Journal of Natural Medicines

, Volume 66, Issue 3, pp 435–446 | Cite as

Withania somnifera extract attenuates stem cell factor-stimulated pigmentation in human epidermal equivalents through interruption of ERK phosphorylation within melanocytes

  • Hiroaki Nakajima
  • Katsunori Fukazawa
  • Yuki Wakabayashi
  • Kazumasa Wakamatsu
  • Genji Imokawa
Original Paper


We previously demonstrated that mitogen-activated protein kinase (MAPK) signaling, including microphthalmia-associated transcription factor (MITF) and cAMP response element-binding protein (CREB) phosphorylation, is a major pathway involved in up-regulating melanogenesis within human melanocytes in several hyperpigmentary disorders such as UVB melanosis and lentigo senilis. Recently, a redox imbalance was shown to be closely linked to a variety of altered cellular responses in which the precise balance between levels of oxidizing and reducing equivalents that reflect the intracellular redox condition profoundly affects intracellular signaling pathways, especially the MAPK pathway. To elucidate the effects of redox balance regulation on epidermal pigmentation, we used an antioxidant-rich extract of the herb Withania somnifera to assess its effect on stem cell factor (SCF)-stimulated pigmentation in human epidermal equivalents and analyzed its biological mechanism of action. Addition of the W. somnifera extract (WSE) caused a marked reduction in SCF-stimulated pigmentation in a dose-dependent manner after 14 days of treatment, which was accompanied by a significant decrease in eumelanin content. In WSE-treated human epidermal equivalents, melanocyte-specific proteins (including tyrosinase) were significantly suppressed at the gene and protein levels by WSE. Signaling analysis with immunoblots revealed that in human melanocytes or human melanoma cells treated with WSE, there was a marked deficiency in SCF-stimulated phosphorylation of ERK, MITF and CREB, but not of Raf-1 and MEK. Since WSE had no direct inhibitory effect on tyrosinase activity and no melano-cytotoxic effect on melanocytes present in the human epidermal equivalents or on cultured human melanocytes, the sum of these findings indicates that WSE attenuates SCF-stimulated pigmentation by preferentially interrupting ERK phosphorylation within melanocytes and can serve as a therapeutic tool for SCF-associated hyperpigmentary disorders.


Withania somnifera Melanocytes Stem cell factor MAPK Pigmentation 



Withania somnifera extract


Stem cell factor




Endothelin B receptor


Mitogen-activated protein kinase


Microphthalmia-associated transcription factor


Normal human melanocytes


Protein kinase C


Reactive oxygen species


Tyrosine kinase


Tyrosinase-related protein-1



We thank Dr. Hiroshi Murata of the Department of Dermatology, School of Medicine, Shinshu University for providing us with acral lentigo malignant melanoma (SM2-1) cells.

Conflict of interest

The authors have declared no conflict of interest.


