Food Chemistry 135 (2012) 993–998
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
The functional property of egg yolk phosvitin as a melanogenesis inhibitor
Samooel Jung a, Dong Hee Kim b, Jun Ho Son b, Kichang Nam c, Dong Uk Ahn d,e, Cheorun Jo a,⇑
a
Department of Animal Science and Biotechnology, Chungnam National University, Daejeon 305-764, South Korea
Product Development Team, Korea Institute for Oriental Medicine Industry, Gyeongsan 712-260, South Korea
c
Department of Animal Science and Technology, Sunchon National University, Suncheon 540-742, South Korea
d
Major in Biomodulation, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea
e
Department of Animal Science, Iowa State University, Ames, IA 50011-3150, USA
b
a r t i c l e
i n f o
Article history:
Received 20 February 2012
Received in revised form 2 May 2012
Accepted 28 May 2012
Available online 7 June 2012
Keywords:
Phosvitin
Melanogenesis
Tyrosinase
Microphthalmia-associated transcription
factor
a b s t r a c t
Phosvitin is a phosphoglycoprotein present in egg yolk. More than half of the amino acids in phosvitin
molecule are serine, of which >90% are phosphorylated. Therefore, phosvitin has a strong metal binding
capability. The aim of this study was to investigate the effect of phosvitin on the inhibition of melanogenesis in melanoma cells. The results showed that phosvitin inhibited the activity of mushroom tyrosinase.
Addition of phosvitin at a concentration of 50 lg/ml, to B16F10 melanoma cells inhibited tyrosinase
activity by approximately 42% and melanin synthesis by 17% compared to those in a control without
phosvitin. Phosvitin inhibited the expression of tyrosinase, tyrosinase-related protein 1 (TRP-1), TRP-2,
and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells. In addition, phosvitin reduced the cellular cAMP concentration in B16F10 melanoma cells. These results indicate that
phosvitin has the potential to be used as a melanogenesis inhibitor in the food and cosmetics industry.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Melanin is an important factor that affects the color of skin and
hair. It is synthesized in melanocytes in the innermost layer of the
epidermis to protect the skin from damage by ultraviolet (UV) radiation (Gilchrest & Eller, 1999). However, excessive accumulation of
melanin can raise aesthetic problems due to hyperpigmentation
such as melasma and age spots, and can be associated with an increased risk for malignant melanoma (Kim & Uyama, 2005). Therefore, finding natural compounds that can regulate melanogenesis
would be valuable for medical and cosmetic industry since proteins that regulate or inhibit melanogenesis is noteworthy in the
medical and cosmetic fields.
Tyrosinase is a key enzyme required for eumelanin and pheomelanin synthesis. Eumelanin is the typical form of biological melanin, a brown–black pigment, whereas pheomelanin is a yellow–
red pigment. Other enzymes such as tyrosinase-related protein 1
and 2 (TRP 1 and TRP 2), regulate eumelanogenesis (Kobayashi
et al., 1995). Many studies were carried out to identify melanogenesis inhibitors to control excessive melanin synthesis in the melanocytes of animals and human skin. As a result, several
melanogenesis inhibitors from natural and synthetic sources, such
as hydroquinone, kojic acid, arbutin, and ethyl-(4-hydroxyphenyl)
oxamate sodium salt, have been identified and used (Cho & Shin,
⇑ Corresponding author. Tel.: +82 42 821 5774; fax: +82 42 825 9754.
E-mail address: cheorun@cnu.ac.kr (C. Jo).
0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.05.113
2011). Several compounds that possess the ability to chelate metals were also reported as melanogenesis inhibitors (Cabanes, Chazarra, & García-Carmona, 1994; Kubo & Kinst-Hori, 1999). Sericin,
which is present in silk, is composed of high levels of serine (30–
33% of total amino acid) and has an ability to inhibit tyrosinase
activity (Kato et al., 1998). However, safer, more stable, and effective melanogenesis inhibitors are still required.
