November 2007 Biol. Pharm. Bull.

30(11) 2141—2145 (2007) 2141

Depigmentation of Melanocytes by Isopanduratin A and

4-Hydroxypanduratin A Isolated from Kaempferia pandurata ROXB.
Ji-Hoon YOON,a Jae-Seok SHIM,a Yumi CHO,a Nam-In BAEK,b Chan-Woo LEE,c Han-Sung KIM,c and
Jae-Kwan HWANG*,a
a
Department of Biotechnology, Yonsei University; 134 Shinchon-dong, Seodaemun-gu, Seoul 120–749, South Korea:
b
Department of Oriental Medicinal Materials and Processing, Kyunghee University; 1 Seocheon-dong, Giheung-gu,
Yongin-si, Gyeonggi-do 446–701, South Korea: and c R&D Center, Amore-Pacific Corporation; 314–1, Bora-dong,
Kiheung-gu, Yongin-si, Kyounggi-do 449–729, South Korea. Received April 11, 2007; accepted July 20, 2007

This study was carried out to investigate the in vitro effects of isopanduratin A and 4-hydroxypanduratin A
isolated from Kaempferia pandurata ROXB. on melanin biosynthesis and tyrosinase activity. Two chalcone com-
pounds, isopanduratin A and 4-hydroxypanduratin A, were isolated from the ethyl acetate fraction of ethanol ex-
tract as the active principles. Compared with phenylthiourea (IC50
34.3 m M) as a positive control, the depigmen-
tation IC50 values for isopanduratin A and 4-hydroxypanduratin A were 10.6 m M and 23.2 m M, respectively. The
compounds also significantly inhibited the activity of tyrosinase, the enzyme that converts DOPA (3,4-dihydroxy-
phenylalanine) to dopachrome in the biosynthetic process of melanin. The IC50 values of isopanduratin A and 4-
hydroxypanduratin A for tyrosinase were 10.5 m M and
30 m M, respectively, while that of phenylthiourea was
47.6 m M. The tyrosinase protein level was also significantly decreased by isopanduratin A and 4-hydroxypan-
duratin A. The results indicate that isopanduratin A and 4-hydroxypanduratin A isolated from K. pandurata
ROXB. are promising compounds that could be useful for treating hyperpigmentation as skin-whitening agents.
Key words melanin; tyrosinase; Kaempferia pandurata; isopanduratin A; 4-hydroxypanduratin A; skin whitening

Melanin is the phenolic biopolymer that is responsible for
pigmentation. Although melanin plays a crucial role in ab-
sorbing free radicals from the cytoplasm and shielding from
UV light, the overproduction and accumulation of melanin in
the skin could be a serious skin disorder.1) Current therapies
for skin pigmentation disease are unsatisfactory. Thus the
search for natural and synthetic chemical agents to modulate
the metabolism of pigmentation is of great interest.2)
Melanin biosynthesis occurs in a cascade of enzymatic
and spontaneous reactions that convert tyrosine to melanin
pigments.3) The initial and rate-limiting step in melanin syn-
thesis, the hydroxylation of tyrosine to dihydroxyphenylala-
nine (DOPA), is controlled by tyrosinase that is the key en-
zyme in the process.4) Since the 1980s, skin cancer re-
searchers have studied the melanin biosynthetic pathways,
which led to the development of whitening cosmetics and
medicines.5) Arbutin, kojic acid, hydroquinone and its deriva-
tives were developed in 1980s.6) However, the clinical effects
of those chemicals are unsatisfactory because of their low ac-
tivity and cell toxicity.7) Therefore there is a need for safe
and effective whitening agents.
Kaempferia pandurata ROXB. is a perennial herb of the
Zingiberaceae family mainly cultivated in tropical countries,
including Indonesia and Thailand. The fresh rhizome has
been used as a food ingredient and as a folk medicine for the
treatment of colic, dry cough, rheumatism, musle pain, and
as an aphrodisiac.8) Several studies reported various activities
of K. pandurata, including antiinflammatory, antitumor, an-
tidiarrhea, antidysentery, antiflatulence, and antiepidermo-
phytid effects.9) Isopanduratin A and 4-hydroxypanduratin A
(Fig. 1) are the typical chalcone compounds found in K. pan-
durata. Chalcones belong to a group of phenolic compounds
in the flavonoid family and widely occur in nature as pig-
ments. This category of flavonoids has a broad bioactive
spectrum such as anticancer, antibacterial, antifungal activi-
ties, etc.10—12) In previous studies, chalcone compounds in K.
pandurata showed anticancer, antioxidative, antimutagenic,
and antibacterial activities.13—15) However, their depigment-
ing effect has not yet been examined. Therefore we focused
on evaluating the inhibitory effects of chalcone compounds
isolated from K. pandurata on melanin biosynthesis and ty-
rosinase.
MATERIALS AND METHODS
Plant Material Dried rhizomes of K. pandurata were
collected in Jakarta, Indonesia, and identified by Dr. Nam-In
Baek, Department of Oriental Medicinal Materials and Pro-
cessing, Kyunghee University (Yongin, Korea). A voucher
specimen is deposited in the Department of Biotechnology,
Yonsei University (Seoul, Korea).
Instrumentation NMR spectra were recorded on a
Bruker Avance-600 spectrometer (Rheinstein, Germany) at
600 MHz for 1H- and 13C-NMR in CDCl3 with TMS as an in-
ternational standard. Complete proton and carbon assign-
ments were based on 1D (1H-, 13C-, 13C-DEPT) and 2D
(1H–1H COSY, 1H–13C HSQC, 1H–13C HMBC) NMR experi-
ments. Mass spectra (FAB-MS) were measured using JMS-
700 (JEOL Ltd., Tokyo, Japan). All instrumental data are
available upon request.
Isolation of Isopanduratin A The ground K. pandurata
Fig. 1. Chemical Structure of (A) Isopanduratin A and (B) 4-Hydoxypan-
duratin A

