Daedalin A, a metabolite of daedalea dickinsii, inhibits melanin synthesis in an in vitro human skin model

Article abstract of DOI:10.1271/bbb.80695

The culture broth of Daedalea dickinsii was found to predominantly contain the tyrosinase inhibitor, (2R)-6- hydroxy-2-hydroxymethyl-2-methyl-2H-chromene, daedalin A (1). Ongoing research into bioactive metabolites resulted in the identification of two new 2H-chromenes, 6-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H- chromene (3) and 6-hydroxy-2-hydroxymethyl-5-methoxy-2-methyl- 2H-chromene (4), together with 6-hydroxy-2,2-dimethyl- 2H-chromene (2). Comparative studies of isolated compounds 1-4 and related compounds (±)-1 and 1a-1c showed 1 to have the strongest tyrosinase inhibitory activity. These results suggest that the hydroxyl groups at positions 6 and 9 of 1 were important for the potent activity. A Lineweaver-Burk plot for a kinetic analysis indicates that 1 competed with L-tyrosine for tyrosinase. Compound 1 also suppressed melanogenesis in a human skin model (up to 49% at 2.8μmol/tissue application) without affecting the cell viability. Compounds 1, 1b and 1c also showed 1,1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity comparable to that of α-tocopherol.

Full text of DOI:10.1271/bbb.80695

Biosci. Biotechnol. Biochem., 73 (3), 627–632, 2009
Daedalin A, a Metabolite of Daedalea dickinsii, Inhibits Melanin Synthesis
in an in Vitro Human Skin Model
1
;2
3
3
1;4
Keiji MORIMURA, Kenji HIRAMATSU, Chihiro YAMAZAKI, Yasunao HATTORI,
5
3;y
Hidefumi MAKABE, and Mitsuru HIROTA
1
Interdisciplinary Graduate School of Science and Technology, Shinshu University,
304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
Geol Chemical Co., Ltd., 111-1 Shimura, Katsuragi, Nara 639-2121, Japan
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University,
8
2
3
8
304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
4
Satellite Venture Business Laboratory, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan
Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University,
5
8
304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan
Received October 1, 2008; Accepted November 20, 2008; Online Publication, March 7, 2009
[doi:10.1271/bbb.80695]
The culture broth of Daedalea dickinsii was found to
tyrosinase inhibitor, (2R)-6-hydroxy-2-hydroxymethyl-
2-methyl-2H-chromene (1) which we named as daedalin
predominantly contain the tyrosinase inhibitor, (2R)-6-
hydroxy-2-hydroxymethyl-2-methyl-2H-chromene, dae-
dalin A (1). Ongoing research into bioactive metabolites
resulted in the identification of two new 2H-chromenes,
5
)
A. Several bioactive 2H-chromenes have been isolated
and shown to have antioxidant, antiviral, antifungal,
6
–10)
cytotoxic and anti-inflammatory activities.
We have
6
(
-hydroxy-5,7-dimethoxy-2,2-dimethyl-2H-chromene
3) and 6-hydroxy-2-hydroxymethyl-5-methoxy-2-meth-
reported that the tyrosinase inhibitory activity of 1 was
stronger than that of arbutin. Furthermore, 1 also showed
potent antioxidant activity. It would therefore be of
interest to study the inhibition of melanin synthesis by
daedalin A.
An analysis of the mycelial culture broth of D. dick-
insii by thin-layer chromatography revealed the presence
of several compounds in the broth. Our interest in the
analogs of 1 isolated from D. dickinsii led to the
synthesis of several compounds, and we then studied
the structure/activity relationship of 1 and the analog
compounds. In comparative studies, the most potent
tyrosinase inhibitory compound, 1, also inhibited mel-
anin biosynthesis in human skin.
yl-2H-chromene (4), together with 6-hydroxy-2,2-di-
methyl-2H-chromene (2). Comparative studies of iso-
lated compounds 1–4 and related compounds (Æ)-1 and
1
a–1c showed 1 to have the strongest tyrosinase
inhibitory activity. These results suggest that the
hydroxyl groups at positions 6 and 9 of 1 were
important for the potent activity. A Lineweaver-Burk
plot for a kinetic analysis indicates that 1 competed with
L-tyrosine for tyrosinase. Compound 1 also suppressed
melanogenesis in a human skin model (up to 49% at
2
.8 ꢀmol/tissue application) without affecting the cell
viability. Compounds 1, 1b and 1c also showed 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical scavenging
activity comparable to that of ꢀ-tocopherol.
