Induction of Melanin Synthesis by <i>Pueraria lobata</i> Leaves in B16 Murine Melanoma Cells and Three-Dimensional Human Skin Equivalent

Puerariae Radix, the root of Pueraria lobata (Willd.) Ohwi is primarily used as a traditional herbal medicine in East Asia. Although phytochemical and biological investigations of the roots have been well-reported, those of the leaves are limited. To explore the potential value of P. lobata leaves, this study aimed to evaluate their effect on melanin synthesis in B16-F1 murine melanoma cells and three-dimensional (3D) human skin equivalents and investigate the underlying molecular mechanism. The leaf extract increased the melanin content in B16-F1 cells, with stronger induction potency than that of the vine and root extracts. Among the 4 fractions prepared from the leaf extract, the hexane fraction significantly induced melanin synthesis. Both the leaf extract and hexane fraction also increased melanin content in a 3D human skin equivalent model. Of the 7 fractions separated from the hexane fraction, fraction 1 (fr. 1) strongly induces melanin synthesis and intracellular tyrosinase activity. Further analysis indicated that fr. 1 may induce melanin synthesis via cAMP-responsive element-binding protein (CREB) phosphorylation and microphthalmia-associated transcription factor (MITF) expression, leading to the expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. These results suggest that P. lobata leaves may be a useful medicinal resource for treating hypopigmentation.