∗ To whom correspondence should be addressed. e-mail: jkhwang@yonsei.ac.kr © 2007 Pharmaceutical Society of Japan
2142
ROXB. (100 g) was extracted with 95% ethanol (400 ml), and
the extract (11.95 g) was further fractionated with ethyl ac-
etate. The ethyl acetate fraction was applied to a silica gel
column (60, 70—230 mesh, Merck & Co., Whitehouse Sta-
tion, NJ, U.S.A.) and eluted with n-hexane, chloroform and
ethyl acetate solution (15 : 5 : 1.5, v/v/v) to give seven frac-
tions (fr. 1 to fr. 7). Fraction 4 (1.09 g) was eluted with 90%
methanol on Rp-18 column chromatography (LiChropep,
25—40 m m, Merck & Co.), yielding fr. 4-B (0.81 g). Then
fraction 4-B was eluted with chloroform and methanol solu-
tion (10 : 0.2, v/v), yielding fr. 4-B-2 (0.65 g). Fr. 4-B-2 was
eluted with n-hexane and ethyl acetate solution (10 : 3, v/v),
yielding the single compound 4-B-2-2 (0.57 g). Careful com-
parison of several spectral data of compound 4-B-2-2 includ-
ing 13C-NMR, 1H-NMR, 13C-DEPT, 1H–1H COSY, 1H–13C
HSQC, 1H–13C HMBC, and FAB-MS with those in the litera-
ture16,17) suggested the chemical structure to be isopanduratin
A (Fig. 1A) or (2-methoxy-4,6-dihydroxyphenyl)-[3-methyl-
2-(3-methybut-2-enyl)-6-phenylcyclohex-3-enyl]-
methanone.
Isolation of 4-Hydroxypanduratin A The ground K.
pandurata ROXB. (100 g) was extracted with 95% ethanol
(400 ml), and the extract (11.95 g) was further fractionated
with ethyl acetate. The ethyl acetate fraction was applied to a
silica gel column (60, 70—230 mesh, Merck & Co.) and
eluted with n-hexane, chloroform, and ethyl acetate solution
(15 : 5 : 1.5, v/v/v) to give seven fractions (fr. 1 to fr. 7). Fr. 6
(0.16 g) was eluted with methylene chloride and methanol
solution (19 : 1, v/v) using a silica gel column, yielding fr. 6-
B (0.08 g). The fraction 6-B yielded the single compound 6-
B-2 (0.03 g), when eluted with chloroform and methanol so-
lutions (20 : 1, v/v). Compound 6-B-2 was finally purified by
preparative HPLC (column: W-252, 20.0 mm i.d.500 mm l,
Japan Analytical Industry Co., Ltd., Tokyo, Japan) using
99.8% methanol as a eluent. Careful comparison of several
spectral data of compound 6-B-2 including 13C-NMR, 1H-
NMR, 13C-DEPT, 1H–1H COSY, 1H–13C HSQC, 1H–13C
HMBC, and FAB-MS with those in the literature9) suggested
the chemical structure to be 4-hydroxypanduratin A (Fig. 1B)
or 2,4,6-trihydroxyphenyl-[3-methyl-2-(3-methylbut-2-
enyl)-6-phenylcyclohex-3-enyl]methanone.
Cell Culture Murine melan-a melanocytes were origi-
nally derived from C57BL/6 J (black, a/a) mice, a kind gift
from Prof. Dorothy C. Bennett (St. George’s Hospital, Lon-
don, U.K.). Melan-a cells were cultured in RPMI 1640 con-
taining 10% heat-inactivated FBS, 100 units/ml of penicillin,
100 m g/ml of streptomycin, and 200 nM of phorbol 12-myris-
tate 13-acetate (TPA) at 37 °C in 10% CO2. The culture
medium was changed twice every week, and the cells were
subcultured once a week.
Cell Viability Determination To evaluate the effects of
isopanduratin A and 4-hydroxypanduratin A on cell viability,
percentages of viable melan-a cells were measured using a
modified crystal violet assay.18) After removing the medium
from each well, the cells were washed with PBS and stained
with 0.1% crystal violet in 10% ethanol for 5 min at room
temperature. The cells were then rinsed four times with dis-
tilled water, and crystal violet retained by adherent cells was
extracted with 95% ethanol at room temperature for 10 min.
Crystal violet absorption was measured at 590 nm (Molecu-
lar Devices Co., San Francisco, CA, U.S.A.).
Vol. 30, No. 11
Measurement of Melanin Content Measurement of
melanin content in melan-a melanocytes was performed as
described previously with minor modification.19) The cells
(2105 cells/ml) were seeded into 24-well plate and the test
compounds were treated in triplicate. The medium was
changed daily, and after 4 d of culture, the cells were lysed
with 1 ml of 1 N NaOH. Then, 200 m l of each crude cell ex-
tracts was transferred into 96-well plates. The relative
melanin content was measured at 400 nm with a microplate
reader (Molecular Devices).
Measurement of Tyrosinase Activity Tyrosinase activ-
ity was measured using the method of Martinez-Esparza et
al.20) Cells were pretreated with 1—30 m M of 3 compounds
for 72 h. The cells were then washed twice with ice-cold PBS
