Methyl p-coumarate, a melanin formation inhibitor in B16 mouse melanoma cells

Article abstract of DOI:10.1016/j.bmc.2004.07.052

p-Coumaric acid and methyl p-coumarate inhibit the oxidation of l-tyrosine catalyzed by mushroom tyrosinase. However, both were oxidized as monophenol substrate analogues at an extremely slow rate. Methyl p-coumarate significantly suppressed the melanin formation in B16 mouse melanoma cells, whereas p-coumaric acid did not show this activity. p-Coumaric acid (4-hydroxycinnamic acid) and methyl p-coumarate (methyl 4-hydroxycinnamate) inhibit the oxidation of l-tyrosine catalyzed by mushroom tyrosinase. However, both were oxidized as monophenol substrate analogues at an extremely slow rate. This oxidation was significantly accelerated as soon as catalytic amounts (0.01 mM) of l-3,4-dihydroxyphenylalanine (l-DOPA) became available as a co-factor. Methyl p-coumarate significantly suppressed the melanin formation in B16 mouse melanoma cells, whereas p-coumaric acid did not show this activity.

Full text of DOI:10.1016/j.bmc.2004.07.052

Bioorganic & Medicinal Chemistry 12 (2004) 5349–5354
Methyl p-coumarate, a melanin formation inhibitor in B16
mouse melanoma cells
a
b
Isao Kubo,a, Ken-ichi Nihei and Kazuo Tsujimoto
*
aDepartment of Environmental Science, Policy and Management, University of California, Berkeley, CA 94720-3112, USA
bSchool of Materials Science, Japan Advanced Institute of Science and Technology (JAIST), 1-1 Asahidai, Tatsunokuchi,
Nomi-gun, Ishikawa 923-1292, Japan
Received 13 February 2004; revised 20 July 2004; accepted 24 July 2004
Available online 23 August 2004
In honor of Professor Tadao KamikawaÕs 70th birthday
Abstract—p-Coumaric acid (4-hydroxycinnamic acid) and methyl p-coumarate (methyl 4-hydroxycinnamate) inhibit the oxidation
of L-tyrosine catalyzed by mushroom tyrosinase. However, both were oxidized as monophenol substrate analogues at an extremely
slow rate. This oxidation was significantly accelerated as soon as catalytic amounts (0.01mM) of L-3,4-dihydroxyphenylalanine
(L-DOPA) became available as a co-factor. Methyl p-coumarate significantly suppressed the melanin formation in B16 mouse mel-
anoma cells, whereas p-coumaric acid did not show this activity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
p-coumarate (3) from the flower of Trixis michuacana
var longifolia (Compositae) were reported4 and hence,
Arbutin, hydroquinone-O-b-D-glucopyranoside (1), has
been widely used as a depigmenting agent in cosmetics
(Fig. 1).1 In our previous paper, arbutin was reported
to inhibit the oxidation of L-tyrosine catalyzed by mush-
room tyrosinase (monophenolase activity). However,
arbutin itself was oxidized as a monophenol substrate
at an extremely slow rate. This oxidation was acceler-
ated as soon as catalytic amounts (0.01mM) of L-3,4-
dihydroxyphenylalanine (L-DOPA) became available
as a co-factor.2 In addition, the recent report indicates
that o-phenylphenol, which is known as a depigmenting
agent, has little depigmenting action while its metabo-
lite, phenylhydroquinone is a potent depigmenting agent
affecting melanocytes.3 Hence, it may not be illogical
to assume that depigmenting action involves quinone
formation catalyzed by tyrosinase. This prompted us
to search for monophenol substrate analogues, which
may possess depigmenting activity. In previous papers,
the isolation of p-coumaric acid (2) from the leaves
OGlc
HO
1
R2
COOR3
R1O
2: R1 = R2 = R3 = H
3: R1 = R2 = H, R3 = CH3
9: R1 = R3 = H, R2 = OCH3
COOH
HO
8
CHO
OH
of
Panax
ginseng
(Araliaceae)
and
methyl
Keywords: Mushroom tyrosinase; Monophenolase; p-Coumaric acid;
Methyl p-coumarate; B16 mouse melanoma cell.
