Curcumin-like diarylpentanoid analogues as melanogenesis inhibitors

Article abstract of DOI:10.1007/s11418-011-0568-0

Anti-melanogenesis screening of 47 synthesized curcumin-like diarylpentanoid analogues was performed to show that some had a potent inhibitory effect on the melanogenesis in B16 melanoma cells. Their actions were considered to be mostly due to tyrosinase inhibition, tyrosinase expression inhibition, and melanin pigment degradation. The structure-activity relationships of those curcumin-like diarylpentanoid analogues which inhibited the melanogenesis and tyrosinase activity were also discussed. Of those compounds assayed, (2E,6E)-2,6-bis(2,5- dimethoxybenzylidene)cyclohexanone showed the most potent anti-melanogenesis effect, the mechanism of which is considered to be the degradation of the melanin pigment in B16 melanoma cells, affecting neither the tyrosinase activity nor tyrosinase expression. The Japanese Society of Pharmacognosy and Springer 2011.

Full text of DOI:10.1007/s11418-011-0568-0

J Nat Med (2012) 66:166–176
DOI 10.1007/s11418-011-0568-0
ORIGINAL PAPER
Curcumin-like diarylpentanoid analogues as melanogenesis
inhibitors
•
Takahiro Hosoya Asami Nakata Fumie Yamasaki
•
•
•
•
•
Faridah Abas Khozirah Shaari Nordin Hj Lajis
Hiroshi Morita
Received: 23 June 2011 / Accepted: 14 July 2011 / Published online: 10 August 2011
Ó The Japanese Society of Pharmacognosy and Springer 2011
Abstract Anti-melanogenesis screening of 47 synthe-
sized curcumin-like diarylpentanoid analogues was per-
formed to show that some had a potent inhibitory effect on
the melanogenesis in B16 melanoma cells. Their actions
were considered to be mostly due to tyrosinase inhibition,
tyrosinase expression inhibition, and melanin pigment
degradation. The structure–activity relationships of those
curcumin-like diarylpentanoid analogues which inhibited
the melanogenesis and tyrosinase activity were also dis-
cussed. Of those compounds assayed, (2E,6E)-2,6-bis(2,5-
dimethoxybenzylidene)cyclohexanone showed the most
potent anti-melanogenesis effect, the mechanism of which
is considered to be the degradation of the melanin pigment
in B16 melanoma cells, affecting neither the tyrosinase
activity nor tyrosinase expression.
Introduction
The essential role of melanins in the skin cells is the
absorption of free radicals, and melanogenesis is thus a
process of producing photoprotective agents against the
damaging effect of ultraviolet [1]. The intracellular pig-
mentation or melanogenesis can be prevented at various
stages in which tyrosinase is a major key enzyme involved
[2]. Tyrosinase is a copper-containing enzyme that cata-
lyzes two reactions: the hydroxylation of L-tyrosine to 3,4-
dihydroxyphenylalanine (DOPA), and the conversion of
DOPA to DOPA quinones [3]. These quinones then pro-
duce pigment deposition in the skin via enzymatic and
nonenzymatic reactions [4]. Thus, for the treatment of skin
pigmentations or for the whitening of skin, inhibition of
melanin synthesis is essential [5], and tyrosinase inhibitors
and antioxidants are often used for the prevention of cel-
lular pigmentation [6].
Keywords Curcumin-like diarylpentanoids ꢀ
Anti-melanogenesis ꢀ Tyrosinase activity ꢀ
Tyrosinase expression ꢀ Melanin degradation
Curcumin,
1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-
heptadiene-3,5-dione, is a diarylheptanoid isolated from
the rhizome of Curcuma longa (Zingiberaceae), which is
used as a yellow spice or pigment in foods [7]. Besides
various biological activities including anti-inflammatory,
anticancer, antioxidative, antiangiogenesis, and anti-HIV
activities [8–11], curcumin is also known to inhibit tyros-
inase activity as do other diarylheptanoids such as yaku-
chinones A and B [12]. In our previous preliminary paper
[13], we reported that some diarylpentanoid analogues
inhibited tyrosinase enzyme activity. In the present study,
we synthesized 47 diarylpentadiene-3-one curcumin-type
analogues(Fig. 1) and evaluated their anti-melanogenesis
activity by using B16 melanoma cells. The modes of their
actions on tyrosinase activity, tyrosinase protein expres-
sion, and/or melanin pigment degradation were also dis-
cussed in detail.
T. Hosoya ꢀ A. Nakata ꢀ F. Yamasaki ꢀ H. Morita (&)
Faculty of Pharmaceutical Sciences, Hoshi University,
Ebara 2-4-41, Shinagawa-ku, Tokyo 142-8501, Japan
e-mail: moritah@hoshi.ac.jp
F. Abas ꢀ K. Shaari ꢀ N. H. Lajis
Laboratory of Natural Products, Institute of Bioscience,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
F. Abas
Department of Food Science, Faculty of Food Science
and Technology, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
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J Nat Med (2012) 66:166–176
167
Fig. 1 Synthesis of the
diarylpentanoid analogues. The
group joining the two aromatic
rings is pentadiene-3-one in
type A (1–19), 2,6-
dibenzyldienecyclohexanone in
type B (20–37), 2,5-
dibenzyldienecyclopentanone in
type C (38–47)
Synthesis of the curcumin analogues
1–47 and 3-isobutyl-1-methylxanthine (IBMX), an inhib-
itor of phosphodiesterase [14]. Table 1 shows the assay
results, or the precentage melanin content in cells along
with the viability of the cells treated with the test com-
pounds. Table 1 shows only the results of active ana-
logues. Many of the type-A and -B compounds were
shown to be active, whereas those of type C were found to
have much less effect on the melanin content in B16
melanoma cells and the cell viability. Compounds 4, 11,
20, 23, 24, 30, 31, and 32 markedly decreased the melanin
contents (\50%) in the range of high cell viability of B16
melanoma cells ([80%) in a dose-dependent manner.
Compounds 2, 6, 7, 12, 28, 35, 37, 38, 42, and 44 showed
moderate anti-melanogenesis activity. The cyclohexanone-
type analogues 30 and 32 (type B) showed more effective
anti-melanogenesis activity in comparison with 4 and 6
(type A), and analogues such as 44 and 45 (type C),
respectively, in which each of them has the same sub-
stituent pattern on the benzene rings. Thus, type-B ana-
logues possessing methoxy groups on the aromatic rings
showed the most efficient anti-melanogenesis activity
among the 47 synthesized curcumin-like diarylpentanoid
analogues.