Materials and Methods
General. (R)-trolox, L-tyrosine, L-dopa, tyrosinase and arbutin were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). A three-
dimensional cultured human skin model, Melanoderm (MEL312B),
was purchased from Kurabo Co. (Osaka, Japan). The growth medium
for the human skin model was a long-life maintenance medium
(LLMM), supplied with the MEL312B kit. Other chemicals were
purchased from Wako Pure Chemical Industries (Osaka, Japan).
Mycelia of D. dickinsii were induced from fruiting bodies collected in
Nara, Japan, and have been deposited in the Riken Bioresource Center
Key words: Daedalea dickinsii; chroman; tyrosinase;
antioxidant
Tyrosinase is a copper-containing enzyme which
catalyzes the oxidation of L-tyrosine and L-3,4-dihy-
1
)
droxyphenylalanine (L-dopa). These oxidation reac-
tions are necessary for melanocytes to synthesize
melanin, the compound largely responsible for hair
and skin color in mammals. However, an excessive
accumulation of melanin in the epidermis causes
(
deposit number JCM12697, Saitama, Japan).
Instruments. Optical rotation values were measured with a JASCO
DIP 1000 polarimeter, and mass spectra were measured by a JEOL
JMS 700 mass spectrometer. 1H- and 13C-NMR spectra were recorded
abnormal hyperpigmentation of the skin such as freckles
and lentigines.2) There has therefore been considerable
effort to find an effective tyrosinase inhibitor. Some
by a Bruker DRX 500 FT-NMR spectrometer, operated at 500.1 MHz
for protons and at 125.1 MHz for carbons. TMS was used as the
internal standard. IR spectra were taken with a JASCO FT-IR 480 Plus
spectrometer.
3
)
4)
tyrosinase inhibitors such as arbutin and kojic acid
have been used as pharmaceutical constituents in order
to prevent hyperpigmentation.
We have recently isolated from a mycelial culture
broth of the Polyporaceae fungus, Daedalea dickinsii, a
Isolation of 2, 3 and 4. Daedalea dickinsii mycelia were cultured in
a liquid broth (10 liters) containing glucose (3%), polypeptone (0.5%),
y
To whom correspondence should be addressed. Fax: +81-265-77-1629; E-mail: hirotam@shinshu-u.ac.jp

628
K. MORIMURA et al.
yeast extract (0.2%), potassium dihydrogenphosphate (0.1%) and
magnesium sulfate (0.05%). After 40 d, the culture broth was filtered to
provide broth and mycelia. The filtered broth was extracted with
EtOAc, and the extract (5.8 g) was chromatographed on silica gel
Tyrosinase inhibitory test. The tyrosinase inhibitory activities were
determined by using L-tyrosine or L-dopa as the substrate, essentially
as previously described.12) The tyrosinase inhibitory test using L-
tyrosine as the substrate was conducted as follows: L-tyrosine (400 ml
of 5 mM) and 1460 ml of a 50 mM phosphate buffer (pH 6.8) were
mixed with 100 ml of a sample in dimethyl sulfoxide. After adding
40 ml of an aqueous solution of mushroom tyrosinase (1000 U/ml,
(Wakogel C300, 150 g) with EtOAc/hexane (450 ml each), using a
10% stepwise elution gradient.
