Original Research Article

Modulation of Melanogenesis by Aloesin: A Competitive Inhibitor

of Tyrosinase

KEN JONES, JENNIFER HUGHES, MEI HONG, QI JIA and STEVE ORNDORFF

Univera Pharmaceuticals Inc., Broomfield, CO, USA*Address reprint requests to Ken Jones, Univera Pharmaceuticals Inc., 100 Technology Drive, Suite 325, Broomfield, CO 80021.E-mail: ken@aloecorp.com

Received 7 February 2002; in final form 24 June 2002

Aloesin, [2-acetonyl-8-b-D-glucopyranosyl-7-hydroxy-5-meth-ylchromone], a compound isolated from the Aloe plant, is

shown in these studies to modulate melanogenesis via compet-

itive inhibition of tyrosinase. Aloesin inhibits purified tyrosin-

ase enzyme and specifically inhibits melanin production in vitro.

Enzyme kinetics studies using normal human melanocyte

cell lysates and cell-based melanin production demonstrated

that aloesin is a competitive inhibitor of tyrosinase from

mushroom, human and murine sources. Tyrosine hydroxylase

and 3,4-dihydroxyphenylalanine (DOPA) oxidase activities of

tyrosinase from normal human melanocyte cell lysates were

inhibited by aloesin in a dose dependent manner. In a

percutaneous absorption study a finite dose of aloesin pene-

trated the skin slowly and was recovered primarily in the

surface wash. Aloesin shows promise as a pigmentation-

altering agent for cosmetic or therapeutic applications.

Key words: Tyrosinase, Melanin, Melanocytes, Aloesin,

Kinetics, Melanogenesis

INTRODUCTION

Melanin is a nitrogenous polymer produced and concentrat-ed in melanosomes within melanocytes located in the basallayer of the skin. Depending on skin type, melanocytes

produce varying ratios of eumelanin, a brown to blackmelanin, or pheomelanin, a red to yellow sulfur containingmelanin. As humans have approximately the same number of

melanocytes in the skin, differences in skin color are dueprimarily to the ratio of eumelanin to pheomelanin, theirdistribution in keratinocytes, degree of melanocyte activity,

and environmental factors such as solar radiation thatstimulates melanin production.Eumelanin absorbs and scatters UV light, attenuating its

penetration in the skin, reducing the harmful effects of thesun. Over-production of melanin as a result of chronic sunexposure, melasma or other hyperpigmentation diseases canlead to undesirable skin discoloration for which a number of

depigmenting agents have been developed. However, manyof the most popular depigmenting agents in use today exhibittoxicity to melanocytes and are known to produce adverse

side-effects (1–3). Inhibitors that target tyrosinase activity,the rate limiting enzyme in melanin production, promise tobe safer alternatives than melanocytolytic compounds. Con-

versely, one well-known tyrosinase inhibitor, kojic acid, hasbeen shown to be highly sensitizing to the skin and cases ofcontact dermatitis have been reported (4–7).

Tyrosinase catalyzes three steps in melanin biosynthesis:the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine(DOPA), oxidation of DOPA to DOPAquinone and the

oxidation of DHI(5,6-dihydroxyindole) to indolequinone (8).Because of its central role in melanogenesis, tyrosinase is akey target for screening and discovery of new inhibitors.

Aloe, a member of the Lily family, has been used forcenturies in cosmetics and Aloe extracts have been reportedto inhibit l-3,4,-dihydroxyphenylalanine (l-DOPA) oxida-tion by mushroom tyrosinase (9). Aloesin, a natural hydrox-

ymethyl chromone compound isolated from aloe extracts, isdemonstrated in this report to be a competitive inhibitor oftyrosinase.

Abbreviations – DHI, 5,6-dihydroxyindole; bFGF, basic fibroblast growth factor; a-MSH, a-melanocyte stimulating hormone; TEWL, transepidermalwater loss

PIGMENT CELL RES 15: 335–340. 2002 Copyright � Pigment Cell Res 2002

Printed in UK—all rights reserved ISSN 0893-5785

Pigment Cell Res. 15, 2002 335

MATERIALS AND METHODS

Chemicals

Mushroom tyrosinase, l-DOPA, dimethyl sulfoxide (DMSO),trypsin ⁄EDTA, insulin, human recombinant basic fibroblastgrowth factor (bFGF), transferrin, a-tocopherol, a-melano-cyte stimulating hormone (a-MSH), IGEPAL CA-630,arbutin, kojic acid and endothelin 1 were purchased fromthe Sigma Chemical Company (St Louis, MO, USA).