Curcumin analogues 1–47 were prepared by the base-cat-
alyzed aldole condensation between an appropriate aro-
matic aldehyde and ketone in a ratio of 2:1 (Fig. 1). The
ketone used for the synthesis of 1–19 (type A) was acetone,
that for the synthesis of 20–37 (type B) was cyclopenta-
none, and that for the synthesis of 38–47 (type C) was
cyclohexanone. Compounds 1–47 were used in the mela-
nogenesis assay as a dimethyl sulfoxide (DMSO) solution.
All the compounds were characterized by the analysis of
their spectroscopic data and by the comparison of these
data with those of related compounds. Detailed spectro-
scopic data of the synthesized compounds can be obtained
from us on request.
Results and discussion
Effect on melanin production in B16 melanoma cells
The effect on melanin production was assayed by incu-
bating B16 melanoma cells in the presence of compounds
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J Nat Med (2012) 66:166–176
Effect on tyrosinase activity (anti-tyrosinase activity)
13, 24, 35, and 37 were much weaker. Curcumin itself
showed a weak anti-tyrosinase activity (IC50 75.5 lM).
Compound 2, possessing the same substitution pattern on
the benzene rings (3-methoxy and 4-hydroxyl) as that of
curcumin, showed a higher inhibition effect than curcumin,
whereas compound 23 with the same aromatic rings did not
show significant inhibitory activity. The result suggests that
the acyclic moiety in type-A compounds plays an impor-
tant role in the anti-tyrosinase activity. Furthermore, the
five-carbon length between the two aromatic rings seems to
dramatically increase the anti-tyrosinase activity (cf. the
results of 2 vs. curcumin), suggesting the primary impor-
tance of the structure between the two aromatic rings in the
tyrosinase inhibition. As regards the substituents on the
aromatic rings, of compounds 3 (no anti-tyrosinase activ-
ity), 11, and 12, which have a hydroxyl group at the 2-, 4-,
and 3-position, respectively, 11 showed potent activity,
suggesting the importance of the presence of the 4-hydro-
xyl group. Of compounds 11, 13, and 16 (no anti-tyrosinase
Tyrosinase is a key enzyme in the melanin synthesis in
melanogenesis. Tyrosinase catalyzes the hydroxylation of
tyrosine to DOPA, the oxidation of DOPA to DOPA qui-
none, and the oxidation of 5,6-dihydroxyindole (DHI) to
indole-quione [3]. Therefore, with the expectation of pro-
ducing a skin-whitening effect, melanogenesis can be
inhibited by the activity of tyrosinase enzyme and/or
expression of tyrosinase in cells. Kojic acid, which is
known to be a tyrosinase inhibitor [15, 16], has been used
in cosmetics as a skin-whitening agent with some success.
The anti-tyrosinase effect of curcumin-like diarylpentanoid
analogues 1–47 on the melanogenesis by tyrosinase was
examined by using an in vitro mushroom tyrosinase assay
[17, 18]. As shown in Table 1, some type-A and -B com-
pounds inhibited tyrosinase, but scarcely any of type C did.
The anti-tyrosinase activities of 2, 11, and 12 in this assay
were about the same as that of kojic acid, whereas those of
Table 1 Melanin content in B16 melanoma cells after 4 days incubation with test compound and 50% inhibition concentration (IC50, lM)
values of anti-tyrosinase activity
Type
Compound
Melanin content in B16 melanoma cells (%)
Anti-tyrosinase
50 lM
25 lM
12.5 lM
6.3 lM
3.1 lM
1.6 lM
(IC50, lM)
c
c
c
c
A
2
4
–
–
–
–
63a
47a
56a
58a
53a
81a
82a
76a
57a
86a
83a
93a
16.3
e
c
c
c
c
–
–
–
–
–
c
c
c
e
–
6
–
–
–
14a
17b
43a
62a
87a
91a
11a
51b
c
c
c
e
–
7
–
–
–
c
c
11
12
13
20
23
24
28
30
31
32
35
37
38
42
44
–
–
17a
16a
70a
70a
6.4
c
c
–
–
12.7
90.4
c
c
d
d
–
–
–
–
c
d
d
e
–
B
–
39a
c
–
–
c
c
e
–
–
–
–
42a
64a
54a
88a
c
c
–
–
9a
43.5
15a
c
65a
c
69a
23a
70a
52a
80a
–
–
–
d
d
d
e
–
e
–
–
–
68a
d
78a
87a
35a
2a
7a
55a
66a
47a
55a
60a
17a
22a
88a
80a
–
–
–
d
d
e
–
63a
d
74a
79a
e
–
d
d
–
–
–
98.9
66.1
d
d
d
d
–
–
–
–
d
d
d
d
e
–
C
–
–
–
–
d
d
d
d
d
e
–
–
–
–
–
–
d
d
d
d
d
e
–
–
–
–
–
–
d
d
d
d
d
d
Curcumin
Arbutin
Kojic acid
a
–
–
–
–
–
–
75.5
73a
d
80a
d
–
–
–
–
d
d
d
d
e
–
d
d
d
d
–
–
–
–
–
–
12.0
Cell viability greater than 80%
b
c
d
e
60–80%
Less than 60%
Not determined
Not active at 100 lM
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J Nat Med (2012) 66:166–176
169
activity), possessing 4-OH, 4-Cl, and 4-F functions,
respectively, only 11 inhibited the tyrosinase activity. The
present findings agree with previous results reported [12],
implying that the anti-melanogenesis effect exerted by
compounds 2, 11, and 12 is at least partly due to inhibition
of the endogenous tyrosinase enzymatic activity in B16
melanoma cells.
Compound 32 inhibited neither the tyrosinase activity nor
the tyrosinase expression in B16 melanoma cells (Fig. 3).
When the cells were treated with IBMX, compound 32
decreased the melanin pigment content considerably, i.e.,
the whitening effect of 32 on the B16 melanoma cells is
apparently tyrosinase-independent (Figs. 4, 5a, b). Almost all
compounds reported as having anti-melanogenesis activity
havebeencorrelatedwithtyrosinase enzyme inhibitionand/or
the expression inhibition, and subsequently effective whit-
ening. However, the mechanism of 32 might be different from
these cases. Thus, the mechanism of the whitening effect by
32 is unique, and considered to be due to the degradation of
melanin pigment in B16 melanoma cells, which suggests that
compound 32 is a good candidate as a skin-whitening agent.