and lysed in lysis buffer (80 mM PO4 buffer1% Triton X-
100100 m g/ml phenylmethanesulphonylfluoride (PMSF))
for 2 h in a deep freezer. Lysates were centrifuged at
12500 rpm for 15 min to remove insoluble material. The pro-
tein concentration was determined using the Bradford
method (Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.)
using BSA as a standard. A 50 m l aliquot of the samples was
mixed with 100 m l of sodium phosphate 100 mM (pH 7.0) at
30 °C for 5 min, and then 50 m l of 100 mM catechol was
added. Activity was assayed at 405 nm for 1 h using a mi-
croplate reader (Molecular Devices).
Western Blot Analysis For tyrosinase immunoblot de-
tection, melan-a cells (2105 cells/ml) were plated on 60 mm
culture dishes and incubated in the presence of 200 nM TPA.
After 24 h incubation, the cells were treated with various
concentrations (1—30 m M) of isopanduratin A and 4-hydrox-
ypanduratin A for 72 h. The cells were lysed with radio-im-
munoprecipitation assay (RIPA) buffer (Sigma-Aldrich Co.,
St. Louis, MO, U.S.A.). The supernatant was collected and
protein concentrations were then determined with protein
assay reagents (Bio-Rad Laboratories Inc., Hercules, CA,
U.S.A.). For Western blotting, equal amounts of proteins
were boiled for 3 min and chilled on ice, subjected to 8—
10% sodium dodecylsulfate polyacrylamide gel elec-
trophoresis, and electrophoretically transferred to a nitrocel-
lulose membrane (Amersham International, Little Chalfont,
U.K.). The membranes were saturated with 5% powdered
skim milk in a saline buffer, and tyrosinase was detected
with the polyclonal anti-tyrosinase antibody (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, U.S.A.) diluted to
1 : 250 in the saturation buffer, and then incubated with the
secondary antibody horseradish peroxidase-conjugated rabbit
anti-goat IgG antibody at a 1 : 1000 dilution. Blotted anti-
body was visualized with a chemiluminescence (ECL) detec-
tion system (Amersham International). The densities of
bands were measured using the RFLPscan version 2.1 soft-
ware program (Scanalytics Inc., Fairfax, VA, U.S.A.).
Statistical Analysis All data are expressed as mean
standard deviation (S.D.). The evaluation of statistical signifi-
cance was performed using one-way analysis of variance
(ANOVA) with a standard software package. The criteria for
statistical significance were set at ∗ p0.05 and ∗∗ p0.01.
RESULTS AND DISCUSSION
To investigate the cytotoxicity of the two chalcone com-
pounds isopanduratin A and 4-hydroxypanduratin A on cell
November 2007
Table 1. Effects of Isopanduratin A and 4-Hydroxypanduratin A on Melanin Synthesis and Tyrosinase Activity of Melan-a Cells
Melanin synthesis
Compounds
IC50a) (m M)
Isopanduratin A 10.64
4-Hydroxypanduratin A 23.25
Phenylthioureab) 34.32
a) 50% inhibitory concentration; b) a positive control.
Fig. 2. Effects of Isopanduratin A and 4-Hydroxypanduratin A on
Melanin Synthesis in Melan-a Cells
(A) Cells (2105 cells/ml) were preincubated for 24 h and then treated with 1—
15 m M of isopanduratin A and (B) 1—30 m M of 4-hydroxypanduratin A for 72 h. Data
are expressed as percentage of control values, and each column represents the
meanS.D. of three determinations. Asterisks indicate a significant difference com-
pared with the control group, ∗ p0.05, ∗∗ p0.01.
proliferation, melan-a cells were treated with various concen-
trations (1—100 m M) of the compounds for 72 h. The IC50
values of isopanduratin A and 4-hydroxypanduratin A for cy-
totoxicity were 35.9 m M and 56.4 m M, respectively (Table 1).
The change in the melanin contents in the cells treated
with isopanduratin A and 4-hydroxypanduratin A was evalu-
ated to determine depigmentation activity. The melanin con-
tents of cells were significantly attenuated by isopanduratin
A and 4-hydroxypanduratin A in a dose-dependent manner
(Fig. 2). The IC50 values for melanin biosynthesis in cultured
melan-a cells are summarized in Table 1. The IC50 values of
isopanduratin A and 4-hydroxypanduratin A were 10.64 m M
and 23.25 m M, respectively, which were about 1.5—3 fold
more potent than that of phenylthiourea (IC5034.32 m M)
used as a positive control.
A number of studies on melanogenic inhibitory com-
pounds are underway for preventing or curing hyperpigmen-
2143
Tyrosinase activity Cell viability
IC50 (m M) IC50 (m M)
10.5 35.9

