Evaluation of in vitro and in vivo anti-melanogenic activity of a newly synthesized strong tyrosinase inhibitor (E)-3-(2,4 dihydroxybenzylidene) pyrrolidine-2,5-dione (3-DBP)

Article abstract of DOI:10.1016/j.bbagen.2012.03.018

Background: Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). Methods: The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the effects of 3-DBP in vivo. Results: The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC₅₀ = 0.53 μM), which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC₅₀ = 8.2 μM). When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value) of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated mice that were not treated with 3-DBP. General significance: The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.

Full text of DOI:10.1016/j.bbagen.2012.03.018

Biochimica et Biophysica Acta 1820 (2012) 962–969
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbagen
Evaluation of in vitro and in vivo anti-melanogenic activity
of a newly synthesized strong tyrosinase inhibitor
(E)-3-(2,4 dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP)
Ki Wung Chung a,b, Yun Jung Park c, Yeon Ja Choi a,b, Min Hi Park a,b, Young Mi Ha a,b, Yohei Uehara a,b
,
Jung Hyun Yoon b, Pusoon Chun d, Hyung Ryong Moon c, , Hae Young Chung a,b,⁎⁎
⁎
a
MRCA, Department of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
Laboratory of Medicinal Chemistry, College of Pharmacy, Pusan National University, Busan 609-735, Republic of Korea
College of Pharmacy, Inje University, Gimhae, Gyeongnam 621-749, Republic of Korea
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Background: Tyrosinase inhibitors have become increasingly important because of their ability to inhibit the
synthesis of the pigment melanin. A search for new agents with strong tyrosinase activity led to the synthesis
of the tyrosinase inhibitor (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP).
Methods: The inhibitory effect of 3-DBP on tyrosinase activity and melanin production was examined in murine
melanoma B16F10 cells. Additional experiments were performed using HRM2 hairless mice to demonstrate the
effects of 3-DBP in vivo.
Received 17 February 2012
Received in revised form 23 March 2012
Accepted 26 March 2012
Available online 3 April 2012
Keywords:
Results: The novel compound, 3-DBP, showed an inhibitory effect against mushroom tyrosinase (IC50=0.53 μM),
which indicated that it was more potent than the well-known tyrosinase inhibitor kojic acid (IC50=8.2 μM).
When tested in B16F10 melanoma cells treated with α-melanocyte stimulating hormone (α-MSH), 3-DBP also
inhibited murine tyrosinase activity, which in turn induced a decrease in melanin production in these cells. The
anti-melanogenic effect of 3-DBP was further verified in HRM2 hairless mice. The skin-whitening index (L value)
of HRM2 hairless mice treated with 3-DBP before irradiation with UVB was greater than that of UVB-irradiated
mice that were not treated with 3-DBP.
Melanogenesis
(E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,
5-dione (3-DBP)
Tyrosinase inhibitor
HRM2 hairless mouse
General significance: The newly synthesized 3-DBP has a potent inhibitory effect on tyrosinase. In addition to an in
vitro investigation of the effects of 3-DBP on tyrosinase, in vivo studies using an HRM2 hairless mouse model
demonstrated the anti-melanogenic potency of 3-DBP. Our newly synthesized 3-DBP showed efficient tyrosinase
inhibitory effect in vivo and in vitro. Our finding suggests that 3-DBP can be an effective skin-whitening agent.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
concerns have prevented the commercialization of most of these
inhibitors. There are many tyrosinase inhibitors such as hydroqui-
Tyrosinase inhibitors have become increasingly important be-
cause they inhibit synthesis of the pigment melanin [1,2]. They are
important in various fields, including the cosmetic industry [3,4], as
medications [5], and in the food industry [6,7] for their ability to
decrease pigmentation. Although melanin provides effective protec-
tion against harmful ultraviolet radiation, abnormal melanin accu-
mulation can cause esthetic problems such as melasma, freckles, and
senile lentigines [8,9]. Although diverse tyrosinase inhibitors can be
obtained from both naturally occurring and synthetic sources, safety
none [10], ascorbic acid derivatives [11], azeleic acid [12], retinoids
[13], arbutin [14], and kojic acid [6]. However, some well-known
whitening agents, such as hydroquinone and kojic acid, are considered
as harmful agents because of their undesirable side effects such as
cytotoxicity, skin cancer, and dermatitis. Therefore, safe and effective
whitening agents are needed. Newly synthesized agents with a fully
different structure moiety may address these issues and serve as a
better solution for treating dermatological disorders associated with
skin pigmentation.
