Alleviation effect of arbutin on oxidative stress generated through tyrosinase reaction with l-tyrosine and l-DOPA
© Tada et al.; licensee BioMed Central Ltd. 2014
Received: 12 August 2014
Accepted: 3 October 2014
Published: 9 October 2014
Hydroxyl radical that has the highest reactivity among reactive oxygen species (ROS) is generated through l-tyrosine-tyrosinase reaction. Thus, the melanogenesis might induce oxidative stress in the skin. A rbutin (p-hydroxyphenyl-β-d-glucopyranoside), a well-known tyrosinase inhibitor has been widely used for the purpose of skin whitening. The aim of the present study was to examine if arbutin could suppress the hydroxyl radical generation via tyrosinase reaction with its substrates, l-tyrosine and l-DOPA.
The hydroxyl radical, which was determined by an electron spin resonance-spin trapping technique, was generated by the addition of not only l-tyrosine but l-DOPA to tyrosinase in a concentration dependent manner. Arbutin could inhibit the hydroxyl radical generation in the both reactions.
It is presumed that arbutin could alleviate oxidative stress derived from the melanogenic pathway in the skin in addition to its function as a whitening agent in cosmetics.
Native human melanin consists of eumelanin and pheomelanin, and eumelanin is found in almost every type of human skin [1, 2]. In the skin, melanin synthesized in melanocytes, which are located in the basal layer and hair bulbs, transfers to keratinocytes. Melanin in keratinocytes acts as a photoprotector through body coloration and scavenging reactive oxygen species such as superoxide anion and singlet oxygen [3–8]. Despite the photoprotective role of melanin, many cosmetics have been developed to prevent melanin formation in the skin because of aesthetic satisfaction by whitening ability. Of these, inhibitor of tyrosinase, which is a pivotal enzyme for melanin synthesis , has been used as a major ingredient of cosmetics [10–14]. Tyrosinase, an enzyme which contains dinuclear copper ions at the active site [15–17], catalyzes two distinct reactions of melanin synthesis , the hydroxylation of a monophenol and the conversion of an o-diphenol to the corresponding o-quinone, indicating that l-tyrosine is hydroxylated to l-DOPA, which is in turn converted to dopaquinone. Our previous study applying electron spin resonance (ESR)-spin trapping method revealed that hydroxyl radicals are generated through L-tyrosine-tyrosinase reaction , so that we assumed that dicopper-peroxide intermediates formed during the catalytic process of l-tyrosine to dopaquinone possibly decay to produce the hydroxyl radical through an internal electron transfer from the ligand. This suggests that tyrosinase inhibitors might contribute to alleviate the oxidative damage of the skin by inhibiting hydroxyl radical generation via the enzyme reaction.
A rbutin (p-hydroxyphenyl-β-d-glucopyranoside), a well-known tyrosinase inhibitor, which can be extracted from plants, has been widely used for the purpose of skin whitening . Regarding the molecular base mechanisms of arbutin, it was reported that arbutin inhibits not only the oxidation of l-DOPA but the hydroxylation of l-tyrosine [21, 22]. With regard to skin-whitening effect of arbutin in relation to melanogenesis, it was reported that arbutin showed no effect on the differentiation of melanocytes while hydroquinone used as a skin-whitening agent downregulated the differentiation . Besides tyrosinase inhibition, it was reported that arbutin has anti-inflammatory effect , it is expected that arbutin could alleviate inflammation in the skin exposed to ultraviolet (UV) light.
The purpose of the present study was to examine if a tyrosinase inhibitor could suppress the hydroxyl radical generation via tyrosinase reaction with its substrates, l-tyrosine and l-DOPA. In the study, arbutin was used as a representative tyrosinase inhibitor.
Results and discussion
The hydroxyl radical generation via tyrosinase reaction with either L-tyrosine or L-DOPA was reduced by a tyrosinase inhibitor, arbutin. Thus, it is expected that tyrosinase inhibitors such as arbutin could alleviate oxidative stress derived from the melanogenic pathway in the skin in addition to its function as a whitening agent in cosmetics.
Test materials and reagents
Reagents were purchased from the following sources: l-Tyrosine, l-DOPA and phosphate buffer solution (PB, pH 6.5) from Wako Pure Chemicals (Osaka, Japan); tyrosinase (from mushroom) from Sigma-Aldrich Corp. (St. Louis, MO); DMPO from Labotec (Tokyo, Japan); arbutin from LKT Laboratories, Inc. (St. Paul, MN). All other reagents used were of analytical grade.