  1. 1.
    Yada Y, Higuchi K, Imokawa G (1991) Effects of endothelins on signal transduction and proliferation in human melanocytes. J Biol Chem 266:18352–18357PubMedGoogle Scholar
  2. 2.
    Imokawa G, Yada Y, Miyagishi M (1992) Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes. J Biol Chem 267:24675–24680PubMedGoogle Scholar
  3. 3.
    Imokawa G, Miyagishi M, Yada Y (1995) Endothelin-1 as a new melanogen: coordinated expression of its gene and the tyrosinase gene in UVB-exposed human epidermis. J Invest Dermatol 105:32–37PubMedCrossRefGoogle Scholar
  4. 4.
    Hachiya A, Kobayashi A, Ohuchi A, Takema Y, Imokawa G (2001) The paracrine role of stem cell factor/c-kit signaling in the activation of human melanocytes in ultraviolet B-induced pigmentation. J Invest Dermatol 116:578–586PubMedCrossRefGoogle Scholar
  5. 5.
    Hachiya A, Kobayashi A, Yoshida Y, Kitahara T, Takema Y, Imokawa G (2004) Biphasic expression of two paracrine melanogenic cytokines, stem cell factor and endothelin-1, in ultraviolet B-induced human melanogenesis. Am J Pathol 65:2099–2109CrossRefGoogle Scholar
  6. 6.
    Suzuki I, Cone RD, Im S, Nordlund J, Abdel-Malek ZA (1996) Binding of melanotropic hormones to the melanocortin receptor MC1R on human melanocytes stimulates proliferation and melanogenesis. Endocrinology 137:1627–1633PubMedCrossRefGoogle Scholar
  7. 7.
    Imokawa G, Yada Y, Kimura M (1996) Signaling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes. Biochem J J314:305–312Google Scholar
  8. 8.
    Imokawa G, Kobayashi T, Miyagishi M, Higashi K, Yada Y (1997) The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis. Pigment Cell Res 10:218–228PubMedCrossRefGoogle Scholar
  9. 9.
    Imokawa G, Kobayashi T, Miyagishi M (2000) Intracellular signaling mechanisms leading to synergistic effects of endothelin-1 and stem cell factor on proliferation of cultured human melanocytes: cross-talk via trans-activation of the tyrosine kinase c-kit receptor. J Biol Chem 275:33321–33328PubMedCrossRefGoogle Scholar
  10. 10.
    Abdel-Malek Z, Swope VB, Suzuki I, Akcali C, Harriger MD, Boyce ST, Urabe K, Hearing VJ (1995) Mitogenic and melanogenic stimulation of normal human melanocytes by melanotropic peptides. Proc Natl Acad Sci USA 92:1789–1793PubMedCrossRefGoogle Scholar
  11. 11.
    Sato-Jin K, Nishimura EK, Akasaka E, Huber W, Nakano H, Miller A, Du J, Wu M, Hanada Sawamura D, Fisher DE, Imokawa G (2008) Epistatic connections between MITF and endothelin signaling in Waardenburg syndrome and other pigmentary disorders. FASEB J 22:1155–1168PubMedCrossRefGoogle Scholar
  12. 12.
    Bentley NJ, Eisen T, Goding CR (1994) Melanocyte-specific expression of the human tyrosinase promoter: activation by the microphthalmia gene product and role of the initiator. Mol Cell Biol 14:7996–8006PubMedGoogle Scholar
  13. 13.
    Fang D, Setaluri V (1999) Role of microphthalmia transcription factor in regulation of melanocyte differentiation marker TRP-1. Biochem Biophys Res Commun 256:657–663PubMedCrossRefGoogle Scholar
  14. 14.
    Du J, Miller AJ, Widlund HR, Horstmann MA, Ramaswamy S, Fisher DE (2003) MLANA/MART1and SILV/PMEL17/GP100 are transcriptionally regulated by MITF in melanocytes and melanoma. Am J Pathol 163:333–343PubMedCrossRefGoogle Scholar
  15. 15.
    Mizutani Y, Hayashi N, Kawashima M, Imokawa G (2010) A single UVB exposure increases the expression of functional KIT in human melanocytes by up-regulating MITF expression through the phosphorylation of p38/CREB. Arch Dermatol Res 302:283–294PubMedCrossRefGoogle Scholar
  16. 16.
