Anti-melanogenic Effect of Lotus Seed and Seedpod Extracts via Downregulation of Tyrosinase Stability in B16F10 Murine Melanoma Cells

Abstract

Purpose: This study investigates the anti-melanogenic effects of lotus seed and seedpod extract in B16F10 murine melanoma cells, and demonstrates the possible mechanisms involved in anti-pigmentation.Methods: The lotus seed and seedpod extracts were prepared using 70% ethanol as solvent. The irritation potential of the extracts was investigated using water-soluble tetrazolium salt (WST-1)-based cytotoxicity assay and expression analysis of the pro-inflammatory cytokine IL1β in B16F10 cells and HaCaT keratinocytes, respectively. The anti-melanogenic effects of the extracts were analyzed using intracellular melanin contents and tyrosinase activity assays. The effects of the extracts on tyrosinase expression were analyzed at the mRNA and protein levels. The effects of those extracts on the stability of tyrosinase protein were analyzed by evaluating the protein level after cycloheximide treatment.Results: WST-1-based cytotoxicity assay indicated that the concentration of ≤1.0% for lotus seed and seedpod extracts did not exhibit any cytotoxicity in B16F10 cells. Also, qRT-PCR analysis showed that the expression of IL-1β mRNAs was not increased by those concentrations of the extracts in HaCaT keratinocytes. Additionally, intracellular melanin contents assay showed that those extracts significantly inhibited α-MSH-induced melanin synthesis. Furthermore, cellular tyrosinase activity was significantly inhibited by the extracts. Additional investigations revealed that tyrosinase activity reduction was independent of its mRNA expression level, but dependent on its protein expression level. These findings were further confirmed by the results of the cycloheximide experiments stating that the protein stability of tyrosinase was reduced after the treatment with those extracts.Conclusion: Lotus seed and seedpod extracts showed anti-pigmentation effects by accelerating degradation of tyrosinase protein at the post-translation level in B16F10 cells.