10
*
Figure 1. Chemical structures of p-coumaric acid and its related
phenylpropanoids.
Corresponding author. Tel.: +1 510 643 6303; fax: +1 510 643
0215; e-mail: ikubo@uclink.berkeley.edu
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.052

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I. Kubo et al. / Bioorg. Med. Chem. 12(2004) 5349–5354
these phenylpropanoids were re-examined if both exhi-
bit depigmenting activity.
2. Results and discussion
Tyrosinase (EC 1.14.18.1) contains a strongly coupled
binuclear copper active site and functions both as a
monophenolase and as an o-diphenolase.5,6 The enzy-
matic reaction can be detected by spectrophotometrical
(dopachrome formation) and polarographical (oxygen
consumption) methods. Since p-coumaric acid is classi-
fied as a monophenol analogue like arbutin, which has
a possibility to be oxidized by oxy-tyrosinase, the incu-
bation experiments were performed by measuring oxy-
gen consumption up to 20min (Fig. 2, line 1).
Although p-coumaric acid consumed a small amount
of oxygen (20lM), its UV–vis absorption spectrum
did not noticeable change up to 20min (data not illus-
trated). In addition, HPLC analysis confirmed that
p-coumaric acid remained without any change up to
20min as shown in Figure 3. However, oxygen con-
sumption was significantly accelerated as soon as a cat-
alytic amount (0.01mM) of L-DOPA became available
(Fig. 2, line 4). HPLC analysis indicates that p-coumaric
acid started being oxidized, presumably to caffeic acid
(4) at an extremely slow rate. This was consistent with
the result from co-injection with an authentic sample
of caffeic acid.7 As soon as a co-factor became available,
HPLC analysis demonstrated that p-coumaric acid
decreased and the peak ÔbÕ corresponding to caffeic acid
increased, and the changes in its UV–vis absorption
spectrum started in increasing absorbance at 260 and
360nm, characteristic of o-quinone (Figs. 4and 5 ). It
appears that p-coumaric acid was oxidized as a substrate
to caffeic acid, followed further oxidation to the corre-
sponding o-quinone (5) (Scheme 1).7,8 Similar to arbu-
tin,2 p-coumaric acid seems to possess tyrosinase
inhibitory activity by following the enzymatic reaction
at dopachrome formation. However, an observation
by spectrophotometer led to an unavoidable considera-
tion when these compounds could act as monophenol
Figure 3. HPLC analysis of the reaction medium with 0.5mM of p-
coumaric acid and tyrosinase. Sampling time was chosen at 0min (left),
10min (middle), and 20min (right), respectively. The HPLC operating
conditions was described in Section 3 in details.
Figure 4. HPLC analysis of the reaction medium with 0.5mM of p-
coumaric acid and tyrosinase. The catalytic amount of L-DOPA
(10lM) was added at 5min. Sampling time was chosen at 0min (left),
10min (middle), and 20min (right), respectively. The HPLC operating
conditions was described in Section 3 in details.
substrates and the corresponding products showed insig-
nificant absorption at 475nm. The result suggests that
p-coumaric acid eventually inhibited dopachrome for-
mation from L-tyrosine catalyzed by mushroom tyrosin-
ase if a co-factor is not available, but it does not inhibit
the oxidation of L-DOPA.
Subsequently, oxygen consumption of methyl p-couma-
rate (3) was also measured for comparison. The results
obtained are similar to those observed for p-coumaric
acid. Methyl p-coumarate did not significantly consume
Figure 2. Oxygen consumption of p-coumaric acid (1,4) and methyl p-
coumarate (2,3) in presence of tyrosinase. The concentration was
selected at 0.5mM. The arrow (›) indicated the time when catalytic
amount (10lM) of L-DOPA was added (3,4).

