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2008 Wiley Periodicals, Inc. Journal of Cosmetic Dermatology, 7, 189193 189

Original Contribution

BlackwellPublishingIncORIGINAL CONTRIBUTION

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 ingre-dient. Compared with arbutin, hydroquinone is a more potent skin lightening agent,but shows cytotoxicity, nephrotoxicity, and genotoxicity. To evaluate whether skinmicroflora can hydrolyze arbutin to hydroquinone, we measured the hydrolytic activity

of the main skin microflora: Staphylococcus epidermidis

and Staphylococcus aureus

. Allstrains hydrolyzed arbutin, with activities of 0.164.51 nmol/min/mg. The hydrolyzedhydroquinone showed more potent 1,1-diphenyl-2-picrylhydrazyl radical scavengingactivity and tyrosinase inhibition than arbutin. These findings suggest that normal skinmicroflora may increase the skin lightening effect of arbutin due to the antioxidantaction of hydroquinone.

Keywords

:

arbutin, cosmetic, hydroquinone, skin microflora, Staphylococcus epidermidis

Introduction

Arbutin, a plant-derived

-

d

-glucopyranoside of hydroquin-one, is an ingredient in skin care products

1,2

for treatingcutaneous hyperpigmentation and works by inhibitingmelanogenesis in melanoma cells.

3,4

Normal microflorareside in human skin and intestines,

5

with Staphylococcusepidermidis

and Staphylococcus aureus

common in theskin.

68

Bacterial growth is dependent on host immunityand skin status, such as sweat, and skin care productsdesigned for moisturizing may stimulate the growth of skinmicroflora,

9

which may in turn metabolize skin careingredients such as arbutin. However, there are no studies

on the arbutin-hydrolytic activity of normal skin microflora.We therefore analyzed the ability of the normal micro-

flora, S. epidermidis

and S. aureus

, to hydrolyze arbutinand change its bioactivity.

Materials and methods

Materials

Arbutin, hydroquinone, and p

-nitrophenyl-

-

d

-glucopyranoside were purchased from Sigma Co. (StLouis, MO, USA).

Staphylococcus epidermidis

and S. aureus

were obtainedfrom Kyung-Hee Medical Center. Twenty-six microbes(13 S. aureus

and 13 S. epidermidis

) were clinically iso-lated from 20 healthy women according to the method ofJeong et al

.

10

The isolated Staphylococcus

species wereidentified according to whether they produced coagulase

and deoxyribonuclease and used mannitol salt agar test,but S. epidermis

was determined to be negative usingMicroScan Walk-Away 96 (Dade Behring, Marburg,Germany).

Escherichia coli

HGU-3, Lactobacillus acidophilus

, Bifido-bacterium longum

H-1, and Bacterioides stercoris

HJ-15were isolated from human feces according to ourreported method.

11,12

Correspondence: Professor Dong-Hyun Kim, College of Pharmacy, Kyung

Hee University, 1, Hoegi, Dongdaemun-ku, Seoul 130-701, South Korea. E-

mail: [email protected]

Accepted for publication February 18, 2008

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Hydrolysis of arbutin to hydroquinone by human skin bacteria

S-H Bang

et al.

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2008 Wiley Periodicals, Inc.

Journal of Cosmetic Dermatology

, 7

, 189193

Arbutin-hydrolyzing activity assay

Cultured bacteria were collected and suspended in50 m

m

phosphate buffer (pH 7.0). The reaction mixturecontaining 0.5 mL of 1 m

m

arbutin, 0.5 mL of bacterialsuspension (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 reactionmixture was extracted with ethyl acetate and evaporated.The extracted compounds dissolved in methanol wereanalyzed by a high-performance liquid chromato-graphy system consisting of a Younglin SP930Dquaternary pump and a Younglin UV730D detector. Theinstrument was controlled, and the data were processedby a Younglin AutoChro-3000 (1.0 build no. 5). Theanalytical 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 solventwas 10% methanol. Separations were performed withan isocratic over a period of 15 min at a flow rate of1.0 mL/min and detection at 287 nm. A sample volumeof 15

L was used for injection. The retention times of

arbutin and its metabolite, hydroquinone, were 6.32 and8.95 min, respectively.