ESR-spin trapping determinations of hydroxyl radicals generated by tyrosinase reaction with l-tyrosine and l-DOPA
Tyrosinase was dissolved in PB to be 100 U/ml. l-Tyrosine was dissolved in PB to be 200 mM. Then 1 mM l-tyrosine solution was prepared by mixing 5 μl of 200 mM l-tyrosine solution with 5 μl of 1 M NaOH and 990 μl of 0.2 M PB. l-DOPA was also dissolved in 1 M HCl to be 200 mM. Then 1 mM l-DOPA solution was prepared by mixing 5 μl of 200 mM l-DOPA solution with 5 μl of 1 M NaOH and 990 μl of 0.2 M PB. Formulated concentrate of DMPO (8.9 M) was used. The reaction mixture was prepared to contain different volume of substrate (1 mM l-tyrosine or 1 mM l-DOPA), 10 μl of 8.9 M DMPO, 4 μl of 100 U/μl tyrosinase and 0.2 M PB which was added to adjust a total volume of 200 μl. Immediately after mixing the mixture was transferred to an ESR spectrometry cell, and the ESR measurement was started after 45 s. The measurement conditions of ESR (JES-FA-100, JEOL, Tokyo, Japan) were as follows: field sweep, 330.80-340.80 mT; field modulation frequency, 100 kHz; filed modulation width, 0.07 mT; amplitude, 400; sweep time, 1 min; time constant, 0.1 s; microwave frequency, 9.430 GHz; microwave power, 5 mW. In the study where the effect of arbutin on the hydroxyl radical generation was examined, 100 mM arbutin dissolved in ultrapure water was diluted 10 times with 0.2 M PB. The reaction mixture was prepared to contain different volume of 10 mM arbutin, 60 μl of substrate (1 mM l-tyrosine or 1 mM l-DOPA), 10 μl of 8.9 M DMPO, 4 μl of 100 U/μl tyrosinase and 0.2 M PB which was added to adjust a total volume of 200 μl. The concentrations of arbutin used in the study were decided by the enzyme assay for tyrosinase where dopachrome formation was monitored at 475 nm. Arbutin at a concentration of 1.5 mM or more clearly inhibited dopachrome formation (data not shown). To further examine if arbutin has an ability to scavenge directly hydroxyl radicals, effect of arbutin on the hydroxyl radical generated by a Fenton reaction. The reaction mixture was prepared to contain 3 mM arbutin, 0.5 mM H2O2, 0.05 mM FeSO4, and 1.78 mM or 445 mM DMPO, and was subjected to ESR analysis.
In the experiments where the effect of arbutin was examined, statistical significances (p < 0.05) in the yield of DMPO-OH were assessed by Dunnett’s multiple comparison test.
This research was supported by the Ministry of Education, Science, Sports and Culture, Japan, Grant-in-Aid for Exploratory Research, 24655159, 2012, and Strategic Research Foundation Grant-in-Aid for Private Universities, S1312001, 2013.
