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.
KeywordsHydroxyl radical Tyrosinase reaction Arbutin
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.
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