I. Kubo et al. / Bioorg. Med. Chem. 12(2004) 5349–5354
5351
sumably, methyl p-coumarate is oxidized to the corre-
sponding o-diol (6) and then to o-quinone (7) when a
co-factor is available (Scheme 1). The resulting quinone
is unstable molecules susceptible of undergoing a variety
of self-driven reactions. The result is consistent with the
previous report that methyl p-coumarate did not inhibit
the oxidation of L-DOPA catalyzed by mushroom tyro-
sinase.4 However, the slow enzymatic oxidation of
methyl p-coumarate possibly indicates that this com-
pound coordinates to one copper like a monophenol
substrate analogue and positions over the binuclear ac-
tive site competitively.
Oxygen consumption of dihydrocoumaric acid (8) and
ferulic acid (9) were also monitored in the presence of
tyrosinase for comparison. In the case of
dihydrocoumaric acid, it consumed oxygen immediately
even without a co-factor (Fig. 7, inset). In the UV–vis
absorption spectrum, the changes started with increas-
ing absorbance at 260, 280, 330, 400, and 470nm as
shown in Figure 7. Evidently, dihydrocoumaric acid
was easily oxidized as a substrate. Its carboxylate is no
longer conjugated into the aromatic ring indicating that
this conjugation is important to elicit the inhibitory
property as a substrate analogue toward met-tyrosinase.
The structure of 8 resembles p-cresol, which has been
frequently employed as an experimental substrate.9,10
In contrast, ferulic acid hardly consumed oxygen and
its UV–vis absorption spectrum was inconsequential
change (data not illustrated). The results obtained indi-
cate that this phenolic acid may not be easy to bind with
the active site because of steric hindrance by an adjacent
methoxy group.
Figure 5. Consecutive spectra obtained in the oxidation of 0.5mM of
p-coumaric acid by tyrosinase for 20min. The catalytic amount of L-
DOPA (10lM) was added at 5min. Scan speed was at 2min intervals
for 30s. The arrows (›) designate the evolution of the peak.
HO
HO
COOH
O
O
COOH
2
3
5
7
4
6
HO
HO
COOCH3
O
O
COOCH3
Scheme 1. Oxidation of p-coumaric acid or methyl p-coumarate to
corresponding compounds by tyrosinase.
oxygen up to 20min as shown in Figure 2. The UV–vis
absorption spectrum did not change up to 20min (data
not illustrated). However, as soon as a catalytic amount
(0.01mM) of L-DOPA became available, oxygen con-
sumption was accelerated but this rate was slower than
that by p-coumaric acid. Moreover, addition of
L-DOPA induced UV–vis spectrum change (Fig. 6). Pre-
The discussion previously reported4 was mainly on the
basis of the experiment using L-DOPA as a substrate.
Therefore, the activity described is o-diphenolase inhib-
itory activity of mushroom tyrosinase. It should be
noted that the lag time is known for the oxidation of
monophenolic substrates such as L-tyrosine to L-DOPA.
This lag can be shortened or abolished by the presence
Figure 6. Consecutive spectra obtained in the oxidation of 0.5mM of
methyl p-coumarate by tyrosinase for 20min. The catalytic amount of
L-DOPA (10lM) was added at 5min. Scan speed was at 2min intervals
for 30s. The arrows (›) designate the evolution of the peak.
Figure 7. Consecutive spectra obtained in the oxidation of 0.5mM of
dihydrocoumaric acid by tyrosinase for 20min. The arrows (›)
designate the evolution of the peak. The inset shows oxygen
consumption of dihydrocoumaric acid (0.5mM) by tyrosinase.

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I. Kubo et al. / Bioorg. Med. Chem. 12(2004) 5349–5354
Figure 8. Inhibitory effect on the rate of dopachrome formation from
L-tyrosine (0.125mM) catalyzed by tyrosinase. The concentration of
methyl p-coumarate (1), p-coumaric acid (2), 4-methoxycinnamic acid
(3), cinnamic acid (4), and ferulic acid (5) were 0.25mM. Curve 6
represents the oxygen consumption without inhibitor.