The 30% EtOAc/hexane eluate (21.3 mg) was purified in a silica
ꢃ
gel column (Wakogel C300, 4 g) with 10% (10 ml ꢀ 5), 15%
Sigma Chemical Co.), the mixture was incubated at 37 C for 60 min,
(
10 ml ꢀ 5) and 20% EtOAc/hexane (5 ml ꢀ 10) giving 20 fractions.
and then the absorbance at 475 nm was measured to estimate the
activity. Tyrosinase inhibition was calculated as inhibition (%) ¼
100 ꢀ ðA ꢁ BÞ=A, where A is the absorbance of the control, and B is
the difference in absorbance before and after incubating the test
sample. The tyrosinase inhibitory test using L-dopa as the substrate was
conducted as follows: L-dopa (400 ml of 5 mM) and 1480 ml of a 50 mM
phosphate buffer (pH 6.8) were mixed with 100 ml of a sample in
dimethyl sulfoxide. After adding 20 ml of an aqueous solution of
Fractions 14–16 were combined (7.7 mg) and purified by HPLC (ODS-
UG-5 column, Nomura Chemical; flow rate, 2.0 ml/min; eluent, 60%
methanol/water). The fraction which eluted at tR 12.1 min was
collected to give 2 (3.2 mg) as a colorless oil. Fraction no. 6
(
12.5 mg) was purified by HPLC (ODS-UG-5 column; flow rate,
.0 ml/min; eluent, 50% methanol/water). The fraction that eluted at
tR 19.8 min was collected to give 3 (4.3 mg) as a colorless oil.
-Hydroxy-2,2-dimethyl-2H-chromene (2). The spectral data for 2
2
ꢃ
6
mushroom tyrosinase (1000 U/ml), the mixture was incubated at 25 C
are consistent with 6-hydroxy-2,2-dimethyl-2H-chromene which had
previously been isolated from Aplidium californicum by Cotelle et al.6)
for 30 min, and then the absorbance at 475 nm was measured to
estimate the activity. Tyrosinase inhibition was calculated as
inhibition (%) ¼ 100 ꢀ ðA ꢁ BÞ=A, where A is the absorbance of the
control, and B is the difference in absorbance before and after
incubating the test sample. The extent of inhibition by the addition of
the sample is expressed as the percentage necessary for 50% inhibition
(IC50). The Michaelis constant (Km), maximum velocity (Vmax) and
inhibition constant (Ki) for tyrosinase were determined by Lineweaver-
Burk plots, using various concentrations of L-tyrosine.
6
-Hydroxy-5,7-dimethoxy-2,2-dimethyl-2H-chromene (3). IR (KBr)
ꢀ
max cm 1: 3446, 1490, 1203, 912, 853; 1H- and 13C-NMR: see
Table 1.
The 50% EtOAc/hexane (853 mg) fraction was purified in a silica
gel column (Wakogel C300, 35 g) with 15% (20 ml ꢀ 10), 20%
ꢁ
(
20 ml ꢀ 5), 25% (20 ml ꢀ 5), 30% (20 ml ꢀ 5) and 40% acetone/
hexane (20 ml ꢀ 5), giving thirty fractions. Fractions 24–30 were
combined (14.5 mg) and purified by HPLC (ODS-UG-5 column; flow
rate, 2.0 ml/min; eluent, 30% methanol/water). The fraction that
eluted at tR 15.6 min was collected and provided 4 (1.5 mg) as a
colorless oil.
Melanogenesis inhibitory assay using a three-dimensional human
skin model. The melanogenesis inhibitory assay using a three-dimen-
sional human skin model was performed as previously described, with
slight modifications.13) The three-dimensional cultured human skin
model was placed in 6-well plates and incubated with 5 ml of LLMM
6
-Hydroxy-2-hydroxymethyl-5-methoxy-2-methyl-2H-chromene (4).
ꢃ
2
1
ꢁ1
½
ꢁꢂ D ¼ ꢁ2:6 (c 0.20, EtOH); IR (KBr) ꢀmax cm : 3446, 2923, 2852,
13
360, 1477, 1248, 1058, 952, 867; 1H- and C-NMR: see Table 1.
ꢃ
2
at 37 C under 5% CO . An aqueous solution (70 ml) of a test sample
2
was applied to the surface of the tissue. The tissue was incubated for
14 d, with LLMM being replaced by a fresh medium on alternate days.
