422 Notes Biol. Pharm. Bull. 27(3) 422—425 (2004) Vol. 27, No.

Inhibitory Effects of Esculetin on Melanin Biosynthesis

Yukimitsu MASAMOTO,a Yoshiyuki MURATA,b Kimiye BABA,c Yasuaki SHIMOISHI,b Mikiro TADA,a and
Kyoya TAKAHATA*, b
a
Graduate School of Natural Science and Technology, Okayama University; b Faculty of Agriculture, Okayama University;
1–1–1 Tsushima-Naka, Okayama 700–8530, Japan: and c Faculty of Phamacognosy, Osaka University of Pharmaceutical
Sciences; 4–20–1 Nasahara, Takatsuki, Osaka 569–1195, Japan.
Received September 22, 2003; accepted December 2, 2003

To investigate the structure–activity relationship of coumarins for the inhibitory activity on mushroom ty-
rosinase, the 50% inhibitory concentration (IC50 values) of 18 coumarins and four cinnamic acid derivatives
were measured. Among these compounds, esculetin had the strongest inhibitory activity (IC50543 m M) on mush-
room tyrosinase. Introduction of a hydroxy group to the C6 and C7 positions of the coumarin ring and no substi-
tution on the lactone ring played an important role in the expression of the strong inhibitory activity of esculetin.
We performed further studies to estimate the in vitro inhibitory effects of esculetin on melanogenesis. Esculetin
5 m M significantly suppressed melanin production in murine B16 melanoma cells without affecting cell growth.
Furthermore, the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the split-epidermal
sheets treated with 0.05% or 0.1% esculetin was significantly lower than that in the control. From these results, it
is suggested that esculetin has inhibitory effects on tyrosinase activity in vitro. However, further detailed studies
are necessary to understand the inhibitory mechanism of esculetin.
Key words esculetin; tyrosinase activity; melanogenesis; 3,4-dihydroxyphenylalanine (DOPA) reaction; coumarin

Coumarins are widely distributed in plants and are espe-
cially abundant in the bark, leaves, and roots of Umbelliferae
and Rutaceae plants. So far more than 1300 types of
coumarin have been identified as natural or synthesized com-
pounds. Coumarins have recently been reported to have inter-
esting pharmacologic and biochemical properties such as an-
tioxidative,1,2) antiinflammatory, and antiallergic effects,3,4)
inhibition of platelet aggregation5) and protein kinase,6) in-
duction of apoptosis,7) and antiviral,8) antidifferentiative,9)
and antimutagenic activity.10)
Melanin pigment is a heteropolymer of indole compounds
synthesized within melanocytes in the epidermis. Inhibitory
compounds on melanogenesis are useful as skin-whitening
agents used in cosmetics and as treatment of hyperpigmenta-
tion. Tyrosinase is known to play a critical regulatory role in
melanin biosynthesis.11) Therefore, many tyrosinase inhibitors
that suppress melanogenesis in epidermal layers have been
actively studied in cosmetics and pharmaceuticals.12—15)
These observations led us to search for naturally occurring
tyrosinase inhibitors.
Recently, we have isolated esculetin from the seeds of Eu-
phorbia lathyris L. as a mushroom tyrosinase inhibitory
compound.16) To the best of our knowledge, this is the first
report of the tyrosinase-inhibitory effects of esculetin. In this
study, 18 coumarins and four cinnamic acid derivatives were
examined for antityrosinase activity and study of the struc-
ture–activity relationship. We evaluated further the inhibitory
effect on melanin synthesis in B16 melanoma cells and
guinea pig epidermal sheets of esculetin, which showed the
strongest inhibitory activity among these compounds.
MATERIALS AND METHODS
Materials Coumarin, 4-hydroxycoumarin, umbellifer-
one, 7-methoxycoumarin, 6-hydroxy-4-methylcoumarin, 7-
hydroxy-4-methylcoumarin, esculin, esculetin, scopoletin, 7-
hydroxycoumarin-4-acetic acid, and 7-hydroxycoumarin-3-
∗ To whom correspondence should be addressed. e-mail: baiyou-1@cc.okayama-u.ac.jp
carboxylic acid were purchased from Aldrich Chemical Co.
(Milwaukee, WI, U.S.A.). 4-Methylesculetin, 5,7-dihydroxy-
4-methylcoumarin, isoscopoletin, and fraxetin were obtained
from Extrasynthese Co. (Genay, France). Daphnetin, daph-
nin, and daphnetin-8-glucoside were isolated from Daphne
odora. The chemicals purchased were used as received.
Assay of Tyrosinase-Inhibitory Activity The assay
was performed as previously described.16) One milliliter
of a 1.5 mM L-3,4-dihydroxyphenylalanine (DOPA) solution,
0.1 ml of dimethyl sulfoxide (DMSO) with or without sam-
ple, and 1.8 ml of 1/15 M phosphoric acid buffer solution (pH
6.8) were mixed. The mixtures were preincubated at 25 °C
for 10 min. Then, 0.1 ml of the aqueous solution of mush-
room tyrosinase (1000 U/ml, Sigma Chemical Co., St. Louis,
MO, U.S.A.) was added, and the reaction was monitored at
475 nm. A control reaction was conducted with DMSO. The
percentage of inhibition of tyrosinase activity was calculated
as follows: inhibition (%)5(A2B)/A3100, where A repre-
sents the difference in the absorbance of the control sample
between the incubation time of 0.5 and 1.0 min, and B repre-
sents the difference in the absorbance of the test sample be-
tween the incubation time of 0.5 and 1.0 min. The results
were the mean of the three concurrent readings.
Inhibitory Effect on Melanogenesis Using Cultured B16
Melanoma Cells B16 murine melanoma cells were cul-
tured in Dulbecco’s MEM medium supplemented with 5%
heat-inactivated (56 °C, 30 min) fetal bovine serum at 37 °C
in a humidified atmosphere containing 5% CO2. Ten micro-
liters of DMSO with or without esculetin was added to the
culture medium (10 ml) 24 h after cell seeding (2.03104
cells/90-mm dish). After 6 d of culture, the cells were har-
vested and the cell counts were determined. The degree of
whitening of pelleted cells was observed with the naked eye.
Melanin content was measured using a modified version of
the methods of Oikawa and Nakayasu17) and Hosoi et al.18)
Approximately 53106 cells were pelleted by centrifugation
at 15003g for 5 min and then washed twice with phosphate-
© 2004 Pharmaceutical Society of Japan
March 2004 423

