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Action of ellagic acid on the melanin biosynthesis pathway

Carmen Vanessa Ortiz-Ruiza , Jose Bernab , Jose Tudelaa , Ramon Varonc ,
Francisco Garcia-Canovasa,*
a
GENZ-Group of Research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare
Nostrum”, University of Murcia, E-30100 Espinardo, Murcia, Spain1
b
Synthetic Organic Chemistry Group, Department of Organic Chemistry Regional Campus of International Excellence “Campus Mare Nostrum”, University of
Murcia, E-30100 Espinardo, Murcia, Spain
c
Department of Physical Chemistry, Technical School of Industrial Engineering, University of Castilla La Mancha, Avda. España s/n. Campus Universitario,
E-02071 Albacete, Spain

A R T I C L E I N F O A B S T R A C T

Article history: Background: Tyrosinase is an enzyme involved in the first steps of the melanogenesis process. It catalyzes
Received 12 November 2015 the hydroxylation of monophenols to o-diphenols and the oxidation of the latter to o-quinones. Ellagic
Received in revised form 7 January 2016 acid (EA) is a phenolic compound which has been described as a tyrosinase inhibitor and is used in the
Accepted 10 February 2016
cosmetic industry as a whitening agent. However, it has hydroxyl groups in ortho position and could act as
a substrate rather than inhibitor. This aspect should be taken into consideration when using this
Keywords: compound as a cosmetic ingredient due to the reactive character of o-quinones.
ellagic acid
Objective: To determine whether ellagic acid is a substrate or an inhibitor of tyrosinase, to characterize it
tyrosinase
melanogenesis
kinetically and interpret its role in the melanogenesis process.
inhibition Methods: UV–vis spectrophotometry was used to follow the action of tyrosinase on typical substrates and
kinetic characterization ellagic acid. A chronometric method was chosen for the kinetic characterization of ellagic acid.
Results: Ellagic acid is not an inhibitor per se but an alternative substrate of tyrosinase. It is oxidized by the
enzyme to an unstable o-quinone. Its kinetic characterization provided low Michaelis and catalytic
M = 138
13 mM and kcat = 0.47
0.02 s
EA 1
constants (K EA ). Furthermore, ellagic acid, which is a powerful
antioxidant, may chemically reduce the o-quinones (o-dopaquinone) and semiquinones, in this way
inhibiting the melanogenesis.
Conclusion: Ellagic acid is oxidized by tyrosinase, producing reactive o-quinones. As an antioxidant it can
inhibit the melanogenesis process. This first aspect should be taken into consideration in its application
as a cosmetic ingredient due to the toxicity of o-quinones and its ability to modify the redox status of the
cell.
ã 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights
reserved.

1. Introduction hydroxylation of monophenols to o-diphenols (monophenolase

activity) and the oxidation of the latter to o-quinones (diphenolase
Tyrosinase (E.C [Link]) is a copper monooxygenase widely activity), which are unstable molecules. Subsequently, o-quinones
distributed in nature, which catalyzes two types of reaction, the evolve to melanins [1].

Abbreviations: Em, met-tyrosinase; Ed, deoxy-tyrosinase; Eox, oxy-tyrosinase; D, o-diphenol; EA, ellagic acid; [EA]0, initial concentration of ellagic acid; EmEA, met-
tyrosinase/ellagic acid complex; EoxEA, oxy-tyrosinase/ellagic acid complex; Q, o-quinone; QEA, o-quinone from ellagic acid; AH2, ascorbic acid; [AH2]0, initial concentration of
ascorbic acid; A, dehydroascorbic acid; [E]0, initial concentration of tyrosinase; TBC, tert-butylcatechol; [TBC]0, initial concentration of tert-butylcatechol; o-TBQ, o-tert-
butylquinone; [L-dopa]0, initial concentration of L-dopa; DOMA, 3,4-dihydroxymandelic acid; [DOMA]0, initial concentration of 3,4-dihydroxymandelic acid; DOBA, 3,4-
EA Q EA
dihydroxybenzaldehide; [NaIO4]0, initial concentration of sodium periodate; K EAM , Michaelis constant of ellagic acid; kcat , catalytic constant of ellagic acid; V 0 , initial
EA
formation rate of the o-quinone from ellagic acid; V Q max , maximum action rate of tyrosinase on ellagic acid; t, time; t , lag period; Tyr, tyrosinase; ABTS 
, radical
from 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid); ARP, antiradical or antioxidant power value; EC50, ratio of the antioxidant concentration necessary to
decrease the initial concentration of the ABTS to 50%.
* Corresponding author. Fax: +34 868 883963.
E-mail address: canovasf@[Link] (F. Garcia-Canovas).
1
[Link]/genz.