After cultivation, the tissue was washed twice with Dulbecco’s
phosphate-buffered saline (DPBS) to remove the aqueous contami-
nants, and the melanin content and viability of the tissue cells were
measured. To measure the melanin content, the tissue cells were
rendered soluble by incubating with 400 ml of aqueous 1 N NaOH at
Synthesis of 1a, 1b, 1c and (ꢄ)-1.
(
2R)-2-Hydroxymethyl-6-methoxy-2-methyl-2H-chromene (1a). 1a
5
)
was synthesized from 1 as previously described.
2R)-6-Hydroxy-2-hydroxymethyl-2-methyl-chroman (1b). To
(
a
solution of 1 (21 mg, 0.11 mmol) in EtOH (5.0 ml) was added 10%
palladium on carbon (0.1 mg, 0.01 mmol) under a H2 atmosphere. The
mixture was stirred for 8 h, filtered and then concentrated. The residue
was purified by silica gel column chromatography (hexane:
EtOAc = 1:1) to give 1b (21 mg, 0.11 mmol, 100%) as a colorless
ꢃ
80 C for 2 h. The solution was centrifuged at 1000 ꢀ g for 10 min,
filtered, and the absorption of the supernatant was determined at
470 nm. The melanin content of the solution was calculated by
comparing with the absorbance of synthetic melanin. Cell viability of
the human skin model was evaluated by an MTT assay. After
cultivating for 14 d, the tissue was placed in a 24-well plate, 3 ml of an
MTT solution (MTT diluted with LLMM at 1 mg/ml) was added to
1
9
ꢃ
ꢁ1
oil, ½ꢁꢂ D ¼ ꢁ9:9 (c 0.21, acetone). IR (film) ꢀmax cm : 3353, 2930,
1
1
494, 1454, 1222, 1050, 809; H-NMR (CDCl3) ꢂH: 1.24 (3H, s), 1.67
(
1H, ddd, J ¼ 4:5, 6.2, 14.0 Hz), 1.99 (1H, ddd, J ¼ 6:2, 11.0,
1
4.0 Hz), 2.27 (OH, br. s), 2.68 (1H, dt, J ¼ 5:2, 17.0 Hz), 2.79 (1H,
ꢃ
each well, and the tissue was incubated at 37 C under 5% CO2. After
ddd, J ¼ 6:2, 11.0, 17.0 Hz), 3.60 (1H, d, J ¼ 12:0 Hz), 3.64 (1H, d,
J ¼ 12:0 Hz), 5.40 (OH, br. s), 6.56 (1H, d, J ¼ 2:9 Hz), 6.59 (1H, dd,
3 h of incubation, the tissue was washed twice with DPBS. Two
milliliters of the MTT extract, isopropyl alcohol containing 40 mM
HCl, and 40 ml of sodium dodecyl sulfate were then added to each well,
and the plate was gently shaken at room temperature for 2 h. The
absorbance of the extract was measured at 570 nm.
13
J ¼ 2:9, 8.7 Hz), 6.66 (1H, d, J ¼ 8:7 Hz); C-NMR (CDCl3) ꢂC:
2
0.4, 21.8, 27.5, 69.1, 76.2, 114.6, 115.4, 117.7, 121.9, 147.1, 149.0.
þ
HREIMS m=z (M ): Calcd. for C11H14O3: 194.0943. Found: 194.0949.
(2R)-6-Hydroxy-2-hydroxymethyl-2,5,7,8-tetramethyl-chroman
(
1c). To a solution of (R)-trolox (50 mg, 0.20 mmol) in THF (5.0 ml)
.
was added dropwise BH3 SMe2 in THF (a 10 M solution, 0.10 ml,
Result and Discussion
ꢃ
1
.0 mmol) at 0 C. The mixture was stirred for 10 min at that
temperature, and then diluted with water. The mixture was extracted
with EtOAc, washed with brine, filtered and concentrated. The residue
was purified by silica gel column chromatography (hexane:EtOAc =
Structure of the isolated compounds 2–4
Compound 2 was identified as 6-hydroxy-2,2-dimeth-
yl-2H-chromene by comparing the spectral data for 2
with the reference data.