buffered saline. After further centrifugation, the supernatant
was removed by carefully decanting and the precipitated cells
were solubilized with 1 ml of 1 M NaOH at 80 °C for 30 min
in a capped test tube. The absorbance was measured at
400 nm, and the melanin content per cell was calculated.
Data are expressed as a percentage of control, and are mean
values6S.D. Student’s t-test was used for the statistical
analysis of the data.
Measurement of DOPA-Positive Melanocytes Skin
biopsy specimens (5 mm2) were taken from the backs of
black guinea pigs. Tissues were rinsed in 0.1 M phosphate
buffer (pH 6.8) and incubated in 16.8 mM ethylenediamine
tetraacetic acid tetrasodium salt solution for 2 h at 37 °C.19)
Epidermal sheets were carefully separated from the dermis
and were fixed in 10% formaldehyde neutral buffer solution
(Nacalai Tesque, Kyoto, Japan) for 30 min at 0 °C, washed
twice with 0.1 M phosphate buffer (pH 6.8), and incubated in
0.1% L-DOPA in 0.1 M phosphate buffer (pH 6.8) with or
without esculetin for 3 h at 37 °C. Distribution of the DOPA-
positive melanocytes was observed under microscopy. The
number of melanocytes per square millimeter was calculated
in each specimen by averaging the numbers found in more
than 30 different microscope fields at a magnification of
3200. Data are expressed as the number of DOPA-positive
melanocytes per square millimeter, and are mean values6
S.D. Student’s t-test was used for the statistical analysis of
the data.
RESULTS AND DISCUSSION
Coumarins have many interesting pharmacologic and bio-
chemical properties. However, there are few reports on the
inhibitory effect of coumarins against melanogenesis and
there has been no systematic research.16,20)
First, to investigate the structure–activity relastionship of
coumarins, the IC50 values on mushroom tyrosinase of 18
coumarins and four cinnamic acid derivatives were mea-
sured. Table 1 summarizes the IC50 values of test compounds
used in this study. Among these compounds, esculetin (11),
which has hydroxyl groups at the C6 and C7 positions of the
coumarin ring, exhibited the strongest inhibitory activity
(IC5050.043 mM), followed by umbelliferone (4) (IC5050.42
mM), which has only one hydroxy group at C7. Daphnetin
(10), scopoletin (12), and 6,7-dihydroxy-4-methylcoumarin
(14) showed a weak inhibitory effect and the other com-
pounds were ineffective.

Table 1. Inhibitory Effect of the Test Compounds on Mushroom Tyrosinase

A) Simple coumarins R3 R4 R5 R6 R7 R8 IC50 (mM)

1. Coumarin H H H H H H 8.1
2. 4-Hydroxycoumarin H OH H H H H .10
3. 6-Hydroxy-4-methylcoumarin H Me H OH H H .3.5a)
4. Umbelliferone H H H H OH H 0.42
5. 4-Methyl-umbelliferone H Me H H OH H .3.5a)
6. 7-Hydroxycoumarin-4-acetic acid H OAc H H OH H .10
7. 7-Hydroxycoumarin-3-carboxylic acid COOH H H H OH H .10
8. 7-Methoxycoumarin H H H H OMe H 7.5
9. Fraxetin H H H OMe OH OH .10
10. Daphnetin H H H H OH OH 4.5
11. Esculetin H H H OH OH H 0.043
12. Scopoletin H H H OMe OH H 2.6
13. Isoscopoletin H H H OH OMe H .3.0a)
14. 6,7-Dihydroxy-4-methylcoumarin H Me H OH OH H 5.1
15. 5,7-Dihydroxy-4-methylcoumarin H Me OH H OH H .0.27a)