[Link]
0923-1811/ ã 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: C.V. Ortiz-Ruiz, et al., Action of ellagic acid on the melanin biosynthesis pathway, J Dermatol Sci (2016), http://
[Link]/10.1016/[Link].2016.02.004
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Tyrosinase is involved in the first steps of the melanogenesis (Madrid, Spain) and dimethyl sulfoxide (DMSO) from Merk
process in human skin pigmentation [2]. Although melanin is an (Madrid, Spain). Stock solutions of substrates were prepared in
essential pigment for the protection of skin against UV-induced 0.15 mM phosphoric acid to prevent auto-oxidation. Ellagic acid
damage, excessive melanin production causes hyperpigmentation was dissolved in DMSO due to its low solubility.
such as melasma, freckles, ephelide and solar lentigines [3–5].
Furthermore, this enzyme is responsible for the browning of fruit, 2.3. Spectrophotometric assays
vegetables, fungi and crustaceans, producing important losses in
the organoleptic properties of food and therefore a decrease in Absorption spectra were recorded in a visible-ultraviolet
commercial value [6–9]. PerkinElmer Lambda 35-spectrophotometer, on-line interfaced
One strategy to avoid these undesirable effects is to inhibit this with a compatible PC 486DX microcomputer, with a 60 nm/s
enzyme. In recent years large number of compounds have been scanning speed controlled by the UV-Winlab software. The
identified and characterized as inhibitors of tyrosinase [10–12]. temperature was maintained at 25  C using a Haake
Measuring the enzymatic activity of tyrosinase is complicated D16 circulating water bath with a heater/cooler, and checked
because the products of the reaction are o-quinones. These are using a Cole–Palmer digital thermometer with a precision of 0.1  C.
unstable and extremely reactive molecules, which has led to many All the assays were carried out under saturating conditions of
phenolic compounds being wrongly identified as inhibitors of tyrosinase by molecular oxygen, 0.26 mM in the assay medium
tyrosinase, when really they are alternative substrates of the same. [41]. The activity of tyrosinase on ellagic acid was measured at
Such is the case of the hydroquinone [13,14], rhododendrol 470 nm, which is the maximum absorption wavelength of its
[15–19], 4-hexylresorcinol [20] and arbutin [21] among others. oxidation product. The action of the enzyme on TBC in the absence
The risk of using these compounds as depigmenting agents is that and presence of different concentrations of ellagic acid was
they may be converted by tyrosinase into o-quinones, which are measured at 400 nm, the maximum absorption wavelength of
very reactive molecules and may react with nucleophilic com- o-tert-butylquinone (o-TBQ) [42,43].
pounds such as cysteine and glutathione, diminishing the In order to determine the role of ellagic acid as an antioxidant in
antioxidant capacity of the cell in which tyrosinase acts [15,22–24]. the melanogenesis process, 600 nm was chosen as the wavelength
Ellagic acid is a naturally occurring phenolic compound found to follow product formation because the oxidation product of
in many natural sources. It has anticarcinogenic [25,26], anti- ellagic acid absorbs at this wavelength, while the absorbance of the
fibrosis [27], and antioxidative [28–30] properties, apart from o-tert-butylquinone (from TBC), dopachrome (from L-dopa, the
inhibiting the melanogenesis process [12,31–37]. This inhibition physiological substrate) and 3,4-dihydroxybenzaldehide (DOBA,
has been described as being due to the inhibiting action of ellagic from DOMA) is very low or null.
acid on tyrosinase [12,31–34] and it may also be produced by its
antioxidant power [31]. 2.4. Kinetic data analysis
In a recent work an experimental design was established which
demonstrated that many of the phenolic compounds described as In order to characterize the action of tyrosinase on ellagic acid
inhibitors of tyrosinase are, in fact, alternative substrates. as a substrate, due to the instability of its oxidation product, a
However, in the specific case of ellagic acid, no enzymatic activity chronometric method was used to determine the initial rate values
was observed [19]. Although our research group characterized this (V Q
EA

