J. Pharm. Pharmacol.

1994,46:982-985 Received September 1993 30, Accepted Apl22,1994

J. @)1994 Pharm.Pharmacol.

Kojic Acid, a CosmeticSkin WhiteningAgent, is a Slow-binding Activity Inhibitor of Catecholase

OI I VTOS1NASE
JUANA CABANES, SOLEDAD CHAZARRA Ai.D FRANCISCO GARCIA.CARMONA Departarnento de Bioquimca y Biologa Molecular A, Unidad docente de Biologia, Facultad de Veterinari, Universidad de Murca, 30071 Espinardo, Murcia, Spain
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Abstract-It wasfound that kojic acid,which is usedin cosmetics its excellent for whiteningeffect,inhibits catecholase actity oftyrosinasein a non-classical in manner.A decrease the initial velocity to a steadystateinhibited velocitycan be observed which is unalteredby over a few minutes.This time-dependence, prior incubation of the enzymewith the inhibitor, is consistent with a first-order transition. The kinetic data obtained correspondto thosefor a postulatedmechanism that involvesthe rapid formation of an enzymeinhibitor complex that subsequently undergoesa relatively slow reversiblereaction. Kinetic parameterscharacterizingthis type of inhibition were evaluatedby means of nonlinear regressionof product accumulationcurves.

Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one) is an antibiotic producedby many species Aspergillusand of Penicillum in an aerobic processfrom a wide range of (Lee et al 1986;Kwak & Rhee 1992).Kojic carbon sources acid has beenextensively usedas a cosmeticagentwith an excellentwhitening effect(Obara et al 1985;Ohyama 1990) (Saruno etal 1979;Chen et al because inhibits tyrosinase it 1 9 9 1 a ,) . b Tyrosinase, polyphenoloxidase (EC. 1.14.18.1), a or is bifunctional copper protein complexwidely distributed on the phylogeneticscaleand responsible melanizationin for animals and browning in plants. Three different forms of binuclear copper in the active site are involved in the reactions(Lerch 1981;Robb 1981). The enzymecatalyses two different reactions:cresolase activity, or hydroxylation of monophenolsto o-diphenols and catecholase activity, or oxidation of o-diphenols o-quinone. to The expression the cresolase of actity of the enzymein the presenceof its substrate(monophenol) shows a lag period @omerantz1966;Garcia-Carmonaet al 1979)that has recently been explained by taking into account the chemicalstepsinvolved in the tyrosinase reaction(Cabanes et al 1987b;Garcia-Carmona al 1982,1987,' et 1988),while catecholase phenomena. actity showsno slow-transition Both the lag period of the cresolaseactivity and the steady-staterate are affected by kojic acid acting as a competitive and mixed-type inhibitor, depending on the phenolic substratesas has been describedby Chen et al (1991a). Thoseauthorsincludekojic acid in an important group of classical inhibitors formed of compoundsstructurally analogousto phenolicsubstrates, towards which they generallyshow competitiveinhibition, although this inhibition varies dependingon the enzymesourceand substrate used(Walker 1975;Mayer & Harel 1979;Vmos-Yigyn Correspondence: F. Garcia-Carmona, Departamento Bioquide

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Tiopolone

)'n"" til )o-Diohenolic substrate

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o-Ouinone oroducts

of Frc. 1. Stuctures the inhibitors,a normal orthodiphenolic products tyrosinase. of substrate orthoqnonic and Khan & Andrawis 1985). t--Mimosine and tropolone 1981; have been describedby us as competitive, slow-binding accordet Valeroet al 1991), inhibitors(Cabanes al 1987a; ing to the classificationof reversible enzyme inhibitors established Morrison (1982).Theseinhibitors have an by oxo group ortho to the hydroxyl group, thus presenting structureswhich are intermediatebetweenthe diphenolic of substrate the enzymeand the enzymaticproduct (Fig. 1). actity that is They result in an inhibition of catecholase by charactenzed a long transition phase. to In view of the above,we thought it would be interesting test whether kojic acid is also an effective inhibitor of accordingto the aboveclassification. tyrosinase Materials and Methods from SigmaChemicalCo. (St Louis, l-Dopa was purchased MO). All other chemicalsused were of analytical grade.

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mica y BiologaMolecularA, Unidad docente Biologia, Facultad e de Veterinaria,Universidadde Murcia, 30071Espinardo,Murcia, Soain.

