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Phytomedicine 18 (2011) 1244– 1249

Contents lists available at ScienceDirect

Phytomedicine

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itrus flavanone naringenin enhances melanogenesis through the activation ofnt/�-catenin signalling in mouse melanoma cells

u-Chun Huang ∗, Chao-Hsun Yang, Yi-Ling Chiouepartment of Cosmetic Science, Providence University, 200 Chung-Chi Rd., Shalu, Taichung 43301, Taiwan, ROC

r t i c l e i n f o

eywords:aringeninelanogenesis

hosphatidylinositol 3-kinase-Cateninlycogen synthase kinase 3�

a b s t r a c t

Citrus fruits are the major source of flavonoids for humans, and flavanones are the main flavonoids inthe Citrus species. Among the Citrus flavanones, the glycoside derivatives of naringenin, naringin andnarirutin, are the most abundant in grapefruit. The present study aimed to investigate the molecularevents of melanogenesis induced by naringenin in murine B16-F10 melanoma cells. Melanin content,tyrosinase activity and Western blot analysis were performed to elucidate the possible underlying mech-anisms. Exposure of melanoma cells to naringenin resulted in morphological changes accompanied bythe induction of melanocyte differentiation-related markers, such as melanin synthesis, tyrosinase activ-

ity, and the expression of tyrosinase and microphthalmia-associated transcription factor (MITF). We alsoobserved an increase in the intracellular accumulation of �-catenin as well as the phosphorylation ofglycogen synthase kinase-3� (GSK3�) protein after treatment with naringenin. Moreover, the activity ofphosphatidylinositol 3-kinase (PI3K) was up-regulated by naringenin since the phosphorylated level ofdownstream Akt protein was enhanced. Based on these results, we concluded that naringenin inducedmelanogenesis through the Wnt-�-catenin-signalling pathway.

ntroduction

The colour of mammalian skin and hair is determined by a num-er of factors, the most obvious phenotypical characteristics ofhich is the distribution of the melanin pigment. Melanin synthe-

is is a complex process that occurs within specialized intracellularrganelles named melanosomes in melanocytes. Melanogenesisan be stimulated by stress, including UV radiation, inflamma-ion, and hormones (Costin and Hearing 2007). Furthermore,-melanocyte-stimulating hormone (�-MSH) and cAMP-elevatinggents, such as forskolin and IBMX, through the activation of pro-ein kinase A (PKA) and cAMP-related element binding proteinCREB) transcription factor, promote an increase in the expressionf microphthalmia-associated transcription factor (MITF), a masteregulator of the development and differentiation of melanocytes.ITF transcriptional regulates the expression of tyrosinase and

yrosinase-related protein that control the conversion of tyrosine toelanin pigments (Vachtenheim and Borovansky 2010). Previous

apers have linked up-regulated melanogenesis to melanoma, and

his viewpoint enhances the importance of further melanogenetictudies.

∗ Corresponding author. Tel.: +886 4 2631 1167; fax: +886 4 2631 1167.E-mail address: ychuang@pu.edu.tw (Y.-C. Huang).

944-7113/$ – see front matter © 2011 Elsevier GmbH. All rights reserved.oi:10.1016/j.phymed.2011.06.028

© 2011 Elsevier GmbH. All rights reserved.

The Wnt-signalling pathways play an important role inmelanocyte development, melanoma genesis and pigment cellformation (O’Connell and Weeraratna 2009). Glycogen synthasekinase 3 (GSK3) is one of the few signalling mediators that playa central role in a diverse range of signalling pathways, includingthose activated by Wnts, growth factors, and G protein-coupledligands (Wu and Pan 2010). When WNT proteins bind to theirreceptors, they inactivate GSK3�, an enzyme that phosphorylates�-catenin and specifically targets its destruction in the proteasome(Bienz 2005). Then, �-catenin accumulates in the cytoplasm andtranslocates to the nucleus. Increased levels of nuclear �-cateninincrease the expression of MITF, and in turn increase the survivaland proliferation of melanoma cells (Miller and Mihm 2006). How-ever, GSK3� which is a negative regulator of Wnt signalling, iscapable of activating MITF function through phosphorylation atSer 298 (Takeda et al. 2000a). A recent study has demonstratedthat inhibiting GSK3� increases melanogenesis both in murine B16cells and human melanocytes (Bellei et al. 2008). It is therefore pos-sible that GSK3� contributes to maintaining the levels of MITF inmelanogenesis.