- Hunt G, Kyne S, Ito S, Wakamatsu K, Todd C, Thody A: Eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes. Pigment Cell Res. 1995, 8 (4): 202-208. 10.1111/j.1600-0749.1995.tb00664.x.PubMedView ArticleGoogle Scholar
- Vincensi MR, d’Ischia M, Napolitano A, Procaccini EM, Riccio G, Monfrecola G, Santoianni P, Prota G: Phaeomelanin versus eumelanin as a chemical indicator of ultraviolet sensitivity in fair-skinned subjects at high risk for melanoma: a pilot study. Melanoma Res. 1998, 8 (1): 53-58. 10.1097/00008390-199802000-00009.PubMedView ArticleGoogle Scholar
- Kvam E, Dahle J: Pigmented melanocytes are protected against ultraviolet-A-induced membrane damage. J Invest Dermatol. 2003, 121 (3): 564-569. 10.1046/j.1523-1747.2003.12418.x.PubMedView ArticleGoogle Scholar
- Yamazaki F, Okamoto H, Miyauchi-Hashimoto H, Matsumura Y, Itoh T, Tanaka K, Kunisada T, Horio T: XPA gene-deficient, SCF-transgenic mice with epidermal melanin are resistant to UV-induced carcinogenesis. J Invest Dermatol. 2004, 123 (1): 220-228. 10.1111/j.0022-202X.2004.22710.x.PubMedView ArticleGoogle Scholar
- Wagner JK, Parra EJ, Norton LH, Jovel C, Shriver MD: Skin responses to ultraviolet radiation: effects of constitutive pigmentation, sex, and ancestry. Pigment Cell Res. 2002, 15 (5): 385-390. 10.1034/j.1600-0749.2002.02046.x.PubMedView ArticleGoogle Scholar
- Ortonne JP: Photoprotective properties of skin melanin. Br J Dermatol. 2002, 146 (Suppl 61): 7-10.PubMedView ArticleGoogle Scholar
- Yamaguchi Y, Takahashi K, Zmudzka BZ, Kornhauser A, Miller SA, Tadokoro T, Berens W, Beer JZ, Hearing VJ: Human skin responses to UV radiation: pigment in the upper epidermis protects against DNA damage in the lower epidermis and facilitates apoptosis. FASEB J. 2006, 20 (9): 1486-1488. 10.1096/fj.06-5725fje.PubMedView ArticleGoogle Scholar
- Tada M, Kohno M, Niwano Y: Scavenging or quenching effect of melanin on superoxide anion and singlet oxygen. J Clin Biochem Nutr. 2010, 46 (3): 224-228. 10.3164/jcbn.09-84.PubMedPubMed CentralView ArticleGoogle Scholar
- Ito S, Wakamatsu K: Chemistry of mixed melanogenesis–pivotal roles of dopaquinone. Photochem Photobiol. 2008, 84 (3): 582-592. 10.1111/j.1751-1097.2007.00238.x.PubMedView ArticleGoogle Scholar
- Khazaeli P, Goldoozian R, Sharififar F: An evaluation of extracts of five traditional medicinal plants from Iran on the inhibition of mushroom tyrosinase activity and scavenging of free radicals. Int J Cosmet Sci. 2009, 31 (5): 375-381. 10.1111/j.1468-2494.2009.00503.x.PubMedView ArticleGoogle Scholar
- Momtaz S, Mapunya BM, Houghton PJ, Edgerly C, Hussein A, Naidoo S, Lall N: Tyrosinase inhibition by extracts and constituents of Sideroxylon inerme L. stem bark, used in South Africa for skin lightening. J Ethnopharmacol. 2008, 119 (3): 507-512. 10.1016/j.jep.2008.06.006.PubMedView ArticleGoogle Scholar
- Ng LT, Ko HH, Lu TM: Potential antioxidants and tyrosinase inhibitors from synthetic polyphenolic deoxybenzoins. Bioorg Med Chem. 2009, 17 (13): 4360-4366. 10.1016/j.bmc.2009.05.019.PubMedView ArticleGoogle Scholar
- Nugroho A, Choi JK, Park JH, Lee KT, Cha BC, Park HJ: Two new flavonol glycosides from Lamium amplexicaule L. and their in vitro free radical scavenging and tyrosinase inhibitory activities. Planta Med. 2009, 75 (4): 364-366. 10.1055/s-0028-1112216.PubMedView ArticleGoogle Scholar
- Wang KH, Lin RD, Hsu FL, Huang YH, Chang HC, Huang CY, Lee MH: Cosmetic applications of selected traditional Chinese herbal medicines. J Ethnopharmacol. 2006, 106 (3): 353-359. 10.1016/j.jep.2006.01.010.PubMedView ArticleGoogle Scholar
- Solomon EI, Chen P, Metz M, Lee SK, Palmer AE: Oxygen binding, activation, and reduction to water by copper proteins. Angew Chem Int Ed Engl. 2001, 40 (24): 4570-4590. 10.1002/1521-3773(20011217)40:24<4570::AID-ANIE4570>3.0.CO;2-4.PubMedView ArticleGoogle Scholar