0 ) at different substrate concentrations [42,43]. This method

compound as a substrate of tyrosinase by means of oxymetric
uses ascorbic acid, which is capable of reducing the generated o-
methods [28], its role in the melanogenesis process is still
quinones, and, once the ascorbic acid is consumed, an absorbance
controversial. Thus, the aim of the present work is to take another
signal appears due to the presence of the products. The time
look at the action of tyrosinase on ellagic acid, using spectropho-
necessary to consume the quantity of ascorbic acid in the medium
tometric methods to demonstrate that ellagic acid, a natural EA

compound used in the cosmetic industry as a depigmenting agent (t), can be measured and is used to calculate V Q
0 values. The
EA
[36,37], is not an inhibitor of mushroom tyrosinase but an resulting V Q
0 versus [EA]0 (the initial concentration of ellagic acid)
alternative substrate, and due to the structural analogy of data were fitted by non-linear regression to the Michaelis-Menten
tyrosinases from different organisms [38], it could also act as a equation through the Sigma Plot 9.0 program for Windows [44],
substrate of human tyrosinase. We propose a mechanism of action EA
thus obtaining the maximum rate (V Q
max ) and the Michaelis
of the enzyme on this compound, whose analysis permits its
kinetic characterization as a substrate. This aspect should be taken constant (K EA
M ).

into consideration by the cosmetics and pharmaceutical industries All assays were made in triplicate and, unless otherwise stated,
when using ellagic acid as a whitening agent. Furthermore, we using 30 mM phosphate buffer pH 7.0.
study the action of this compound on the melanin biosynthesis
pathway, taking into consideration its high antioxidant power. 3. Results and discussion

2. Material and methods Ellagic acid is a natural phenolic compound which has been
described as an inhibitor of the melanogenesis process [12,31–37].
2.1. Enzyme source This compound inhibits the first steps of the melanin biosynthesis
pathway which are catalyzed by tyrosinase and is used as an
Mushroom tyrosinase (3130 U/mg) was purchased from Sigma ingredient in whitening creams and other cosmetics [36,37]. In
(Madrid, Spain) and purified as previously described [39]. The order to check the action of ellagic acid as an inhibitor of tyrosinase
protein content was determined by Bradford’s method [40]. kinetically, measurements were made of the activity of the enzyme
on TBC as substrate in the absence and presence of different
2.2. Reagents concentrations of ellagic acid (Fig. 1SI, see Supplementary
material). As can be seen in Fig. 1SI, the slope of the recordings
Ellagic acid, L-dopa and 3,4-dihydroxymandelic acid (DOMA) decreased when the ellagic acid concentration increased, which
were purchased from Sigma (Madrid, Spain), 4-tert-butylcatechol means that the initial o-quinone formation rate values are lower in
(TBC) and sodium periodate (NaIO4) were from Acros Organics the presence of this compound, confirming its interaction with the

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conditions to assay the activity of the enzyme on this compound

