Original Contribution

O R I GInc INAL CONTRIBUTION Blackwell Publishing

Hydrolysis of arbutin to hydroquinone by human skin bacteria and its effect on antioxidant activity
Seo-Hyun Bang, MS, Sang-Jun Han, PhD, & Dong-Hyun Kim, PhD
Department of Life and Nanopharmaceutical Sciences and College of Pharmacy, Kyung Hee University, Seoul, South Korea

Summary

Arbutin, the -d-glucopyranoside of hydroquinone, is a skin whitening cosmetic ingredient. Compared with arbutin, hydroquinone is a more potent skin lightening agent, but shows cytotoxicity, nephrotoxicity, and genotoxicity. To evaluate whether skin microora can hydrolyze arbutin to hydroquinone, we measured the hydrolytic activity of the main skin microora: Staphylococcus epidermidis and Staphylococcus aureus. All strains hydrolyzed arbutin, with activities of 0.164.51 nmol/min/mg. The hydrolyzed hydroquinone showed more potent 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and tyrosinase inhibition than arbutin. These ndings suggest that normal skin microora may increase the skin lightening effect of arbutin due to the antioxidant action of hydroquinone. Keywords: arbutin, cosmetic, hydroquinone, skin microora, Staphylococcus epidermidis

Introduction
Arbutin, a plant-derived -d-glucopyranoside of hydroquinone, is an ingredient in skin care products1,2 for treating cutaneous hyperpigmentation and works by inhibiting melanogenesis in melanoma cells.3,4 Normal microora reside in human skin and intestines,5 with Staphylococcus epidermidis and Staphylococcus aureus common in the skin.68 Bacterial growth is dependent on host immunity and skin status, such as sweat, and skin care products designed for moisturizing may stimulate the growth of skin microora,9 which may in turn metabolize skin care ingredients such as arbutin. However, there are no studies on the arbutin-hydrolytic activity of normal skin microora. We therefore analyzed the ability of the normal microora, S. epidermidis and S. aureus, to hydrolyze arbutin and change its bioactivity.
Correspondence: Professor Dong-Hyun Kim, College of Pharmacy, Kyung Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, South Korea. Email: dhkim@khu.ac.kr Accepted for publication February 18, 2008

Materials and methods

Materials

Arbutin, hydroquinone, and p-nitrophenyl--dglucopyranoside were purchased from Sigma Co. (St Louis, MO, USA). Staphylococcus epidermidis and S. aureus were obtained from Kyung-Hee Medical Center. Twenty-six microbes (13 S. aureus and 13 S. epidermidis) were clinically isolated from 20 healthy women according to the method of Jeong et al.10 The isolated Staphylococcus species were identied according to whether they produced coagulase and deoxyribonuclease and used mannitol salt agar test, but S. epidermis was determined to be negative using MicroScan Walk-Away 96 (Dade Behring, Marburg, Germany). Escherichia coli HGU-3, Lactobacillus acidophilus, Bidobacterium longum H-1, and Bacterioides stercoris HJ-15 were isolated from human feces according to our reported method.11,12

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Hydrolysis of arbutin to hydroquinone by human skin bacteria S-H Bang et al.

Figure 1 Metabolic pathway of arbutin to hydroquinone by skin microora. (A) The reaction of arbutin metabolism by Staphylococcus epidermidis isolated from normal skin ora. (B) High-performance liquid chromatography (HPLC) chromatogram of the reaction mixture of arbutin and S. epidermidis: (a) arbutin standard; (b) hydroquinone standard; (c) 10 min before incubation; (d) 30 min after incubation; (e) 1 h after incubation of arbutin with S. epidermidis; (f ) 1 h after incubation of arbutin alone without S. epidermidis. The HPLC system consisted of a Younglin SP930D quaternary pump and a Younglin UV730D detector: column, Develosil C30-UG-5 (250 4.6 mm i.d., 5 m, 100 ; Nomura Chemical); elution solvent, 10% methanol; ow rate, 1.0 mL/min; detection, 287 nm; and sample volume, 15 L.

Arbutin-hydrolyzing activity assay

Cultured bacteria were collected and suspended in 50 mm phosphate buffer (pH 7.0). The reaction mixture containing 0.5 mL of 1 mm arbutin, 0.5 mL of bacterial suspension (50 mg), and 1 mL of 0.1 m phosphate buffer (pH 7.0) was incubated for 5 h at 37 C. Saline was used as the blank instead of bacterial suspension. The reaction mixture was extracted with ethyl acetate and evaporated. The extracted compounds dissolved in methanol were analyzed by a high-performance liquid chromatography system consisting of a Younglin SP930D quaternary pump and a Younglin UV730D detector. The instrument was controlled, and the data were processed by a Younglin AutoChro-3000 (1.0 build no. 5). The analytical column was a Develosil C30-UG-5 (250 4.6 mm

inter-diameter (i.d.), 5 m, 100 ; Nomura Chemical, Seto, Japan) protected by a C18 Security Guard Cartridge (Phenomenex, Torrance, CA, USA). The elution solvent was 10% methanol. Separations were performed with an isocratic over a period of 15 min at a ow rate of 1.0 mL/min and detection at 287 nm. A sample volume of 15 L was used for injection. The retention times of arbutin and its metabolite, hydroquinone, were 6.32 and 8.95 min, respectively.
-Glucosidase activity assay