Flavanones occur almost exclusively in citrus fruits. Citrusflavanones exhibit wide ranges of biological activities, such as

antioxidant, anti-inflammatory and anti-tumour activities, whichindicate that these compounds may exert beneficial effectsagainst cardiovascular diseases or cancers (Benavente-García andCastillo 2008). The main flavonoids in grapefruit are naringin

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naringenin-7-neohesperoside) (70%) and narirutin (naringenin-7-utinoside) (20%) (Kawaii et al. 1999). Most studies on naringeninave reported its possible roles in grapefruit juice-drug interac-ions (Fuhr 1998). Recently, several reports have focused on theotential use of flavonoids for preventing oxidative skin damageMortimer 1997; Proteggente et al. 2003). Bioflavonoids with fla-anone structures, such as hesperidin, have been found to inhibityrosinase activity in human primary melanocytes (Zhu and Gao008). In a previous study, it has been shown that naringenin

ncreases the melanin content and tyrosinase activity by increas-ng the expression of melanogenic enzymes (Ohguchi et al. 2006).he mechanisms underlying the activities of naringenin and its gly-osides on melanogenesis have not yet been well elucidated. Theresent study aimed to evaluate whether the flavanones in grape-ruit juice affect melanogenesis in melanoma cells and to elucidatehe possible underlying related signalling.

aterials and methods

aterials

Naringenin, l-DOPA, 3-[4,5-dimethylthiazol-2-yl]-2,5-iphenyltetrazolium bromide (MTT), melanin and IBMX werebtained from Sigma–Aldrich (St. Louis, MO, USA). Naringin andarirutin were purchased from Acros Organics and ECHO Chem-

cal Co., respectively. Antibodies for tyrosinase, �-catenin, andhospho-Akt (Ser 473) were obtained from Epitomics (Burlingame,A, USA). Actin and phospho-GSK3� (Ser 9) antibodies were sup-lied by Millipore (Temecula, CA, USA). Anti-MITF antibody wasbtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Naringenin, narirutin, or naringin was dissolved in dimethyl-ulfoxide (DMSO) and further diluted in culture medium. The finalMSO concentration in the medium was 0.1% and did not affectellular function or the assay systems used in this study.

ell cultureThe B16-F10 murine melanoma cells were purchased from

he Bioresource Collection and Research Center (Hsinchu, Taiwan)nd maintained in Dulbecco’s modified Eagle’s medium (DMEM;yclone, Logan, UT, USA) supplemented with 10% fetal bovine

erum (Biological Industries, Kibbutz Beit Haemek, Israel), 50 U/mlenicillin, and 50 �g/ml streptomycin in a humidified incubator at7 ◦C in 5% CO2/air.

ell viability assayBriefly, cells were seeded at a density of 7 × 104/ml on 96-well

lates and cultured overnight as described above. The medium washen replaced with fresh medium containing flavanones at vari-us concentrations. After incubation for 48 h at 37 ◦C in 5% CO2/air,TT (final concentration, 0.5 mg/ml) was added, and the cells were

hen incubated at 37 ◦C for 2 h. Finally, the cells were lysed andbsorbance was detected at 550 nm. For cell number determina-ion, a standard correlation between the known cell numbers andhe absorbance density values was constructed for measuring theell number from various detected absorbance density values.

