.ALPHA.-Lipoic Acid Inhibits Melanogenesis in Three-dimensional Human Skin Model

Abstract

The overproduction of pigmentary melanin in the skin causes an undesirable cosmetic appearance. The present study was undertaken to evaluate the inhibitory effect of α-Iipoic acid (LA) on melanogenesis using a three-dimensional human skin model with ultraviolet (UV) irradiation. It is evident that melanin biosynthesis in human skin cells was decreased by the treatment with LA under the concentration range from 0.01 to 0.1% (w/v) in a concentration-dependent fashion, reflecting no effect on cell viability.

Full text

Food Sci. Technol. Res., 15 (5), 537–540, 2009
Note
α
-Lipoic Acid Inhibits Melanogenesis in Three-dimensional Human Skin Model
Tomonori unno
*,†
, Fumihiro ito and Yuko M. SageSaka
Central Research Institute, ITO EN, Ltd., 21 Mekami, Makinohara-shi, Shizuoka 421-0516, Japan
†
Present afliation: Faculty of Home Economics, Tokyo Kasei-Gakuin University, 2600 Aihara-machi, Machida-shi, Tokyo 194-0292,
Japan
Received November 20, 2008; Accepted May 8, 2009
The overproduction of pigmentary melanin in the skin causes an undesirable cosmetic appearance.
The present study was undertaken to evaluate the inhibitory effect of
α
-lipoic acid (LA) on melanogenesis
using a three-dimensional human skin model with ultraviolet (UV) irradiation. It is evident that melanin
biosynthesis in human skin cells was decreased by the treatment with LA under the concentration range
from 0.01 to 0.1% (w/v) in a concentration-dependent fashion, reecting no effect on cell viability.
Keywords:
α
-lipoic acid, melanin, tyrosinase, three-dimensional human skin model
*To whom correspondence should be addressed.
E-mail: tunno@kasei-gakuin.ac.jp
Introduction
α
-Lipoic acid (LA), chemically designated 1,2-dithio-
lane-3-pentanoic acid, is a natural substance found in plant
and animal foods. It is known that LA functions as a mito-
chondrial cofactor in multienzyme complexes that catalyze
the oxidative decarboxylation of
α
-keto acids (Bunik, 2003).
Apart from its biological role in the body, LA has an impres-
sive list of beneficial health effects, such as powerful anti-
oxidant activity (Biewenga et al., 1997), removal of several
toxic metals from the body (Patrick, 2002), suppression of
diabetes complications (Packer et al., 2001) and prevention
of obesity by suppressing hypothalamic AMP-activated pro-
tein kinase activity (Kim et al., 2004).
Topical application of LA to the skin to protect against
a variety of skin damage, including photoaging and hyper-
pigmentation, has recently drawn considerable interest. Ex
vivo pig skin models have demonstrated that LA decreases
UVB radiation-induced cell death or apoptosis (Rijnkels et
al., 2003). Animal studies using dark-skinned Yucatan swine
have also shown that topical treatments with LA for 8 wk
suppressed hyperpigmentation of skin (Lin et al., 2002).
However, there is little evidence of a link to a melanin-
suppressing effect by LA in human skin cells. The present
study aimed to assess the inhibitory effects of LA on mela-
nin biosynthesis in a cultured epidermal human skin model.
Three-dimensional human skin equivalent is a useful tool to
investigate cell-cell interactions in the regulation of in vivo
melanogenesis (Monteiro-Riviere et al., 1997), and the ef-
fects of LA have not been examined using this model to date.
Materials and Methods
Materials
dL-
α
-lipoic acid and kojic acid were pur-
chased from Wako Pure Chemical Industry, Ltd. (Osaka, Ja-
pan). Proteinase K, synthetic melanin and thiazoyl blue tetra-
zolium bromide (MTT) were obtained from Sigma Chemical
Co. (St. Louis, MO, USA). All other chemicals and organic
solvents were of reagent grade.
Cultured skin model Three-dimensional tissue culture
model of human epidermis from an Asian donor (MEL-
300A, Lot. 5765) was purchased from Kurabo Industries,
Ltd. (Osaka, Japan). Skin cultures were kept at 4
℃
until use,
usually within 24 h of arrival. Before treatment, skin cultures
were transferred to 6-well plastic culture plates containing
0.9 mL of pre-warmed long-life maintenance medium, which
is supplied with the MEL-300A kit, and were equilibrated in
a 5% CO
2
incubator at 37
℃
for 1 h. After the 1-h pre-equil-
ibration period, the medium was aspirated off, and the skin
model was exposed to 31.5 mJ/cm
2
UVB for 30 s using a UV
irradiator (Model DT-20MP, ATTO, Co., Tokyo, Japan). Five
milliliters of fresh medium was replaced beneath the skin
c
ultures, and then a 100-μL aliquot of tested solution was
applied directly onto the corneum of the skin tissue. LA was
rst dissolved in dimethyl sulfoxide (DMSO), and was then