Effect on tyrosinase protein expression in B16
melanoma cells
The cellular tyrosinase protein content in the B16 mela-
noma cells, assayed by the Western blotting method,
showed that type-A (4 and 11) and type-B (20, 23, 24, 30,
31, and 32) compounds had higher anti-melanogenesis
activities in the first screening. As shown in Fig. 2, only 4,
20, 24, and 30 decreased the tyrosinase expression in B16
melanoma cells, resulting in reduction of the melanin con-
tent in B16 melanoma cells, although 11, 23, 31 (data not
shown), and 32 (Fig. 3) did not affect the expression levels.
Compound 11, which showed a potent anti-tyrosinase
activity, had no effect on the tyrosinase protein content.
These results showed that 11 decreased the melanin content
in B16 melanoma cells by inhibiting tyrosinase enzyme but
not tyrosinase expression in B16 melanoma cells. Com-
pounds 4 and 30, possessing two methoxy groups at 2- and
3-position on the benzene rings, were able to inhibit the
expression of tyrosinase in B16 melanoma cells.
Conclusion
Forty-seven curcumin-like analogues of three types (A–C),
each with a different group linking the two aromatic rings,
were assayed for their anti-melanogenesis activity by using
B16 melanoma cells. The first screening of anti-melanogen-
esis in B16 melanoma cells indicated that compounds of type
A and B had a more potent anti-melanogenesis effect than
those of type C. Of those compounds, compound 11 exhibited
the most potent anti-tyrosinase activity, although it showed
scarcely any effect on the tyrosinase expression in B16 mel-
anoma cells. Of those compounds assayed, compound 32 was
shown to be the most desirable skin-whitening agent. The
mechanism was considered to be the degradation of the
melanin pigment in B16 melanoma cells, affecting neither
the tyrosinase activity nor tyrosinase expression. Compound
32 might be a promising cosmetic skin-whitening agent.
Experimental
Chemistry
Fig. 2 Effects of compounds 4, 20, 24, and 30 on tyrosinase protein
expression in B16 melanoma cells
All chemicals and reagents are of research grade and
purchased from either Sigma-Aldrich, Merck, or Acros
Organics. All solvents used in experiment were either
HPLC-grade or dried and distilled prior to use. The typical
Fig. 3 Effects of compound 32 on tyrosinase protein expression in
B16 melanoma cells. 32 did not affect the expression level of
tyrosinase in B16 melanoma cells
Fig. 4 B16 melanoma cells treated with 32 with or without IBMX
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J Nat Med (2012) 66:166–176
Fig. 5 Melanin content in B16
melanoma cells treated with 32
with (a) or without IBMX (b)
workup included washing with brine and drying the organic
layer with magnesium sulfate (anhydrous) before concen-
tration in vacuo. Analytical TLC was carried out on silica gel
F254 precoated (0.2 mm thickness, Merck) aluminum TLC
sheets. The TLC plates were spotted with samples using a
fine glass capillary tube and developed in a chromatographic
tank saturated with solvent vapor at room temperature.
Melting points were determined using a hot stage melting
point apparatus equipped with a microscope, XSP-12 model
500X, and are uncorrected. IR spectra were recorded on a
Perkin Elmer RXI FT-IR spectrometer as KBr discs or thin
films. 1H NMR spectra were recorded on a Varian Unity 500
spectrometer (Varian Inc., Palo Alto, CA, USA) and mea-
sured at 500 MHz. Deuterated chloroform (CDCl3) and
acetone-d6 were used as solvents. Chemical shifts (d) are
reported in ppm. Mass spectra (MS) were recorded on a
Polaris Q ThermoFinnigan (San Jose, CA, USA) with ioni-
zation-induced by electron impacts (EIMS) at 70 eV.
General procedure for synthesis of analogues (1–47)
The bis-aldol adducts were prepared the dropwise addition of
aqueous NaOH (20 wt%, 15 mL, 75 mmol) into a vigorously
stirred solution of the appropriate benzaldehyde (51 mmol)
and ketone (25 mmol) in absolute EtOH (20 mL). The reac-
tion was stirred at room temperature for 48 h, and 100 mL
distilled water was then added. The resulting solution was
neutralized by gently bubbling CO2 gas through it and the
yellow precipitate produced was filtered off, washed with
distilled water, and dried under vacuum. The products were
purified by recrystallization from an appropriate solvent,
typically a MeOH–water mixture.
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J Nat Med (2012) 66:166–176
171
1
1,611 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.98 (d, 2H, J = 15.5 Hz), 6.88 (d, 2H, J = 15.5 Hz), 6.84
(d, 4H, J = 8.0 Hz), 6.82 (s, 2H), 3.96 (s, 6H), 3.85 (s,
6H); MS (EI): m/z 354 [M]?.
(1E,4E)-1,5-Diphenylpenta-1,4-dien-3-one (1)
Mp 112–114°C; UV (MeOH): kmax 326 (e 28,700), 326
(15,500), 202 (37,600) nm; IR (KBr): mmax 1,574, 1,593,
1
1,651 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.83 (m, 5H), 7.50 (m, 5H), 7.35 (d, 2H, J = 16.5 Hz); MS
(EI): m/z 234 [M]?.
(1E,4E)-1,5-Bis(2,6-dimethoxyphenyl)penta-1,4-dien-3-
one (7)
(1E,4E)-1,5-Bis(4-hydroxy-3-methoxyphenyl)penta-1,4-
dien-3-one (2)
Mp 153–155°C; UV (MeOH): kmax 366 (e 28,100), 213
(42,900) nm; IR (KBr): mmax 1,568, 1,604, 1,637 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 8.17 (d, 2H,
J = 16.3 Hz), 7.59 (d, 2H, J = 16.3 Hz), 7.36 (t, 2H,
J = 8.5 Hz), 6.75 (d, 4H, J = 8.5 Hz), 3.90 (s, 12H); MS
(EI): m/z 354 [M]?.
Mp 82–83°C; UV (MeOH): kmax 388 (e 28,900), 260
(12,700), 203 (31,200) nm; IR (KBr): mmax 1,513, 1,639,
1
3,398 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.70 (d, 2H, J = 16.0 Hz), 7.40 (d, 2H, J = 1.5 Hz), 7.24
(dd, 2H, J = 8.5, 1.5 Hz), 7.14 (d, 2H, J = 16.0 Hz), 6.91
(d, 2H, J = 8.5 Hz), 3.95 (s, 6H); MS (EI): m/z 326 [M]?.