elanin content determinationThe melanin content was measured by a previously described

ethod (Kim et al. 2005) with slight modifications. Cells wereeeded at a density of 9 × 105/ml in 60-mm dishes and cultured asescribed above. After overnight incubation, cells were then cul-ured for 48 h with or without flavanones in either the absence

r presence of LY294002. The medium was then removed, andhe cells were washed twice with phosphate-buffered saline (PBS)nd harvested by trypsinisation using 0.05% trypsin/0.02% EDTA.he harvested cells were centrifuged, and the pellet was dissolved

ne 18 (2011) 1244– 1249 1245

by adding 1 N NaOH, followed by incubation at 60 ◦C for 1 h. Theamount of melanin in the solution was determined by measur-ing the absorbance at 470 nm using the microplate reader (BioTek,Synergy HT). The total melanin content was estimated using thestandard curve of synthetic melanin.

Tyrosinase assayTyrosinase enzyme activity was estimated spectrophotometri-

cally as described earlier (Bellei et al. 2008), using l-DOPA as thesubstrate. B16-F10 cells cultured with or without naringenin for48 h were solubilised with 0.1 M sodium phosphate buffer (pH 6.8)containing 1% Triton X-100. The cells were then disrupted, and cen-trifuged at 10,000 × g for 30 min. After protein quantification andadjustment, 90 �l of cell lysate (each containing the same amountof protein) was incubated in duplicate with 10 �l of 10 mM l-DOPAat 37 ◦C for 90 min. The absorbance was then monitored at 475 nm.In order to assess the direct activity of tyrosinase, naringenin wasadded to cell lysate at the highest concentration and incubated for5 min at room temperature. The cell lysates were then mixed withl-DOPA solution and incubated at 37 ◦C for 2 h, as described above.

Western blot analysisWhole cell lysates were prepared using RIPA buffer (50 mM

Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM PMSF,1 mM Na3VO4, 1 mM NaF, 1 �g/ml aprotinin, 1 �g/ml pepstatin, and1 �g/ml leupeptin). Aliquots of cell lysates were separated by elec-trophoresis on sodium dodecyl sulphate-polyacrylamide gels andtransferred onto polyvinylidene difluoride membranes and thenblotted with the appropriate antibodies. Finally, the proteins weredetected using an enhanced chemiluminescence kit (AmershamBiosciences, Buckinghamshire, UK). Quantitative analysis was per-formed using ImageQuant analysis software (GE Healthcare).

Statistical evaluationData are expressed as mean ± S.E.M. of the indicated num-

ber of separate experiments. A one-way analysis of variance wasperformed for multiple comparisons, and if there was significantvariation between treatment groups, the mean values were com-pared with the respective control using Student’s t-test. P valuesless than 0.05 were considered significant.

Results and discussion

As a first step towards determining the effects of naringeninand its related glycosides on melanogenesis, we measured the cellviability and melanin content in B16-F10 melanoma cells. Cellstreated with various concentrations of the flavanones (3–100 �M)were estimated using the mitochondria MTT reduction assay. Theresults demonstrated that the three structure-related flavanones– naringenin, naringin and narirutin – had no cytotoxic effects atconcentrations ranging from 3 to 50 �M (data not shown). Thecells were then exposed to the flavanones (50 �M) for 48 h, andcellular melanin contents were examined. As shown in Fig. 1a,naringenin increased melanin synthesis apparently. Narirutin ornaringin, the rutinose or neohesperidose glycoside of naringenin,did not show the melanogenic effects as potential as naringenindid. Although narirutin also enhanced the melanin production,it simultaneously increased the cell number, indicating that theeffects of narirutin on melanogenesis may occur due to cell growth.The cell proliferation and membrane integrity of 50 �M narin-genin was also evaluated by a trypan blue exclusion assay after48 h treatment. Naringenin did not exert the proliferative effect

in B16-F10 cells since no statistically significant differences wereobserved between naringenin-treated cells and DMSO-treated con-trol cells (data not shown). Here, we confirmed that only naringeninenhanced melanin synthesis in B16-F10 cells, but not its derivatives