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INHIBITION

OF ryROSINASE

BY KOJIC ACID L-DoPa r met L \k't

-

983
L-Dopa orthodopaquinone

ridibunda)were obtained from local Frogs (Ranaesculenta suppliersfrom Novemberto March. Epidermiswas separated from dermisafter incubationwith a 2 v NaBr for 24 h timeswith bidistilledwater, at0-4'C. After washingseveral negativebromide reaction took place and the epidermis a was lyophilizedand kept at 0-4'C until used.Frog epidermis protyrosinasewas extracted,partly purifled and actiofLozano et al (1975). vatedby the procedure activity was determined spectrophotoCatecholase metrically at 475nm by the appearanceof dopachrome in (e :3700r"r-'cm-r) using r,-dopa(5'6mru)as substrate buffer (pH 6'5). One unit of enzymewas sodium phosphate taken as the amount of enzymethat producedO'5pmol dopachromemin-l, since this involves the production of et l pmol dopaquinone(Garcia-Cnovas al 1982).Protein by was determined the methodof Lowry et al concentration fl951). The progresscurveswer fitted by nonlinear regression to the following equation, using Marquardt's algorithm (Marquardt 1963): P : Czt+ (C, - Cr)(l - e-c"C:. Co + (l)

orthodopaquinon"

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postulated Cabanes al (1987b) the for by et 1. Scner\4 Mechanism E-",, tyrosinase l-mimosine. by slowinhibitionof frog epidermis Eo"rI*, Eo*",oxy form ol tyrosinase, met form of tyrosinase; of complex foried by slowisomerization E"-rl ::r,r{frj;:"ntbt,.r decrease with a progressive was observed biphasicresponse in initial activity followed by a constantrate (Fig. 2, curves as B-D); both the initial and the constantrate decreased the inhibitor concentrationincreased. This resultindicatesthat the inhibition producedby kojic slowly, thus behavingas a slow-binding acid is expressed inhibitor, according to the definition given by Morrison is tightly (1982). The inhibition expressed not necessarily in sincekojic acidconcentration the bound or stoichiometric progresscurve experiments was always much higher than the enzymeconcentration(Williams & Morrison 1979). The progresscurved'obtainedcan be describedby the integratedform of Frieden'sequation (Frieden 1970)for a flrst-order process: -.-tuct)/k"pp [P]: v,t + (v. vo)(l (2)

Equation I is equivalentto equation 2 (seebelow), Ca representing experimentaluncertainty at the zero-time the absorbance,caused by addition of enzyme to start the reaction.

Resultsand Discussion was assayed with r-dopa as When frog epidermistyrosinase rate was immediatelyattained;the a substrate, steady-state werelinearup to at least0'3 absorbance changes absorbance units (Fig. 2, crrve A). When the reaction was started by addition of the enzyme in the presenceof kojic acid, a

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0.5

1.O Time (minl

1.5

2.O

Frc. 2. Progress curvesfor the inhibition of frog epidermistyrosiwere A 0, B 0'32, nase by kojic acid. Kojic acid concentrations C 0.94;D ['57 ru. The ieaction was startedby the addition of the enzvme.

respectively, initial and the represent, were Ve, V, and kunn the steady-staterates and the apparent first-order rate constant (the meaning of which dependson the mechancurves ism). Data analysisof theseproduct-accumulation can be performed by making an overall fit of the experias mental data to equation 2 by nonlinear regression, has by beendescribed Morrison (1982). To study this effect of kojic acid on frog tyrosinase, were performedin which the enzymewas preexperiments at incubatedwith differentkojic acid concentrations various times, at the end of which the reactionwas started by the progress curvessimilar to thosein addition of the substrate; Fig. 2 were obtained(data not shown).This result indicates that kojic acid doesnot bind to the free form of the enzyme (met form) and can only be interpreted by taking into activity of account the internal mechanism the catecholase of tyrosinase(Schemel) (Galindo et al 1983;Lerch 1983; postulates successive This scheme the et Cabanes al 1987b). to binding of two diphenolic substrates completethe cataof lytic cycle with the appearance an enzymaticoxy form, which, in comparison with the met form, has a greater affinity for the substrate(Galindo et al 1983).Thus, the with the binding of the secondmolecule inhibitor competes of r,-dopa,and so kojic acid mustbind to the oxy form of the enzyme.This form is an obligatory intermediate in the of catalytic turnover, and thus the presence the substrate for (and therefore,of'the catalytic activity) is necessary the slow binding of kojic acid to the enzymeto be observed. Thus, kojic acid is differentfrom other inhibitors previously

