연자육과 연자방 추출물을 이용한 세포 내 미백 효능 유효성 평가
Anti-melanogenic Effect of Lotus Seed and Seedpod Extracts via Downregulation of Tyrosinase Stability in B16F10 Murine Melanoma Cells
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목적: 본 연구는 B16F10 세포 내에서 연꽃(Nelumbo nucifera Gaertner)의 부위인 연자육과 연자방 추출물의 anti-melanogenesis 기전을 입증하여 그 효능을 조사하고, 화장품의 새로운 소재로써 사용하는 것을 목표로 하였다. 방법: 연자육과 연자방 추출물은70% 에탄올 용액으로 추출하였다. 각 추출물의 자극성은 B16F10 세포와 HaCaT 세포에서 WST-1을 기반으로 세포 독성 분석 및염증성 cytokine인 IL-1β분석을 통해 확인하였다. 해당 추출물들의 미백 효과는 세포 내 멜라닌 함량 및 tyrosinase 활성 분석을 통해 확인하였다. Tyrosinase 발현에 미치는 영향은 mRNA와 단백질 수준에서 분석되었다. Cycloheximide 처리 후 단백질 수치를 평가하여 해당 추출물이 tyrosinase 단백질 안정성에 미치는 영향을 분석하였다. 결과: WST-1 세포 독성 평가를 통해 ≤1.0% 농도의 연자육과 연자방 추출물은 B16F10 세포에서 독성을 나타내지 않았다. 또한 qRT-PCR 분석에 의해 HaCaT 세포 내에서 IL-1β mRNA 발현은 해당 추출물 처리에 의해 증가하지 않았다. 세포 내 멜라닌 함량 측정을 통해 연자육과 연자방 추출물이 α-MSH 로부터 유도된 멜라닌 합성을 현저하게 저해함을 확인했다. 세포 내 tyrosinase 활성은 추출물에 의해 유의하게 감소하였다. 추가 연구를 통해 tyrosinase 활성의 감소는 mRNA 발현 수준과 무관하며 단백질 발현 수준에 의존함을 확인했다. 이러한 결과는cycloheximide를 사용한 tyrosinase 안정성 평가에서 해당 추출물들에 의해 감소한 것을 재확인하였다. 결론: 연자육과 연자방 추출물은 B16F10 세포에서 번역 후 과정에서 tyorsinase 단백질 분해를 가속화하여 색소 침착 방지 효과를 보였다
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 IL- 1β 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.
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