Figure 9. Inhibitory effect on the rate of oxygen uptake by L-tyrosine
(0.125mM) catalyzed by tyrosinase. The concentration of methyl p-
coumarate (1), p-coumaric acid (2), 4-methoxycinnamic acid (3),
cinnamic acid (4), and ferulic acid (5) were 0.25mM. Curve 6
represents the oxygen consumption without inhibitor.
The inhibition activity exerted by p-coumaric acid and
methyl p-coumarate could be on the basis of the
assumption that both bind as a monophenol substrate
analogue (M) to the met-form of tyrosinase (Emet), and
the resulting EmetM complex is inactive. This postulate
is consistent with the fact that monophenols can only re-
act with the oxy-form of tyrosinase in an ordered
sequential mechanism.13 Thus, the EmetM is the dead-
end complex.5 However, the commercial tyrosinase is
known to contain a small amount of the oxy-form
(Eoxy). If this is so, the Eoxy would start the turnover
by acting on M, which is hydroxylated to generate
EmetD. At this point the enzyme may oxidize o-diphenol
(D) to the corresponding o-quinone (Q) generating
Edeoxy, or release D producing Emet as illustrated in
Scheme 2. In the case of p-coumaric acid, D is caffeic
acid, which is known to act as a co-factor. If D is
released, it can be recycled to the reaction medium
through intramolecular cyclation and further redox
steps. Similar to arbutin,2 both p-coumaric acid and its
methylester were oxidized as monophenol substrates to
the corresponding o-diols at an extremely slow rate.
Once oxidized to o-diols, they were further oxidized to
the corresponding o-quinone rather quickly.
of reducing agents (co-factors), especially o-diphenols
(such as L-DOPA and caffeic acid). On the other hand,
inhibitors for monophenolase activity are known to
extend the lag phase. Thus, inhibitory effects on the
enzymatic oxidation of L-tyrosine by several coumaric
acid derivatives and their benzoic acid counterparts were
compared by spectrophotometric and polarographic
methods. Interestingly, p-coumaric acid and methyl
p-coumarate significantly lengthened this lag phase,
indicating that both act as effective inhibitor with regard
to dopachrome formation. Those inhibitory effects can
be placed in decreasing order methyl p-coumarate >
p-coumaric acid > 4-methoxycinnamic acid = cinnamic
acid, at the result as shown in Figure 8. In connection
with cinnamic acid, its methyl ester was noted to be an
inhibitor with an IC50 of 2.4mM without being oxidized,
indicating that the general presence of phenolic hydroxyl
group is not a prerequisite for the inhibition. Although
this activity is weak, the result may provide a hint to
search for tyrosinase inhibitors without being oxidized.
However, at polarographic observation, p-coumaric
acid showed gradually slow acceleration after the reac-
tion was started (Fig. 9). These differences between
spectroscopic and polarographic experiments were pos-
sibly originated by facile oxidation property of p-cou-
maric acid, and can be considered from the conversion
of met-tyrosinase to oxy-tyrosinase by a diphenol that
its product emerged in the assay system.11 However,
methyl p-coumarate inhibited effectively the enzymatic
oxidation of L-tyrosine, which was confirmed spectro-
photomerically as well as polarographically, although
this methyl ester is slightly oxidized by generated oxy-
tyrosinase. Notably, methyl coumarate was found to
be a most potent monophenolase inhibitor in the cou-
maric acids tested. Hence, esterification of p-coumaric
acid resulted in increasing activity. In the case of dipheno-
lase inhibitory activity, esterification of the carboxylic
group decreased the inhibitory strength,12 and more
importantly, the ester is no longer an inhibitor but a
substrate.
Subsequently, the effects of p-coumaric acid and methyl
p-coumarate on melanin formation in B16 mouse mel-
anoma cells were examined. Arbutin was also tested
Eoxy
M
Eoxy
M
O2
Edeoxy
Q
M
Emet
D
Emet
Emet M
D
Dead-end pathway
Scheme 2. Action mechanism of tyrosinase on monophenol substrates.