(1E,4E)-1,5-Bis(2,3,4-trimethoxyphenyl)penta-1,4-dien-3-
one (8)
(1E,4E)-1,5-Bis(2-hydroxyphenyl)penta-1,4-dien-3-one (3)
Mp 89–91°C; UV (MeOH): kmax 366 (e 15,200), 203
1
(43,800) nm; IR (KBr):mmax 1,496, 1,590, 1,611 cm-1; H
Mp 156°C; UV (MeOH): kmax 371 (e 22,600), 312 (15,400),
256 (8,700), 211 (31,500) nm; IR (KBr): mmax 1,555, 1,604,
NMR (500 MHz, acetone-d6) d in ppm: 7.89 (d, 2H,
J = 16.0 Hz), 7.47 (d, 2H, J = 16.0 Hz), 7.20 (d, 2H,
J = 8.5 Hz), 6.88 (d, 2H, J = 8.5 Hz), 3.91 (s, 6H), 3.87
(s, 6H), 3.84 (s, 6H); MS (EI): m/z 414 [M]?.
1
3,207 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
8.09 (d, 2H, J = 16.0 Hz), 7.72 (dd, 2H, J = 8.0, 1.5 Hz),
7.34 (d, 2H, J = 16.0 Hz), 7.27 (td, 2H, J = 8.5, 1.5 Hz),
6.99 (d, 2H, J = 8.5 Hz), 6.93 (t, 2H, J = 7.5 Hz); MS
(EI): m/z 248 [M-H2O]?.
(1E,4E)-1,5-Bis(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-
one (9)
(1E,4E)-1,5-Bis(2,3-dimethoxyphenyl)penta-1,4-dien-3-
one (4)
Mp 209–211°C; UV (MeOH): kmax 384 (e 10,400), 254
(7,100), 201 (32,100) nm; IR (KBr): mmax 1,561, 1,602,
1
1,629 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
Mp 106–108°C; UV (MeOH): kmax 333 (e 36,000), 204
(60,700) nm; IR (KBr): mmax 1,577, 1,619, 1,737 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 8.07 (d, 2H,
J = 16.0 Hz), 7.41 (t, 2H, J = 4.5 Hz), 7.31 (d, 2H,
J = 16.0 Hz), 7.14 (d, 4H, J = 4.5 Hz), 3.91 (s, 6H), 3.89
(s, 6H); MS (EI): m/z 354 [M]?.
8.13 (d, 2H, J = 16.0 Hz), 7.43 (d, 2H, J = 16.0 Hz),
6.33 (s, 4H), 3.97 (s, 12H), 3.90 (s, 6H); MS (EI): m/z 414
[M]?.
(1E,4E)-1,5-Bis(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-
one (10)
(1E,4E)-1,5-Bis(2,4-dimethoxyphenyl)penta-1,4-dien-3-
one (5)
Mp 105–107°C; UV (MeOH): kmax 366 (e 23,200), 200
(38,100) nm; IR (KBr): mmax 1,504, 1,582, 1,620 cm-1; 1H
NMR (500 MHz, acetone-d6) 7.71 (d, 2H, J = 16.0 Hz),
7.24 (d, 2H, J = 16.0 Hz), 7.09 (s, 4H), 3.92 (s, 12H), 3.80
(s, 6H); MS (EI): m/z 414 [M]?.
Mp 138–140°C; UV (MeOH): kmax 392 (e 30,400), 251
(16,200), 301 (50,100) nm; IR (KBr): mmax 1,504, 1,588,
1,645 cm-1; 1H NMR (500 MHz, acetone-d6) d in ppm: 8.00
(d, 2H, J = 15.5 Hz), 7.71 (d, 2H, J = 8.5 Hz), 7.17 (d, 2H,
J = 15.5 Hz), 6.66 (d, 2H, J = 2.0 Hz), 6.62 (dd, 2H, J = 8.5,
2.0 Hz), 3.96 (s, 6H), 3.89 (s, 6H); MS (EI): m/z 354 [M]?.
(1E,4E)-1,5-Bis(4-hydroxyphenyl)penta-1,4-dien-3-one
(11)
Mp 244–246°C; UV (MeOH): kmax 375 (e 34,200), 242
(16,400), 201 (28,500) nm; IR (KBr): mmax 1,504, 1,582,
(1E,4E)-1,5-Bis(2,5-dimethoxyphenyl)penta-1,4-dien-3-
one (6)
1
1,620, 3,321 cm-1; H NMR (500 MHz, acetone-d6) d in
ppm: 7.71 (d, 2H, J = 16.0 Hz), 7.63 (d, 4H, J = 8:0 Hz),
7.10 (d, 2H, J = 16.0 Hz), 6.93 (d, 4H, J = 8.0 Hz); MS
(EI): m/z 266 [M]?.
Mp 105–106°C; UV (MeOH): kmax 396 (e 20,000), 316
(21,000), 207 (38,300) nm; IR (KBr): mmax 1,493, 1,578,
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J Nat Med (2012) 66:166–176
(1E,4E)-1,5-Bis(3-hydroxyphenyl)penta-1,4-dien-3-one
(12)
(1E,4E)-1,5-Bis(2-fluorophenyl)penta-1,4-dien-3-one (18)
Mp 68–70°C; UV (MeOH): kmax 312 (e 24,100), 201
(37,300) nm; IR (KBr): mmax 1,230, 1,486, 1,626 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.83 (d, 2H,
J = 16.0 Hz), 7.63 (dd, 4H, J = 7.5 Hz, 2.0 Hz), 7.57 (m,
2H), 7.38 (d, 2H, J = 16.0 Hz), 7.26 (td, 2H, J = 7.5,
2.0 Hz); MS (EI): m/z 270 [M]?.
Mp 198–200°C; UV (MeOH): kmax 329 (e 27,300), 254
(111,200), 212 (39,300) nm; IR (KBr): mmax 1,581, 1,620,
1
3,387 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.70 (d, 2H, J = 16.0 Hz), 7.29 (t, 2H, J = 7.6 Hz), 7.22
(d, 2H, J = 16.0 Hz), 7.21 (d, 2H, J = 7.6 Hz), 7.11
(s, 2H), 6.93 (dd, 2H, J = 8, 2.4 Hz); MS (EI): m/z 266
[M]?.
