Ki-Won Choi
    • Department of Biological Sciences, College of Natural SciencesPusan National University
  • Min Kyung Jang
    • Department of Bioscience and Biotechnology, College of EngineeringSilla University
  • Hwan Yul Kang
    • Amaranth Cosmetics
  • Sang-Hyeon Lee
    • Department of Bioscience and Biotechnology, College of EngineeringSilla University
  • Kwan-Hwa Park
    • Department of Food Science and Biotechnology, School of Agricultural BiotechnologySeoul National University
    • Department of Biological Sciences, College of Natural SciencesPusan National University
Original Research Paper

DOI: 10.1007/s10529-007-9605-1

Cite this article as:
Jun, S., Park, K., Choi, K. et al. Biotechnol Lett (2008) 30: 743. doi:10.1007/s10529-007-9605-1

Abstract

To develop a new skin whitening agent, arbutin-β-glycosides were synthesized and evaluated for their melanogenesis inhibitory activities. Three active compounds were synthesized via the transglycosylation reaction of Thermotoga neapolitana β-glucosidase and purified by recycling preparative HPLC. As compared with arbutin (IC50 = 6 mM), the IC50 values of these compounds were 8, 10, and 5 mM for β-d-glucopyranosyl-(1→6)-arbutin, β-d-glucopyranosyl-(1→4)-arbutin, and β-d-glucopyranosyl-(1→3)-arbutin, respectively. β-d-Glucosyl-(1→3)-arbutin also exerted the most profound inhibitory effects on melanin synthesis in B16F10 melanoma cells. Melanin synthesis was inhibited to a significant degree at 5 mM, at which concentration the melanin content was reduced to below 70% of that observed in the untreated cells. Consequently, β-d-glucopyranosyl-(1→3)-arbutin is a more effective depigmentation agent and is also less cytotoxic than the known melanogenesis inhibitor, arbutin.

Keywords

ArbutinArbutin-β-glycosidesMelanoma cellTransglycosylationTyrosinase

Introduction

Melanins are the skin pigments in humans. They perform a primary role in photoprotection (Riley 2003; Ahn et al. 2006). Melanins are secreted by melanocyte cells which are distributed throughout the basal layer of the dermis (Spritz et al. 1994). One of the roles of melanin is to protect the skin and underlying tissues from UV-induced skin injury. However, excessive melanin production in the skin has negative hyper-pigmentation effects, inducing melasma, freckles, and senile lentigines (Ha et al. 2005; Min et al. 2004; Unver et al. 2006). Epidermal and dermal hyper-pigmentation can be dependent on either an increased number of melanocytes or the actions of melanogenic enzymes (Griffiths et al. 1993; Kanwar et al. 1994).

Tyrosinase (EC 1.14.18.1) is one of the key enzymes in melanin synthesis (Kubo et al. 2000; Perez-Gilabert and Garcia-Carmona 2001). The enzyme catalyzes the first two steps in melanin synthesis: the hydroxylation of tyrosine to 3-(3,4-dihydroxyphenyl)-alanine (DOPA) and the oxidation of DOPA to dopaquinone (Shin et al. 1998). A number of tyrosinase inhibitors have been reported from both natural and synthetic sources, but only a few of these have been utilized as skin-whitening agents, principally due to a variety of safety concerns (Maeda and Fukuda 1991). For example, linoleic acid, hinokitol, kojic acid, naturally occurring hydroquinones, and catechols have all been reported to inhibit enzyme activity, but they also evidence deleterious side effects (Seo et al. 2003). Among them, 4-hydroxyphenyl β-d-glucopyranoside (arbutin) exhibits prominent tyrosinase inhibitory activity. In previous studies, 4-hydroxyphenyl α-d-glucopyranoside (α-arbutin) and α-arbutin-α-glycosides were synthesized enzymatically (Sugimoto et al. 2003, 2005), and their inhibitory effects on melanogenesis were evaluated. The inhibitory activities of α-arbutin and α-arbutin-α-glycosides against several tyrosinases from different sources have also been compared. α-Arbutin and α-arbutin-α-glycosides evidenced more profound inhibitory activities than arbutin against mammalian tyrosinases (Sugimoto et al. 2005). Tyrosinase inhibition studies with arbutin derivatives have shown that α-glucosidic linkages in hydroquinone-glycosides may perform an important function in the inhibition of human tyrosinase.