using spectrophotometric methods.
In Fig. 2SI (see Supplementary material) the scan spectrum of
the ellagic acid is shown. As can be seen, the level of absorbance of
this compound between 230 and 400 nm is very high and detecting
any variation of absorbance in these conditions can be difficult.
This led us to follow the product formation from a minimum
wavelength of 400 nm, in order to be able to detect enzymatic
activity if it existed.
Taking into account that o-diphenols are oxidized by sodium
periodate [45], we spectrophotometrically monitored the oxida-
tion product of ellagic acid by registering the scan spectra of its o-
quinone with time (Fig. 2). In this assay, a substoichiometric
amount of sodium periodate was used to oxidize the compound
Fig. 1. A. Kinetic mechanism of the action of tyrosinase on ellagic acid [28,48]. under study. The decrease in absorbance with time (Fig. 2) shows
Em = met-tyrosinase, Ed = deoxy-tyrosinase, Eox = oxy-tyrosinase, EA = ellagic acid that this o-quinone is unstable and evolves to other products.
and QEA = o-quinone from ellagic acid. B. Reduction of QEA in the presence of In order to evaluate the ability of tyrosinase to act on ellagic acid
ascorbic acid (chronometric method). AH2 = ascorbic acid and A = dehydroascorbic
as a substrate, the scan spectra of this compound with time were
acid.
recorded in the presence of enzyme (Fig. 2 Inset). As can be seen,
the level of absorbance increased with time, meaning that ellagic
acid was being oxidized by tyrosinase. Note that the scan spectra of
ellagic acid in the presence of sodium periodate in default and
enzyme. However, the chemical structure of the target molecule
those corresponding to the action of tyrosinase on it are similar.
with hydroxyl groups in ortho position (Fig. 1A) led us to check its
The only difference between these spectra is the fact that the
possible action as enzyme substrate.
enzyme produces the o-quinone from ellagic acid gradually while
sodium periodate acts immediately, so the instability of the
3.1. Identification of ellagic acid as a tyrosinase substrate
generated o-quinone could be observed more easily in the latter
case. All enzymatic reactions show a dependence of the initial
In a previous work, our research group already identified ellagic
product formation rate on the substrate and the enzyme
acid as a substrate of tyrosinase [28], using oxymetric methods due
concentrations. Fig. 3 depicts the recordings of the action of
to the instability of the generated o-quinones and the high
tyrosinase on EA at 470 nm, the maximum absorbance wavelength
absorbance of the target molecule. However, in another extensive
of the ellagic acid oxidation product. As can be seen in Fig. 3, the
study using spectrophotometric methods that included a series of
initial rate depends on the enzyme (Fig. 3) and substrate (Fig. 3
phenolic compounds, the enzyme was not seen to act on this
Inset) concentrations, which can be regarded as typical behavior of
compound [19], which makes the actual role of ellagic acid in the
enzymatic reactions. Furthermore, we recorded the stability of
enzymatic reaction of tyrosinase controversial. Thus, in order to
ellagic acid in the absence of enzyme (Fig. 3, recording a) without
shed light on this duality we optimized the experimental
obtaining any variation in the absorbance, confirming that the
obtained oxidation product was only produced by the action of the
enzyme on ellagic acid. Fig. 3 also reveals that the product of the

0.3
0.12
d

0.08
A470

0.2 f
0.04
A470

a
0.00
0 200 400 600

time (s)
0.1

b a
0.0
0 100 200 300 400 500 600
time (s)

Fig. 3. Action of different concentrations of tyrosinase on ellagic acid. The

Fig. 2. Scan spectra of ellagic acid (a) and its oxidation product with time (b-k). The experimental conditions were: [EA]0 = 100 mM, DMSO (5%) and [E]0 (nM): (a) 0, (b)
experimental conditions were: [EA]0 = 280 mM, [NaIO4]0 = 100 mM and DMSO (5%). 285, (c) 425, (d) 570, (e) 710 and (f) 850. Inset: Action of tyrosinase on different
Recordings were made every 40 s. Inset: Scan spectra of the action of tyrosinase on concentrations of ellagic acid. The experimental conditions were: [E]0 = 425 nM,
ellagic acid with time. The experimental conditions were: [EA]0 = 280 mM, DMSO (5%) and [EA]0 (mM): (a) 10, (b) 20, (c) 40 and (d) 60.
[E]0 = 600 nM and DMSO (5%). Recordings were made every 40 s.

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In the presence of AH2, the measurable concentration of o-quinone

EA
at a given time is V Q
0 t minus the amount of o-quinone reduced by
the AH2 present initially:
EA
½Q EA  ¼ V Q
0 t  ½AH2 0 ð2Þ