diluted with phosphate-buffered saline (PBS), with the nal
concentration of DMSO being 0.5% (v/v). The skin cultures
were again maintained in the 5% CO
2
incubator in a humidi-
fied atmosphere at 37
℃
for 7 d. In the meantime, sample
solutions were changed on the rst and fourth day.
Melanin extraction Skin tissue was homogenized in 0.45
mL of 10 m
M Tris-HCl buffer (pH 6.8) containing 1% so-
dium dodecyl sulfate and 50 μ
M ethylenediaminetetraacetic
acid. To each homogenate, 20 μL of 205 units/mL Proteinase
K was added, and digestion proceeded overnight at 45
℃
. An
a
dditional 20-μL aliquot of Proteinase K was then added, and
further incubation was performed for 4 h. Samples were then
mixed
with 50 μL of 500 m
M sodium carbonate and 10 μL of
30% hydrogen peroxide, and were maintained at 80
℃
for 30
min. After cooling to room temperature, the mixture was ex-
tracted
with 100 μL of chloroform:methanol (2:1, v/v). After
centrifuging at 10,000 × g and room temperature for 10 min,
the optical density of the upper layer was read at 405 nm.
Using a commercial melanin reagent, a standard curve was
constructed so that the melanin contents of unknowns could
be determined.
MTT assay Skin tissue was washed with 0.01
M PBS
three times, Skin models were transferred to well plates
lled with 300 µL of 1 mg/mL MTT solution. After 3-h in-
cubation at 37
℃
in a humidied 5% CO
2
atmosphere, they
were washed with 0.01
M PBS. Extraction was carried out
by adding 2 mL of isopropanol containing 0.04
n hydrogen
chloride. After leaving to stand for 2 h at room temperature,
absorbance of the extract was determined using a spectro-
photometer at 560 nm. The absorbance at 655 nm was used
as a reference.
Statistical analysis Results are represented as means ±
SeM. One-way analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test was used to assess the sta-
tistical signicance of differences, with a p value of < 0.05
used as a measure of significance. Data calculations were
performed using GraphPad Prism for Windows version 4.0
(GraphPad Software, San Diego, CA, USA).
Results
Inhibition of melanogenesis Figure 1 shows typical
photographs of cultured skin samples 7 d after UV irradia-
tion. The apparent darkening was veried in the control skin
model (Fig. 1a). Supplementation with 0.1% (w/v) LA in the
culture media reduced the biosynthesis of melanin in human
skin cells (Fig. 1b). Quantitative evaluation of its reducing
effects was performed by spectrophotometric analysis of
the melanin extracted from the cultured skin model. When
compared with phosphate-buffered saline, exposure of hu-
man epidermal skin cells to 0.5% (v/v) DMSO tended to
stimulate the melanin biosynthesis, although not to a signi-
cant level. Treatment with LA resulted in the reduction of the
melanin biosynthesis in a dose-dependent manner (Fig. 2).
Tukey’s multiple comparison test applied to the data showed
signicant differences with exposure to LA solutions at nal
concentrations of 0.1% (4.8 m
M) versus the control (0.5%
DMSO only). Meanwhile, kojic acid at 0.1% (7.0 m
M) was
less active than LA.
Cell viability To exclude the possibility that LA inu-
ences cell damage to a significant degree, MTT assay of
cultured human epidermis was performed 7 d after treatment
with LA. The results demonstrated that LA did not show de-
tectable cytotoxicity in the concentration range from 0.01 to
0.1% (w/v) (data not shown).
Discussion
The three-dimensional cultured skin model is valuable
for dermatological research as an in vitro model for assess-
ing the pharmacological potency, toxicity and metabolism of
Fig. 1. Microscopic observation of three-dimensional reconstructed
skin tissue after treatment without (A) and with 0.1% LA (B). The
skin model was irradiated with UVB at 31.5 mJ/cm
2
for 30 min, and
was incubated for 7 d with test agents. Magnication: ×100.
Fig. 2. Effects of LA on melanin production in cultured human
skin cells. The amount of melanin in the cells was equivalent for
commercial synthetic melanin. Results are means ±
SeM of triplicate
samples. *p < 0.05 vs. control.
t.
u
nno et al.
A B
PBS Control 0.01% 0.025% 0.05% 0.1% 0.1%
Melanin (µg/culture
）
*
Kojic
acid
α-Lipoic acid
0
10
20
30
40
538

