Withania somnifera Extract/Withaferin A as a Prospective Anti-pigmenting Agent

Abstract

A screening test was used to assess more than 400 kinds of herbal extracts and related chemicals for their ability to interrupt the endothelin (EDN)1- and/or stem cell factor (SCF)-activated interacellular signaling that leads to the increased activity of tyrosinase in normal human melanocytes (NHMs). A Withania somnifera extract (WSE) and one of its active compounds withaferin A (WFA) showed distinct abrogating effects on the EDN1- and SCF- stimulation of tyrosinase activity without any direct inhibitory effect on tyrosinase, which suggests that the WSE and WFA interrupt signaling pathways upstream of tyrosinase expression. In addition, in a co-culture system using UVB-exposed normal human keratinocytes (NHKs) and NHMs, WFA has a potent ability to abrogate the UVB-stimulated tyrosinase activity in NHMs by abolishing the increased secretion of the intrinsic melanogenic cytokine EDN1 in UVB-exposed NHKs as well as by interrupting signaling pathways upstream of tyrosinase expression in NHMs. Consistently, signaling analysis with immunoblots revealed that in WSE-treated human melanoma cells or NHMs in culture, there is a marked deficiency in the EDN1-stimulated phosphorylation of Raf-1, MEK, ERK, CREB and MITF at 15 min after EDN1 treatment. The absence of an inhibitory effect on EDN1-induced intracellular calcium mobilization in NHMs suggests that the WSE inhibits EDN1-triggered PKC activity and/or abolishes its activation. In NHMs treated with SCF, the WSE also has a distinct potential to abrogate the stimulated phosphorylation of ERK, MITF and CREB, but not of Raf-1 and MEK, which indicates that the WSE attenuates the SCF-triggered phosphorylation of ERK by inhibiting MEK kinase activity. In WFA-treated NHMs, there is a marked deficiency in the SCF-stimulated series of phosphorylations of c-KIT, Shc, Raf-1, MEK, ERK, CREB and MITF. Taken together, the analysis of SCF binding and the effect of dithiothreitol (DTT) revealed that WFA attenuates the SCF-induced activation of c-KIT in NHMs by interrupting the auto-phosphorylation of c-KIT through DTT-suppressible Michael addition thioalkylation reactions without interrupting the binding of SCF to the c-KIT receptor. In human epidermal equivalents consisting of multilayered NHKs and NHMs, addition of the WSE or WFA elicits a marked depigmenting effect on EDN1- or SCF-stimulated pigmentation 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 tyrosinase and microphthalmia associated transcription factor (MITF), is significantly suppressed at days 7–10 of culture by the WSE and by WFA. The sum of these findings strongly suggests that the WSE and WFA can serve as potent melanogenic signaling-interruption type anti-pigmenting agents without any risk of hypopigmentation, thus they are prospective therapeutic candidates for treating hyperpigmentary disorders such as UVB-melanosis, solar lentigo and melasma where the over-expression of SCF and/or EDN1 is involved in the hyperpigmentation mechanism.