In an effort to evaluate the effects of the β-glucosidic linkages in arbutin on tyrosinase inhibitory activity, we enzymatically synthesized new arbutin β-glycosides, β-d-glucosyl-(1→6)-arbutin, β-d-glucosyl-(1→4)-arbutin, and β-d-glucosyl-(1→3)-arbutin via the transglycosylation of Thermotoganeapolitana β-glucosidase using arbutin and cellobiose as an acceptor and donor molecule, respectively (Park et al. 2005; Fig. 1). The inhibitory activities of the β-glycosides on mushroom tyrosinase and the melanin production activities of melanoma cells are addressed in this paper.
https://static-content.springer.com/image/art%3A10.1007%2Fs10529-007-9605-1/MediaObjects/10529_2007_9605_Fig1_HTML.gif
Fig. 1

Chemical structures of arbutin derivatives (compound 1, 2, and 3) synthesized by the transglycosylation of Thermotoga neapolitana β-glucosidase

Materials and methods

Materials

α-Melanocyte stimulating hormone (MSH), arbutin, cellobiose, mushroom tyrosinase, and l-tyrosine were purchased from the Sigma. α-d-Glucosyl-(1→4)-arbutin was generously provided by Dr. Cheon-Seok Park of the KyungHee University, Republic of Korea.

Preparation and purification of arbutin β-glycosides

Arbutin β-glycosides were prepared as described by Park et al. (2005). A 5 ml reaction mixture containing 17.5 μg Thermotoga neapolitana β-glucosidase, 15% (w/v) cellobiose and 15% (w/v) arbutin in 100 mM sodium phosphate buffer (pH 7.0) was incubated for 12 h at 80°C. The transglycosylated products were separated via recycling preparative HPLC: 3 ml transglycosylated products were applied to a JAIGEL W-251 (JAI Korea) column (2 × 50 cm) and eluted with deionized water at 3 ml/min. The fractions containing the arbutin β-glycosides were collected and freeze-dried. The purity of each of the separated arbutin β-glycosides was confirmed via TLC and high-performance anion exchange chromatography (HPAEC).

Enzymatic assay of tyrosinase

Tyrosinase activity was assayed in accordance with the previously described method, with slight modifications (Miyazawa et al. 2007). The reaction mixture containing 100 μl 0.03% l-tyrosine and 640 μl 67 mM phosphate buffer (pH 7.0), and 50 μl test sample solution were preincubated for 10 min at 37°C. Ten μl of 60 U mushroom tyrosinase solution dissolved in 67 mM phosphate buffer was then added, and the reaction mixtures were incubated for 30 min at 37°C in 96-well plates. The enzymatic activity was quantified via measurements of the absorbance at 475 nm. Tyrosinase activity was determined via the following formula:
$$ {\text{tyrosinase activity }}(\% )\, = \,{\text{ }}\left[ {(A - B)/(C_{\text{p}} - C_{\text{n}} )} \right] \times {\text{ }}100 $$
in which A is the absorbance of the test sample (67 mM phosphate buffer, l-tyrosine, sample solution, and tyrosinase); B is the absorbance of the blank (67 mM phosphate buffer, distilled water, sample solution, and tyrosinase); Cp is the absorbance of the positive control (67 mM phosphate buffer, l-tyrosine, and tyrosinase); and Cn is the absorbance of the negative control (67 mM phosphate buffer, distilled water, and tyrosinase).

Inhibition of melanin production in B16F10 melanoma cells

The B16F10 murine melanoma cell line (Korean Cell Line Bank, Seoul, Republic of Korea) was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (BioWhittaker, Walkersville, MD, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (BioWhittaker) at 37°C in a humidified atmosphere containing 5% CO2. B16F10 cells were cultured in 60 mm tissue culture dishes (Iwaki, Japan), and were maintained in 2 ml DMEM containing 10% (v/v) FBS. After 48 h, the cells were washed twice with 2 ml phosphate-buffered saline (PBS) (BioWhittaker), fed 2 ml fresh media, and treated with α-MSH at 200 nM. The samples were distributed to the dishes in a dilution series (final concentrations: 1, 5, 10, 20 mM). The cells were harvested after 48 h and were then washed twice with 2 ml PBS. The cells were collected by centrifugation (5 min at 1,700 × g) at 4°C, and the supernatant was discarded. 200 μl of 1 M NaOH (10% DMSO) was added to the cells and incubated for 1 h at 80°C. After the melanin was dissolved by vortexing, the melanin content was determined at 405 nm with a microplate reader.