Phosvitin is a phosphoglycoprotein present in egg yolk and represents about 7% of yolk proteins (Mecham & Olcott, 1949). Phosvitin has a specific amino acid composition comprised of 50%
serine, and 90% of which are phosphorylated (Clark, 1985). This
specific structure makes phosvitin a very strong metal chelator
(Grizzuti & Perlmann, 1975; Vieira, 2007). Ishikawa, Yano, Arihara,
and Itoh (2004) and Ishikawa, Ohtsuki, Tomita, Arihara, and Itoh
(2005) reported that phosvitin inhibited iron-catalyzed oxidation
by decreasing hydroxyl radicals formation from the Fenton reactions, and thus inhibited lipid oxidation in a mouse dorsal skin
homogenate in the presence of ferric nitrilotriacetate or UV light
conditions. Furthermore, the metal-chelating ability of phosvitin
was not influenced by 90 °C heat treatment or 600 MPa high pressure (Castellani, Guérin-Dubiard, David-Briand, & Anton, 2004).
However, the ability of phosvitin as a melanogenesis inhibitor
has not yet been studied.
The aim of this study was to evaluate the inhibition of tyrosinase and melanin biosynthesis activity of phosvitin using mushroom tyrosinase and B16F10 melanoma cells. Additionally, the
protein levels of melanogenic enzymes and MITF in melanoma
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S. Jung et al. / Food Chemistry 135 (2012) 993–998
cells were also determined to elucidate the potential use of phosvitin as a melanogenesis inhibitor.
2. Materials and methods
2.1. Chemicals
Tyrosine, mushroom tyrosinase, dimethyl sulfoxide (DMSO), L3,4-dihydroxyphenyl-alanine (L-DOPA), 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl-tetrazoliumbromide (MTT), sodium phosphate
monobasic, sodium phosphate dibasic, sodium chloride, potassium
chloride, phenylmethylsulfonyl fluoride (PMSF), aprotinin, leupeptin, nonyl phenoxypolyethoxylethanol (NP-40), acrylamide, N,N0 methylene-bis-acrylamide, tris (hydroxymethyl) aminomethane
(Tris), sodium dodecyl sulfate (SDS), ammonium persulfate,
N,N,N0 ,N’-tetramethyle-ethylenediamine (TEMED), glycine, glycerin, tetrasodium dehydrate (EDTA), nonfat dried milk, and Tween
20 were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA).
Dulbecco’s modified eagle medium (DMEM), foetal bovine serum
(FBS), penicillin/streptomycin, trypsin 250, and 0.4% trypan blue
stain were purchased from Gibco BRL Co. (Grand Island, NY,
USA). Sodium chloride and ethanol for phosvitin extraction were
of first grade, and other chemicals used in this study were guaranteed reagents.
2.2. Preparation of phosvitin from egg yolks
Phosvitin was prepared according to the method of Ko, Nam, Jo,
Lee, and Ahn (2011). Chicken eggs were obtained from a local market (Daejeon, Korea). Egg yolk was separated from egg white, and
egg yolk membrane and chalaza were removed by filtration
through a testing sieve (2 mm). The filtered egg yolk was homogenized with two volumes of distilled water and centrifuged at 4070g
for 30 min for elimination of yolk plasma. The precipitate (yolk
granules) was collected, homogenized with four volumes of 85%
ethanol and centrifuged at 4070g for 30 min to remove phospholipids. The lipid-free precipitate was then homogenized with five
volumes of 12% NaCl solution to extract phosvitin and centrifuged
at 4070g for 30 min. The supernatant was concentrated and desalted using an ultrafiltration system (Quixstand Benchtop System
using a membrane column with a 10 kDa molecular weight cut-off,
GE Healthcare, Waukesha, WI, USA). At the end of ultrafiltration,
the pH of the solution was adjusted to 4.0, centrifuged at 4070g
for 30 min to remove insoluble particles and then lyophilized.