If t is the time required for the enzyme to produce a

concentration of o-quinone equal to [AH2]0 then
EA

0 t ¼ ½AH2 0
VQ ð3Þ

and so
EA ½AH2 0
VQ ¼ ð4Þ
0 t
EA
In order to obtain the K EA Q
M and V max values for this compound, we
measured the lag period in the action of tyrosinase on different
concentrations of ellagic acid and in the presence of a fixed
concentration of ascorbic acid (Fig. 4 Inset). From the lag period,
EA
the V Q
0 values were calculated according to Eq. (4), which were
then represented versus [EA]0 (the initial concentration of ellagic
acid) and fitted by non linear regression to Eq. (10) through the
Fig. 4. Initial rate values calculated from the spectrophotometric recordings of the
action of tyrosinase on ellagic acid in the presence of ascorbic acid (chronometric Sigma Plot 9.0 program for Windows (Fig. 4) [44].
method) shown in Fig. 4 Inset versus the ellagic acid concentration. Inset: The mechanism proposed to explain the action of tyrosinase on
Spectrophotometric recordings of the action of tyrosinase on different concentra- ellagic acid is described in Fig. 1A [28,48]. Taking into account the
tions of ellagic acid in the presence of ascorbic acid (chronometric method). The approximation to the steady-state, the kinetic analysis of this
experimental conditions were: [AH2]0 = 35 mM, [E]0 = 450 nM, DMSO (5%) and [EA]0
mechanism provides the following equation for the initial o-
(mM): (a) 0.08, (b) 0.10, (c) 0.20, (d) 0.30, (e) 0.40 and (f) 0.60.
quinone formation rate:
EA
EA VQ
max ½EA0 ½O2 0
VQ
0 ¼ ð5Þ
K OS 2 K EA O2 EA
M þ K M ½EA0 þ K M ½O2 0 þ ½EA0 ½O2 0

enzymatic reaction evolves showing a very patent lag period. Note where
that the lag period decreases when the concentration of enzyme EA
EA
increases, as can be clearly seen in Fig. 3SI (see Supplementary VQ
max ¼ 2kcat ½E0 ð6Þ
material). This behavior might correspond to the coupling of a
second order chemical reaction of the oxidation product of ellagic
acid [46,47]. However, the variation of the substrate concentration k8
KO
S ¼
2
ð7Þ
produces a deviation from this behavior, as shown in Fig. 4SI of the k8
Supplementary material. These assays reveal the instability of the
enzymatic reaction product, and so, to characterize ellagic acid
EA
kinetically, as indicated below, a chronometric method was used, kcat
KO
M ¼
2
ð8Þ
thus avoiding the evolution of the o-quinone through the action of k8
ascorbic acid.

3.2. Kinetic characterization of ellagic acid as a tyrosinase substrate kcat

EA
K EA
M ¼ ð9Þ
k6
Having confirmed that ellagic acid is a substrate of the enzyme,
we proceeded to characterize it kinetically, using a chronometric As the initial concentration of molecular oxygen is saturating
assay due to the instability of its o-quinone and its tendency to ([O2]0 = 0.26 mM) and taking into account Eq. (8) and references
evolve to other products [42,43]. This method uses a small quantity [28,41], Eq. (5) is simplified to:
of ascorbic acid to reduce the o-quinones generated by the enzyme, EA
EA VQ
max ½EA0
until all the ascorbic acid is consumed and the reaction products VQ
0 ¼ ð10Þ
start to be accumulated in the medium. Fig. 1A depicts the K EA
M þ ½EA0
proposed mechanism of the action of tyrosinase on ellagic acid EA
The fitting of V Q versus [EA]0 to Eq. (10), and Eq. (6) gave a
and, in general, on o-diphenols [28,48], while Fig. 1B shows the 0

reduction of the o-quinone from this compound in the presence of M = 138
13 mM and a catalytic
Michaelis constant value of K EA
constant of kcat = 0.47
0.02 s1.
EA
ascorbic acid.
A lag period (t ) is induced in the chronometric assay when AH2
is present, the value of which depends on the initial AH2 3.3. Ellagic acid oxidation rate as function of pH
concentration. In the absence of AH2, the measurable concentra-
tion of o-quinone at a given time is The influence of pH on the action of tyrosinase on ellagic acid as
EA EA substrate was studied spectrophotometrically by the chronometric
½Q ¼ VQ
0 t ð1Þ method describe above. Fig. 5SI (see Supplementary material)
shows the typical bell shape behavior of tyrosinase substrates
which have a negative charge in the work zone, defining a

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Fig. 5. A. Action of ellagic acid on o-quinones and semiquinones generated in the oxidation of o-diphenols (L-dopa, DOMA and TBC) by tyrosinase. Tyr = tyrosinase, EA = ellagic
acid, QEA = o-quinone from ellagic acid, EA = free radical from ellagic acid, Q = semiquinone, O2 = superoxide anion, DOBA = 3,4-dihydroxybenzaldehide, o-TBQ = o-tert-
butylquinone. B. Enzymatic action of tyrosinase on o-diphenols (e) and non-enzymatic reactions (a)–(d) that explain the semiquinone, superoxide anion and hydrogen
peroxide formation in the melanogenesis pathway. D = o-diphenol, Q = o-quinone [49].