1246 Y.-C. Huang et al. / Phytomedicine 18 (2011) 1244– 1249

Fig. 1. Effect of citrus flavanones on melanin synthesis in B16-F10 melanoma cells.(A) Cells were treated with 50 �M of naringenin, narirutin, or naringin for 48 h.Then, the cellular melanin content and cell viability were determined. Each valuewas expressed as a percentage relative to the DMSO-treated control. Data representthe mean ± S.E.M. of at least three independent experiments. *P < 0.05, **P < 0.01,amm

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Fig. 2. Effect of naringenin on melanin synthesis and tyrosinase activity in B16-F10melanoma cells. Cells were treated with various concentrations of naringenin for48 h, and the cellular melanin content (A) and tyrosinase activity (B) were deter-mined. IBMX (30 �M) was used as a positive control for melanin synthesis. Eachvalue was expressed as a percentage relative to the DMSO-treated control. Data

nd ***P < 0.001 compared with the control. (B) Representative morphology ofelanoma cells treated with or without naringenin for 48 h was assessed by lighticroscopy. Original magnification 400×.

ith the rutinose or neohesperidose glycone. The result observedrom naringenin and its glycosides on melanogenesis was similar touercetin and its glycoside. Quercetin rather induced melanogen-sis in human melanoma cells and three-dimensional epidermalodel, as has been reported (Nagata et al. 2004; Takeyama et al.

004). However, the glycoside derived from quercetin, i.e., rutin,id not show the same activities (Nitoda et al. 2008). Naringeninas thus a more promising candidate for causing alterations in theechanisms of melanogenesis, and was therefore investigated in

ater experiments.Melanocyte differentiation is normally characterized as an

ncrease in melanocyte dendrite production (Buscà and Ballotti000). B16-F10 cells have short dendrites under normal cultureonditions. However, cells treated with naringenin exhibited ausiform shape associated with a parallel cell arrangement (Fig. 1b).he number of elongated cells was notably increased. Therefore, weonsidered it worthy to elucidate the effects of naringenin carefully.

Upon naringenin treatment, the melanin content increased in concentration-dependent manner. The effect of 50 �M narin-enin on melanogenesis was comparable to that of 30 �M IBMXFig. 2a). Since tyrosinase is known to play a key role in melano-enesis, the effect of naringenin on tyrosinase activity in cellsas determined using an l-DOPA oxidation assay. The results

btained displayed that tyrosinase activity was up-regulated in aoncentration-dependent manner by naringenin in B16-F10 cellsFig. 2b). The increased tyrosinase activity caused by naringeninas similar to the increase in melanin content. To exclude a direct

nfluence of naringenin on tyrosinase activity, we directly addedaringenin to an untreated cell lysate and measured the in vitroyrosinase activity (data not shown). It was found that naringenin

id not directly regulate tyrosinase activity in an in vitro system,ut did stimulate tyrosinase activity in B16-F10 cells.

We observed that naringenin enhanced melanogenesis by acti-ating tyrosinase. We hypothesized that the modulation of the

represent the mean ± S.E.M. of at least three independent experiments. *P < 0.05,**P < 0.01, and ***P < 0.001 compared with the control.

melanogenic signalling pathway may be responsible for the stim-ulatory activity of naringenin on the melanin synthesis. Therefore,the cell lysates obtained after treatment with different doses ofnaringenin were subjected to Western blot analysis in order todetermine the expression of melanogenesis-related proteins. It wasnoted that the levels of tyrosinase and MITF protein was augmentedby naringenin (Fig. 3a). Quantification of the signal detected usingthe ImageQuant program revealed a 1.4-fold increase in the levelof tyrosinase following treatment with 50 �M naringenin (P < 0.01,n = 4) as compared to the DMSO-treated control value. Moreover,the expression of �-catenin, one of the factors that control MITFtranscription, was increased gradually by the increasing concen-trations of naringenin (Fig. 3a). After a period of 6 h, naringeninaugmented the expression �-catenin to about 1.3-fold, and thisphenomenon was sustained until the end of the treatment (Fig. 3b).According to the above data, naringenin induced the accumula-tion of intracellular �-catenin while promoting melanogenesis byup-regulating the expression of MITF and tyrosinase, resulting inincreased melanin content.