High-resolution EIMS of 3 showed a molecular ion
peak at m=z 236.1047, consistent with the molecular
1
:1) to give compound 1c (43 mg, 0.18 mmol, 90%) as a colorless
6
)
solid, ½ꢁꢂ D ¼ ꢁ1:1ꢃ (c 0.10, CHCl3). IR (film) ꢀmax cm : 3388,
1
9
ꢁ1
1
2929, 1454, 1419, 1257, 1086, 1047; H-NMR (CDCl3) ꢂH: 1.22
(
3H, s), 1.73 (1H, dt, J ¼ 13:5, 6.0 Hz ), 1.93 (OH, br. s), 2.00 (1H, m),
1
2
4
2
.11 (3H, s), 2.12 (3H, s), 2.16 (3H, s), 2.68 (2H, m), 3.62 (2H, m),
.28 (OH, br. d); 13C-NMR (CDCl3) ꢂC: 11.3, 11.8, 12.2, 20.3, 20.4,
7.9, 69.4, 75.1, 117.3, 118.7, 121.3, 122.6, 144.9, 145.1. HREIMS
formula C H O (Calcd.: 236.1049). The H- and
1
3
16
4
13
C-NMR spectra resembled those of 2. Comparison of
13
1
the H- and C-NMR data for 3 and 2 revealed that 3
differed from 2 by the presence of two methoxy groups
þ
m=z (M ): Calcd. for C14H20O3: 236.1412. Found: 236.1417.
(
ꢄ)-6-Hydroxy-2-hydroxymethyl-2-methyl-2H-chromene ((ꢄ)-1).
(ꢂ 61.3, ꢂ 3.87, ꢂ 56.2, ꢂ 3.84). The positions of the
C H C H
(ꢄ)-1 was synthesized from 2,5-dihydroxybenzaldehyde as previously
described.
11)
methoxy groups at C-5 and C-7 were determined on the
basis of NOESY and HMBC correlations (Fig. 1). Thus,
3 was elucidated as 6-hydroxy-5,7-dimethoxy-2,2-di-
methyl-2H-chromene.
DPPH radical scavenging test. The DPPH radical scavenging
activities were evaluated as previously described.5)

Inhibitory Effect of Daedalin A on Melanin Synthesis
629
J = 8.6 Hz
H C
3
H
H
H2
C
O
O
H
CH3
CH3
H
H
O
H
OH
CH3
H
HO
HO
O
O
J = 10.0 Hz
J = 9.9 Hz
CH3
CH3
3
4
Selected H-H correlations from COSY
Selected H-C correlations from HMBC
Selected H-H correlations from NOESY
Fig. 1. Selected H–H and C–H Correlations from COSY, HMBC and NOESY Analyses of Compounds 3 and 4.
of L-dopa by tyrosinase at a concentration of 800 mM.
Based on these results, the tyrosinase inhibitory activity
of 1 was probably caused by competitive inhibition with
L-tyrosine. This was confirmed by Lineweaver-Burk
plots, using L-tyrosine as a substrate for tyrosinase with
Table 1. 1H-NMR and 13C-NMR Data for 3 and 4
3
4
ꢂC
ꢂH
ꢂC
ꢂH
2
3
75.7 C
128.1 CH
78.6 C
127.8 CH
1
the same V
7
(Fig. 4). Compound 1 at 50 mM and 100 mM exhibited
5.51 d
J ¼ 10:0 Hz)
6.53 d
J ¼ 10:0 Hz)
5.67 d
(J ¼ 9:9 Hz)
6.69 d
ꢁ
2
value of 1:5 ꢀ 10 and Km values of
max
(
:5 ꢀ 10ꢁ ꢄ 4:6 ꢀ 10 (n ¼ 3) and 1:0 ꢄ 4:2 ꢀ 10
1
ꢁ2 ꢁ2
4
116.9 CH
119.2 CH
(
(J ¼ 9:9 Hz)
(n ¼ 3) mM. Therefore, daedalin A was proved to be a
4
5
6
7
a
108.2 C
142.7 C
132.3 C
147.6 C
114.6 C
142.9 C
145.9 C
115.2 CH
competitive inhibitor of L-tyrosine hydroxylation by
ꢁ1
tyrosinase, with a Ki value of 4:6 ꢀ 10 ꢄ 9:3 ꢀ 10
n ¼ 3) mM.