2.3. Measurement of mushroom tyrosinase activity
Tyrosinase inhibition activity was determined by the degree of
inhibition during tyrosinase-catalyzed oxidation of L-dihydroxyphenylalanine (L-DOPA) to dopachrome following the method described by Yagi, Kanbara, and Morinobu (1986). The reaction
mixture was consisted of 0.5 ml of 0.175 M sodium phosphate buffer (pH 6.8), 0.1 ml sample (phosvitin or ascorbic acid in deionized
distilled water at concentrations of 5, 10, 50, 100, 500, and
1000 lg/ml), 0.2 ml of 10 mM L-DOPA, and 0.2 ml mushroom
tyrosinase (110 U/ml), and was reacted for 2 min at 25 °C. The
accumulation of dopachrome in the reaction mixture was measured with a spectrophotometer (DU530, Beckman Instruments
Inc., Fullerton, CA, USA) at 475 nm. The control used deionized distilled water instead of phosvitin. Tyrosinase inhibitory activity was
calculated and presented the remaining tyrosinase activity (nmol/
min/mg of protein) as % of control. Tyrosinase activity (%) = (activity of the samples/activity in the control) 100.
2.4. Measurement of cell viability
Cell viability was assessed with the MTT assay following the
method described by Carmichael, DeGraff, Gazdar, Minna, and
Mitchell (1987). B16F10 melanoma cells were cultured in DMEM
containing 10% FBS and 1% penicillin/streptomycin (100 U/ml) in
a 5% CO2 incubator at 37 °C. Cultured cells (5 104 cells/well) were
seeded in 96-well plates, and then 20 ll of phosvitin in deionized
distilled water at a concentration of 5, 10, 50, 100, 500, and
1000 lg/ml were added to the wells. Deionized distilled water
was substituted in the control. After a 48 h incubation at 37 °C under 5% CO2, 20 ll of MTT solution (5 mg/ml) was added to each
well. The reaction was terminated by adding 150 ll of DMSO after
4 h incubation. The absorbance of each well was measured at
540 nm using an immunoassay reader (Packard Instrument Co.,
Downers Grove, IL, USA). Cell viability was determined by the following equation: Cell viability (%) = (absorbance in the samples/
absorbance in the control) 100.
2.5. Measurement of intracellular tyrosinase activity
Tyrosinase activity in B16F10 melanoma cells was measured
following the method described by Martinez-Esparza, JimenezCervantes, Solano, Lonzano, and Carcía-Borrón (1998). The cells
were cultured as above (2.4). After incubation, the cells were
washed with potassium phosphate buffered saline (PBS, pH 7.4)
and disrupted in PBS containing 1% Triton X-100. Then, the cells
were lysed by freeze–thawing, and the lysates were centrifuged
at 10,000g for 10 min. Protein content was determined using a
Bio-Rad protein assay kit (Hercules, CA, USA). Each well of a 96well plate contained approximately 40 lg protein, 2.5 mM L-DOPA,
and 0.1 M PBS. The 20 ll of phosvitin or ascorbic acid in deionized
distilled water was added to the culture dish at a concentration of
5, 10, and 50 lg/ml. After 1 h incubation at 37 °C, absorbance was
measured at 475 nm in an immunoassay reader. The control used
deionized distilled water instead of phosvitin. Data were presented
as the remaining tyrosinase activity. Tyrosinase activity
(%) = (activity of the samples/activity in the control) 100.
2.6. Measurement of intracellular melanin level
Intracellular melanin level in B16F10 melanoma cells was measured following the modified method described by Hosoi, Abe,
Suda, and Kuroki (1985). B16F10 melanoma cells were cultured
as above (2.4). Cells were seeded in 100 mm culture dishes
(1 105 cells/dish) and incubated for 24 h. Then, the 2 ml of phosvitin or ascorbic acid in deionized distilled water was added to culture dishes at a concentration of 5, 10, and 50 lg/ml. The control
used deionized distilled water instead of phosvitin. After 48 h incubation, the dishes were washed with PBS (pH 7.4), and the cells
were harvested using 0.25 M trypsin–EDTA. Harvested cells were
mixed with 5% trichloroacetic acid (1 106 cells/ml) and centrifuged at 2090g for 20 min. The obtained melanin was washed with
PBS followed by centrifugation at 2090g for 20 min after adding
1 ml of ether:ethanol (1:3) and then dried. The dried melanin
was dissolved in 1 ml of 1 N NaOH at 80 °C for 1 h, and the melanin
level measured by spectrophotometer at 405 nm. Inhibition of
melanin synthesis was determined with the following equation:
Melanin level (%) = (absorbance in the samples/absorbance in the
control) 100.