Cytotoxicity assay

B16F10 cells were cultured in 96-well tissue culture plates (Nunc, USA) and maintained in 200 μl of DMEM containing 10% (v/v) FBS. After 48 h, the cells were washed twice in PBS, fed 200 μl fresh media, and treated with α-MSH at 200 nM. The samples were then dissolved in PBS and distributed to a plate in a dilution series (final concentrations: 1, 5, 10, 20 mM). After 48 h, the cells were washed twice in PBS and replaced with fresh media. 20 μl MTT solution (5 mg/ml in PBS) was then added to each well, and the cells were incubated for an additional 12 h at 37°C. The media were removed and the cells were dissolved in 100 μl DMSO. The conversion of MTT to formazan was quantified at 570 nm using a microplate reader.

Results and discussion

The transglycosylation reaction of β-glucosidase from Thermotoga neapolitana was successfully employed for the production of arbutin β-glycosides in this study. Arbutin and cellobiose were utilized as the acceptor and donor molecules, respectively. The synthesis of arbutin β-glycosides was confirmed using TLC and HPAEC. Purification via recycling preparative HPLC yielded compounds 1, 2, and 3 as white crystallines (8.1 mg, 8 mg, and 5.3 mg) in a 2.8% total yield based on the donor added. The NMR analyses of three purified arbutin β-glycosides showed that the structure of the three compounds was β-d-glucosyl-(1→6)-arbutin (compound 1), β-d-glucosyl-(1→4)-arbutin (compound 2), and β-d-glucosyl-(1→3)-arbutin (compound 3), respectively (Park et al. 2005).

To investigate the tyrosine inhibitory effects of purified arbutin β-glycosides, the IC50 values of the three arbutin β-glycosides against mushroom tyrosinase were evaluated using l-tyrosine as a substrate. Among the tested arbutin glycosides, β-d-glucosyl-(1→3)-arbutin evidenced the most profound inhibitory effect on mushroom tyrosinase (Fig. 2). The IC50 value of β-d-glucosyl-(1→3)-arbutin was 5 mM, while that of arbutin was 6 mM (Table 1). When the IC50 values of β-d-glucopyranosyl-(1→4)-arbutin and β-d-glucopyranosyl-(1→6)-arbutin were compared with those of arbutin and α-d-glucosyl-(1→4)-arbutin, which were utilized as controls, they evidenced less profound inhibitory effects on mushroom tyrosinase than were observed with arbutin and α-d-glucosyl-(1→4)-arbutin.
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Fig. 2

Inhibitory effects of arbutin and arbutin derivatives on mushroom tyrosinase. Tyrosinase activity was assessed using 0.03% l-tyrosine as the substrate. The results are expressed as the percentage of activity of (•) arbutin, (◯) β-d-glucopyranosyl-(1→6)-arbutin, (▼) β-d-glucopyranosyl-(1→4)-arbutin, (▽) β-d-glucopyranosyl-(1→3)-arbutin, or (■) α-d-glucopyranosyl-(1→4)-arbutin with regard to the untreated controls. Each value is expressed as the mean ± standard error of experiments conducted in triplicate

Table 1

Effects of arbutin derivatives on mushroom tyrosinase

Compound

Inhibition at 10 mM (%)

IC50 (mM)

Arbutin

66

6

β-d-Glucopyranosyl-(1→6)-arbutin

54

8

β-d-Glucopyranosyl-(1→4)-arbutin

52

10

β-d-Glucopyranosyl-(1→3)-arbutin

74

5

α-d-Glucopyranosyl-(1→4)-arbutin

69

5

The inhibitory effects of α-arbutin and arbutin α-glycosides on human tyrosinase were previously assessed (Sugimoto et al. 2003, 2005). In a previous study, the α-arbutin and arbutin α-glycosides were shown to evidence much stronger inhibitory activities as compared to arbutin, which indicates that the α-glucosidic linkages of the hydroquinone-glycosides were essential to their inhibitory effects on human tyrosinase. The density functional theory calculation on arbutin derivatives also suggested that the molecular size and electrostatic potential around the benzene ring of the inhibitors influenced the inhibitory effects of the tyrosinase inhibitors (Sugimoto et al. 2005).

The results of our tyrosinase inhibition assay showed that β-d-glucosyl-(1→3)-arbutin evidenced inhibitory activity similar to that of α-d-glucosyl-(1→4)-arbutin, which harbors an α-glucosidic linkage, whereas the other two arbutin β-glycosides, β-d-glucosyl-(1→4)-arbutin and β-d-glucosyl-(1→6)-arbutin, evidenced weaker inhibitory activities. Therefore, we presume that the regioselectivity of the glycosidic linkage of arbutin glycosides is also involved in the inhibitory effects of the tyrosinase inhibitors.