Bicinchoninic acid (BCA) protein assay kit was purchasedfrom Pierce (Rockford, IL, USA). Fetal calf serum waspurchased from Gemini Bio-Products (Woodland, CA,USA). L-[3,5–3H] Tyrosine and 3H Thymidine were pur-

chased from Amersham Pharmacia Biotech (Piscataway, NJ,USA). Norit SX 2 activated charcoal was purchased fromNORIT (Amersfoort, the Netherlands).

Aloesin Purification

Aloesin (>99.5% purity) was manufactured at UniveraPharmaceuticals by a method previously described (10).Briefly, aloesin is chromatographically separated and purified

from Aloe ferox leaf and crystallized from the concentratedchromatographic pool. The process is monitored for purity,moisture content and color with standard (Lot no. K2200).

Mushroom Tyrosinase Purified Enzyme Assay

Tyrosinase activity was measured by a method modified fromPomerantz (11). Tyrosinase was prepared in 50 mM potassi-um phosphate buffer, pH 6.8 (assay buffer) at 2000 U ⁄ml andstored at )20�C in 1 ml aliquots prior to use. For use inassays, stock enzyme solutions were thawed and diluted to200 U ⁄ml with assay buffer. A 2 mM working solution of

substrate, l-DOPA, was prepared in assay buffer for eachassay. Samples were dissolved in 10% DMSO (0.5 ml) anddiluted to 5 ml with assay buffer. The reaction mixture

consisted of 0.050 ml 2 mM l-DOPA, 0.050 ml 200 U ⁄mlmushroom tyrosinase and 0.050 ml inhibitor. Reaction vol-ume was adjusted to 0.200 ml with assay buffer. Assays wereperformed in 96 well Falcon 3097 flat-bottom microtiter

plates (Beckton Dickinson, NJ, USA). Appearance of dopa-chrome was measured with a Bio-Tek Instruments EL311microplate reader (Winooski, VT, USA). Average velocity

was determined from linear enzyme rate as measured bychange in absorbance (DA450) at 450 nm ⁄min. Percentinhibition of tyrosinase by test samples was determined by

comparison of DA450 of samples vs. control using theformula:

%Inhibition ¼ ½ðDA450 Control� DA450 SampleÞ=DA450Control� � 100

Cell Culture

Murine B16 F1melanoma cells, strain C57BL ⁄ 6J, werepurchased from the American Type Culture Collection

(Manassas, VA, USA) and grown in a humidified atmo-sphere with 5% CO2 at 37�C. Cells were subcultured inModified Eagle Medium (Gibco, Rockville, MD, USA)

supplemented with 1% MEM non-essential amino acids and1.5% vitamin solution, 5% heat-inactivated fetal calf serum,100 U ⁄ml penicillin, 0.100 mg ⁄ml streptomycin, 2 mM

l-glutamine. Cells were passaged by brief treatment withtrypsin ⁄EDTA and resuspended in growth medium.Primary cultures of normal human melanocytes from

neonatal foreskin of African American descent (a generous

gift from Raymond E. Boissy, University of Cincinnati) weregrown in Medium 154 (Cascade Biologics, Inc., Portland,OR, USA) supplemented with 4% fetal calf serum, 5 ·10)3 mg insulin ⁄ml, 1.8 · 10)6 mg human recombinantbFGF ⁄ml, 1 · 10)3 mg transferin ⁄ml, 1.0 lg a-toco-pherol ⁄ml, 10)8 M a-MSH ⁄ml, 10)9 M endothelin-1.

Murine Melanoma Melanin Assay

Murine melanin was measured by a method modified fromSiegrist (12). Murine melanoma cells were seeded at12 500 cells ⁄ml, 0.20 ml ⁄well, in 96 well flat-bottom plates

(Falcon) overnight for attachment. Cell treatments wereadded in triplicate in fresh medium and equivalent volumesadded to duplicate control wells for a final volume of

0.20 ml ⁄well. Cells were treated for 5 days with sample orequivalent volume of growth medium for control and1.5 · 10)7 mM a-MSH to stimulate melanin production.