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INHIBITION

OF TYROSINASE BY KOJIC ACID

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of presence the inhibitor. Thus, incorrect rate values are is obtained if the product increase followed over a limited time and so different patterns of velocity vs substrateare on obtaineddepending the level ofinhibitor used. In agreementwith the kinetic mechanism for tyrosinase(Cabaneset al 1987b),only the oxy form, of the three different forms of the enzyme, acts on r,-tyrosine. hydroxylating it to r--dopa.On the other hand, both oxy and met forms act on l-dopa to give dopaquinone,which evolvesto give L-dopa and dopachrome.Thus, the inhibiand tion produced by kojic acid affects both catecholase activity through the oxy form. As mentioned cresolase previously, cresolaseactivity shows a complex kinetic et i..pottt" (Cabanes al 1987b),with a slow transition the phase,which basically expresses accumulation of the to maintain this activity. Control of the l-dopa necessary level of r-dopa produced in theseconditions has recently for as beenestablished an important mechanism the regulaactivity of tyrosinase(Garciation of the monophenolase Riley 1993). Carmonaet al 1987,1988; of The presence a slow transition phasesuch as the lag period in the cresolaseactivity of tyrosinase makes it impossibleto quantify the kinetic constantsof the interactlon of kojic acid and the enzyme. However, since the ernymatic intermediaries,oxy and met' are the same in activity, it might reasonably and both cresolase catecholase be assumedthat K, k6, and k-6 are the same for both activities,since theseconstantsexclusivelyreflect the pre1). ofthe oxy form and kojic acid (Scheme sence

F', F., Garcia-Cnovas, Garcia-Carnona, Vera, J', Iborra, J' L', Lozano,J. A. (1982)The ole of pH in the melaninbiosynthesis J. DathwaY. Biol' Chem.25'7:8738-8744 E., F., Grcia-Carmona, Pedreo, Galindo, J' D'' Garcia-Cnovas, method for the determinaF. (1979)A new spectrophotometric Anal' Biochem' tyrosinase' activity of epidermis tion of ciesolase