Melanin is the primary agent responsible for skin color and plays
an important role in preventing skin injury under normal physiolog-
ical conditions. The photochemical properties of melanin make it an
excellent photo-protectant. It absorbs harmful UV-radiation, trans-
forming this energy into harmless heat through a process referred to
as ‘ultrafast internal conversion’ [15]. Despite its advantages, melanin
can also cause abnormal pigmentations such as freckles, age spots,
⁎
Correspondence author. Tel.: +82 51 510 2815; fax: +82 51 513 6754.
⁎⁎ Correspondence to: H.Y. Chung, Molecular Inflammation Research Center for Aging
Intervention (MRCA), College of Pharmacy, Pusan National University, Kumjeong-Gu,
Busan 609-735, Republic of Korea. Tel.: +82 51 510 2814; fax: +82 51 518 2821.
E-mail addresses: mhr108@pusan.ac.kr (H.R. Moon), hyjung@pusan.ac.kr
(H.Y. Chung).
0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagen.2012.03.018

K.W. Chung et al. / Biochimica et Biophysica Acta 1820 (2012) 962–969
963
and melanoma, which can be serious skin problems. Therefore,
modulating melanogenesis is an important strategy for treating
abnormal skin pigmentation.
2.2. Synthesis of (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione
(3-DBP)
Melanogenesis is significantly affected by tyrosinase, which cata-
lyzes the hydroxylation of L-tyrosine to 3,4-dihydroxyphenylalanine
(L-DOPA) and L-DOPA to dopaquinone [16]. Tyrosinase is a multi-
functional type-3 binuclear copper-containing enzyme; the copper-
containing site plays an important role in the activity of tyrosinase
[17]. The best activator and substrate for human tyrosinase is L-DOPA
and not L-tyrosine [18,19]. The substrate L-tyrosine and the activator
L-DOPA were shown to have separate binding sites on tyrosinase [20].
Tyrosinase is a rate-limiting enzyme that catalysis steps in melanin
biosynthesis and causes the abnormal accumulation of melanin pig-
ments under unregulated conditions [21,22]. Because of its key role
in melanogenesis, tyrosinase is an attractive target in the search for
various types of depigmenting agents [23]. Thus, the present study
focused on inhibition of tyrosinase and melanin production; to this
end, a novel compound with a new type of scaffold was synthesized.
The purpose of this study was to identify and characterize a
new tyrosinase inhibitor. In a search for tyrosinase inhibitors from
natural sources and synthesized compounds, we synthesized the
novel compound (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-
dione (3-DBP, Fig. 1a) containing a pyrrolidine-2,5-dione skeleton
with powerful tyrosinase inhibitory effects. 3-DBP has not been
synthesized previously or used as a potent tyrosinase inhibitor.
The inhibitory effects of 3-DBP on murine tyrosinase activity and
melanogenesis were evaluated using an in vitro model with B16F10
mouse melanoma cells. Moreover, using an HRM2 hairless in vivo
mouse model, 3-DBP was identified to be a powerful skin-whitening
agent that may be used for treating skin hyperpigmentation without
perceptible cytotoxicity.
A suspension of 2,4-dihydroxybenzaldehyde (100 mg, 0.72 mmol)
and triphenyl phosphoranylidene succinimide (260 mg, 0.72 mmol)
in MeOH (5 mL) was refluxed. Before reaching the boiling point of
methanol, the reaction mixture became a clear solution. After cooling
the solution to room temperature, precipitates were filtered through a
Buchner funnel. The filter cake was washed with methanol, methylene
chloride, and water to remove the remaining starting materials, 2,4-
dihydroxybenzaldehyde and triphenylphosphoranylidene succini-
mide, and obtain the final product.