A colored product, not observed in purified enzyme assays,is produced by treatment with aloesin in cell based in vitroassays that interferes with spectrophotometric readings at450 nm. The source of this colored product is under inves-

tigation. For this reason we used 14C thiouracil, which isselectively incorporated in the formation of melanin (13–15),to quantify the effect of aloesin on tyrosinase in B16 murine

melanoma cell based in vitro assays. Briefly, B16 murinemelanoma cells were pulsed with 1.8 · 10)5 mCi 14C thio-uracil per well of a 96 well plate for 24 h. Cell supernatant was

collected and exogenous melanin was precipitated by treat-ment with 5% trichloroacetic acid and pelleted by centrifu-gation. The pellet was solubilized in 0.5 N KOH with heatingat 90�C for 1 h. Radioactivity of incorporated 14C thiouracil

was counted in a Beckman LS 6500 multipurpose scintillationcounter. These data (not shown) demonstrated that spectro-photometric analysis at 570 nm correlated well with incor-

porated 14C thiouracil for detection of melanin. To reduce theexpense of routine melanin detection in human DOPAoxidase assays, where melanin production is significantly less

than that of murine melanoma cells, spectrophotmetricanalysis at 570 nm was utilized.

Human Tyrosine Hydroxylase Assay

Cultured human primary melanocytes were harvested and

solubilized in 0.5 ml of 0.5% IGEPAL CA-630 ⁄phosphate-buffered saline (PBS) by sonication. Cell lysate was centri-fuged (3000 · g) at 4�C for 10 min and protein concentration

of the supernatant was determined by BCA protein assay kit.The reaction mixture consisted of 50 mM potassium phos-phate buffer, pH 6.8, 2 · 10)3 mCi [3,5-3H] tyrosine,

5 · 10)3 mM l-DOPA and 0.10 mg ml)1 human melanocytelysate in the presence or absence of tyrosinase inhibitors.Reaction mixtures were incubated for 1 h at 37�C. Reactions

336 Pigment Cell Res. 15, 2002

were stopped by addition of 1 ml of 0.1 N HCl containing100 mg Norit SX 2 activated charcoal to capture unreactedradiolabeled tyrosine and incubated for another hour at

25�C. Volume of control wells was adjusted with phosphatebuffer replacing inhibitor or lysate (blank). Followingincubation, reaction mixtures were centrifuged at 21 000 · gfor 15 min. The supernatant (0.5 ml) was collected and the

radioactivity of tritiated water released by hydroxylation oftyrosine by enzymatic action was determined by liquidscintillation. The data was expressed as percent inhibition

of tyrosinase by aloesin compared with control.

DOPA Oxidase Assay

Cultured human primary melanocytes were harvested andsolubilized by the method described for the tyrosine hydrox-

ylase assay. The reaction mixture consisted of 1 mM

l-DOPA, 0.10 mg ⁄ml lysate protein and tyrosinase inhibitorsin 50 mM potassium phosphate buffer, pH 6.8. Inhibitor

volume was replaced with assay buffer for control wells.Reaction mixtures were incubated for 30 min at 37�C. Thechange in absorbance at 570 nm was measured with a Bio-

Tek Instruments EL311 microplate reader. Absorbance at570 nm was used for reasons described in the murinemelanoma melanin assay above. Inhibitory activity of test

samples was expressed as percent inhibition. All tests wereperformed in triplicate.

Percutaneous Absorption Study

Percutaneous absorption was measured in vitro using human

cadaver skin. Briefly, cryopreserved, split-thickness(� 0.25 mm) leg skin was obtained from a skin bank andstored in water-impermeable plastic bags at )70�C until used.Immediately prior to the experiment, skin was placed in 37�Cwater for 5 min, then cut into sections large enough to fit on0.8 cm2 Franz cells (Crown Glass Co., Somerville, NJ, USA).The dermal chamber was filled with PBS, pH 7.3–7.4, and the

epidermal chamber (chimney) left open to ambient condi-tions. All cells were mounted in a diffusion apparatus inwhich the dermal bathing solution was stirred magnetically

and maintained at 37�C. The integrity of each skin sectionwas checked by determining the rate of transepidermal waterloss (TEWL) using an electronic evaporimeter (Courage-

Khazaka Electronic GmbH Tewlameter, model no. Tm210,Koln, Germany). Just prior to dosing the receptor solutionwas replaced with a 1:10 dilution of PBS and 0.05 ml of 2%aloesin in ethanol was applied to the skin. At various times

after dosing, the receptor solution was removed in itsentirety, replaced with fresh solution, and a 4.0-ml aliquotsaved for analysis. Following the last sample in the study the

skin surface was washed twice with 0.5 ml water, then theskin removed and separated into dermis and epidermis. Eachtissue was extracted overnight in 1 ml water. All samples

were analyzed by a Hitachi M-8000 LC mass spectrometer(Hitachi Instruments, Inc., San Jose, CA, USA). Pharmac-okinetic data were expressed as a percentage of dose. The

replicate data for each donor were averaged to give a perdonor mean. Subsequently, the mean data per donor wereaveraged to give a grand mean across both donors.