�-Catenin is known to directly interact with the MITF pro-tein and then activate MITF-specific target genes (Schepsky et al.2006). Both MITF and �-catenin are mediators of Wnt signals dur-ing melanocyte differentiation (O’Connell and Weeraratna 2009;

Takeda et al. 2000b). The inhibition of GSK3-mediated �-cateninphosphorylation is known to be the key event in Wnt-�-cateninsignalling (Wu and Pan 2010). The amount of phospho-GSK3� in

Y.-C. Huang et al. / Phytomedicine 18 (2011) 1244– 1249 1247

Fig. 3. Effect of naringenin on the expression of melanogenic proteins in B16-F10 melanoma cells. Cells were treated with 3–50 �M of naringenin for 48 h (A) or 50 �Mo to Wp

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f naringenin for the indicated time periods (B). Whole cell lysates were subjectedhospho-GSK3� and phospho-Akt. Equal protein loading was confirmed by actin.

esponse to naringenin was also up-regulated, thus causing thenactivation of GSK3� (Fig. 3a).

GSK3 has constitutive kinase activity; further, its activity isignificantly reduced by phosphorylation at Ser 9 in GSK3� ander 21 in GSK3�. Several kinases can phosphorylate these serineesidues, such as Akt, PKA, protein kinase C, and MAPK-activatedrotein kinase-1 (MAPKAP-K1, also called p90rsk) (Jope and Johnson004; Stambolic and Woodgett 1994). Phosphatidylinositol 3-inase (PI3K/Akt) signalling has been suggested to be involvedn the regulation of melanogenesis. Therefore, we estimated therotein expression of phospho-Akt. The results showed that thectivation of Akt was induced by naringenin from early timeoints (Fig. 3b). After treated with naringenin for 1 h, the lev-ls of phospho-Akt (Ser 473) were increased. The Akt activationccurred prior to �-catenin accumulation. These results indicatedhat in naringenin-induced melanogenesis stimulation, the activityf tyrosinase increased along with up-regulation of the upstreamignalling pathway related to its activity and expression.

In this respect, we observed a naringenin-induced increase inhospho-Akt prior to the elevation in the level of �-catenin. It waseasonable to speculate that the activation down-stream target ofI3K by naringenin was associated with the protein stabilizationf �-catenin. In addition, the increase in phospho-GSK3� levelsorresponded to that in �-catenin levels. In other words, the phos-horylation of GSK3� by naringenin inactivated the kinase activityf GSK3�, resulting in the intracellular accumulation of �-catenin.herefore, naringenin elicited cellular responses associated withhe activation of the Wnt pathway (Fig. 4). To the best of ournowledge, this is the first time that a Citrus flavonoid has been

emonstrated to induce melanogenesis by activating the Wnt sig-alling pathway.

Naringenin via the direct activation of PI3K has been proved toave an insulin-like effect on apolipoprotein B secretion by HepG2

estern blot analysis using specific antibodies against tyrosinase, MITF, �-catenin,

cells (Borradaile et al. 2003). In the present study, it was unclearwhether naringenin activated PI3K directly to stimulate melano-genesis since the increase in melanin content following naringenintreatment was not abolished by the PI3K inhibitor LY294002.On the contrary, the melanin production induced by naringeninwas potentiated by LY294002 (data dot shown). LY294002 hasbeen noted to up-regulate the expression of melanogenic enzymesthrough a transcriptional mechanism (Khaled et al. 2003). In a pre-vious report, it was also shown that the phosphorylation of Akt aswell as of GSK3� was attenuated by forskolin, a cAMP-elevatingagent. Correspondingly, the activation of GSK3� is known to stim-ulate the expression of melanogenic enzymes (Khaled et al. 2002).These data possibly indicate that PI3K/MAPK pathways are consti-tutively active and negatively regulate melanogenesis in B16-F10cells (Singh et al. 2005). Since naringenin did not repress the acti-vation of Akt nor GSK3� as did forskolin, we hypothesize thatcAMP-involved regulatory activity in melanogenesis did not con-tribute to the effects of naringenin.