(1E,4E)-1,5-Bis(2-fluoro-4-methoxyphenyl)penta-1,4-dien-
3-one (19)
(1E,4E)-1,5-Bis(4-chlorophenyl)penta-1,4-dien-3-one (13)
Mp 115–117°C; UV (MeOH): kmax 357 (e 33,900), 325
(17,600), 200 (37,800) nm; IR (KBr): mmax 1,105, 1,508,
Mp 184–186°C; UV (MeOH): kmax 322 (e 26,800), 225
(20,100), 201 (32,300) nm; IR (KBr): mmax 1,091, 1,491,
1
1,612 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
1
1,589, 1,651 cm-1; H NMR (500 MHz, acetone-d6) d in
7.84 (d, 2H, J = 15.5 Hz), 7.81 (m, 2H), 7.24 (d, 2H,
J = 15.5 Hz), 6.90 (m, 4H), 3.91 (s, 6H); MS (EI): m/z 330
[M]?.
ppm: 7.80 (d, 2H, J = 15.9 Hz), 7.52 (d, 4H, J = 8.5 Hz),
7.37 (d, 4H, J = 8.5 Hz), 7.34 (d, 2H, J = 15.9 Hz); MS
(EI): m/z 303 [M]?.
(2E,6E)-2,6-Bis(2-chlorobenzylidene)cyclohexanone (20)
(1E,4E)-1,5-Bis(3-chlorophenyl)penta-1,4-dien-3-one (14)
Mp 90–92°C; UV (MeOH): kmax 313 (e 21,700), 203
(46,000) nm; IR (KBr): mmax 1,051, 1,576, 1,603 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.85 (s, 2H), 7.56
(m, 4H), 7.45 (m, 4H), 2.87 (t, 4H, J = 6.5 Hz), 1.78 (q,
2H, J = 6.5 Hz); MS (EI): m/z 343 [M]?.
Mp 123–126°C; UV (MeOH): kmax 316 (e 22,300), 202
(45,600) nm; IR (KBr): mmax 1,077, 1,594, 1,655 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.83 (s, 2H), 7.78
(d, 2H, J = 15.9 Hz), 7.73 (d, 2H, J = 7.0 Hz), 7.54 (m,
4H), 7.42 (d, 2H, J = 15.9 Hz); MS (EI): m/z 303 [M]?.
(2E,6E)-2,6-Bis(3-fluorobenzylidene)cyclohexanone (21)
(1E,4E)-1,5-Bis(2-chlorophenyl)penta-1,4-dien-3-one (15)
Mp 73–75°C; UV (MeOH): kmax 323 (e 29,500), 229
(16,200), 201 (39,000) nm; IR (KBr): mmax 1,170, 1,582,
Mp 114–116°C; UV (MeOH): kmax 321 (e 21,600), 203
(40,500) nm; IR (KBr): mmax 1,099, 1,587, 1,618 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 8.11 (d, 2H,
J = 16.1 Hz), 7.97 (dd, 2H, J = 7.5, 1.5 Hz), 7.56 (dd,
2H, J = 7.5, 1.5 Hz), 7.47 (m, 4H), 7.34 (d, 2H,
J = 16.1 Hz); MS (EI): m/z 303 [M]?.
1
1,612 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.67 (s, 2H), 7.54 (m, 2H), 7.39 (d, 2H, J = 8.0 Hz), 7.33
(d, 2H, J = 10.0 Hz), 7.20 (m, 2H), 3.00 (t, 4H,
J = 6.5 Hz), 1.84 (q, 2H, J = 6.5 Hz); MS (EI): m/z 310
[M]?.
(1E,4E)-1,5-Bis(4-fluorophenyl)penta-1,4-dien-3-one (16)
(2E,6E)-2,6-Bis(2-hydroxybenzylidene)cyclohexanone (22)
Mp 90–92°C; UV (MeOH): kmax 324 (e 23,000), 225
(15,900), 201 (28,800) nm; IR (KBr): mmax 1,237, 1,508,
Mp 146–148°C; UV (MeOH): kmax 356 (e 19,200), 257
(13,700), 206 (47,900) nm; IR (KBr): mmax 1,550, 1,604,
1
1
1,634 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
3,365 cm–1; H NMR (500 MHz, acetone-d6) d in ppm:
7.86 (d, 2H, J = 15.9 Hz), 7.52 (d, 4H, J = 8.5 Hz), 7.27
(d, 4H, J = 8.5 Hz), 7.17 (d, 2H, J = 15.9 Hz); MS (EI):
m/z 270 [M]?.
7.98 (s, 2H), 7.39 (d, 2H, J = 8.0 Hz), 7.24 (t, 2H,
J = 8.0 Hz), 6.97 (d, 2H, J = 7.5 Hz), 6.93 (t, 2H,
J = 7.5 Hz), 2.92 (t, 4H, J = 5.5 Hz), 1.75 (quintet, 2H,
J = 5.5 Hz); MS (EI): m/z 288 [M-H2O]?.
(1E,4E)-1,5-Bis(3-fluorophenyl)penta-1,4-dien-3-one (17)
(2E,6E)-2,6-Bis(4-hydroxy-3-
methoxybenzylidene)cyclohexanone (23)
Mp 132–134°C; UV (MeOH): kmax 316 (e 23,900), 201
(38,900) nm; IR (KBr): mmax 1,248, 1,581, 1,594,
1
1,655 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
Mp 187–189°C; UV (MeOH): kmax 383 (e 37,900), 253
(17,300), 201 (54,500) nm; IR (KBr): mmax 1,513, 1,577,
7.83 (d, 2H, J = 15.5 Hz), 7.59 (m, 6H), 7.38 (d, 2H,
J = 15.5 Hz), 7.23 (m, 2H); MS (EI): m/z 270 [M]?.
1
3,375 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
123

J Nat Med (2012) 66:166–176
173
1
7.65 (s, 2H), 7.16 (s, 2H), 7.10 (d, 2H, J = 8.2 Hz), 6.93 (d,
2H, J = 8.2 Hz), 3.92 (s, 6H), 2.98 (t, 4 H, J = 5.5 Hz),
1.81 (q, 2H, J = 5.5 Hz); MS (EI): m/z 366 [M]?.
1,661 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.72 (s, 2H), 7.56 (d, 4H, J = 8.0 Hz), 7.49 (t, 4H,
J = 8.0 Hz), 7.39 (dd, 2H, J = 1.5 Hz, 8.0 Hz), 2.99 (m,
4H), 1.83 (quintet, 2H, J = 6.0 Hz); MS (EI): m/z 274
[M]?.