30 56.4
47.6
1000
Fig. 3. Effects of Isopanduratin A and 4-Hydoxypanduratin A on Tyrosi-
nase Activity in Melan-a Cells
Effects on tyrosinase activity are examined with (A) 1—15 m M of isopanduratin A
and (B) 1—30 m M of 4-hydroxypanduratin A in melan-a cells for 72 h. Data are ex-
pressed as percentage of control values, and each column represents the meanS.D. of
three determinations. Asterisks indicate a significant difference compared with the con-
trol group, ∗∗ p0.01.
tary disorders. Compounds such as hydroquinone, ascorbic
acid derivatives, kojic acid, azelaic acid, corticosteroids,
retinoids, arbutin, and others have been reported to show in-
hibitory efficacy.21—27) Most known depigmenting agents
with small molecular weights are phenolic compounds and
usually they show high cytotoxicity against melanocytes.28)
Phenols such as hydroquinone have been reported to induce
irreversible depigmenting effect in melan-a melanocytes.29)
The first steps in the biosynthetic pathway of melanin are
the hydroxylation of monophenol tyrosine to o-diphenol
DOPA and the oxidation of DOPA to DOPAquinone, both
using molecular oxygen. Tyrosinase catalyzes these steps,
and antioxidants may inhibit these oxidation steps.30) Thus
changes in tyrosinase activity in cells treated with isopan-
duratin A and 4-hydroxypanduratin A are a useful indication
of their depigmentation activity. The two compounds signifi-
cantly inhibited tyrosinase activity in melan-a cells (Fig. 3).
2144
Fig. 4. Effects of Isopanduratin A and 4-Hydroxypanduratin A on the Expression of Tyrosinase Protein
Melan-a cells were seeded at 2105 cells/ml. The cells were treated with 5—15 m M of isopanduratin A and 10—30 m M of 4-hydroxypanduratin A for 72 h. The samples were re-
solved on a 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Specific detection of tyrosinase was performed with the antibody polyclonal tyrosinase.
The IC50 values for tyrosinase inhibitory activities in cultured Korea 21 Project.
melan-a cells are summarized in Table 1. The IC50 value of
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