Naringenin is known to play an inhibitory role on cyclicnucleotide phosphodiesterases (PDEs), such as PDE1, PDE4, andPDE5, to increase the production of cGMP and cAMP (Oralloet al. 2005). Therefore, we evaluated whether naringenin inducesalterations in cAMP levels in B16-F10 cells. In our unpublisheddata, cell incubation with 50 �M naringenin for 30 min did notalter the cAMP levels, while a 4.2-fold increase was observed incAMP levels following IBMX treatment. In addition, naringenin-induced a fusiform shape rather than the dendritic phenotypeas forskolin did. Hence, our results suggest that naringeninmediates its melanogenic effects through a cAMP-independent

pathway. However, our present data are insufficient for exclud-ing the possibility of cGMP-mediated signalling participation innaringenin-induced melanogenesis. Moreover, PKC-related sig-nalling has also been implicated in melanocyte differentiation (Park

1248 Y.-C. Huang et al. / Phytomedicine 18 (2011) 1244– 1249

Fig. 4. Model of signalling pathways involved in naringenin-induced melanogenesis. Naringenin enhances phosphorylation of Akt and GSK3� protein. The phosphorylationo ing inb tyros

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f GSK3� on serine 9 by naringenin inactivated the kinase activity of GSK3�, resulty up-regulating the melanocyte differentiation-related proteins, such as MITF and

t al. 2009). Whether naringenin acts via the activation of PKC forxecuting its melanogenic effects requires further clarification.

The most abundant Citrus flavonoids are flavanones (Benavente-arcía and Castillo 2008). Citrus flavanones are present in thelycoside or aglycoside forms. Grapefruit juice contains quite highmounts of these compounds, and therefore the intake from dietan be relatively high. Erlund et al. (2000) studied the bioavail-bility and pharmacokinetics of flavanones after single ingestionf grapefruit juice (8 ml/kg). The resulting plasma concentra-ions of naringenin were comparatively high (up to 14.8 �mol/l).

hen single oral administration of 135 mg of naringenin, theean peak plasma concentration was 7.39 ± 2.83 �mol/l (Kanaze

t al. 2007). Upon the limited information, the concentrationshat were required for obtaining the melanogenetic effects in vitron the B16-F10 cell model were comparatively higher than thelasma concentrations obtained after oral ingestion of naringenin

n human.Topically applied dihydroxyacetone and melanins have been

hown to provide some photoprotection. The skin pigmentationnhancers have the potential to reduce both photodamage andkin cancer incidence. Bicyclic monoterpenes (BMPs) are abun-ant in plants and foods, and BMP diols when applied to humankin have been found to be efficacious in the induction of pigmen-ation (Brown 2001). Naringenin possesses a flavanone backbonend belongs to polyphenolic structure. GSK3 inhibitors stimulatedelanin synthesis both in murine melanoma B16 and normal

uman melanocytes (Bellei et al. 2008). Further studies are neededo determine if naringenin have the resembling effects in human

elanocytes.In conclusion, naringenin enhances melanin synthesis and

yrosinase activity in B16-F10 cells by promoting the expres-

ion of tyrosinase, MITF, and �-catenin along with the enhancedhosphorylation of Akt or GSK3�. These results suggest that narin-enin induces melanogenic signalling by activating the PI3K/Aktr Wnt/�-catenin pathways. This is of great cosmeceutical impor-

the elevation of intracellular �-catenin levels. �-Catenin promotes melanogenesisinase.

tance for designing tanning products with potential to reduce skincancer risks.