I. Kubo et al. / Bioorg. Med. Chem. 12(2004) 5349–5354
5353
site by increasing its acidity. This assumption can be
applicable to explain the inhibitory behaviors by p-cou-
maric acid and methyl p-coumarate since both possess
the conjugated carboxyl group. On the other hand, di-
rect interaction with oxy-tyrosinase has been observed
spectroscopically in the absence of the phenolic sub-
strate, and coordination of the carboxylate group to
one copper of the binuclear site was suggested.13 Methyl
p-coumarate possesses an affinity toward active site by
its phenolic hydroxyl group whereas p-coumaric acid
binds to copper site for being two moieties, carboxylate
and phenolic hydroxyl. The latter bidentate coordina-
tion is possibly attributable to the inhibitory activity
of p-coumaric acid less than that of methyl p-coumarate.
Since dihydrocoumaric acid is oxidized easily by tyrosin-
ase, a valance of the electron density and structural fea-
ture by phenolic hydroxyl and styrene is important in
order to conserve high affinity on active site with pre-
venting its easy dissociation and oxidation.
Figure 10. Inhibitory effect on melanin formation using B16 mouse
melanoma cell. Inhibition (%) was estimated by comparison of
absorbance at 475nm with drug-free experiment. The concentrations
of arbutin (1) and p-coumaric acid (2) were 0.1mM, respectively. The
concentration of methyl p-coumarate (3) was 0.01mM.
The results obtained so far may indicate a hint to their
interaction with the enzyme but this remains unclear
since the structure of mushroom tyrosinase used for this
study has not yet been established. However, safety is a
primary consideration for tyrosinase inhibitors, espe-
cially for those in cosmetic products, which may be uti-
lized in unregulated quantities on a regular basis.
Compared to other depigmenting agents, p-coumaric
acid and its methyl ester are almost colorless and odor-
less, and therefore are superior cosmetic additive, partic-
ularly to inhibit the oxidation of L-tyrosine.
Interestingly, one molecule of p-coumaric acid scavenges
one molecule of 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical at an extremely slow rate, while methyl p-couma-
rate does not have this scavenging activity. In addition,
substituted derivatives of cinnamic acid are predominant
phenolic acids present in many edible plants.18 The phen-
ylpropanoids play important roles in UV protectants.19
for comparison. The melanin production was signifi-
cantly suppressed when the cells were cultured with
10lM of methyl p-coumarate (Fig. 10). This cellular
depigmenting activity was about 10-fold more potent
than that of arbutin. The precise explanation for the
depigmenting activity remains to be clarified but it likely
acts similar to arbutin.14 Notably, p-coumaric acid did
not suppress the melanin formation in cultured melano-
cytes at 100lM. This difference likely comes from the
fact that deprotonated p-coumaric acid may not enter
the cells. The cellular depigmenting activity of methyl
p-coumarate was observed without inhibiting cell
growth. The possibility that the depigmenting activity
observed might be caused in part by its cytotoxicity15
may not be entirely ruled out. However, the cytotoxic
activity of methyl p-coumarate against B16 mouse mel-
anoma cells and several solid tumor cell lines was
observed at 500lM. Hence, the depigmenting activity
of the cells by methyl coumarate may be expected at
dose levels causing no cytotoxicity.
3. Experimental
3.1. Materials
It is not clear if the inhibition of the melanin formation
is caused either methyl p-coumarate inhibits tyrosinase
or the oxidized metabolites is involved in the melano-
cyte. The latter case is more like for methyl p-coumarate
because chamaecin (2-hydroxy-4-isopropylbenzalde-
hyde) (10) has recently been reported as a potent tyro-
sinase inhibitor16 but it did not suppress the melanin
formation in B16 mouse melanoma cells at all at the
concentration of 100lM. It should be noted, however,
that the relevance of the results of in vitro experiments
in simplified systems to the cellular depigmenting activ-
ity need to be carefully considered. The further study is
currently under investigation.