-Glucosidase activity assay

We measured the

-glucosidase activity for a synthetic

-

d

-glycoside, p

-nitrophenyl-

-

d

-glucopyranoside. Thereaction mixture containing 200

L of 1 m

m

p

-nitrophenyl-

-

d

-glucopyranoside, 400

L of 50 m

m

phosphate buffer,

Figure 1 Metabolic pathway of arbutin tohydroquinone by skin microflora. (A) Thereaction of arbutin metabolism byStaphylococcus epidermidisisolated fromnormal skin flora. (B) High-performanceliquid chromatography (HPLC)chromatogram of the reaction mixtureof arbutin and S. epidermidis: (a) arbutinstandard; (b) hydroquinone standard;(c) 10 min before incubation; (d) 30 minafter incubation; (e) 1 h after incubationof arbutin with S. epidermidis; (f ) 1 h afterincubation of arbutin alone withoutS. epidermidis. The HPLC system consistedof a Younglin SP930D quaternary pumpand a Younglin UV730D detector: column,

Develosil C30-UG-5 (250 4.6 mm i.d.,5 m, 100 ; Nomura Chemical); elutionsolvent, 10% methanol; flow rate,1.0 mL/min; detection, 287 nm; andsample volume, 15 L.

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and 200

L of the cultured microbial suspension (orsaline as blank) was incubated for 15 min at 37

C in ashaking water bath. The reaction was stopped by adding1.0 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

To evaluate skin lightening effect, the inhibitory effectsof arbutin and its metabolite hydroquinone against theformation of hyperpigmented product, dopachrome, bytyrosinase was measured. The activity assay of tyrosinasewas performed according to the modified method of Curto

et

al

.

13

The reaction mixture containing 0.1 mL of testagents and 0.8 mL of l

-DOPA (0.05 mg) in 0.05 m

phosphate buffer (pH 7.0) was preincubated at 25

C for10 min, and then 0.1 mL tyrosinase solution (10 units/mL) in phosphate buf fer (pH 7.0) was added and

incubated at 25

C for 15 min. Kojic acid was used as apositive agent. The amount of dopachrome produced inthe reaction mixture was measured at an absorbance of595 nm.

Antioxidant activity assay

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

scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH)according to the method of Xiong et

al

.

14

DPPH inethanol (0.5 mL) was added to a 75% ethanolic samplesolution (0.5 mL), incubated at 37

C for 15 min, andthen the absorbance at 530 nm was measured. Caffeicacid was used as a positive agent.

Results

We evaluated the ability of normal skin bacteria,

S. epidermidis

and

S. aureus

, to hydrolyze arbutin, acosmetic ingredient, to hydroquinone (Fig. 1). Theseisolates 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 bacteriapotently hydrolyzed arbutin to hydroquinone as previouslyreported.

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 skinisolates also hydrolyzedp

-nitrophenyl-

-

d

-glucopyranoside,but with lower potency than arbutin. The averageactivities of S. epidermis

and S. aureus

were 0.23 0.04and 0.23 0.06 nmol/min/mg, respectively.

We next investigated the free radicalscavengingactivity, tyrosinase inhibition, and cytotoxicity of arbutinand hydroquinone (Fig. 3). Hydroquinone showed more

Figure 2 Arbutin- (a) andp-nitrophenyl--d-glucopyranoside-hydrolyzing activities (b) of bacteria isolated from skin and intestinalmicroflora. These activities were assayed for 13 Staphylococcus aureus and 13 Staphylococcus epidermidisisolates from human skin;S. aureus andS. epidermispurchased from KCTC,Escherichia coli, Bacterioides stercoris HJ-15, andBifidobacterium longum H-1 isolated fromhuman feces. Activities of isolated strains indicate mean standard deviation.