RESULTS

Tyrosinase Inhibition by Aloesin

The effect of aloesin on tyrosinase activity was studied in vitrousing a purified mushroom tyrosinase assay for comparisonwith the known tyrosinase inhibitors, arbutin and kojic acid.

Using l-DOPA as the substrate the IC50 of various tyrosinaseinhibitors was determined. Aloesin (Fig. 1), arbutin and kojicacid (data not shown) each inhibited mushroom tyrosinase in

a dose dependent manner. Kojic acid was the most potentinhibitor of mushroom tyrosinase (IC50 ¼ 0.010 mM) fol-lowed by aloesin (IC50 ¼ 0.193 mM) and arbutin (IC50¼11.13 mM) (Table 1).

Inhibition of tyrosinase enzyme activity and subsequentmelanin formation was confirmed in a cell-based assay usingB16 F1 murine melanoma cells. Melanin biosynthesis was

inhibited by aloesin (IC50 ¼ 0.167 mM) (Fig. 2) in a dosedependent manner. Aloesin had no effect on 3H-thymidineincorporation, suggesting that it did not significantly alter

cell viability or replication.Inhibition of tyrosinase from three primary sources,

fungal, murine and human, was determined for aloesin incomparison with two leading depigmenting ingredients,

arbutin and kojic acid (Table 1). The relative rank of theseinhibitors was accurately predicted from any assay, despitedifferences in the origin and composition of each species of

Fig. 1. Aloesin Inhibits Mushroom Tyrosinase. The ability of aloesin toinhibit the enzymatic activity of purified mushroom tyrosinase wasmeasured using l-DOPA as substrate. Percent inhibition was calculatedin comparison to controls (h). Average velocity (s) was determined bymonitoring the change in absorbance at 450 nm.

Table 1. In vitro inhibition of various tyrosinase enzymes by aloesin andother putative pigment modulators. A summary of results from in vitro(mushroom), cell lysate (human melanocytes) and in situ (murine) assays

IC50 (mM)inhibitor Mushrooma Murineb Humanc

Arbutin 11.13 0.205 3.02Aloesin 0.193 0.167 0.71Kojic acid 0.010 0.131 0.41

a Mushroom, one unit tyrosinase activity ¼ DA280 of 0.001 ⁄min atpH 6.5 at 25� in 3 ml reaction mix containing l-tyrosine (Sigma ChemicalCo.).b Murine B16 melanoma cells (ATCC no. CRL 6323).c Human primary melanocytes (lysate), African–American donor (Uni-versity of Cincinnati).

Pigment Cell Res. 15, 2002 337

tyrosinase. Cell based assays generally gave lower IC50 valuesthan the purified fungal enzyme assay, and kojic acid showedthe greatest inhibitory activity due in large part to its high

cytolytic activity. Aloesin activity was significantly betterthan arbutin, the most widely used cosmetic, non-bleachingdepigmenting agent.

Kinetics of Human Tyrosinase Inhibition by Aloesin

To better understand the mechanism of action of thesetyrosinase inhibitors, extensive enzyme kinetic analysis wasperformed on human tyrosinase. Data from these studies

were used to generate a Lineweaver–Burke plot (Fig. 3) todetermine whether aloesin acted as a competitive inhibitor ofthe enzyme. The y-intercept is the same in the presence andabsence of aloesin with l-DOPA as substrate demonstrating

that aloesin is a competitive inhibitor of human tyrosinase.Based on these kinetic data, human tyrosinase oxidizesl-DOPA with an apparent Km of 0.588 mM at a concentra-

tion of 0.0313 mM aloesin. In the presence of 0.0625 mM

aloesin the Km is increased to 0.690 and 0.889 mM at

0.125 mM aloesin. The dissociation constant, Ki, for thetyrosinase-aloesin enzyme-inhibitor complex is 0.152 mM.

