Objective

In this study, we examined the effect of C. japonicum flower extract (CFE) on melanogenesis and its mechanism in vitro and ex-vivo.

Methods

The effect of CFE on melanogenesis was investigated with lightly (HEMn-LP) and moderately (HEMn-MP) pigmented normal human melanocytes, reconstituted 3-dimensional skin (3D skin) model and ex vivo human hair follicles. The melanogenesis-inducing effect of CFE was evaluated using melanin content and intracellular tyrosinase activity assay. The amount and type of eumenlanin and pheomelanin was analyzed by using HPLC method. The mechanism involved in the effect of CFE on hyperpigmentation was explored by cyclic adenosine monophosphate (cAMP) immunoassay and western blot analysis for tyrosinase, microphthalmia-associated transcription factor (MITF), and phosphorylated CRE binding protein (pCREB) expression. The degree of pigmentation in 3D skin , L-values were measured using a CR-300 chroma meter. The amount of dissolved melanin was measured using a spectrophotometer. The content of melanin in the hair follicles was evaluated by Fontana Masson staining.

Results

CFE significantly increased the melanin content and cellular tyrosinase activity in both HEMn-LP and HEMn-MP cells. The markers of pheomelanin and eumelanin in HEMn-LP and HEMn-MP were also increased by CFE. We observed CFE treatment on melanocytes increased intracellular cAMP with inducing pCREB and up-regulating the protein levels of TYR and MITF. Furthermore, CFE considerably increased the melanin content in a 3D skin model and ex vivo human hair follicles.

Conclusions

These results suggest that CFE exerts hyper-pigmentation activity through cAMP signaling in human melanocytes that it can improve follicular depigmentation and vitiligo by stimulating the melanin synthesis.