The solid product was very pale and brown in color; the reaction
time was 24 h, the yield was 82%, and the melting point was >300 °C.
1H NMR (500 MHz, DMSO-d6) showed peaks at δ 11.18 (s, 1H, NH),
10.04 (s, 1H, OH), 9.92 (s, 1H, OH), 7.65 (s, 1H, vinyl H), 7.28 (d, 1H,
J=8.5 Hz, 6′-H), 6.37 (d, 1H, J=2.0 Hz, 3′-H), 6.31 (dd, 1H, J=2.0,
8.5 Hz, 5′-H), 3.50 (s, 2H, CH2); 13C NMR (100 MHz, DMSO-d6)
showed peaks at δ 176.8 (C5), 173.2 (C2), 161.3 (C4′), 159.5 (C2′),
131.0 (benzyl C), 127.2 (C6′), 121.2 (C3), 113.5 (C1′), 108.4 (C5′), 103.0
(C3′), 35.5 (C4); low-resolution mass spectrometry-electrospray ioniza-
tion (LRMS–ES) m/z, expected: 218 (M–H)−, found: 218 (M–H)−
.
2.3. Cell culture system
Murine melanoma B16F10 cells were obtained from the American
Type Culture Collection (Manassas, VA, USA). Cells were cultured in
Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal
bovine serum (FBS, Gibco, NY, USA), and penicillin/streptomycin
(100 IU/50 μg/mL) in a humidified atmosphere containing 5% CO2 in
air at 37 °C. B16F10 cells were cultured in 24-well plates for cell
viability (MTT) assay and a 60π dish for a melanin content assay and
tyrosinase activity assay. All experiments were performed at least 3
times to ensure reproducibility.
2. Materials and methods
2.1. Materials
2.4. Cell viability assay
3-DBP containing a pyrrolidine-dione moiety was synthesized
using a Wittig reaction in our laboratory. Mushroom tyrosinase,
L-tyrosine, α-melanocyte stimulating hormone (α-MSH), and other
chemical reagents were purchased from Sigma (St. Louis, MO, USA).
Antibodies against tyrosinase, microphthalmia-associated transcrip-
tion factor (MITF), and β-actin were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA).
The cell viability assay was carried out using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma). Next, 5×104 cells
were plated in each well of a 24-well plate. After the cells were treated
with 3-DBP at concentrations ranging from 10 to 200 μM for 24 h,
MTT solutions were added and the insoluble derivative formed by
cellular dehydrogenase was solubilized in a mixture of ethanol and
Fig. 1. Rationale for the design and synthesis of (E)-3-(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP). (a) Chemical structure of 3-DBP and explanation of the rationale
for design of the desired compound. (b) Brief scheme of the synthetic process of 3-DBP. Reagents and conditions were as follows: a. anhydrous acetone, reflux, 1 h, 92%; b. methanol,
reflux, 24 h, 82%.

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dimethylsulfoxide (EtOH–DMSO, 1:1 mixture); the absorbance of each
well was determined at 560 nm using a microplate reader.
4 °C overnight, followed by incubation with horseradish peroxidase-
conjugated anti-mouse antibody (Santa Cruz, 1:10,000), an anti-
rabbit antibody (Santa Cruz, 1:10,000), or an anti-goat antibody
(Santa Cruz, 1:10,000) at 25 °C for 1 h. Antibody labeling was detected
using West-zol Plus and chemiluminescence FluorchemTMSP (Alpha
Innotech Corporation, San Leandro, CA, USA). Pre-stained protein
markers were used for molecular weight determination.
2.5. Measurement of mushroom tyrosinase activity
To evaluate the inhibitory action of 3-DBP on tyrosinase, tyrosinase
isolated from mushrooms was utilized as described previously with
some modifications [25]. Briefly, 20 μL of aqueous solution of
mushroom tyrosinase (1000 units) was added to a 96-well microplate
with 200 μL of a reaction mixture containing 1 mM L-tyrosine solution,
50 mM phosphate buffer (pH 6.5), and the test material 3-DBP (0.5
to 10 μM). The assay mixture was incubated at 25 °C for 30 min.