2.7. Western blot analysis
Western blot analysis of MITF, TRP-1, TRP-2, and tyrosinase
measured following the method described by Kim et al. (2011).
The medium containing B16F10 melanoma cells was added with
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S. Jung et al. / Food Chemistry 135 (2012) 993–998
phosvitin in deionized distilled water at a concentration of 5, 10,
and 50 lg/ml, followed by 48 h incubation. The control used deionized distilled water instead of phosvitin. The cells were harvested
by washing with PBS, lysed with 100 ll of RIPA buffer (pH 7.4)
composed of 50 mM Tris, 0.1% SDS, 50 mM NaCl, 1% NP-40, 1 mM
PMSF, 10 lg/ml aprotinin, and 10 lg/ml leupeptin, and then centrifuged at 10,000g for 20 min. Protein content in supernatant was
quantified using a Bio-Rad protein assay kit. Samples containing
equal amounts of protein (20 lg) were analyzed using 10% SDS–
PAGE. Proteins in the SDS–PAGE gel were transferred to PVDF
membranes (Bio-Rad) using the Trans-Blot apparatus (Bio-Rad).
After transfer, the membranes were blocked in a solution of 5%
non-fat dried milk (w/v) in TBS-T (pH 7.4) composed of 24.8 mM
Tris, 137 mM sodium chloride, 2.7 mM potassium chloride, and
0.05% Tween 20 for 1 h at room temperature. After six washes with
TBS-T, the membranes were incubated with primary goat tyrosinase antibody, goat TRP-1 polyclonal antibody, goat TRP-2 polyclonal antibody, and mouse MITF monoclonal antibody from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) diluted (1:1000)
with TBS-T for 12 h at 4 °C. Membranes were washed and incubated at room temperature for 2 h with the mouse anti-rabbit
IgG HRP or bovine anti-goat IgG HRP secondary antibody from Santa Cruz Biotechnology (diluted 1:1000). After washing, proteins
were detected in a dark room using an Enhanced Chemiluminesence kit (Amersham Bioscience UK, Ltd., Little Chalfont, UK). The
detection reagent was poured onto the membrane and incubated
for 1 min, and the band density was quantified with a LAS4000 image analyzer (Fujifilm Life Science, Tokyo, Japan).
2.8. Measurement of intracellular cAMP levels
B16F10 melanoma cells were incubated for 1 h at 37 °C in the
absence or presence of phosvitin at a concentration of 5, 10, and
50 lg/ml. The control used deionized distilled water instead of
phosvitin. Cellular cAMP concentrations were determined using a
cAMP immunoassay kit (Cayman Chemical Company, Ann Arbor,
MI, USA) according to the manufacturer’s instructions as follows.
B16F10 melanoma cells (3 105) were lysed in 0.1 M HCl to inhibit the phosphodiesterase activity. The supernatants were then
collected, neutralized, and diluted. After neutralization and dilution, a fixed amount of cAMP conjugate was added to compete with
cAMP in the cell lysate for sites on rabbit polyclonal antibody
immobilized on a 96 well plate. After washing to remove excess
conjugated and unbound cAMP, substrate solution was added to
the wells to determine the activity of the bound enzyme. The color
development was then stopped, after which the absorbance was
read at 415 nm. The concentration of cAMP in the cell lysate was
expressed as a percentage of the value measured in untreated cells.
Fig. 1. Effect of phosvitin on mushroom tyrosinase activity.
among samples indicate significant differences (P < 0.05).
a–i
Different letters
tyrosinase activity was inhibited by phosvitin or ascorbic acid at
a concentration of 5, 10, 50, 100, 500, and 1000 lg/ml (P < 0.05).
However, phosvitin had a lower inhibitory effect than that of ascorbic acid.