    Kadono S, Manaka I, Kawashima M, Kobayashi T, Imokawa G (2001) The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis. J Invest Dermatol 116:571–577PubMedCrossRefGoogle Scholar
  17. 17.
    Hattori H, Kawashima M, Ichikawa Y, Imokawa G (2004) The epidermal stem cell factor is over-expressed in lentigo senilis: Implication for the mechanism of hyperpigmentation. J Invest Dermatol 122:1256–1265PubMedCrossRefGoogle Scholar
  18. 18.
    Wang ZQ, Si L, Tang Q, Lin D, Fu Z, Zhang J, Cui B, Zhu Y, Kong X, Deng M, Xia Y, Xu H, Le W, Hu L, Kong X (2009) Gain-of-function mutation of KIT ligand on melanin synthesis causes familial progressive hyperpigmentation. Am J Hum Genet 84:672–677PubMedCrossRefGoogle Scholar
  19. 19.
    Matsuzawa A, Ichijo H (2008) Redox control of cell fate by MAP kinase: physiological roles of ASK1–MAP kinase pathway in stress signaling. Biochim Biophys Acta 1780:1325–1336PubMedCrossRefGoogle Scholar
  20. 20.
    Agarwal R, Diwanay S, Patki P, Patwardhan B (1999) Studies on immunomodulatory activity of Withania somnifera (Ashwagandha) extract in experimental immune inflammation. J Ethnopharmacol 67:27–35PubMedCrossRefGoogle Scholar
  21. 21.
    Gupta SK, Mohanty I, Talwar KK, Dinda A, Joshi S, Bansal P, Saxena A, Arya DS (2004) Cardioprotection from ischemia and reperfusion injury by Withania somnifera: a hemodynamic, biochemical and histopathological assessment. Mol Cell Biochem 260:39–47PubMedCrossRefGoogle Scholar
  22. 22.
    Ahmad M, Saleem S, Ahmad AS, Ansari MA, Yousuf S, Hoda MN, Islam F (2005) Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol 24:137–147PubMedCrossRefGoogle Scholar
  23. 23.
    Owais M, Sharad KS, Shehbaz A, Saleemuddin M (2005) Antibacterial efficacy of Withania somnifera (Ashwagandha) an indigenous medicinal plant against experimental murine salmonellosis. Phytomedicine 12:229–235PubMedCrossRefGoogle Scholar
  24. 24.
    Rasool M, Varalakshmi P (2006) Immunomodulatory role of Withania somnifera root powder on experimental induced inflammation: an in vivo and in vitro study. Vascul Pharmacol 44:406–410PubMedCrossRefGoogle Scholar
  25. 25.
    Singh D, Aggarwal A, Maurya R, Naik S (2007) Withania somnifera inhibits NF-kB and AP-1 transcription factors in human peripheral blood and synovial fluid mononuclear cells. Phytother Res 21:905–913PubMedCrossRefGoogle Scholar
  26. 26.
    Ichikawa H, Takada Y, Shishodia S, Jayaprakasam B, Nair MG, Aggarwal BB (2006) Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through suppression of nuclear factor-nB (NF-nB) activation and NF-nB-regulated gene expression. Mol Cancer Ther 5:1434–1445PubMedCrossRefGoogle Scholar
  27. 27.
    Kaileh M, Vanden Berghe W, Heyerick A, Horion J, Piette J, Libert C, De Keukeleire D, Essawi T, Haegeman G (2007) Withaferin a strongly elicits IkappaB kinase beta hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282:4253–4264PubMedCrossRefGoogle Scholar
  28. 28.
    Aalinkeel R, Hu Z, Nair BB, Sykes DE, Reynolds JL, Mahajan SD, Schwartz SA (2010) Genomic analysis highlights the role of the JAK-STAT signaling in the anti-proliferative effects of dietary flavonoid ‘Ashwagandha’ in prostate cancer cells. Evid Based Complement Altern Med 7:177–187PubMedCrossRefGoogle Scholar
  29. 29.
    Green H (1978) Cyclic AMP in relation to proliferation of the epidermal cell: new view. Cell 15:801–811PubMedCrossRefGoogle Scholar
  30. 30.
    Rheinwald JG, Green H (1975) Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331–344PubMedCrossRefGoogle Scholar
  31. 31.