The tyrosinase inhibitory activities of the isolated
(
6.73 d
(
J ¼ 8:6 Hz)
6.51 d
J ¼ 8:6 Hz)
compounds (1–4) and synthesized compounds (1a–1c,
and (ꢄ)-1) are shown in Table 2. Compound 1 inhibited
the tyrosinase activity by up to 60.7% at 400 mM, whereas
6-methoxy-daedalin A (1a) and 9-deoxy-daedalin A (2)
did not show activity at 400 mM. These results suggest
that the hydroxyl groups at positions 6 and 9 might have
played an important role in the tyrosinase inhibitory
activity of chromans. Interestingly, a racemer of 1 ((ꢄ)-
1) had weaker activity (IC50, 289 mM) than 1 (IC50,
8
96.2 CH
6.23 s
1.40 s
112.3 CH
(
8
9
a
146.1 C
27.5 CH3
142.7 C
68.4 CH2
3.60 d
J ¼ 11:5 Hz)
.68 d
J ¼ 11:5 Hz)
1.34 s
(
3
(
1
0
27.5 CH3
1.40 s
3.84 s
3.87 s
22.0 CH3
5
6
6.2 OCH3
1.3 OCH3
62.3 OCH3 3.81 s
194 mM). These results also support the competitive
inhibitory mechanism for 1 on tyrosinase which was
elucidated by the kinetic study of 1 and L-tyrosine.
Yu has reported the tyrosinase inhibitory effect of
High-resolution EIMS of 4 showed a molecular ion
peak at m=z 222.0890, consistent with the molecular
formula C12H14O4 (Calcd.: 222.0892). The IR spectrum
1
4)
(R)-trolox bearing methyls at positions 5, 7 and 8.
However, the activity was extremely weak (IC ,
of
4 showed absorption bands for OH groups
ꢁ1
50
ꢁ1
(
3446 cm ) and a benzene ring (867 cm ). As shown
6100 mM), about 30 times lower than that of 1.
Compound 1c did not show any activity at 800 mM,
whereas the activity of 1b was stronger (IC , 345 mM)
in Table 1, the H- and 13C-NMR data for 4 were similar
to those for 1, except for the presence of a methoxy
group (ꢂC 62.3, ꢂH 3.81) at position 5. We thus
concluded that 4 was 5-methoxy-daedalin A; this was
substantiated by C-H correlations between the methoxy
proton (ꢂH 3.81) and C-5 (ꢂC 142.9), and between C-5
and H-4 (ꢂH 6.69) in the HMBC spectrum (Fig. 1).
1
5
0
than that of arbutin. Structurally, 1b and 1c differ by the
presence of methyl groups at positions 5, 7 and 8. In
addition, 4, which has a methoxy group at position 5,
did not show activity at a concentration of 400 mM,
and 3, which was substituted with methoxy groups at
position 5 and 7, did not show activity at 800 mM.
Therefore, the tyrosinase inhibitory activities of the
chroman analogs were substantially decreased by methyl
and methoxy groups on the benzene ring, possibly due to
restricted binding to the enzyme. A tyrosinase inhibitory
test revealed compound 1 to show the strongest activity
among our tested chroman analogs.
Tyrosinase inhibitory activities
As shown in Fig. 3, a hydroxyl and carboxyl group of
L-tyrosine could adopt conformations similar to that of
the two hydroxyl groups of 1. Compound 1 potently
suppressed the hydroxylation of L-tyrosine by tyrosinase
at low concentrations, but did not suppress the oxidation

630
K. MORIMURA et al.
9
R4
1
R4
7
O
R1
R1
8
5
R3
O
2
6
10
3
R O
3
4
HO
R2
R2
R1
R2
R3
R4
R1
R2
R3
R4
Daedalin A (1) OH
H
H
OCH3
H
H
H
H
CH3
H
H
OCH3
H
(R)-Trolox COOH
CH3 CH3 CH3
2
3
4
H
H
1b
1c
CH OH
H
H
H
2
CH OH
CH3 CH3 CH3
2
*
OH OCH3
OH
1
a
H
H
Fig. 2. Structures of Compounds 1–4, 1a–1c, and (R)-Trolox.