Following incubation, the amount of dopachrome produced in the
reaction mixture was determined spectrophotometrically at 492 nm
2.9. Determinations of depigmenting activity in HRM2 hairless mouse
The in vivo depigmenting efficacy of 3-DBP was assessed through
animal experiments that were performed in accordance with the
guidelines for animal experimentation of Pusan National University.
Six-week-old male HRM-2 melanin-possessing hairless mice were
obtained from Hoshino Laboratory Animals (Yashino, Saitama, Japan)
and housed in a controlled room (23 °C 1 °C, 55 5% humidity,
12-h light/dark cycle) with ad libitum access to water and standard
laboratory diet. After an acclimation period (2 weeks), mice were
randomly divided into 5 groups of 6 animals. 3-DBP was prepared at 3
concentrations (0.4, 2, and 10 μM) in a solution of propylene glycol
and ethanol (3:7). Control solution and 3-DBP-dissolved solution
(200 μL) were topically applied to a designated site (3 cm×3 cm) on
the dorsal skin of the animal once per day. Animals were exposed
to UVB in a CROSSLINKER (BEX-800, Ultra-Lum, Inc., Claremont, CA,
USA) at 150 mJ/cm2. Colors of skin sites were measured using a CR-10
spectrophotometer (Konica Minolta Sensing, Inc., Sakai, Osaka, Japan)
in which the colors are described by L*, a*, and b* values according to
the Commission Internationale de l'Eclairage color system.
(OD492) in a microplate reader. The inhibitory concentration-50 (IC50
)
is the concentration of a substance that inhibits a standard response by
50%. To determine the inhibitory mechanism of 3-DBP, a tyrosinase
kinetic assay was carried out. Various concentrations of L-tyrosine
(16, 8, 4, 2, and 1 mM) were used for the inhibition assay. After
examination, the reciprocal of each value was calculated to construct
Lineweaver–Burk plots. The plot shows the inverse of reaction velocity
(1/V) versus the inverse of substrate concentration (1/[S]). On the
basis of the point of convergence of lines on the plot, an inhibitory
mechanism could be determined.
2.6. Determination of melanin content
The amount of melanin present was used as an index of
melanogenesis in the current study. Briefly, B16 cells were plated on
a 60π-dish and incubated in the presence or absence of 100 μM
α-MSH. Cells were then incubated for 48 h with or without 3-DBP at
concentrations ranging from 10 to 100 μM. After washing twice with
PBS, samples were dissolved in 500 μL of 1 N NaOH. The samples were
incubated at 60 °C for 1 h and mixed to solubilize the melanin. The
absorbance at 405 nm was compared with that derived from a
standard curve of synthetic melanin.
2.10. Fontana–Masson staining
Skins were fixed in 4% paraformaldehyde overnight at room
temperature and stained for melanin by using a Fontana–Masson
staining kit from American Mastertech, Inc. (Lodi, CA, USA) according
to the manufacturer's instructions. Briefly, sliced skins were stained
with ammoniacal silver solution for 60 min at 60 °C followed by
incubation in 0.1% gold chloride and then in 5% sodium thiosulfate.
2.7. Measurements of cellular tyrosinase activity
2.11. Statistical analysis
Tyrosinase activity in B16F10 cells was examined by measuring
the rate of oxidation of L-DOPA. Cells were plated in 60π-well dishes
at a density of 5×104 cells/mL. B16 cells were incubated in the
presence or absence of 100 μM α-MSH and then treated for 48 h with
various concentrations (10–100 μM) of 3-DBP. The cells were lysed in
500 μL of 50 mM sodium phosphate buffer (pH 6.8) containing 25 μL
of 1% Triton X-100 and 25 μL of 0.1 mM phenylmethyl-sulfonyl
fluoride and then frozen at −80 °C for 30 min. After thawing and
mixing, cellular extracts were clarified by centrifuging the samples
at 12,000×g for 30 min at 4 °C. The supernatant (80 μL) and 20 μL
of L-DOPA (2 mg/mL) were placed in a 96-well plate, and the
absorbance at 492 nm was read every 10 min for 1 h at 37 °C using
an ELISA plate reader.