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potent DPPH radicalscavenging activity and tyrosinaseinhibition than arbutin, which also has these activities.

16

Their 50% radicalscavenging concentrations were 0.02and 0.05 m

m

, and their 50% tyrosine-inhibitory concen-trations were 0.06 and 1.6 m

m

, respectively.

Discussion

Arbutin and hydroquinone are plant-derived ingredientsin hair dye, fingernail coatings, and skin lighteningcosmetics.

16

Hydroquinone is a skin lightening agent, butshows cytotoxicity, nephrotoxicity, and genotoxicity.

1618

Arbutin, or hydroquinone-

-d-glucopyranoside, is aherbal medicine that is metabolized to hydroquinone byhuman intestinal microflora.15,18 The effects of arbutinmay be due to the hydrolyzed hydroquinone, but it isunclear whether similar metabolism occurs in normalskin microflora. Two skin isolates, S. epidermidis andS. aureus, potently hydrolyzed arbutin to hydroquinone atsimilar levels (0.164.51 nmol/min/mg) as intestinalbacteria. When the hydrolytic effect of arbutin was com-pared with that of p-nitrophenyl--d-glucopyranoside,the skin isolates also hydrolyzed p-nitrophenyl--d-glucopyranoside, but with lower potency than arbutin.

Bacteria density on skin is between 104 and 107 percm2,19but can vary by skin conditions such as moisture,sweat, nutrition, and body temperature.

The metabolite hydroquinone showed more potentDPPH radicalscavenging activity and tyrosinase inhibi-tion effects than arbutin.20 Hydroquinone also exhibitsan antimelanogenic effect, such as skin lightening, aswell as toxicity.59 These findings suggest that arbutinmay be partially hydrolyzed to hydroquinone by normal

skin microflora, and that skin lightening may be due toarbutin itself as well as its metabolite hydroquinone.

References

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2 Wen AH, Choi MK, Kim DD. Formulation of liposome fortopical delivery of arbutin. Arch Pharm Res2006; 29:118792.

3 Nishimura T, Kometani T, Okada S, Ueno N, Yamamoto T.[Inhibitory effects of hydroquinone-alpha-glucoside onmelanin synthesis]. Yakugaku Zassh1995; 115: 62632(in Japanese).

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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 Materialand Methods (b) The antioxidant effect of arbutin (), hydroquinone () and caffeic acid () measured by using DPPH dissolved in ethanol.These experiments are triplicates.

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2008 Wiley Periodicals, Inc. Journal of Cosmetic Dermatology, 7, 189193 193

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intestinal bacteria and biological activities of theirtransformants. Arch Pharm Res2004; 27: 617.12 Kim DH, Hong SW, Kim BT, Bae EA, Park HY, Han MJ.

Biotransformation of glycyrrhizin by human intestinalbacteria and its relation to biological activities. Arch PharmRes2000; 23: 172277.

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15 Blaut M, Braune A, Wunderlich S, Sauer P, Schneider H,Glatt H. Mutagenicity of arbutin in mammalian cells afteractivation by human intestinal bacteria. Food Chem Toxicol2006; 44: 19407.

16 Westerhof W, Kooyers TJ. Hydroquinone and its analoguesin dermatology a potential health risk. J Cosmet Dermatol

2005; 4: 559.17 Maeda K, Fukuda M. Arbutin: mechanism of itsdepigmenting action in human melanocyte culture.

J Pharmacol Exp Ther1996; 276: 7659.18 Gaskell M, McLuckie KI, Farmer PB. Genotoxicity of the

benzene metabolites para-benzoquinone and hydroquinone.Chem Biol Interact2005; 153154: 26770.

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20 McDonald TA, Holland NT, Skibola C, Duramad P, SmithMT. Hypothesis: phenol and hydroquinone derived mainlyfrom diet and gastrointestinal flora activity are causalfactors in leukemia. Leukemia2001; 15: 1020.

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