Acknowledgements

This study was supported by grants from the National ScienceCouncil of the Republic of China (NSC98-2320-B-126-001-MY3)and Providence University (PU97-11100-B15).

References

Bellei, B., Flori, E., Izzo, E., Maresca, V., Picardo, M., 2008. GSK3beta inhibitionpromotes melanogenesis in mouse B16 melanoma cells and normal humanmelanocytes. Cell. Signal. 20, 1750–1761.

Benavente-García, O., Castillo, J., 2008. Update on uses and properties of citrusflavonoids: new findings in anticancer, cardiovascular, and anti-inflammatoryactivity. J. Agric. Food Chem. 56, 6185–6205.

Bienz, M., 2005. beta-Catenin: a pivot between cell adhesion and Wnt signalling.Curr. Biol. 15, R64–R67.

Borradaile, N.M., de Dreu, L.E., Huff, M.W., 2003. Inhibition of net HepG2 cellapolipoprotein B secretion by the citrus flavonoid naringenin involves activationof phosphatidylinositol 3-kinase, independent of insulin receptor substrate-1phosphorylation. Diabetes 52, 2554–2561.

Brown, D.A., 2001. Skin pigmentation enhancers. J. Photochem. Photobiol. B 63,148–161.

Buscà, R., Ballotti, R., 2000. Cyclic AMP a key messenger in the regulation of skinpigmentation. Pigment Cell Res. 13, 60–69.

Costin, G.E., Hearing, V.J., 2007. Human skin pigmentation: melanocytes modulateskin color in response to stress. FASEB J. 21, 976–994.

Erlund, I., Kosonen, T., Alfthan, G., Mäenpää, J., Perttunen, K., Kenraali, J., Parantainen,J., Aro, A., 2000. Pharmacokinetics of quercetin from quercetin aglycone and rutinin healthy volunteers. Eur. J. Clin. Pharmacol. 56, 545–553.

Fuhr, U., 1998. Drug interactions with grapefruit juice. Extent, probable mechanismand clinical relevance. Drug Saf. 18, 251–272.

Jope, R.S., Johnson, G.V., 2004. The glamour and gloom of glycogen synthase kinase-3.Trends Biochem. Sci. 29, 95–102.

Kanaze, F.I., Bounartzi, M.I., Georgarakis, M., Niopas, I., 2007. Pharmacokinetics of thecitrus flavanone aglycones hesperetin and naringenin after single oral adminis-tration in human subjects. Eur. J. Clin. Nutr. 61, 472–477.

Kawaii, S., Tomono, Y., Katase, E., Ogawa, K., Yano, M., 1999. Quantitation of flavonoidconstituents in citrus fruits. J. Agric. Food Chem. 47, 3565–3571.

edici

K

K

K

MMN

N

O

O

O

P

P

Wu, D., Pan, W., 2010. GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem.

Y.-C. Huang et al. / Phytom

haled, M., Larribere, L., Bille, K., Aberdam, E., Ortonne, J.P., Ballotti, R., Bertolotto, C.,2002. Glycogen synthase kinase 3beta is activated by cAMP and plays an activerole in the regulation of melanogenesis. J. Biol. Chem. 277, 33690–33697.

haled, M., Larribere, L., Bille, K., Ortonne, J.P., Ballotti, R., Bertolotto, C.,2003. Microphthalmia associated transcription factor is a target of thephosphatidylinositol-3-kinase pathway. J. Invest. Dermatol. 121, 831–836.

im, Y.J., No, J.K., Lee, J.H., Chung, H.Y., 2005. 4,4′-Dihydroxybiphenyl as a new potenttyrosinase inhibitor. Biol. Pharm. Bull. 28, 323–327.

iller, A.J., Mihm Jr., M.C., 2006. Melanoma. N. Engl. J. Med. 355, 51–65.ortimer, P.S., 1997. Therapy approaches for lymphedema. Angiology 48, 87–91.agata, H., Takekoshi, S., Takeyama, R., Homma, T., Yoshiyuki Osamura, R., 2004.