    Nakajima H, Wakabayashi Y, Wakamatsu K, Imokawa G (2011) An extract of Melia toosendan attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes. Arch Dermatol Res 303:263–276PubMedCrossRefGoogle Scholar
  32. 32.
    Ito S, Fujita K (1985) Microanalysis of eumelanin and pheomelanin in hair and melanomas by chemical degradation and liquid chromatography. Anal Biochem 144:527–536PubMedCrossRefGoogle Scholar
  33. 33.
    Winder AJ, Harris H (1991) New assays for the tyrosine hydroxylase and dopa oxidase activities of tyrosinase. Eur J Biochem 198:317–326PubMedCrossRefGoogle Scholar
  34. 34.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  35. 35.
    Amae S, Fuse N, Yasumoto K, Sato S, Yajima I, Yamamoto H, Udono T, Durlu YK, Tamai M, Takahashi K, Shibahara S (1998) Identification of a novel isoform of microphthalmia-associated transcription factor that is enriched in retinal pigment epithelium. Biochem Biophys Res Commun 247:710–715PubMedCrossRefGoogle Scholar
  36. 36.
    Blume-Jensen P, Claesson-Welsh L, Siegbahn A, Zsebo KM, Westermark B, Heldin CH (1991) Activation of the human c-kit product by ligand-induced dimerization mediates circular actin reorganization and chemotaxis. EMBO J 10:4121–4128PubMedGoogle Scholar
  37. 37.
    Cutler RL, Liu L, Damen JE, Krystal G (1993) Multiple cytokines induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cells. J Biol Chem 268:21463–21465PubMedGoogle Scholar
  38. 38.
    Lennartsson J, Blume-Jensen P, Hermanson M, Ponten E, Carlberg M, Ronnstrand L (1999) Phosphorylation of Shc by Src family kinases is necessary for stem cell factor receptor/c-kit mediated activation of the Ras/MAP kinase pathway and c-fos induction. Oncogene 18:5546–5553PubMedCrossRefGoogle Scholar
  39. 39.
    Liu L, Damen JE, Cutler RL, Krystal G (1994) Multiple cytokines stimulate the binding of a common 145-kilodalton protein to Shc at the Grb2 recognition site of Shc. Mol Cell Biol 14:6926–6935PubMedGoogle Scholar
  40. 40.
    Luyendyk JP, Piper JD, Tencati M, Reddy KV, Holscher T, Zhang R (2007) Novel class of antioxidants inhibit LPS induction of tissue factor by selective inhibition of the activation of ASK1 and MAP kinases. Arterioscler Thromb Vasc Biol 27:1857–1863PubMedCrossRefGoogle Scholar
  41. 41.
    Kamata H, Honda S, Maeda S, Chan L, Hirata H, Karin M (2005) Reactive oxygen species promote TNFα-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120:649–661PubMedCrossRefGoogle Scholar
  42. 42.
    Pham CG, Bubici C, Zazzeribu F, Papa S, Jones J, Alvarezm K (2004) Ferritin heavy chain upregulation by NF-kB inhibits TNFα-induced apoptosis by suppressing reactive oxygen species. Cell 119:529–542PubMedCrossRefGoogle Scholar
  43. 43.
    Goldman EH, Chen L, Fu H (2004) Activation of apoptosis signal-regulating kinase 1 by reactive oxygen species through dephosphorylation at serine 967 and 14-3-3 dissociation. J Biol Chem 279:10442–10449PubMedCrossRefGoogle Scholar
  44. 44.
    Hiroaki N, Yuki W, Kazumasa W, Genji I.(2011) An extract of Withania somnifera attenuates endothelin-1-stimulated pigmentation in human epidermal equivalents through the interruption of PKC activity within melanocytes. Phytother Res. doi: 10.1002/ptr.3552

Copyright information

© The Japanese Society of Pharmacognosy and Springer 2011

Authors and Affiliations

  • Hiroaki Nakajima
    • 1
  • Katsunori Fukazawa
    • 1
  • Yuki Wakabayashi
    • 1
  • Kazumasa Wakamatsu
    • 2
  • Genji Imokawa
    • 1
  1. 1.School of Bioscience and BiotechnologyTokyo University of TechnologyHachiojiJapan
  2. 2.School of Health SciencesFujita Health UniversityToyoakeJapan

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