95:433-415 F., F., Garcia-Carmona, Garcia-Cnovas, Iborra, J' L', Lozano, J' A. (1932) Kinetic study of melanizationbetweenr--dopa and Biochim. Biophys. Acta'l 17'.l?4-l3l dopachrome. F' F., Garcia-Carmon, Cabanes,J', Garcia-Cnovas, (1987) Enzymaticoxidation by frog epidermistyrosinase.of4-methylcatecholand p-cresol.Influenceof l-serine. Biochim' Biophys' Acta 914:.198-204 J. F., Garcia-Carmona, Valero, E., Cabanes, (1988) Effect of r-proline on mushroom tyrosinase.Phytochemistry27: 196l1964 tyrosinase Inhibitionof mushroom A. Khan, V., Andrawis, (1985) 24: by tropolone.Phytochemistry 905-908. of J. fwt, . Y., Rh-ee, S. (1992) Cultivation characteristics oryzaefor kojic acid production' BiotecAspergllus immobrlzn nol. Bioeng.39:903-906 depletion T' Lee, L. S., Prrish,F' W., Jacks, J' (1986)Substrate during iormation of aflotoxin and kojic acid on corn inocuiated with spergillus fa'tus. Mycopathologia93: 105-107 tyrosrnaseano Lerch, K. (1981) Copper monooxygenases: In: dopamine-B-monooxygenase' Sigel, H. (ed.) Metal Ions in MacelDekker,Inc.,New York, pp 143-186 Systems. Biiogical structural, spectroscopic Lerch, K. (193)Neurosporatyrosinase: Biochem.52:125-138 Mol. Cel1. and cataiyticproperties. N. Lowry, O. ., Rose-brough, J., Farr, A. L., Randall,R' J' (1951) with the Folin phenol reagent' J' Biol' Protein measurement Chem.193:265-275 E' Lozano,J. A., Monserrat,F., Galindo' J. D., Pedreo, (1975) Revista dopa-oxidase' Activatory action of trypsin on epidermis Espaolade Fisiologia3l:21-28 estimation Marquardt, D. W. (1963)An algorithmfor least-squares J. of nonlinearparameters. Soc.Ind. Appl. Math' 1l:431-441 Acknowledgement in Mayer, A.M., Harel, E' (1979)Polyphenoloxidases plants' This work was supportedin full by a grant from Comision 18: PhytochemistrY 193-215 Cientificay Tecnica(BIO 9l-0790)' de Investigacion Morson, J. E. i1982) The slow-bindingand slow tight binding TrendsBiochem'Sci' 7: reactions. inhibition ofenzyme-catalysed 102-105 References Morrison. J. E., Walsh, C' T. (1989)The behaviorand significance inhibitors. Adv. Enzymol.201-203 of slow-bindingenzyme F', Belda, F. J., Garcia-Carmona,F., Garcia-Cnovas, Gmezskin whiteningby food Obara,Y., Ito, T; Hizu, Y. (1985)Cosmetic Fernndez,J. C., Lozano, J. A. (1983)A kinetic study of the In: conaittingkojic acid and its esters. Jpn. Kokai Tokyo Koho triphosphatase betweenrnitochondrialFl adenosine interaction J.P.60 137,253 and and adenylylimidodiphosphatase quanylyliminodiphosphaof Ohyama, Y. (1990)Melanogenesis-inhibitory-effett kojic acid tase.Biochem.J. 210:72'l-735 J. Fragrance 6: 53-58 and its action mechanism. F., J., Cabanes, Garcia-Cnovas, Tudela,J., Lozano,J' A', GarciaPomerantz, S. H. (1966) The tyrosine hydroxylase activity of Carmona, F. (1987a)r-Mimosine, a slow-bindinginhibitor of J. mammaliantyrosinase. Biol. Chem' 241: 16l-168 26 917-919 Phytochemistry mushroomtyrosinase. of Riley, P. A. (1993)Mechanisticaspects the control of tyrosinase F', J., Cabanes, Garcia-Canovas, Lozano, J. A., Garcia-Carmona, Cell Res.6: 182-185 activity.Pigment F. (1987b)A kinetic study of the melanizationpathwaybetween In: Robb, D. A.11981) Molecula propertiesof plant tyrosinases' and dopachrome.Biochim. Biophys' Actz923: 187r--tyrosine Friend. J., ihod"s, M. (eds)Biochemistryof Fruits and Vege195 London,pp 181-192 Press, Academic tables. M' Chen,J. S.,Wei, Ch., Rolle,R. S., Otwell,W' S', Balaban, O', Saruno, R., Kato, F., Ikeno, T. (1979) Kojic acid' a tyrosrnase Marshall, M. R. (1991a)Inhibitory effectof kojic acid on some albus. Agtic. Biol' Chem' 43:1337inhibitor'from Aspergillus J' polyphenoloxidases. Agric' Food' Chem' plant and crustacean 1339 39: 1936-1401 Valero, E., Garcia-Moreno,M., Varn, R., Garcia-Carmona,F' Chen,J. S., Wei, Ch., Marshall, M. R' (1991b)Inhibition mechanby inhibition ofgrape polyphenoloxidase (1991)Time dependent J. of kojic acid on polyphenoloxidase. Agric' Food' Chem' ism i.opoione.J. Agric. Food Chem.39: 1043-1046 39:1897-1901 L. Vms-Vigyaz, (tSst) Polyphenoloxidaseand peroxidasein Frieden, C. (1970) Kinetic aspectsof regulation of metabolic CRC Crit. Rev.Food' Sci'Nutr' 15:49-127 fruits an vegetables. processes. Biol. Chem. 245:5788-5799 J. (1975) Enzymic browning in foods: a reew' F', E., D., Pedreo, Garca-Carmona, Garcia-Cnovas, Walker. J. R. L. Galindo, J. EnzymeTechnol.Dig. 4: 89-100 study of the F., Solano, F., Lozano, J. A. (1983) Steady-state Williams, J. W., Morriion, J' E. (1979)The kineticsof reversible activity of tyrosinase'Int' J' Biomchanismof dopa-oxidase tight-bindinginhibition. Methods Enzymol. 63: 437-467 l5: chem., 1455-1461