Tyrosinase activity is expressed as a percentage of activity on
the basis of the formula [(A·100)/B], where A=OD492 with a test
sample and B=OD492 without a test sample. Values are presented as
means SEM (Fig. 2a, n=6; Fig. 2b, n=6; Fig. 2c, n=5; Fig. 3a,
n=6; Fig. 3b, n=5; Fig. 3c, n=5, and Fig. 4b and c, n=6 samples per
group). Analysis of variance (ANOVA) was used to analyze differences
among all groups. Differences in the means of individual groups
were assessed using the Fischer's protected least significant differ-
ence post hoc test. Values of Pb0.05 were considered statistically
significant.
3. Results
2.8. Western blotting
3.1. Design and synthesis of 3-DBP
Preparation of cell lysates was carried out as described previously,
with some modifications [25]. Cell lysates (20 μg of protein each)
were boiled for 5 min in gel-loading buffer (0.125 M Tris–HCl, pH 6.8,
4% SDS, 10% 2-mercaptoethanol, and 0.2% bromophenol blue) at a
1:1 ratio. Total protein equivalents for each sample were separated
using sodium dodecyl sulfate–polyacrylamide gel electrophoresis
on 10% acrylamide gels and transferred to polyvinylidene fluoride
(PVDF) membranes. Membranes were immediately placed into
blocking buffer (5% non-fat milk) in 10 mM Tris, pH 7.5, 100 mM
NaCl, and 0.1% Tween 20. Blots were blocked at room temperature for
1 h. Membranes were incubated with specific primary antibodies at
3-DBP was designed by combining the structure characteristics of
resorcinol and pyrrolidine-2,5-dione into 1 compound. 3-DBP (Fig. 1a)
was prepared via the Wittig reaction between 2,4-dihydroxybenzaldehyde
and triphenylphosphorylidenyl succinimide, which was synthesized
from a Michael addition reaction of triphenylphosphine to maleimide
(Fig. 1b). The Wittig reaction proceeded smoothly under reflux con-
ditions in methanol and yielded 3-DBP, a very pale, brownish solid, with
an 82% yield. The isomer with the (E)-configuration was exclusively
obtained from filtration of the precipitates generated from the Wittig
reaction. The structure of 3-DBP was determined using 1H and 13C NMR

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Fig. 2. Effects of 3-DBP on mushroom tyrosinase activity and determination of the inhibitory mechanism in vitro. (a) Various concentrations of 3-DBP (0.5–10 μM) were used to treat
mushroom tyrosinase. Kojic acid and resveratrol were used as positive controls at the same concentrations of 3-DBP. Data are shown as the percent of tyrosinase activity. (3-DBP:
rectangle shape, Kojic acid: diamond shape, resveratrol: triangle shape). A double asterisk (**) denotes the statistical significance between 3-DBP vs. kojic acid, Pb0.001 in a
Student's t test. (b) IC50 values of 3-DBP, resveratrol, and kojic acid for mushroom tyrosinase activity. The IC50 value of 3-DBP was 0.53 μM, which was significantly smaller than that
of kojic acid (8.2 μM) and resveratrol (13.48 μM). (c) Determination of the inhibitory mechanism of 3-DBP. The inhibitory mechanism of 3-DBP was determined on the basis of a
Lineweaver–Burk plot. Data were obtained as mean values of 1/V, the inverse of the absorbance increase at a wavelength 492 nm per min, of 3 independent tests at different
concentrations of L-tyrosine as a substrate. Four different enzyme inhibitor concentrations were used to determine the inhibitory mechanism (10 μM=cross shape, 5 μM=triangle
shape, 1 μM=rectangle shape, and 0 μM=diamond shape). The modified Michaelis–Menten equation used was 1/Vmax =1/Km (1+[S]/Ki). V denotes the velocity of the reaction,
S is the L-tyrosine concentration, and Ki is the inhibitor constant.
and mass spectroscopic analyses. The brief synthetic scheme is shown
in Fig. 1b.
and kojic acid) are shown in Fig. 2b. The low IC50 value of 3-DBP
(IC50 =0.53 0.11 μM) indicates that the potency is significantly
stronger than that of resveratrol (IC50 =13.48 0.96 μM) and kojic
acid (IC50 =8.2 1.3 μM), which were used as positive controls.