(2E,6E)-2,6-Bis(4-hydroxybenzylidene)cyclohexanone (24)
Mp 171–173°C; UV (MeOH): kmax 369 (e 35,400), 243
(18,700), 200 (36,700) nm; IR (KBr): mmax 1,510, 1,594,
(2E,6E)-2,6-Bis(2,3-dimethoxybenzylidene)cyclohexanone
(30)
1
3,348 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.65 (s, 2H), 7.45 (d, 4H, J = 8.5 Hz), 6.93 (d, 2H,
J = 8.5 Hz), 2.93 (t, 4 H, J = 6.0 Hz), 1.79 (quintet, 2H,
J = 6.0 Hz); MS (EI): m/z 306 [M]?.
Mp 107–109°C; UV (MeOH): kmax 326 (e 31,000), 202
(58,400) nm; IR (KBr): mmax 1,573, 1,591, 1,609 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.90 (s, 2H), 7.12
(m, 4H), 7.03 (dd, 2H, J = 1.5 Hz, 7.5 Hz), 3.90 (s, 6H),
3.81 (s, 6H), 2.88 (m, 4H), 1.76 (quintet, 2H, J = 6.0 Hz);
MS (EI): m/z 394 [M]?.
(2E,6E)-2,6-Bis(4-chlorobenzylidene)cyclohexanone (25)
UV (MeOH): kmax 331 (e 40,300), 235 (22,200), 201
(40,300) nm; IR (KBr): mmax 1,092, 1,489, 1,576,
1,606 cm-1; 1H NMR (500 MHz, acetone-d6) 7.67 (s, 2H),
7.57 (d, 4H, J = 8.5 Hz), 7.49 (d, 4H, J = 8.5 Hz), 2.98 (t,
4H, J = 6.0 Hz), 1.83 (quintet, 2H, J = 6.0 Hz); MS (EI):
m/z 343 [M]?.
(2E,6E)-2,6-Bis(2,4-dimethoxybenzylidene)cyclohexanone
(31)
Mp 110–112°C; UV (MeOH): kmax 373 (e 3,700), 247
(3,300), 200 (21,000) nm; IR (KBr): mmax 1,501, 1,573,
1
1,599 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
(2E,6E)-2,6-Bis(3-chlorobenzylidene)cyclohexanone (26)
7.92 (s, 2H), 7.38 (d, 2H, J = 8.5 Hz), 6.64 (d, 2H,
J = 1.5 Hz), 6.64 (dd, 2H, J = 8. 5 Hz, 1.5 Hz) 3.89
(s, 6H), 3.87 (s, 6H), 2.91 (t, 4 H, J = 6.0 Hz), 1.77
(quintet, 2H, J = 6.0 Hz); MS (EI): m/z 394 [M]?.
Mp 136–138°C; UV (MeOH): kmax 323 (e 38,100), 234
(21,400), 207 (58,400) nm; IR (KBr): mmax 1,092, 1,577,
1
1,606 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.65 (s, 2H), 7.56 (s, 2H), 7.51 (t, 4H, J = 5.5 Hz), 7.44
(m, 2H), 2.99 (m, 4H), 1.86 (quintet, 2H, J = 6.0 Hz); MS
(EI): m/z 343 [M]?.
(2E,6E)-2,6-Bis(2,5-dimethoxybenzylidene)cyclohexanone
(32)
Mp 161–163°C; UV (MeOH): kmax 376 (e 8,700), 313
(11,000), 279 (9,400), 202 (30,100) nm; IR (KBr): mmax
(2E,6E)-2,6-Bis(4-fluorobenzylidene)cyclohexanone (27)
1
1,494, 1,582, 1,603 cm-1; H NMR (500 MHz, acetone-
Mp 86–88°C; UV (MeOH): kmax 328 (e 35,400), 230
(21,000), 201 (36,600) nm; IR (KBr): mmax 1,158, 1,507,
d6) d in ppm: 7.89 (s, 2H), 7.02 (d, 2H, J = 8.5 Hz), 6.97
(d, 4H, J = 8.5 Hz), 3.84 (s, 6H), 3.80 (s, 6H), 2.92 (t, 4H,
J = 6.0 Hz), 1.78 (quintet, 2H, J = 6.0 Hz); MS (EI):
m/z 394 [M]?.
1
1,608 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.68 (s, 2H), 7.56 (s, 2H), 7.49 (d, 2H, J = 8.3 Hz), 7.42
(d, 4H, J = 8.3 Hz), 2.99 (t, 4H, J = 5.5 Hz), 1.83
(quintet, 2H, J = 6.0 Hz); MS (EI): m/z 310 [M]?.
(2E,6E)-2,6-Bis(2,6-dimethoxybenzylidene)cyclohexanone
(33)
(2E,6E)-2,6-Bis(2-fluorobenzylidene)cyclohexanone (28)
Mp 128–130°C; UV (MeOH): kmax 328 (e 19,700), 202
(67,600) nm; IR (KBr): mmax 1,583, 1,616, 1,669 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.91 (s, 2H), 7.36
(t, 2H, J = 8.5 Hz), 6.75 (d, 4H, J = 8.5 Hz), 3.87 (s, 6H),
3.81 (s, 6H), 2.90 (t, 4H, J = 6.0 Hz), 1.77 (quintet, 2H,
J = 6.0 Hz); MS (EI): m/z 394 [M]?.
Mp 137–139°C; UV (MeOH): kmax 320 (e 28,200), 227
(15,500), 201 (38,100) nm; IR (KBr): mmax 1,167, 1,483,
1
1,614 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.78 (s, 2H), 7.58 (t, 2H, J = 8.0 Hz), 7.49 (m, 2H), 7.29
(m, 4H), 2.91 (t, 4H, J = 6.0 Hz), 1.81 (quintet, 2H,
J = 6.0 Hz); MS (EI): m/z 310 [M]?.