Arbutin was available from our previous work20 and
also purchased from Sigma Chemical Co. (St. Louis,
MO). Methyl p-coumarate was from our previous
work.4 Caffeic acid, p-coumaric acid, dimethylsulfoxide
(DMSO), 4-methoxycinnamic acid, and trifluoroacetic
acid (TFA) were purchased from Aldrich Chemical
Co. (Milwaukee, WI). Cinnamic acid, dihydrocoumaric
acid, ferulic acid, and mushroom tyrosinase were pur-
chased from Sigma Chemical Co. Acetonitrile (MeCN)
for HPLC was purchased from Alfa Aesar Chemical
Co. (Ward Hill, MA). 4-tert-Butylcatechol was pur-
chased from Fluka Chemical Co. (Milwaukee, WI).
Mushroom tyrosinase was purified by the procedure as
previously reported.21
On the basis of an Huckel theory calculation for tyro-
¨
sinase active site model, it has been shown that ioniza-
tion of hydroxyl group of phenolic compound is a
crucial step in its interaction with positively charged
copper of the active site in monophenolase reaction.17
Accordingly, it is assumed that an electron-withdrawing
group increased the phenolÕs affinity toward an active
3.2. Enzyme assay
The assay was performed as previously reported
with some modifications.22 Oxygen consumption was

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I. Kubo et al. / Bioorg. Med. Chem. 12(2004) 5349–5354
measured by Clark style electrode and an oxygraph
equipped (YSI-53) with a water-jacketed chamber (Yel-
low Springs Instrument Co., Yellow Springs, OH) main-
tained at 30°C. Calibration of an oxygen electrode was
performed by using 4-tert-butylcatechol and excess
tyrosinase according to the previous report.23 Absorp-
tion measurements were recorded by a Spectra MAX
plus spectrophotometer (Molecular Device, Sunnyvale,
CA) at 30°C. All samples were first dissolved in DMSO
at 150mM and used for the experiment with dilution.
The final concentration of DMSO in the test solution
is always 3.3%. For obtaining data of absorbance incre-
ment at 475nm and oxygen consumption, 0.1mL of a
inhibitor solution was mixed with 0.6mL of 0.25M so-
dium phosphate buffer (pH6.8) and 2.2mL of water.
Then, 0.1mL of the 0.05M sodium phosphate buffer
solution (pH6.8) of tyrosinase (1.0lg/mL) was added.
When L-tyrosine was used as monophenol substrate, in-
stead of 2.2mL of water, 0.3mL of 1.25mM L-tyrosine
solution and 1.9mL of water were added. In the exper-
iment by using co-factor, 6lL of DOPA (5mM) was
added at 5min after enzymatic reaction started. Scan
speed was selected for 2min with 30s intervals for
obtaining consecutive spectra (220–500nm).
mation was estimated by the comparison of absorbance
at 475nm with inhibitor-free experiment. The cytotoxic-
ity assay was performed as previously described.24
Acknowledgements
The authors are grateful to Ms. I. Hori for performing
some assays at an earlier stage of the work and Dr. H.
Ushijima for carrying out the cytotoxicity assay.
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HPLC analysis was performed as previously reported
with slight modification.2 The analysis was performed
on an EYELA LPG-1000 with an EYELA UV-7000
detector (EYELA, Tokyo) and Capcell Pak C-18 col-
umn (5lm, 4.6 · 250mm, Shiseido, Tokyo). In general,
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mim, detection; UV at 280nm, injected amount; 10lL
from 3mL assay system. The retention time (tR) of
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The cell culture and melanin assay were performed as
previously reported with slight modification.14 Briefly,
B16 mouse melanoma cells were donated from Institute
of Development, Aging and Cancer, Tohoku University,
Japan, were cultured in RPMI1640 medium in a humid-
ified atmosphere with 10% CO2 at 37°C. Subcultures of
cells were plated at a density of 2.0 · 105 cells/well.
Approximately 24h later, the sample was added, and
cells were harvested for 72h after drug addition. All
samples were dissolved in DMSO and the final DMSO
concentration was 0.1% in all experiments. The har-
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solution containing 10% DMSO, which were sonicated
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