3.2. Effect of phosvitin on B16F10 melanoma cell viability
The MTT assay is generally used for determination of cytotoxicity of medicinal agents and toxic materials. The viable cell can reduce MTT, which is soluble yellow tetrazolium dye, by a
dehydrogenation reaction in the mitocondria during metabolism,
and then resultantly produce on insoluble purple formazan product (Carmichael et al., 1987). The cytotoxicity of a melanogenesis
inhibitor is an important consideration when it is used in cosmetics and medical treatments for humans. Melanoma cells incubated
with phosvitin for 48 h showed no significant decrease in cell viability at 5–500 lg/ml level (Fig. 2). Cell viability decreased significantly (20%) in melanoma cells exposed to 1000 lg/ml phosvitin
(P < 0.05). However, the concentration of 50 lg/ml was used as
the maximum phosvitin concentration in subsequent experiments
because the viability of B16F10 cells was shown decreasing trend
at the concentrations of 100 lg/ml or higher.
3.3. Effect of phosvitin on tyrosinase activity and melanin synthesis in
B16F10 melanoma cells
Both tyrosinase activity and melanin synthesis in B16F10 cells
were inhibited by phosvitin (Table 1). Phosvitin (50 lg/ml) induced 42% reduction in cellular tyrosinase activity and 17% reduction in cellular melanin synthesis (P < 0.05). These results indicated
that phosvitin had weaker inhibitory effect on cellular tyrosinase
2.9. Statistical analysis
This study was performed in triplicate. An analysis of variance was performed using the raw data, and the mean values
and standard deviations were calculated using the Statistical
Analysis System (SAS, 2000). Differences among the means were
determined by Duncan’s multiple range test, and significance at
P < 0.05 level.
3. Results
3.1. Effect of phosvitin on mushroom tyrosinase activity
The effect of phosvitin on mushroom tyrosinase activity was
tested and compared with that of ascorbic acid, which has been
widely used as a skin whitening agent (Fig.1). Mushroom
Fig. 2. Effect of phosvitin on B16F10 melanoma cell viability.
among samples indicate significant differences (P < 0.05).
a–c
Different letters
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S. Jung et al. / Food Chemistry 135 (2012) 993–998
Table 1
Effect of phosvitin on tyrosinase activity and melanin synthesis in B16F10 melanoma
cells.
Concentration
(lg/ml)
Tyrosinase
activity
(nmol/min/mg)
Melanin
content
Controla
Phosvitin
–
5
10
50
100
64.1 ± 4.26a
59.9 ± 0.90bc
57.7 ± 0.61c
100
98.8 ± 0.25a
96.3 ± 1.31a
82.5 ± 1.69b
Ascorbic
acid
5
62.6 ± 2.45ab
83.9 ± 4.72b
10
50
43.8 ± 1.28d
32.5 ± 1.06e
75.9 ± 0.45c
58.0 ± 1.38d
a–e
Values with different letters within the same column differ significantly
(P < 0.05). n = 3.
a
Tyrosinase activity of control (0 lg/ml phosvitin) sample was 100 nmol/min/mg.
and melanin synthesis than those of ascorbic acid, which were 67%
and 42%, respectively (P < 0.05).
3.4. Effect of phosvitin on tyrosinase, TRP-1, TRP-2, and MITF levels in
B16F10 melanoma cells
To clarify the effect of phosvitin on melanogenesis, its effect on
expression of melanogenesis-related enzymes, including tyrosinase,
TRP-1, and TRP-2, and MITF were examined in B16F10 melanoma
cells using Western blot (Fig. 3). The tyrosinase level in melanoma
cells exposed to phosvitin (50 lg/ml) was low as much as 40% when
compared with that of the control (P < 0.05). Phosvitin (50 lg/ml)
inhibited the expression of TRP-1 and TRP-2 as much as 10% and
27%, respectively, when compared to those of the control
(P < 0.05). Additionally, the MITF level in melanoma cells exposed
to phosvitin at concentrations of 5, 10, and 50 lg/ml was 91%, 79%,
and 61% of the control, respectively (P < 0.05).
3.5. Effect of phosvitin on cAMP levels in B16F10 melanoma cells
When compared with control, phosvitin at a concentration of 5,
10, and 50 lg/ml caused a 17%, 18%, and 39% reduction of cAMP
level in B16F10 melanoma cells, respectively (P < 0.05) (Fig. 4).