(2E,6E)-2,6-Bis(2,3,4-
trimethoxybenzylidene)cyclohexanone (34)
(2E,6E)-2,6-Dibenzylidenecyclohexanone (29)
Mp 92–94°C; UV (MeOH): kmax 328 (e 33,800), 231
(16,600), 201 (42,300) nm; IR (KBr): mmax 1,575, 1,607,
Mp 163–165°C; UV (MeOH): kmax 359 (e 37,800), 202
(68,700) nm; IR (KBr): mmax 1,493, 1,573, 1,596 cm-1; 1H
123

174
J Nat Med (2012) 66:166–176
(2E,5E)-2,5-Bis(4-chlorobenzylidene)cyclopentanone (40)
NMR (500 MHz, acetone-d6) d in ppm: 7.89 (s, 2H), 7.20
(d, 2H, J = 8.5 Hz), 6.88 (d, 2H, J = 8.5 Hz), 3.91 (s,
6H), 3.87 (s, 6H), 3.84 (s, 6H), 2.90 (t, 4H, J = 6.5 Hz),
1.77 (quintet, 2H, J = 6.5 Hz); MS (EI): m/z 454 [M]?.
Mp 197–199°C; UV (MeOH): kmax 357 (e 7,700), 235
(4,700), 200 (19,600) nm; IR (KBr): mmax 1,092, 1,585,
1
1,608, 1,623, 1,695 cm-1; H NMR (500 MHz, acetone-
d6) d in ppm: 7.47 (s, 2H), 7.74 (d, 4H, J = 8.0 Hz), 7.55
(d, 4H, J = 8.0 Hz), 3.19 (s, 4H); MS (EI): m/z 328 [M]?.
(2E,6E)-2,6-Bis(2,4,6-
trimethoxybenzylidene)cyclohexanone (35)
(2E,5E)-2,5-Bis(2-fluorobenzylidene)cyclopentanone (41)
Mp 197–199°C; UV (MeOH): kmax 353 (e 8,900), 201
(45,300) nm; IR (KBr): mmax 1,494, 1,584, 1,606 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.90 (s, 2H), 6.86
(s, 4H), 3.97 (s, 12H), 3.90 (s, 6H), 2.89 (t, 4H,
J = 6.0 Hz), 1.78 (quintet, 2H, J = 6.0 Hz); MS (EI):
m/z 454 [M]?.
Mp 180–182°C; UV (MeOH): kmax 340 (e 22,100), 228
(12,700), 201 (29,500) nm; IR (KBr): mmax 1,214, 1,603,
1
1,625, 1,694 cm-1; H NMR (500 MHz, acetone-d6) d in
ppm: 7.82 (t, 2H, J = 8.0 Hz), 7.71 (s, 2H), 7.52 (m, 2H),
7.36 (t, 2H, J = 8.0 Hz), 7.27 (t, 2H, J = 10 Hz), 3.17 (s,
4H); MS (EI): m/z 296 [M]?.
(2E,6E)-2,6-Bis(3,4,5-
trimethoxybenzylidene)cyclohexanone (36)
(2E,5E)-2,5-Bis(3-fluorobenzylidene)cyclopentanone (42)
Mp 147–149°C; UV (MeOH): kmax 357 (e 18,300), 547
(10,400), 200 (42,900) nm; IR (KBr): mmax 1,505, 1,604,
Mp 215–217°C; UV (MeOH): kmax 345 (e 37,100), 230
(15,200), 201 (38,300) nm; IR (KBr): mmax 1,212, 1,578,
1
1,579 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
1
1,601, 1,626 cm-1; H NMR (500 MHz, acetone-d6) d in
7.64 (s, 2H), 6.86 (s, 4H), 3.90 (s, 12H), 3.79 (s, 6H), 2.82
(t, 4H, J = 6.5 Hz), 1.77 (quintet, 2H, J = 6.5 Hz); MS
(EI): m/z 454 [M]?.
ppm: 7.57 (s, 2H), 7.54 (d, 2H, J = 8.5 Hz), 7.48 (d, 4H,
J = 8.5 Hz), 7.24 (m, 2H), 3.22 (s, 4H); MS (EI): m/z 296
[M]?.
(2E,6E)-2,6-Bis(2-fluoro-4-
methoxybenzylidene)cyclohexanone (37)
(2E,5E)-2,5-Bis(4-fluorobenzylidene)cyclopentanone (43)
Mp 135–137°C; UV (MeOH): kmax 353 (e 31,100), 230
(14,000), 200 (31,700) nm; IR (KBr): mmax 1,231, 1,508,
Mp 165–167°C; UV (MeOH): kmax 350 (e 27,600), 231
(22,300), 201 (43,300) nm; IR (KBr): mmax 1,163, 1,504,
1
1,586, 1,595 cm-1; H NMR (500 MHz, acetone-d6) d in
1
1,617 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
ppm: 7.80 (dd, 4H, J = 4.5 Hz, 8.5 Hz), 7.48 (s, 2H), 7.28
(t, 4H, J = 8.5 Hz), 3.17 (s, 4H); MS (EI): m/z 296 [M]?.
7.76 (s, 2H), 7.54 (t, 2H, J = 8.5 Hz), 6.85 (m, 4H), 3.89
(s, 6H), 2.91 (t, 4H, J = 6.5 Hz), 1.82 (t, 2H, J = 6.5 Hz);
MS (EI): m/z 370 [M]?.
(2E,5E)-2,5-Bis(2,3-dimethoxybenzylidene)cyclopentanone
(44)
(2E,5E)-2,5-Bis(2-chlorobenzylidene)cyclopentanone (38)
Mp 155–157°C; UV (MeOH): kmax 356 (e 37,800), 213
(52,400) nm; IR (KBr): mmax 1,574, 1,602, 1,616 cm-1; 1H
NMR (500 MHz, acetone-d6) d in ppm: 7.87 (s, 2H), 7.29
(d, 2H, J = 8.0 Hz), 7.19 (m, 4H), 3.91 (s, 6H), 3.86 (s,
6H), 3.09 (s, 4H); MS (EI): m/z 380 [M]?.
Mp 215–217°C; UV (MeOH): kmax 340 (e 41,900), 235
(17,100), 204 (57,600) nm; IR (KBr): mmax 1,039, 1,601,
1
1,621 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.84 (s, 2H), 7.80 (d, 2H, J = 7.5 Hz), 7.59 (d, 2H,
J = 7.5 Hz), 7.47 (m, 4H), 3.1 (s, 4H); MS (EI): m/z 328
[M]?.