Inhibition of Human Tyrosine Hydroxylase and DOPA

Oxidase Activity by Aloesin

Tyrosinase is a bifunctional enzyme that catalyzes threereactions in the melanin pathway. Measurement of 3H2O,which is released from [3,5-3H] tyrosine hydroxylation, was

used to determine the hydroxylase activity of tyrosinase inthe presence of aloesin. Aloesin inhibited human tyrosinasehydroxylase activity in a dose dependent manner (Fig. 4a)

with an IC50 of 0.92 mM. Assays were performed withprimary human melanocyte lysates in a spectrophotometricassay to compare the activity of aloesin, arbutin and kojicacid. Aloesin inhibited DOPA oxidase activity in a dose

dependent manner (Fig. 4B), with an IC50 ¼ 0.70 mM

compared with kojic acid, IC50 ¼ 0.41 mM, and arbutin,IC50 ¼ 3.02 mM.

Percutaneous Absorption Study

A finite dose of aloesin (1 mg) in ethanol was applied to theoutside surface of human cadaver skin on a Franz cell to

Aloesin (mM)

Fig. 2. Effect of aloesin on melanin formation in situ. Murine melanomacells were treated with aloesin for 5 days. Melanin production wasstimulated with a-MSH.

1/[S] L-DOPA (mM)

0.125mM

0.0625mM

0.0313mM

0mM

Fig. 3. Lineweaver-Burke plot of normal human melanocyte tyrosinase(lysate) inhibition by aloesin. Enzyme activity was measured at 570 nmin the presence of 0, 0.125, 0.063 and 0.031 mM aloesin while thesubstrate, l-DOPA, was varied from 0.25 to 1 mM.

Fig. 4. Effect of aloesin on human tyrosine hydroxylase and DOPAoxidase activities of tyrosinase. Lysate of normal human melanocyteswere treated with aloesin using 14C tyrosine (A) or l-DOPA (B) assubstrate. Results are shown as percent inhibition compared with control.

338 Pigment Cell Res. 15, 2002

determine the percutaneous absorption pharmacokinetics of

the compound. After 32 h, aloesin detected in the receptorsolution, was 0.021 ± 0.027% of the applied dose (Table 2).Aloesin absorption in the dermis was 0.086 ± 0.047% and

1.48 ± 0.393% in the epidermis. The remainder, 88.56 ±14.47%, was recovered in the surface wash. A total of90.15 ± 14.15% of the applied dose was recovered.

DISCUSSION

In this report we have examined the modulating effects ofaloesin on tyrosinase. Aloesin demonstrates a dose-depen-dent inhibition of mushroom, murine and human tyrosinaseactivity. Tyrosinase catalyzes the hydroxylation of tyrosine

to DOPA and oxidation of DOPA to DOPAquinone (16).The tyrosine hydroxylase activity of tyrosinase, the ratelimiting step, requires DOPA as a co-factor (17). We have

shown that aloesin inhibits both activities of tyrosinase withsimilar potency. In contrast, Kojic acid has been reported tobe a slow-binding inhibitor of the hydroxylase activity of

tyrosinase (18). While arbutin is reported to inhibit bothprimary activities of tyrosinase, its affinity for the DOPAoxidase catalytic site on tyrosinase is lower than aloesin(Ki) ¼ 16.48 mM19 vs. 0.152 mM, respectively.

Enzyme kinetics experiments show that aloesin, likearbutin (19), is a competitive inhibitor of DOPA oxidaseactivity. However, in a recent report (20) aloesin was shown

to be a non-competitive inhibitor of tyrosine hydroxylaseactivity. There are data (17, 21–24) to support the hypothesisfor separate binding sites on tyrosinase that may account for

these findings. The oxidation of tyrosine by tyrosinase isdistinguished by a lag period (21) that is affected by enzymeand substrate concentration, pH and hydrogen donors such

as DOPA (22). Hearing et al. (17, 23) proposed that there aretwo catalytic sites on tyrosinase, one for tyrosine hydroxylaseand another site specific for DOPA oxidase activity. Ac-cording to this hypothesis a small amount of DOPA is

produced during the induction period that binds to theDOPA specific site and acts as a positive allosteric effectorfor tyrosine hydroxylation and provides a hydrogen donor

for the reaction. Wood et al. (24) showed that 5,6,7,8,-tetrahydrobiopterin did not inhibit tyrosinase with l-DOPAas substrate but did inhibit tyrosine hydroxylase activity with

l-tyrosine as substrate, further supporting the hypothesis ofseparate catalytic sites for DOPA and tyrosine.An alternative hypothesis comes from studies demonstrat-

ing that the reduced form of iron (25), nickel and cobalt (26)

are capable of stimulating tyrosine hydroxylase activity inthe absence of l-DOPA by reduction of the copper ions at