The inhibitory mechanism of 3-DBP on mushroom tyrosinase for
oxidizing L-DOPA was determined from Lineweaver–Burk double-
reciprocal plots. Fig. 2c showed double-reciprocal plots of enzyme
inhibition by 3-DBP. The 1/V vs. 1/[S] plot showed 4 different lines
with different slopes, which intersected on the same horizontal axis.
Accompanying the increase in the concentration of the compound,
3.2. Determination of anti-melanogenic effects on mushroom tyrosinase
Inhibitory activities of the synthesized compound and control
compounds were examined using mushroom tyrosinase as described
previously with minor modifications [26,27]. As shown in Fig. 2a,
3-DBP inhibited tyrosinase activity in a concentration-dependent
manner. IC50 values of 3-DBP and reference compounds (resveratrol

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Fig. 3. Effects of 3-DBP on B16F10 melanoma cells. (a) The effect of 3-DBP on the viability of B16F10 cells. Cells were treated with various concentrations of 3-DBP (10–200 μM) and
were examined using the MTT assay. Data are expressed as the percent of cell viability. (b) Effect of 3-DBP on melanin content. B16F10 cells were treated with vehicle or 3-DBP
(10–100 μM) and incubated in the absence or presence of 100 μmol of α-MSH for 48 h. Intracellular melanin extracted using 0.1 M NaOH was quantified spectrophotometrically
and normalized for protein content. A sharp (#) denotes the statistical significance between non-treated cells and α-MSH-treated cells; Pb0.001 in the Student's t test. A double
asterisk (**) denotes the statistical significance between α-MSH-treated cells and compound (3-DBP or kojic acid)-treated cells; Pb0.001 in the Student's t test. (c) Tyrosinase
activities were examined in B16F10 cells. B16F10 cells were treated with vehicle or 3-DBP (10–100 μM) and incubated in the absence or presence of 100 μmol of α-MSH for 48 h.
Treated cells were lysed to obtain cell-free extracts, and tyrosinase activity was assayed using L-DOPA as the substrate. Results are expressed as percentages of the control (only in
the α-MSH-treated group). Kojic acid (100 μM) was used as a positive control. A # denotes statistical significance between non-treated cells and α-MSH-treated cells; Pb0.001 in
the Student's t test. A double asterisk (**) denotes statistical significance between α-MSH-treated cells and compound (3-DBP or kojic acid)-treated cells; Pb0.001 in the Student's
t test. (d) Analysis of total tyrosinase protein and nuclear MITF expression change after α-MSH and/or 3-DBP treatment. β-actin was measured as a control.
Vmax decreased, but Km remained the same, suggesting that 3-DBP is a
non-competitive inhibitor of tyrosinase.
the specific melanin content. On the basis of the potent tyrosinase
inhibitory activity of 3-DBP, melanogenesis inhibition by 3-DBP may
be attributed to its ability to suppress tyrosinase.
To investigate whether 3-DBP-mediated depigmenting activity
is involved in modulating tyrosinase gene expression, we further
evaluated tyrosinase gene expression and MITF gene expression. As
shown in Fig. 3d, no changes in expression levels of tyrosinase and
MIFT were observed, indicating that the inhibitory effect of 3-DBP is
limited to inhibition of tyrosinase activity.
3.3. Evaluating depigmenting activity of 3-DBP in cell culture system
We used a B16F10 melanoma cell system to evaluate the depig-
menting activity of 3-DBP. The results for in vitro treatment of B16F10
cells with 3-DBP for cell survival, cellular tyrosinase activity, and
melanin contents are shown in Fig. 3. Results from the cell viability
assay using MTT for B16F10 cells (Fig. 3a) indicate that 3-DBP is
relatively non-cytotoxic to the cells under the experimental condi-
tions used.