- Chen P, Solomon EI: O2 activation by binuclear Cu sites: noncoupled versus exchange coupled reaction mechanisms. Proc Natl Acad Sci U S A. 2004, 101 (36): 13105-13110. 10.1073/pnas.0402114101.PubMedPubMed CentralView ArticleGoogle Scholar
- Inoue T, Shiota Y, Yoshizawa K: Quantum chemical approach to the mechanism for the biological conversion of tyrosine to dopaquinone. J Am Chem Soc. 2008, 130 (50): 16890-16897. 10.1021/ja802618s.PubMedView ArticleGoogle Scholar
- Rob D: Tyrosinase. Copper Proteins and Copper Enzymes. Edited by: Lontie R. 1984, Florida: CRC Press, 207-240. IIGoogle Scholar
- Tada M, Kohno M, Kasai S, Niwano Y: Generation mechanism of radical species by tyrosine-tyrosinase reaction. J Clin Biochem Nutr. 2010, 47 (2): 162-166. 10.3164/jcbn.10-48.PubMedPubMed CentralView ArticleGoogle Scholar
- Maeda K, Fukuda M: Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther. 1996, 276 (2): 765-769.PubMedGoogle Scholar
- Funayama M, Arakawa H, Yamamoto R, Nishino T, Shin T, Murao S: Effects of alpha- and beta-arbutin on activity of tyrosinases from mushroom and mouse melanoma. Biosci Biotechnol Biochem. 1995, 59 (1): 143-144. 10.1271/bbb.59.143.PubMedView ArticleGoogle Scholar
- Hori I, Nihei K, Kubo I: Structural criteria for depigmenting mechanism of arbutin. Phytother Res. 2004, 18 (6): 475-479. 10.1002/ptr.1456.PubMedView ArticleGoogle Scholar
- Inoue Y, Hasegawa S, Yamada T, Date Y, Mizutani H, Nakata S, Matsunaga K, Akamatsu H: Analysis of the effects of hydroquinone and arbutin on the differentiation of melanocytes. Biol Pharm Bull. 2013, 36 (11): 1722-1730. 10.1248/bpb.b13-00206.PubMedView ArticleGoogle Scholar
- Lee HJ, Kim KW: Anti-inflammatory effects of arbutin in lipopolysaccharide-stimulated BV2 microglial cells. Inflamm Res. 2012, 61 (8): 817-825. 10.1007/s00011-012-0474-2.PubMedView ArticleGoogle Scholar
- Hisatomi E, Matsui M, Kubota K, Kobayashi A: Antioxidative activity in the pericarp and seed of Japanese pepper (Xanthoxylum piperitum DC). J Agric Food Chem. 2000, 48 (10): 4924-4928. 10.1021/jf000252j.PubMedView ArticleGoogle Scholar
- Bang SH, Han SJ, Kim DH: Hydrolysis of arbutin to hydroquinone by human skin bacteria and its effect on antioxidant activity. J Cosmet Dermatol. 2008, 7 (3): 189-193. 10.1111/j.1473-2165.2008.00387.x.PubMedView ArticleGoogle Scholar
- Baier J, Maisch T, Maier M, Landthaler M, Baumler W: Direct detection of singlet oxygen generated by UVA irradiation in human cells and skin. J Invest Dermatol. 2007, 127 (6): 1498-1506. 10.1038/sj.jid.5700741.PubMedView ArticleGoogle Scholar
- Pellosi MC, Suzukawa AA, Scalfo AC, Di Mascio P, Martins Pereira CP, de Souza Pinto NC, de Luna MD, Martinez GR: Effects of the melanin precursor 5,6-dihydroxy-indole-2-carboxylic acid (DHICA) on DNA damage and repair in the presence of reactive oxygen species. Arch Biochem Biophys. 2014, 557: 55-64.PubMedView ArticleGoogle Scholar
- Halliwell B, Gutteridge JM: Oxygen free radicals and iron in relation to biology and medicine: some problems and concepts. Arch Biochem Biophys. 1986, 246 (2): 501-514. 10.1016/0003-9861(86)90305-X.PubMedView ArticleGoogle Scholar
- Dhumrongvaraporn A, Chanvorachote P: Kinetics of ultraviolet B irradiation-mediated reactive oxygen species generation in human keratinocytes. J Cosmet Sci. 2013, 64 (3): 207-217.PubMedGoogle Scholar
- Nicolay JF, Levrat B: A keratinocytes-melanocytes coculture system for the evaluation of active ingredients’ effects on UV-induced melanogenesis. Int J Cosmet Sci. 2003, 25 (1–2): 15-19.PubMedView ArticleGoogle Scholar
- Zi SX, Ma HJ, Li Y, Liu W, Yang QQ, Zhao G, Lian S: Oligomeric proanthocyanidins from grape seeds effectively inhibit ultraviolet-induced melanogenesis of human melanocytes in vitro. Int J Mol Med. 2009, 23 (2): 197-204.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.