B) Coumarin glycosides R3 R4 R5 R6 R7 R8 IC50 (mM)

16. Esculin H H H OGlc OH H .10

17. Daphnin H H H H OGlc OH .10
18. Daphnetin-8-glucoside H H H H OH OGlc .10

C) Cinnamic acid derivatives R3 R4 IC50 (mM)

19. 3-Hydroxycinnamic acid OH H 1.4

20. 4-Hydroxycinnamic acid H OH 1.7
21. 4-Hydroxy-3-methoxycinnamic acid OMe OH .10
22. 3,4-Dihydroxycinnamic acid OH OH .10

a) Not tested because insoluble above this concentration in reaction mixture.

424 Vol. 27, No. 3

The mutual comparison of these results indicates that the from black guinea pigs. The number of DOPA-positive
introduction of hydroxyl groups to the ortho position of C6 melanocytes was measured in the esculetin-treated sheets and
and C7 potentiates the inhibitory activity, and conversely, the compared with control sheets. Representative micrographs of
introduction of hydroxyl groups to the ortho position of C7 DOPA-positive melanocytes in split-epidermal sheets incu-
and C8 drastically decreases the activity. Methoxylation or bated with or without esculetin are shown in Fig. 3. In the
glycosylation at C6 and C8, respectively, decreased the in- control epidermal sheets, a large number of DOPA-positive
hibitory activity in spite of the presence of the hydroxyl melanocytes with many dendrites was observed. In contrast,
group at C7 of the coumarin ring. Further, substitution of the distribution of DOPA-positive melanocytes in the 0.05%
any group at the C3 or C4 position of the coumarin ring or 0.1% esculetin-treated sheets was reduced compared with
markedly decreased the inhibitory activity. The four cin-
namic acid derivatives (19—22) were not as potent as the
corresponding umbelliferone (4) and esculetin (11), suggest-
ing that cleavage of the lactone ring of coumarin reduces the
activity. These results indicate that hydroxylation at the C6
and C7 positions of the coumarin ring is the most important
in increasing the activity but any substitution in the lactone
ring is unfavorable to the activity.
We performed further studies to estimate the in vitro in-
hibitory effects of esculetin on melanogenesis. The inhibitory
effects of esculetin on melanogenesis in cultured B16
melanoma cells are shown in Figs. 1 and 2. The growth rate
of B16 melanoma cells was not significantly altered after 6 d Fig. 1. Growth Rates and Melanin Contents of B16 Melanoma Cells after
of incubation in the 1.25 to 5.0 m M concentration range of es- 6 d of Incubation with Esculetin
culetin, indicating that the esculetin-induced inhibitory effect Each value represents the mean6S.D. (n53—4). ∗ p,0.05, ∗∗ p,0.01 compared
on melanogenesis in B16 melanoma cells occurred without with the control (Student’s t-test).
affecting cell proliferation. After 6 d of incubation with es-
culetin 5.0 m M, the melanin content decreased to 65.5% com-
pared with that in control cells (5100%), while esculetin
1.25 and 2.5 m M did not affect melanin production (Fig. 1).
The results are consistent with the visible appearance, as
shown in Fig. 2. At the cellular level, we were unable to
assess the inhibitory effect on melanogenesis at the more
effective concentration because esculetin exhibited strong
cytotoxicity. Therefore, we examined inhibitory effects of Fig. 2. Pellets of B16 Melanoma Cells after 6 d of Incubation with Es-
esculetin on melanogenesis using split-epidermal sheets culetin Compared with Control (Diluent-Treated) Cells

Fig. 3. Representative Photomicrographs Showing Dopa-Positive Melanocytes in Epidermal Sheets of Guinea Pig Treated with or without Esculetin for 3 h
The scale bar is 50 m m. A, control; B, 0.01% esculetin; C, 0.05% esculetin; D, 0.1% esculetin.
March 2004 425

mushroom tyrosinase. Several coumarins and cinnamic acid

derivatives studied inhibited tyrosinase activity. In particular,
esculetin exhibited potent inhibitory activity on tyrosinase
and demonstrated the importance of the 6,7-dihydroxy moi-
ety. Furthermore, esculetin suppressed melanin production in
B16 melanoma cells and epidermal sheets. From these re-
sults, it is concluded that esculetin has inhibitory effects on
tyrosinase activity in vitro.

Acknowledgements The authors wish to thank to Ms.

Yuko Ito and Mr. Hidenobu Okumura of the Kobe Research
Laboratory of Noevir Inc. for their technical assistance in
this study.
Fig. 4. Number of DOPA-Positive Melanocytes in Epidermal Sheets
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