3.4. Effects of 3-DBP on in vivo skin pigmentation
The inhibitory effect of 3-DBP on cellular melanogenesis was
compared to that of kojic acid, which is widely used in cosmetics as a
depigmenting agent [28]. To assess the effect of 3-DBP on the melanin
contents of B16F10 cells, 3-DBP was used to treat B16F10 cells in the
presence of 100 μM α-MSH at various concentrations; no significant
cell cytotoxicity was observed. As shown in Fig. 3b, 3-DBP inhibited
cellular melanogenesis, which was augmented by α-MSH in a dose-
dependent manner. The inhibitory effect of 3-DBP was stronger than
that of kojic acid. To analyze the inhibition mechanism of melanin
by 3-DBP, we examined the inhibitory effect of 3-DBP on cellular
tyrosinase activity by using murine-derived tyrosinase as depicted in
Fig. 3c. Cellular tyrosinase activity exhibited a similar profile to that of
The inhibitory effects of 3-DBP on in vivo skin pigmentation were
examined in melanin-possessing hairless mice that were treated
according to the schedule shown in Fig. 4a. Animals treated with
control vehicle or 3-DBP-condddtaining solution for 3 days prior
to UVB exposure did not show any skin irritation. Repeated UVB
exposure led to visible pigmentation in mice (Fig. 5a). The colors
of the skin sites were measured using a spectrophotometer. UVB
exposure led to a decrease in L* values, which represent pigmentation
(Fig. 4b). The UVB-induced decreases in L* values were significantly
and dose-dependently recovered in the 3-DBP treated animals than in
the control animals treated with vehicle (Fig. 4b and c), demonstrat-
ing the potent depigmenting effect of 3-DBP. UVB exposure of mice

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967
Fig. 4. Effects of 3-DBP on pigmentation in animal skins exposed to UVB. (a) HRM2 melanin-possessing hairless mice were treated with vehicle or 3-DBP on a designated site on
the dorsal skins according to the indicated schedules. UVB irradiation was conducted 8 times at the indicated doses. (b) The upper panel shows the lightness (L* value) of
the designated skin sites, which was measured prior to 3-DBP or vehicle application on each day. Graph shows changes in lightness (L* value). The lower panel shows the redness
(a* value) of the designated skin sites, which was measured prior to 3-DBP or vehicle application on each day. A # denotes statistical significance between control mice and UVB-
treated mice; Pb0.05 in the Student's t test. An asterisk (*) denotes statistical significance between UVB-treated mice vs. 3-DBP-treated mice; Pb0.05 in the Student's t test. (c) The
upper panel shows ΔL* values calculated as the average values after the final UVB exposure (day 16) minus the average baseline values before treatment (day 1). The lower panel
shows Δa* values, which were calculated as the average values after final UVB exposure (day 16) minus the average baseline values before treatment (day 1).
led to an increase in a* values, which represent sunburn induction.
The UVB-induced increase in the a* value was significantly recovered
when the mice were treated with more than 2 μM 3-DBP. Fontana–
Masson staining, which highlights melanin [29], confirmed the effects
of 3-DBP. As shown in Fig. 5b, UVB-irradiated animals showed
increased melanin spots when stained with collected skin. Consistent
with the numerical data collected using the spectrophotometer, 3-DBP-
treated animals showed decreased stained melanin spots compared to
UVB-irradiated controls.
and melanin product inhibition in murine B16 melanoma cells without
cell toxicity and showed better mushroom tyrosinase inhibition than
catechol-containing derivatives [30,31]. Phenolic hydroxyl groups
can act as both hydrogen-bonding donors and acceptors. Therefore,
compounds with a resorcinol moiety on a side of a double bond and a
structure capable of playing a role as both a hydrogen-bonding donor
and acceptor on opposite sides of the double bond can be regarded
as potential tyrosinase inhibitors. The amino group of the imide in
pyrrolidine-2,5-dione can serve as both a hydrogen-bonding donor
and acceptor, similar to the hydroxyl group of phenolic compounds.