We measured the -glucosidase activity for a synthetic -d-glycoside, p-nitrophenyl--d-glucopyranoside. The reaction mixture containing 200 L of 1 mm p-nitrophenyl-d-glucopyranoside, 400 L of 50 mm phosphate buffer,

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Hydrolysis of arbutin to hydroquinone by human skin bacteria S-H Bang et al.

Figure 2 Arbutin- (a) and p-nitrophenyl--d-glucopyranoside-hydrolyzing activities (b) of bacteria isolated from skin and intestinal microora. These activities were assayed for 13 Staphylococcus aureus and 13 Staphylococcus epidermidis isolates from human skin; S. aureus and S. epidermis purchased from KCTC, Escherichia coli, Bacterioides stercoris HJ-15, and Bidobacterium longum H-1 isolated from human feces. Activities of isolated strains indicate mean standard deviation.

and 200 L of the cultured microbial suspension (or saline as blank) was incubated for 15 min at 37 C in a shaking water bath. The reaction was stopped by adding 1.0 mL of 0.5 N sodium hydroxide, centrifuged at 3000 g for 10 min, and the absorbance at 405 nm was measured.
Tyrosinase activity assay

scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) according to the method of Xiong et al.14 DPPH in ethanol (0.5 mL) was added to a 75% ethanolic sample solution (0.5 mL), incubated at 37 C for 15 min, and then the absorbance at 530 nm was measured. Caffeic acid was used as a positive agent.

To evaluate skin lightening effect, the inhibitory effects of arbutin and its metabolite hydroquinone against the formation of hyperpigmented product, dopachrome, by tyrosinase was measured. The activity assay of tyrosinase was performed according to the modied method of Curto et al.13 The reaction mixture containing 0.1 mL of test agents and 0.8 mL of l-DOPA (0.05 mg) in 0.05 m phosphate buffer (pH 7.0) was preincubated at 25 C for 10 min, and then 0.1 mL tyrosinase solution (10 units/ mL) in phosphate buffer (pH 7.0) was added and incubated at 25 C for 15 min. Kojic acid was used as a positive agent. The amount of dopachrome produced in the reaction mixture was measured at an absorbance of 595 nm.
Antioxidant activity assay

Results
We evaluated the ability of normal skin bacteria, S. epidermidis and S. aureus, to hydrolyze arbutin, a cosmetic ingredient, to hydroquinone (Fig. 1). These isolates potently hydrolyzed arbutin to hydroquinone, with average values of 0.164.51 nmol/min/mg (Fig. 2), which were similar levels as intestinal bacteria such as B. stercoris and B. longum. These intestinal bacteria potently hydrolyzed arbutin to hydroquinone as previously reported.15 The average activities of S. epidermis and S. aureus were 1.38 0.40 (mean standard deviation) and 1.38 0.29 nmol/min/mg, respectively. The skin isolates also hydrolyzed p-nitrophenyl--d-glucopyranoside, but with lower potency than arbutin. The average activities of S. epidermis and S. aureus were 0.23 0.04 and 0.23 0.06 nmol/min/mg, respectively. We next investigated the free radicalscavenging activity, tyrosinase inhibition, and cytotoxicity of arbutin and hydroquinone (Fig. 3). Hydroquinone showed more

We measured the antioxidant activity of arbutin and its metabolite, hydroquinone, by assessing their radical

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Hydrolysis of arbutin to hydroquinone by human skin bacteria S-H Bang et al.

Figure 3 Tyrosinase inhibition (a) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of arbutin and hydroquinone (b). (a) The inhibitory effect of arbutin () and hydroquinone () and kojic acid () against tyrosinase was measured as described in the Material and Methods (b) The antioxidant effect of arbutin (), hydroquinone () and caffeic acid () measured by using DPPH dissolved in ethanol. These experiments are triplicates.

potent DPPH radicalscavenging activity and tyrosinase inhibition than arbutin, which also has these activities.16 Their 50% radicalscavenging concentrations were 0.02 and 0.05 mm, and their 50% tyrosine-inhibitory concentrations were 0.06 and 1.6 mm, respectively.

skin microora, and that skin lightening may be due to arbutin itself as well as its metabolite hydroquinone.