(2E,5E)-2,5-Bis(2,5-dimethoxybenzylidene)cyclopentanone
(45)
(2E,5E)-2,5-Bis(3-chlorobenzylidene)cyclopentanone (39)
Mp 169–171°C; UV (MeOH): kmax 410 (e 25,300), 329
(20,300), 281 (11,200), 208 (46,400) nm; IR (KBr): mmax
Mp 216–218°C; UV (MeOH): kmax 340 (e 25,800), 233
(10,800), 202 (36,500) nm; IR (KBr): mmax 1,099, 1,606,
1
1,495, 1,585, 1,615 cm-1; H NMR (500 MHz, acetone-
1
1,625, 1,690 cm-1; H NMR (500 MHz, acetone-d6) d in
d6) d in ppm: 7.89 (s, 2H), 7.20 (d, 2H, J = 2.5 Hz), 7.05
(d, 2H, J = 9 Hz), 7.01 (d, 2H, J = 2.5 Hz), 3.88 (s, 6H),
3.83 (s, 6H), 3.14 (s, 4H); MS (EI): m/z 380 [M]?.
ppm: 7.73(s, 2H), 7.69 (d, 2H, J = 8.0 Hz), 7.56 (t, 2H,
J = 8.0 Hz), 7.48 (d, 4H, J = 12.0 Hz), 3.22 (s, 4H); MS
(EI): m/z 328 [M]?.
123

J Nat Med (2012) 66:166–176
175
(2E,5E)-2,5-Bis(2,3,4-
trimethoxybenzylidene)cyclopentanone (46)
was estimated spectrophotometrically by the absorbance at
400 nm [19]. Arbutin was used as a positive control.
Mp 147–149°C; UV (MeOH): kmax 394 (e 40,400), 248
(14,300), 210 (44,600) nm; IR (KBr): mmax 1,495, 1,586,
Cell viability assay
1
1,615 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
The cell viability was determined by the MTT assay [20]
after the cultivation. MTT solution (5 mg/mL, 15 lL) was
added to each well. After a further 2-h incubation, the
medium was removed, and DMSO (50 lL) was added to
each well to dissolve the formazan crystals formed. The
optical density was measured on a microplate reader
equipped with a two-wavelength system (550 and 700 nm).
In each experiment, three replicates were prepared for each
sample. The percentage of living cells was determined on
the basis of the differences in the absorbance between those
with the test samples and the controls.
7.79 (s, 2H), 7.44 (d, 2H, J = 8.5 Hz), 6.94 (d, 2H,
J = 8.5 Hz), 3.93 (s, 12H), 3.85 (s, 6H), 3.07 (s, 4H); MS
(EI): m/z 440 [M]?.
(2E,5E)-2,5-Bis(3,4,5-
trimethoxybenzylidene)cyclopentanone (47)
Mp 195–197°C; UV (MeOH): kmax 386 (e 24,600), 246
(7,800), 214 (30,000) nm; IR (KBr): mmax 1,505, 1,578,
1
1,622 cm-1; H NMR (500 MHz, acetone-d6) d in ppm:
7.42 (s, 2H), 7.01 (s, 4H), 3.93 (s, 12H), 3.81 (s, 6H), 3.22
(s, 4H); MS (EI): m/z 440 [M]?.
Anti-tyrosinase assay
Biological activity
Tyrosinase activity was spectrophotometrically determined
as in the case of the L-tyrosine oxidase activity assay of
mushroom tyrosinase [17, 18], with some modifications.
Briefly, 0.1 M phosphate buffer (pH 6.8, 500 lL) with or
without test samples, and 2.5 mM L-tyrosine (300 lL)
were mixed in an Eppendorf tube. Part of the mixtures
(200 lL) was transferred to four wells of a 96-well
microtiter plate. The absorbance at 475 nm was read on the
microplate reader (blank value). Then, 200 units/mL
tyrosinase (50 lL) was added to each well. After incuba-
tion (37°C, 15 min), the amount of dopachrome produced
in the reaction mixture was determined spectrophotomet-
rically by reading the absorbance at 475 nm on a micro-
plate reader. The tyrosinase activity of each test sample
was calculated as follows: % inhibition = 100 - [(A -
B)/C] 9 100 where A is the OD475 of a mixture of enzyme
and test sample; B the OD475 of test sample, but without
enzyme; C the OD475 with enzyme, but without test sam-
ple. Kojic acid was used as a positive control.
Materials
Murine melanoma cells (B16) were obtained from the Cell
Resource Center for Biomedical Research Institute of
Development, Aging and Cancer, Tohoku University,
Japan. The culture medium used was Dulbecco’s modified
Eagle medium (DMEM, Sigma), supplemented with fetal
bovine serum (10% FBS, Biowest), penicillin (100 U/mL),
and streptomycin (0.1 mg/mL, Wako). Other reagents,
3-isobutyl-1-methylxanthine (IBMX, Sigma), arbutin
(Sigma), L-(-)-tyrosine (TCI), mushroom tyrosinase
(Sigma-Aldrich), disodium hydrogen phosphate, (Wako),
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT, Sigma), were purchased from the respec-
tive suppliers.
Measurement of melanin content
B16 melanoma cells precultured in DMEM supplemented
with 10% FBS and penicillin/streptomycin (5,000 cells in
100 lL) were added to each well of a 96-well microtiter
plate, and were preincubated for 24 h at 37°C in a
humidified atmosphere containing 5% CO2. Then the cells
were cultured in the fresh DMEM containing IBMX
(100 lM) with DMSO (control) or the DMSO solution of
test compounds of different concentrations (100 lM) for
48 h. Then the medium was replaced by a fresh medium
containing the same concentration of compound, and the
cells were incubated for 48 h. The medium was removed
and the cells were dissolved in 50 lL of 1 N NaOH, which
was heated at 90°C for 1 h. The relative melanin content
Western blotting
Cells treated with test compounds were lysed in lysis
buffer, and the extract was passed through a 7.5% SDS–
PAGE. The protein fraction was transferred to an Immun-
Blot poly(vinylidene difluoride) membrane (PVDF,
Bio-Rad) and blocked for 1 h with 5% skim milk. The
blotted membrane was incubated with primary antibodies
(diluted 1:2,000) for anti-tyrosinase (Santa Cruz) over-
night, or 1:2,000 for b-actin (Sigma) for 1 h, followed by
horseradish peroxide-conjugated rabbit or mouse IgG
(Amersham). Protein bands were visualized with ECL
solution (GE Healthcare).
123

176
J Nat Med (2012) 66:166–176
Acknowledgments This work was supported by a Grant-in-Aid for
Scientific Research from the Ministry of Education, Culture, Sports,
Science, and Technology of Japan and a grant from The Open
Research Center Project in Hoshi University. Authors also thank the
Ministry of Science Technology and Innovation for the funds pro-
vided under the SAGA research funding program.
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