Additionally, pyrrolidine-2,5-dione may provide additional interactions
with several amino acid residues of tyrosinase through hydrogen-
bonding interactions of its 2 carbonyl groups, implying that these
additional interactions with tyrosinase may induce tyrosinase inhib-
itory activity.
4. Discussion
Tyrosinase, which mediates skin pigmentation, is
a copper-
containing enzyme; its substrates are L-tyrosine and L-DOPA [16,17].
Therefore, most inhibitors of tyrosinase are Cu2+ chelators or phe-
nolic compounds that are structurally analogous to L-tyrosine and
L-DOPA. Recently, however, there have been reports stating that
resorcinol-containing derivatives exhibited better tyrosinase inhibition
On the basis of these findings, we designed and synthesized (E)-3-
(2,4-dihydroxybenzylidene)pyrrolidine-2,5-dione (3-DBP) by com-
bining the structural characteristics of resorcinol and pyrrolidine-2,5-

968
K.W. Chung et al. / Biochimica et Biophysica Acta 1820 (2012) 962–969
Fig. 5. Effects of 3-DBP on pigmentation in animal skins exposed to UVB. (a) The photos indicate pigmentation differences between the dorsal skins of tested animals. UVB-
irradiated vehicle-treated mice (day 16) tanned markedly in contrast to control mice that were not irradiated. Changes were observable with the naked eye or with a normal
camera. As shown in the photos, 3-DBP-treated mice showed a significant increase in lightness compared to vehicle-treated mice. Optical data were coincident with numerical data.
(b) Fontana–Masson staining of dorsal skin sections from the mice shown in (a) reveals differences in melanin content.
dione into one compound (Fig. 1a) and evaluated its inhibitory
activity against tyrosinase and melanin production.
An HRM2 melanin-possessing hairless mouse model was used as
an in vivo model. Although many other melanogenesis inhibitors
identified through in vitro studies failed to show in vivo efficacy, likely
due to the inability to enter the stratum corneum barrier [34], 3-
DBP showed significant efficacy in modulating UVB-induced in vivo
melanogenesis (Fig. 4). We are currently conducting studies to examine
how 3-DBP works as a non-competitive inhibitor by using a computa-
tional docking system and to demonstrate the mechanism of 3-DBP
efficacy by using an in vivo model.
Recent studies have focused on the mechanism of tyrosinase
inhibition in different pathways [35]. A suitable compound may be
more potent if it contains 2 mechanisms for reducing melanogenesis,
including direct tyrosinase activity inhibition and the reduction of
tyrosinase expression. However, it may be safer to target a minimum
number of molecules. The inhibitory effect of 3-DBP on melanogen-
esis is attributed to the direct inhibition of tyrosinase activity rather
than suppression of tyrosinase gene expression.
Our recent experiments to synthesize and evaluate new com-
pounds with potent inhibitory effects on tyrosinase revealed several
possibilities for developing new types of tyrosinase inhibitors [24,32,33].
These studies, involving a computational docking system between
tyrosinase and other compounds, showed efficacy for developing new
and potent tyrosinase inhibitors. Although these studies clearly demon-
strated the anti-melanogenic effect of newly synthesized compounds
in melanoma cells, important questions remained regarding its in vivo
efficacy.
Through continuous design and synthesis of potent tyrosinase
inhibitors, the novel 3-DBP was found to be an effective tyrosinase
inhibitor by using in vitro and in vivo models. Although it was difficult
to demonstrate docking of 3-DBP with tyrosinase due to its non-
competitive inhibitory mechanism, significant efficacy was demon-
strated in vitro. The IC50 value of 3-DBP in vitro was 16-fold lower
than that of kojic acid, and the molecule showed no adverse effects on
cell viability.
In summary, we identified 3-DBP as a novel anti-melanogenic
agent in both in vitro and in vivo. The compound appears to decrease

K.W. Chung et al. / Biochimica et Biophysica Acta 1820 (2012) 962–969
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Korea (NRF) grant funded by the Ministry of Education, Science and
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