ꢅ
Possible absolute structure which might be different.
H
H
O
H
H
H
O
H
C
C
H
O
H
H
H
O
O
C
C
H
O
H
C
H
H
C
C
C
C
C
C
H
C
C
N
C
C
C
C
H
C
H
C
H
C
H
H
H
H
H
1
L-Tyrosine
Fig. 3. Optimum Conformation of Compound 1 and Selected Conformation of L-Tyrosine.
The optimum conformation of compound 1 was generated by using MM2 computations of the Chem 3D program.
4
00
300
200
100
-3
-2
-1
0
1
2
4
1
/ [
-tyrosine] (µM)-1
L
Fig. 4. Lineweaver-Burk Plots of Mushroom Tyrosinase and L-Tyrosine without ( ), and with 50 mM ( ) and 100 mM ( ) of 1.
DPPH radical scavenging activities
Daedalin A (1) showed potent free radical scavenging
17.8 mM) and 1c (IC50, 14.0 mM) showed strong activities,
comparable to that of (R)-trolox (IC50, 13.9 mM) and ꢁ-
tocopherol (IC , 14.2 mM). However 1a, which lacked a
phenolic hydroxyl group, showed no activity at 500 mM.
Therefore, the phenolic hydroxyl group was essential for
the antioxidant activity of our tested compounds.
Melanin is also biosynthesized by the auto-oxidative
activity.5 We compared the DPPH radical scavenging
activity of 1 with that of related compounds. As shown
in Table 3, at 100 mM, all the tested compounds except
)
5
0
1
a scavenged more than 50% of the DPPH radicals.
Among the compounds, 1 (IC50, 16.9 mM), 1b (IC50,

Inhibitory Effect of Daedalin A on Melanin Synthesis
631
Table 2. Tyrosinase Inhibitory Activities of 1–4, (ꢄ)-1, 1a–1c, Arbutin and (R)-Trolox
a
Inhibition ꢄ SD (%)
Compound
IC50 (mM)
1
00 mM
200 mM
400 mM
800 mM
Daedalin A (1)
34:9 ꢄ 1:1
23:7 ꢄ 5:7
50:9 ꢄ 6:7
60:7 ꢄ 3:2
NTb
NT
194
289
(
ꢄ)-1
45:5 ꢄ 3:1
55:6 ꢄ 1:1
c
2
3
4
NI
NI
NT
NI
NT
NT
NI
>400
>800
>400
>800
345
NT
NI
NI
NI
NI
NI
NT
NI
1
1
a
NI
15:9 ꢄ 1:9
NI
b
28:5 ꢄ 3:5
NI
58:2 ꢄ 3:7
NI
NT
NI
1c
>800
672
6100
Arbutin
R)-Trolox
22:0 ꢄ 4:1
NI
28:6 ꢄ 9:5
NI
34:7 ꢄ 5:0
NI
51:0 ꢄ 3:1
8:6 ꢄ 2:9
(
a
Each value is expressed as the means ꢄ SD from three tests.
b
NT, not tested
cNI, no inhibition
Table 3. DPPH Radical Scavenging Activities of 1–4, (ꢄ)-1, 1a–1c, ꢁ-Tocopherol and (R)-Trolox
a
Inhibition ꢄ SD (%)
Compound
IC50 (mM)
2
0 mM
40 mM
50 mM
100 mM
Daedalin A (1)
57:4 ꢄ 2:5
44:6 ꢄ 0:8
17:4 ꢄ 1:6
18:0 ꢄ 0:1
32:4 ꢄ 1:4
0:4 ꢄ 2:0
NTb
72:2 ꢄ 0:5
NT
79:9 ꢄ 0:8
NT
83:8 ꢄ 0:2
84:9 ꢄ 0:9
57:0 ꢄ 0:1
81:7 ꢄ 0:1
81:2 ꢄ 0:2
1:9 ꢄ 0:6
16.9
23.9
83.8
57.3
31.1
>500
17.8
14.0
14.2
13.9
(
ꢄ)-1
2
3
4
35:3 ꢄ 0:4
44:5 ꢄ 1:0
NT
NT
64:0 ꢄ 0:6
NT
NT
1
1
a
1:2 ꢄ 2:4
79:5 ꢄ 0:6
87:5 ꢄ 0:3
92:8 ꢄ 2:2
84:0 ꢄ 0:3
b
54:0 ꢄ 1:8
70:2 ꢄ 0:7
72:2 ꢄ 0:4
69:8 ꢄ 1:5
83:9 ꢄ 0:6
87:9 ꢄ 0:1
94:6 ꢄ 0:7
84:0 ꢄ 0:4
1c
NT
NT
ꢁ
-Tocopherol
(
R)-Trolox
85:9 ꢄ 0:3
a
Each value is expressed as the means ꢄ SD from three tests.