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significant pKa in the work zone (pKa1 = 6.3) [28]. The results (a)–(d) (Fig. 5B) described above and reveals the reaction of the
obtained using spectrophotometric methods (Fig. 4 and Fig. 5SI) ellagic acid with the semiquinones (it must be taken into account
are consistent with those previously obtained oxymetrically [28]. that the dopachrome absorbs at the measurement wavelength). As
the o-quinone from L-dopa, o-dopaquinone, is very unstable (since
3.4. Effect of ellagic acid on the melanogenesis pathway it evolves to dopachrome), reaction (d) (Fig. 5B) will be more
displaced towards the left but the ellagic acid reacting with the
Ellagic acid inhibits the melanin biosynthesis pathway, which is semiquinones will partially inhibit the melanogenesis pathway.
why it is used in the cosmetics industry [12,31–37]. We suggest The same occurs when DOMA is oxidized (Fig. 7SI C, Supplemen-
that this depigmenting effect is largely due to the antioxidant tary material)  as the o-quinone is unstable (it evolves to DOBA)
effect of this molecule, which may react with the o-quinones and the reaction of ellagic acid with the semiquinones is hindered and
semiquinones (Q) originated in the melanogenesis pathway [49]. the increase in absorbance is lower. Fig. 5A summarizes the
To test this hypothesis we used three o-diphenolic substrates, TBC, reaction sequences that may lead to the inhibition of the
L-dopa and DOMA, which, through enzymatic oxidation by melanogenesis process by ellagic acid. Note how the key step is
tyrosinase or chemical oxidation by sodium periodate, give rise the formation of o-quinone on the part of the enzyme (Fig. 5B, (e)).
to their corresponding o-quinones (Fig. 5A). In the melanin In the case of L-dopa, the o-quinone basically evolves to
biosynthesis pathway and, in general, in the oxidation of o- dopachrome and in the case of DOMA it evolves to DOBA, and
diphenols, free radicals and hydrogen peroxide are originated [49]. only in the case of TBC is the o-quinone fairly stable (Fig. 5A).
The antiradical or antioxidant power value (ARP), which is defined The reaction between the o-quinone and its o-diphenol (Fig. 5B
as the inverse of the EC50 (ratio of the antioxidant concentration (d)) can be assayed in the case of TBC because its o-quinone is quite
necessary to decrease the initial concentration of the ABTS to stable. Fig. 8SI (see Supplementary material) shows the spectra
50%), is 20 for ellagic acid, which is five times greater than that of obtained for the oxidation of TBC (variable) while keeping the
ascorbic acid, the antioxidant of reference [28,50,51]. In this way, concentrations of sodium periodate (in default) and ellagic acid
the ellagic acid could inhibit melanogenesis, trapping the fixed. In agreement with reaction (d) (Fig. 5B), when the quantity of
o-quinones, semiquinones and superoxide anions that are o-quinone (Q) is fixed, with more o-diphenol (D) in the medium,
produced in the reactions depicted in Fig. 5B [49], where (a) is the reaction is displaced towards the right, leading to the
the spontaneous oxidation of the o-diphenol, (b) and (c) describe formation of semiquinone, which interacts with the ellagic acid.
the reaction of the products of (a), (d) is a key step, whereby the Note that the concentration of sodium periodate is the same, and
semiquinones undergo a disproportion reaction and finally (e) is this is the factor determining the concentration of o-quinone. The
the enzymatic catalysis that generates o-quinones. The o-quinones higher the concentration of TBC, the sooner the same concentra-
and their o-diphenols are in equilibrium with the semiquinones tion of o-quinone is consumed. In Fig. 8SI Inset, Q and D were
through the reaction (d). Ellagic acid could react with the o- varied in order to keep the residual concentration of D constant
quinones and, in even a more reactive manner, with the after Q formation (see Supplementary material). In agreement
semiquinones, displacing the equilibrium (reaction d) towards with reaction (d) Fig. 5B, the higher the concentration of Q (more