tested compounds because of its similarity to actual human
skin having the interaction between dermis and epidermis
(Bernerd and Asselineau, 2008, Duval et al., 2003, Shigeta
et al., 2004). UVB radiation can increase melanin biosyn-
thesis and proliferation of melanocytes in the epidermis by
acting on melanocytes directly (Friedmann and Gilchrest,
1987). The present study aimed to evaluate the suppressive
effects of LA on the magnitude of melanogenesis using the
three-dimensional cultured human skin cell. As compared to
controls (0.5% DMSO), treatment of this cultured cell model
with LA resulted in a dose-dependent reduction of melanin
biosynthesis in a concentration range from 0.01% to 0.1%,
not reecting the relevant marker of cytotoxic activity.
It has been documented that the mechanism underly-
ing skin protection by LA may be specic to its antioxidant
potential to scavenge free radicals generated (Podda et al.,
2001). Melanocytes in the epidermis contain melanosomes,
the activity of which is a major determinant of the skin color
to produce melanin. Melanosomes acquire tyrosinase, which
is the key enzyme catalyzing the conversion of
L-tyrosine
to dopa and dopaquinone, as well as the subsequent auto-
polymerization of melanin. The microphthalmia-associated
transcription factor is implicated in melanocyte development
and also upregulates melanogenesis via transactivation of
tyrosinase gene expression. Lin et al. (2002) showed that LA
reduced the microphthalmia-associated transcription factor
and tyrosinase promoter activities, resulting in depigmenta-
tion. Attention should be focused on the regulative mecha-
nisms of LA, not only a reduction of intracellular tyrosinase
expression but also direct tyrosinase inhibition. The diverse
mechanisms responsible for reducing melanin biosynthesis
in human skin cells are matters for further consideration.
LA can be readily absorbed from after oral dosage and
appears to be readily converted into its reduced form, dihy-
drolipoic acid, in mammalian cells (Takaishi et al., 2007).
Dihydrolipoic acid is capable of enhancing antioxidant
potency by regenerating
L-ascorbic acid vitamin E (Kagan
et al., 1992), and reportedly impacts both the regulation of
tyrosinase gene expression and direct inhibition of tyrosinase
(Lin et al., 2002). Moreover, dihydrolipoic acid is considered
to have superior inhibitory activity in melanin biosynthesis
when compared to parent LA (Tsuji-Naito et al., 2006). To
better understand the detailed mechanisms of orally or topi-
cally administered LA on the inhibition of melanogenesis,
further investigations into the involvement of such LA me-
tabolites and endogenous antioxidants are warranted.
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