4. Discussion
The eumelanogenesis pathway is consisted of two phases,
whereas pheomelanogenesis occurs in a single phase. Tyrosinase
catalyzes two indispensable reactions in the first phase such as
the hydroxylation of tyrosine to L-DOPA by monophenolase and
the subsequent oxidation of L-DOPA to dopaquinone by diphenolase (Kim & Uyama, 2005). Dopaquione forms cysteinyldopa in
5
Phosvitin
10
50
Tyrosinase
TRP-1
TRP-2
MITF
Fig. 3. Effect of phosvitin on melanogenic protein expression in B16F10 melanoma cells.
a–d
Different letters among samples indicate significant differences (P < 0.05).
S. Jung et al. / Food Chemistry 135 (2012) 993–998
Fig. 4. Effect of phosvitin on cAMP level in B16F10 melanoma cells.
letters among samples indicate significant differences (P < 0.05).
a–c
Different
the presence of cysteine and converts it to 1,4-benzothiazinylalanine, which in turn, polymerizes to pheomelanin. Dopaquione is
spontaneously converted to leucodopachrome, which is oxidized
to dopachrome, the final product of the first phase in the eumelanogenesis pathway (Kobayashi et al., 1995). Tyrosinase has two
copper ions bound to six histidines at the active site and is classified into met-, oxy-, and deoxytyrosinase according to the binuclear copper structure. Monophenolase activity is affected only
by oxytyrosinase, whereas diphenolase activity is affected by both
oxy- and mettyrosinase (Wilcox et al., 1985). Because of this structure, some compounds with metal chelating activity have been
suggested as tyrosinase inhibitors. Flavonoids such as kaempferol
and quercetin, and kojic acid, a fungal metabolite, inhibit tyrosinase activity by chelating the copper ions of mettyrosinase and
oxytyrosinase, respectively (Cabanes et al., 1994; Kubo & KinstHori, 1999). Synthetic sources such as tropolone and methimazole,
also inhibit tyrosinase activity by same mechanism (Andrawis &
Kanh, 1986; Kahn & Andrawis, 1985). The ability of phosvitin to
chelate metal ions may be one of the reasons for its inhibitory
activity on mushroom tyrosinase and cellular tyrosinase in
B16F10 melanoma cells.
The other probable reason for why phosvitin inhibits tyrosinase
activity is the amino acid sequence of phosvitin, because the Nand C- terminal parts of phosvitin are composed of relatively rich
hydrophobic amino acids (Byrne et al., 1984). The binuclear copper
at the tyrosinase active site is surrounded by a hydrophobic protein pocket (Kim & Uyama, 2005). A previous study reported that
the hydrophobic group of dodecyl gallate drives the interaction
with the hydrophobic protein pocket at the active site and inhibits
tyrosinase activity (Kim & Uyama, 2005). Additionally, Noh et al.
(2009) suggested that kojic acid conjugated with an amino acid
such as phenylalanine or tryptophan dramatically increases the
tyrosinase inhibitory activity because of restricted access of the
substrate to the tyrosinase active site as a consequence of a hydrophobic interaction between the amino acid aromatic ring and the
hydrophobic protein pocket at the active site.
Our results showed that phosvitin inhibited the expression of
tyrosinase in B16F10 melanoma cells. The inhibitory activity of
mushroom tyrosinase and cellular tyrosinase by phosvitin was significantly lower than that of ascorbic acid, which may be due to
different mechanisms. Tomita, Hariu, Mizuno, and Seiji (1980) reported that tyrosinase inhibitory activity by ascorbic acid occurs
due to its reducing activity in which dopaquinone is immediately
converted to L-DOPA. However, the difference in inhibitory activity
between phosvitin and ascorbic acid decreased when cellular
tyrosinase was compared in vitro. Although, inhibitory activity of
phosvitin was lower than that of ascorbic acid, these results suggested that phosvitin inhibited the first phase of the melanogenesis
pathway by inhibiting tyrosinase activity.