b
NT, not tested
action of dopaquinone, following the oxidation of
L-tyrosine to L-dopa and of L-dopa to dopaquinone by
tyrosinase.1 It has been reported that some antioxidants
prevent melanogenesis. For example, Hayakawa et al.
have reported that an oral intake of ꢁ-tocopherol was an
Conclusion
5)
This study shows that daedalin A had the most
potent tyrosinase inhibitory activity of the compounds
tested. An analysis of structure-activity relationships and
Lineweaver-Burk plots confirmed that the tyrosinase
inhibitory activity of daedalin A was due to competitive
inhibition with L-tyrosine.
Furthermore, daedalin A inhibited the pigmentation
of a three-dimensional cultured human skin model
without affecting the cell viability. In the epidermis,
dermal pigmentation appears after the synthesis of
melanin in melanosomes and the transfer of melanin
pigment to epidermal cells. Melanin transfer is mediated
by the expression of protease-activated receptor 2
1
6)
effective treatment for facial hyperpigmentation, and
Matsuda et al. have reported that an antioxidant extract
1
7)
from Myrica rubra inhibited melanin synthesis. In
addition to the tyrosinase inhibitory activity of 1, the
antioxidant activity might enhance the inhibitory activ-
ity toward melanin synthesis.
Melanogenesis inhibitory activity of 1
We evaluated the melanogenesis inhibitory activity of
on a three-dimensional cultured human skin model
1
1
8,19)
composed of normal human melanocytes and keratino-
cytes, as well as human skin. As shown in Fig. 5A, after
(PAR-2) in keratinocytes, but not in melanocytes.
Paine et al. have reported that a soybean extract
inhibited pigmentation in a three-dimensional cultured
human skin model, although the extract did not show
1
4 d of incubation of the cultured human skin model
with 1, the pigmentation of the model was dose-
dependently suppressed. Figure 5B demonstrates that
melanin synthesis by the model was reduced to 72% ꢄ
2
0)
tyrosinase inhibitory activity,
indicating that the
inhibition of pigmentation was caused by the inhibition
of PAR-2 activation. Since daedalin A inhibited melanin
synthesis as well as tyrosinase activity, its inhibitory
effect on pigmentation was probably due to the direct
inhibition of tyrosinase activity in melanocytes. In
addition to the tyrosinase inhibitory activity, the anti-
oxidant activity might also have contributed to the
melanogenesis inhibitory effect of daedalin A.
4
2
:2 (n ¼ 3) and 49% ꢄ 2:3 (n ¼ 3) by 1 at 1.4 and
.8 mmol per well, respectively. In contrast, the inhib-
itory activity of arbutin was 67% ꢄ 4:1 (n ¼ 3) at
.8 mmol per well. The cell viability of the human skin
model was determined by using an MTT assay. After
4 d incubation with 2.8 mmol of 1 per well, the cell
2
1
viability did not change (102% ꢄ 7:5, n ¼ 3). Thus, 1
inhibited melanin biosynthesis in the human skin model
without affecting the cell viability.
Yoon et al. have reported the melanogenesis inhib-
itory test using a three-dimensional cultured human skin

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B 1
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00
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A, Three-dimensional cultured human skin model without or with
8
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1
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1
1
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2
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ꢁ
(
1981).
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