the active site on tyrosinase. These data suggest that theactivation of hydroxylase activity by DOPA may be the resultof its function as a hydrogen donor rather than as an allosteric

effector. In a mechanism proposed by Karg (27), the rate ofhydroxylation of tyrosine toDOPAandDOPAquinonewouldbe proportional to the amount of tyrosinase in the reducedform. Therefore, DOPA would become both substrate and

hydrogen donor eventually reaching a steady state.The finding that aloesin acts by two different mechanisms

of action on tyrosinase activity supports the hypothesis of

two catalytic sites on tyrosinase. These data also suggest thatinhibition of DOPA oxidation would interfere with thesteady state of DOPA mediated reduction of copper ions at

the hydroxylase catalytic site. In this proposed mechanism ofaction, aloesin inhibits the formation of DOPAquinone bycompetitive inhibition at the DOPA oxidation site, reduction

of copper ions at the hydroxylase site and consequently,tyrosine hydroxylation by non-competitive inhibition.Modulators of melanogenesis may act directly on tyrosin-

ase, tyrosinase gene expression or mechanisms responsible

for transfer of melanosomes from melanocytes to keratino-cytes (28, 29) to reduce pigmentation of the skin. Despite theoften modest inhibitory activity of commercial depigment-

ing agents, most rely on cytolytic activity that can lead toadverse side-effects after chronic use. Hydroquinone hasbeen reported to be highly mutagenic and cytotoxic to V79

Chinese hamster (2). Kojic acid, a popular food ingredient inthe Japanese diet, has been shown to enhance neutrophilphagocytosis and lymphocyte proliferation, therefore sup-porting the immune system (30). Despite these benefits, kojic

acid has been shown to cause allergic contact dermatitis andis considered to have high sensitizing potential (5, 6). Kojicacid was also found to be mutagenic in the Ames assay (6)

and in hamster ovary cells (7). Use of products that arecytotoxic or sensitizing require careful monitoring when usedat concentrations that are effective, and they may need to be

combined with steroids to reduce irritation or dermatitis.Consequently, non-toxic modulators of pigmentation arepreferable. Arbutin has been characterized as an effective

inhibitor at non-cytotoxic concentrations (19). In compari-son, aloesin shows no cytotoxicity in cell-based assays, noskin irritation in preliminary human studies and no geno-toxicity or mutagenicity in the Ames assay (NAMSA,

Northwood, OH, USA). Cultured cells used in tyrosinaseactivity assays show no morphologic abnormalities whentreated with aloesin and human melanocytes appear normal

with multiple dendrites (K.N. Jones, personal observation).We have shown that aloesin is a potent inhibitor of human

tyrosinase. However, because of the hydrophilic nature of the

compound and moderately high molecular weight, penetra-tion of human skin was poor. Our studies show that aloesin,dissolved in ethanol, penetrates the skin slowly with approx-

imately 1.59% of a finite dose penetrating the skin over a 32-hperiod. In an attempt to improve penetration, a hydrophilicpatch was impregnated with 1% aloesin and a preliminaryin vivo test with human volunteers was performed. A linear,

statistically significant decrease in melanin (data not shown)was detected over the trial period and repigmentation of theskin occurred rapidly following the end of the treatment

period.

Table 2. Percutaneous absorption of aloesin by human cadaver skin andFranz diffusion cells. Aloesin applied in a finite dose to human cadaverskin. Results for cumulative penetration in 32 h are shown

Absorption and mass balance (% of dose)

Total absorbed 0.021 ± 0.027Dermis 0.086 ± 0.047Epidermis 1.480 ± 0.393Surface wash 88.56 ± 14.47Total recovery 90.15 ± 14.15

Pigment Cell Res. 15, 2002 339

This study shows that aloesin inhibits human tyrosinase bya unique dual mechanism of action not evident in othercommercial depigmenting agents, and more importantly,

demonstrates an excellent safety profile. Although skinpenetration is poor, new drug delivery technology shouldovercome this problem.

Acknowledgements – Raymond Boissy, University of Cincinnati, provid-ed human primary melanocytes. Teikoku Seiyaku Co., Ltd. of Japanproduced the aloesin hydrophilic patches used for the preliminary humanstudy. Padmapriya Abeysinghe, Tom Farrow and Larry Brown ofUnivera Pharmaceuticals Inc. provided aloesin for these studies. Specialthanks to Tim Nichols, Joel Guthridge, Raymond Boissy and Akira Yagifor their helpful discussions about these studies. This work was supportedby Univera Pharmaceuticals Inc.

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