An Extract of Withania somnifera Attenuates Endothelin‐1‐stimulated Pigmentation in Human Epidermal Equivalents through the Interruption of PKC Activity Within Melanocytes

Redox imbalances have been shown to be closely linked to a variety of altered cellular responses and profoundly affect intracellular signaling pathways, especially the PKC/MAPK pathway which is a major pathway involved in regulating melanogenesis within human melanocytes. To elucidate the effects of redox balance regulation on epidermal hyperpigmentary disorders, an antioxidant‐rich herb extract of Withania somnifera was used to assess its effect on endothelin‐1 (EDN1)‐stimulated pigmentation in human epidermis equivalents and its biological mechanisms analysed. Addition of the Withania somnifera extract (10 µg/mL) elicited a marked depigmenting effect on EDN1 (10 nm)‐stimulated pigmentation which was accompanied by a significant decrease in eumelanin content. Real‐time RT‐PCR and western blotting revealed that the stimulated expression of melanocyte‐specific mRNAs and proteins, including microphthalmia associated transcription factor (MITF), was significantly suppressed at days 7–10 of culture by the Withania somnifera extract (10 µg/mL), suggesting an impairment in intracellular signaling upstream of gene expression. Signaling analysis revealed that in Withania somnifera extract (10 µg/mL)‐treated human melanoma cells in culture, there was a marked deficiency in EDN1 (10 nm)‐stimulated phosphorylation of Raf‐1, MEK, ERK, MITF and Cyclic AMP responsive element binding protein (CREB) at 15 min after EDN1 treatment. Consistently, treatment with withaferin A, a major component of the Withania somnifera extract, at concentrations of 10–50 µm also significantly down‐regulated the EDN1 stimulated phosphorylation of Raf‐1, MEK, ERK, MITF and CREB at 15 min after EDN1 treatment. Since Raf‐1 is phosphorylated by protein kinase C (PKC) activity, these findings indicate that the Withania somnifera extract attenuates EDN1‐stimulated pigmentation by preferentially inhibiting EDN1‐triggered PKC activity. Copyright © 2011 John Wiley & Sons, Ltd.