997
The second phase of eumelanogenesis pathway begins at dopachrome. Dopachrome is spontaneously converted to 5,6dihydroxyindole (DHI) by chemical decarboxylation or by enzymatic conversion to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) by TRP-2 as a dopachrome tautomerase (del Marmol &
Beermann, 1996). Subsequently, DHI is oxidized to indole-5,6-quinone, and DHICA is oxidized to indole-5,6-quinonecarboxylic acid
by TRP-1 and indoles and quinones are polymerized to eumelanin
(Kobayashi et al., 1995). In the present study, 50 lg/ml phosvitin
caused 17% reduction in B16F10 melanoma cell melanin synthesis
because phosvitin inhibited the first phase and the second phase of
melanogenesis pathway. The inhibited expression of TRP-1 and
TRP-2 in B16F10 melanoma cells were verified with that of MITF
when B16F10 melanoma cells were treated with phosvitin. MITF
is a basic-helix-loop-helix (bHLH) structure and bHLH-leucine zipper transcription factors (Park et al., 2006). MITF is related to the
survival of melanocytes and is a major transcription factor regulating the melanogenic enzymes such as tyrosinase, TRP-1, and TRP-2
by binding M-box, which is the melanogenic enzyme gene promoter region (Goding, 2000). Therefore, phosvitin could inhibit
the second phase of the eumelanogenesis pathway by inhibiting
MITF expression. The inhibitory activity of phosvitin on MITF
expression was distinct from other melanogenesis inhibitors, such
as ascorbic acid, arbutin, and kojic acid, which had no effect on
MITF expression (Kim et al., 2004; Choi, Kim, & Chang, 2011; Choi
et al., 2010). This indicated that phosvitin has an excellent potential to be used as an ingredient for functional cosmetics and dermal
medicine because inhibition of MITF expression could increase
melanoma apoptosis by decreasing transcription of the melanoma
apoptosis inhibitor (Dynek et al., 2008).
Regulation of MITF begins with cyclic adenosine-3,5-monophosphate (cAMP) (Goding, 2000). The cAMP-dependent signaling
pathway plays a key role in melanogenesis. When skin is exposed
to UV, melanotrophic hormones such as a-melanocyte-stimulating
hormone (a-MSH), ß-MSH, and adrenocorticotropic hormone bind
to Gs-protein-coupled receptors (MCIR) followed by activation of
adenylyl cyclase (AC). Consequently, AC catalyzes ATP to cAMP. Increased intracellular cAMP activates protein kinase A, which phosphorylates the cAMP responsive element-binding protein (CREB).
CREB induces cellular MITF (Busca & Ballotti, 2000; Im et al.,
1998). The present results showed that 50 lg/ml phosvitin decreased intracellular cAMP levels by 39% in B16F10 melanoma
cells. Therefore, inhibition effect of phosvitin on expression of MITF
in B16F10 melanoma cells may be related to decrease of intracellular cAMP level. These results suggested that phosvitin inhibited
melanogenesis by inhibiting the cAMP-dependent signaling pathway. However, it is difficult to describe the mechanism of decreased cAMP levels by phosvitin in the present study because
the pathway producing cAMP is extremely complex and affected
by many factors. Holst, Elling, and Schwartz (2002) reported that
metal ions, particularly divalent zinc ions, act as an MCIR enhancer.
Tesmer (2005) reported that transmembrane AC produced cAMP
from ATP by two-metal ion catalysis particularly divalent calcium
ion. Therefore, the metal binding ability of phosvitin may influence
MCIR and AS activity. However, further studies are needed for
clearer understanding.
5. Conclusion
Egg yolk phosvitin inhibited not only tyrosinase activity, but
also melanogenic enzyme expression, e.g. tyrosinase, TRP-1, and
TRP-2 by inhibiting MITF in B16F10 melanoma cells. Consequently,
melanin synthesis was inhibited in B16F10 melanoma cells. Furthermore, cAMP levels in B16F10 melanoma cells decreased with
phosvitin treatment. These results suggested that phosvitin has a
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S. Jung et al. / Food Chemistry 135 (2012) 993–998
potential to be used as a natural melanogenesis inhibitor for the
cosmetics industry, which is seeking natural bioactive compounds
as hyper-pigmentation inhibitors for human skin.
Acknowledgment
This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ0081330), Rural Development
Administration, South Korea.
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