A desirable skin-whitening agent would inhibit melanin synthesis in the melanosomes by specifically reducing the synthesis or activity of tyrosinase, and would also exhibit low cytotoxicity and be non-mutagenic. Melanoma cells have been previously used in order to assess the inhibition of melanin synthesis and cell toxicity. After arbutin and arbutin β-glycosides were administered to the cultured B16F10 melanoma cells for 2 days, the degree of inhibition of cultured B16F10 melanoma cell pigmentation as the result of melanin synthesis was assessed and compared to the cytotoxic effects. The inhibition of melanin synthesis is depicted in Fig. 3. The cellular content of melanin was reduced via the addition of arbutin β-glycosides to the medium in a dose-dependent manner, except in the case of β-d-glucosyl-(1→4)-arbutin. Among the four tested arbutin derivatives, β-d-glucosyl-(1→3)-arbutin evidenced the most profound inhibitory effects at concentrations in the range of 1–20 mM. Melanin synthesis was inhibited significantly at 5 mM, at which the melanin content was reduced to less than 70% that observed in the untreated cells. In an effort to exclude the possibility that the above inhibitory effect of β-d-glucosyl-(1→3)-arbutin on melanogenesis might arise from the inhibition of cell growth, we compared the number of cells grown in the presence of this compound. As is shown in Fig. 4, this compound at up to 20 mM did not appear to inhibit cell viability, whereas β-d-glucosyl-(1→6)-arbutin evidenced profound cytotoxicity above 5 mM. Combined with the results of mushroom tyrosinase inhibitory activity and the inhibition of melanin production in the melanoma cell line, β-d-glucosyl-(1→3)-arbutin was determined to be the most effective melanogenesis inhibitor assessed in this study.
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Fig. 3

Inhibitory effects of arbutin and arbutin derivatives on melanin synthesis in B16F10 melanoma cells. The results are expressed as the percentage of inhibition of (•) arbutin, (◯) β-d-glucopyranosyl-(1→6)-arbutin, (▼) β-d-glucopyranosyl-(1→4)-arbutin, (▽) β-d-glucopyranosyl-(1→3)-arbutin, or (■) α-d-glucopyranosyl-(1→4)-arbutin with regard to the untreated control. Each value is expressed as the mean ± standard error of experiments conducted in triplicate

https://static-content.springer.com/image/art%3A10.1007%2Fs10529-007-9605-1/MediaObjects/10529_2007_9605_Fig4_HTML.gif
Fig. 4

Cytotoxicity of arbutin and arbutin derivatives in B16F10 melanoma cells. Results are expressed as the percentage of the reduction of cell viability of (•) arbutin, (◯) β-d-glucopyranosyl-(1→6)-arbutin, (▼) β-d-glucopyranosyl-(1→4)-arbutin, (▽) β-d-glucopyranosyl-(1→3)-arbutin, or (■) α-d-glucopyranosyl-(1→4)-arbutin, with regard to the untreated control. Each value is expressed as the mean ± standard error of experiments conducted in triplicate

Although studies concerning tyrosinase inhibitory activity and the inhibition of melanin synthesis in melanoma cells using arbutin α-glycoside derivatives have been previously conducted (Sugimoto et al. 2004), no study of melanin synthesis inhibition in melanoma cells using arbutin β-glycosides has been performed in the past. Therefore, it is worthy of note that arbutin β-glycosides, along with arbutin α-glycosides, are also potent melanin synthesis inhibitors. The development of this enzyme, which can generate only β-d-glucosyl-(1→3)-arbutin via the enzymatic reaction, will be necessary in order to advance the possibility of the industrialization of this compound as the depigmenting agent. We recently succeeded in generating a mutant, which specifically generates β-d-glucosyl-(1→3)-arbutin via site-directed mutagenesis. The mutant increased the yield of this compound by 3-fold as compared to the wild-type enzyme. Investigations targeted toward the optimization of an enzyme reaction condition for the maximum yield of this compound are currently underway.

Acknowledgements

This study was supported, in part, by the Marine and Extreme Genome Research Center Program, Ministry of Marine Affairs and Fisheries, and by the New University for Regional Innovation, Ministry of Education and Human Resources Development, Republic of Korea.

Copyright information

© Springer Science+Business Media B.V. 2007