References
1 ODonoghue JL. Hydroquinone and its analogues in dermatology a risk-benet viewpoint. J Cosmet Dermatol 2006; 5: 196 203. 2 Wen AH, Choi MK, Kim DD. Formulation of liposome for topical delivery of arbutin. Arch Pharm Res 2006; 29: 1187 92. 3 Nishimura T, Kometani T, Okada S, Ueno N, Yamamoto T. [Inhibitory effects of hydroquinone-alpha-glucoside on melanin synthesis]. Yakugaku Zassh 1995; 115: 62632 (in Japanese). 4 Gallarate M, Carlotti ME, Trotta M, Grande AE, Talarico C. Photostability of naturally occurring whitening agents in cosmetic microemulsions. J Cosmet Sci 2004; 55: 139 48. 5 Sullivan A, Edlund C, Nord CE. Effect of antimicrobial agents on the ecological balance of human microora. Lancet Infect Dis 2001; 1: 10114. 6 OGara JP, Humphreys H. Staphylococcus epidermidis biolms: importance and implications. J Med Microbiol 2001; 50: 5827. 7 Keyworth N, Millar MR, Holland KT. Development of cutaneous microora in premature neonates. Arch Dis Child 1992; 67: 797801. 8 Kozitskaya S, Olson ME, Fey PD, Witte W, Ohlsen K, Ziebuhr W. Clonal analysis of Staphylococcus epidermidis isolates carrying or lacking biolm-mediating genes by multilocus sequence typing. J Clin Microbiol 2005; 43: 47517. 9 Akiyama H, Yamasaki O, Tada J, Arata J. Adherence characteristics and susceptibility to antimicrobial agents of Staphylococcus aureus strains isolated from skin infections and atopic dermatitis. J Dermatol Sci 2000; 23: 15560.

Discussion
Arbutin and hydroquinone are plant-derived ingredients in hair dye, ngernail coatings, and skin lightening cosmetics.16 Hydroquinone is a skin lightening agent, but shows cytotoxicity, nephrotoxicity, and genotoxicity.1618 Arbutin, or hydroquinone--d-glucopyranoside, is a herbal medicine that is metabolized to hydroquinone by human intestinal microora.15,18 The effects of arbutin may be due to the hydrolyzed hydroquinone, but it is unclear whether similar metabolism occurs in normal skin microora. Two skin isolates, S. epidermidis and S. aureus, potently hydrolyzed arbutin to hydroquinone at similar levels (0.164.51 nmol/min/mg) as intestinal bacteria. When the hydrolytic effect of arbutin was compared with that of p-nitrophenyl--d-glucopyranoside, the skin isolates also hydrolyzed p-nitrophenyl--dglucopyranoside, but with lower potency than arbutin. Bacteria density on skin is between 104 and 107 per cm2,19 but can vary by skin conditions such as moisture, sweat, nutrition, and body temperature. The metabolite hydroquinone showed more potent DPPH radicalscavenging activity and tyrosinase inhibition effects than arbutin.20 Hydroquinone also exhibits an antimelanogenic effect, such as skin lightening, as well as toxicity.59 These ndings suggest that arbutin may be partially hydrolyzed to hydroquinone by normal

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10 Jeong J, Chang CL, Park TS, Lee SH, Kim SR, Jeong SH. Early screening of oxacillin-resistant Staphylococcus aureus and Staphylococcus epidermidis from blood culture. J Korean Med Sci 2002; 17: 16872. 11 Bae EA, Han MJ, Kim EJ, Kim DH. Transformation of ginseng saponins to ginsenoside Rh2 by acids and human intestinal bacteria and biological activities of their transformants. Arch Pharm Res 2004; 27: 617. 12 Kim DH, Hong SW, Kim BT, Bae EA, Park HY, Han MJ. Biotransformation of glycyrrhizin by human intestinal bacteria and its relation to biological activities. Arch Pharm Res 2000; 23: 172277. 13 Curto EV, Kwong C, Hermersdrfer H, et al. Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem Pharmacol 1999; 57: 66372. 14 Xiong Q, Kadota S, Tani T, Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 1996; 19: 15805.

15 Blaut M, Braune A, Wunderlich S, Sauer P, Schneider H, Glatt H. Mutagenicity of arbutin in mammalian cells after activation by human intestinal bacteria. Food Chem Toxicol 2006; 44: 1940 7. 16 Westerhof W, Kooyers TJ. Hydroquinone and its analogues in dermatology a potential health risk. J Cosmet Dermatol 2005; 4: 559. 17 Maeda K, Fukuda M. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther 1996; 276: 7659. 18 Gaskell M, McLuckie KI, Farmer PB. Genotoxicity of the benzene metabolites para-benzoquinone and hydroquinone. Chem Biol Interact 2005; 153154: 26770. 19 Selwyn S, Ellis H. Skin bacteria and skin disinfection reconsidered. Br Med J 1972; 1: 13640. 20 McDonald TA, Holland NT, Skibola C, Duramad P, Smith MT. Hypothesis: phenol and hydroquinone derived mainly from diet and gastrointestinal ora activity are causal factors in leukemia. Leukemia 2001; 15: 1020.

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