the right, consuming o-quinones and inhibiting the melanogenesis sodium periodate), the higher the level of absorbance and the rate
process. of the reaction, since a high number of ellagic acid molecules are
The advantage of using TBC as o-diphenol in these experiments being oxidized. Thus, these experiments lend weight to the
is that, when oxidized by sodium periodate or by tyrosinase, it antioxidant character of ellagic acid and explain the inhibition of
produces an o-quinone that is practically stable. In these experi- the melanogenesis pathway fundamentally through its reaction
ments 600 nm was chosen as the wavelength to follow product with the semiquinones.
formation, because the oxidation product of ellagic acid absorbs at When TBC is oxidized by sodium periodate in default and then
this wavelength while the absorbance of o-TBQ (from TBC), ellagic acid is added, absorbance increases to become identical to
dopachrome (from L-dopa) and DOBA (from DOMA) is very low or that obtained for the oxidation of the mixture TBC + EA (with
null. [TBC]0 >> [EA]0). However, in the case of L-dopa and DOMA, there is
Fig. 6SI shows the recordings obtained after the enzymatic no increase in absorbance, the instantaneous formation of
oxidation of TBC by tyrosinase with time (see Supplementary o-quinones leads the system to rapidly evolve towards dopa-
material). Note that the absorbance of the o-quinone (o-TBQ) in the chrome or DOBA (data not shown). When o-quinones from L-dopa
600 nm zone is very low. However, Fig. 6SI Inset shows that when and DOMA are gradually produced by the enzyme, ellagic acid can
the above reaction is carried out in the presence of ellagic acid, this react with them and so, the formation of o-quinone from ellagic
compound is oxidized through the semiquinones originated in acid is observed (Fig. 7SI A and 7SI C, see Supplementary material).
reactions (a), (c) and (d) of Fig. 5B. The oxidation of ellagic acid as In conclusion, this work has demonstrated how ellagic acid acts
enzyme substrate should also be taken into account. as a substrate of tyrosinase. Although the enzyme has a high
A similar effect can be seen in Fig. 7SI B (see Supplementary affinity for this substrate (low value ofK EA
M ) its kinetic constant
material). The action of the enzyme on TBC produces its o- EA
(kcat ) is low. We also propose a mechanism to explain the inhibition
quinone (o-TBC), which reacts with the substrate (Fig. 5B (d))
of the melanogenesis pathway, which may occur because ellagic
producing semiquinones, which, in turn, react with the ellagic
acid acts as alternative substrate to L-tyrosine and L-dopa and as a
acid. By increasing the concentration of ellagic acid and keeping
result of its great antioxidant power, reacting with the o-quinones
the enzyme and TBC concentrations fixed, the formation rate of its
and semiquinones that are formed in the pathway. This first aspect
oxidation product increases, at the same time as the products
should be taken into consideration in the application of ellagic acid
evolves. Note that in these recordings the TBC concentration is
as a cosmetic ingredient due to the toxicity of o-quinones and their
much higher than the Michaelis constant (K M ).
capacity to alter the redox status of cells.
In the case of L-dopa (Fig. 7SI A, Supplementary material),
physiological substrate of the melanogenesis, when it is oxidized
Conflict of interest
enzimatically at a substrate concentration value of 5K M , the
product formation rate increases as the concentration of ellagic
The authors have no conflict of interest to declare.
acid increases. This result agrees with the sequence of reactions

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C.V. Ortiz-Ruiz et al. / Journal of Dermatological Science xxx (2015) xxx–xxx 7

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Spanish organizations: Projects 19545/PI/14, 19304/PI/14 and Tyrosinase-catalyzed hydroxylation of 4-hexylresorcinol, an antibrowning
19240/PI/14 (Fundacion Seneca, CARM, Murcia, Spain); Projects and depigmenting agent: a kinetic study, J. Agric. Food Chem. 63 (2015) 7032–
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