Quercetin enhances melanogenesis by increasing the activity and synthesis oftyrosinase in human melanoma cells and in normal human melanocytes. Pig-ment Cell Res. 17, 66–73.

itoda, T., Isobe, T., Kubo, I., 2008. Effects of phenolic compounds isolated fromRabdosia japonica on B16-F10 melanoma cells. Phytother. Res. 22, 867–872.

’Connell, M.P., Weeraratna, A.T., 2009. Hear the Wnt Ror: how melanoma cellsadjust to changes in Wnt. Pigment Cell Melanoma Res. 22, 724–739.

hguchi, K., Akao, Y., Nozawa, Y., 2006. Stimulation of melanogenesis by the citrusflavonoid naringenin in mouse B16 melanoma cells. Biosci. Biotechnol. Biochem.70, 1499–1501.

rallo, F., Camina, M., Alvarez, E., Basaran, H., Lugnier, C., 2005. Implication ofcyclic nucleotide phosphodiesterase inhibition in the vasorelaxant activity ofthe citrus-fruits flavonoid (+/-)-naringenin. Planta Med. 71, 99–107.

ark, H.Y., Kosmadaki, M., Yaar, M., Gilchrest, B.A., 2009. Cellular mechanisms regu-

lating human melanogenesis. Cell. Mol. Life Sci. 66, 1493–1506.

roteggente, A.R., Basu-Modak, S., Kuhnle, G., Gordon, M.J., Youdim, K., Tyrrell, R.,Rice-Evans, C.A., 2003. Hesperetin glucuronide, a photoprotective agent arisingfrom flavonoid metabolism in human skin fibroblasts. Photochem. Photobiol.78, 256–261.

ne 18 (2011) 1244– 1249 1249

Schepsky, A., Bruser, K., Gunnarsson, G.J., Goodall, J., Hallsson, J.H., Goding, C.R., Ste-ingrimsson, E., Hecht, A., 2006. The microphthalmia-associated transcriptionfactor Mitf interacts with beta-catenin to determine target gene expression. Mol.Cell. Biol. 26, 8914–8927.

Singh, S.K., Sarkar, C., Mallick, S., Saha, B., Bera, R., Bhadra, R., 2005. Human placentallipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma.Pigment Cell Res. 18, 113–121.

Stambolic, V., Woodgett, J.R., 1994. Mitogen inactivation of glycogen synthasekinase-3 beta in intact cells via serine 9 phosphorylation. Biochem. J. 303,701–704.

Takeda, K., Takemoto, C., Kobayashi, I., Watanabe, A., Nobukuni, Y., Fisher, D.E.,Tachibana, M., 2000a. Ser298 of MITF, a mutation site in Waardenburg syn-drome type 2, is a phosphorylation site with functional significance. Hum. Mol.Genet. 9, 125–132.

Takeda, K., Yasumoto, K., Takada, R., Takada, S., Watanabe, K., Udono, T., Saito,H., Takahashi, K., Shibahara, S., 2000b. Induction of melanocyte-specificmicrophthalmia-associated transcription factor by Wnt-3a. J. Biol. Chem. 275,14013–14016.

Takeyama, R., Takekoshi, S., Nagata, H., Osamura, R.Y., Kawana, S., 2004. Quercetin-induced melanogenesis in a reconstituted three-dimensional human epidermalmodel. J. Mol. Histol. 35, 157–165.

Vachtenheim, J., Borovansky, J., 2010. “Transcription physiology” of pig-ment formation in melanocytes: central role of MITF. Exp. Dermatol. 19,617–627.

Sci. 35, 161–168.Zhu, W., Gao, J., 2008. The Use of botanical extracts as topical skin-lightening agents

for the improvement of skin pigmentation disorders. J. Invest. Dermatol. Symp.Proc. 13, 20–24.