항산화와 미백효과를 증진시킨 새로운 Sulforaphane 함유 혼합 조성의 개발

Development of Novel Sulforaphane Contained-composition to Increase Antioxidant and Whitening Effects

Abstract

본 연구는 새로운 항산화 및 미백 기능성 화장품 성분을 개발하기 위해 기존의 항산화와 미백 기능성 성분으로 알려진 두 가지 성분을 혼합한 새로운 조성을 개발하고, 이를 이용해 항산화 및 미백효과를 알아보고자 하였다. 두 가지 성분은 항산화와 멜라닌 생성억제 기능이 알려진 sulforaphane과 멜라닌 생성 억제 효과가 알려진 PF-3758309이다. 각 성분 및 혼합 조성에 대한 세포독성을 조사하여 세포독성이 나타나지 않는 농도를 결정하였다. 단독 처리 시 sulforaphane는 $10{\mu}M$까지, PF-3758309는 100 nM까지 세포독성이 없다는 것을 확인하였다. 두 화합물 $10{\mu}M$ sulforaphane과 1 nM PF-3758309를 병합 처리 시 세포독성은 없었다. LPS 자극에 의해 생성된 일산화질소는 sulforaphane과 PF-3758309의 병합 처리시 현저히 감소되었다. ${\alpha}$-MSH에 의한 멜라닌 생성 역시 병합 처리에 의해 억제되었다. 화장품 소재로 사용 가능성을 확인하기 위해 혼합 조성시료에 대한 인체적용시험을 실시하였다. 혼합조성 시료는 피부첩포에 의한 안전성 평가는 무자극으로 판정되었고, 피부밝기 증가효과와 피부 멜라닌 침착을 억제함을 확인하였다. 이러한 결과들로 혼합 조성물이 항산화와 미백 기능이 있는 새로운 기능성 화장품 조성으로 개발될 가능성을 확인할 수 있었다.

In this study, we analyzed two components, sulforaphane and PF-3758309 to prove that the mixed composition of them has more effective antioxidant and whitening functions compared to each component. We analyzed the cellular toxicity of each component and also the mixed composition to find the safe concentration level for cell viability. From the single component treatment, we discovered that sulforaphane was safe up to $10{\mu}M$, and PF-3758309 up to 100 nM. Combination treatment of $10{\mu}M$ sulforaphane and 1 nM PF-3758309 did not affect the cell viability. The LPS-stimulated NO generation was significantly reduced by the mixed composition of sulforaphane and PF-3758309. Melanogenesis by ${\alpha}$-melanocyte stimulating hormone (${\alpha}$-MSH) was also inhibited by the mixed composition. In order to confirm the possibility as the cosmetic material, we carried out clinical studies for the mixed composition samples. Skin safety evaluation using patch test was judged to be unstimulated, skin whitening effect was increased, and melanin deposition was suppressed by treatment of mixed composition samples. These results provide us with the opportunity for applying it into the development of new functional cosmetics.

Figure 1. Effect of sulforaphane, PF-3758309 or sulforaphane+ PF-3758309 on cell viability in RAW 264.7 cells. RAW 264.7 cells (1 × 10⁴) were seeded into 96-well plates and cultured for 24 h. Cells were then treated with increasing concentrations of sulforaphane (SFN) (A), PF -3758309 (PF) (B) or the indicated concentrations of sulforaphane+PF-3758309 (C) for 24 h. Cell viability was measured by the MTT assay.

Figure 3. Effect of sulforaphane, PF-3758309 or sulforaphane+ PF-3758309 on melanin synthesis in B16F10 cells. B16F10 melanoma cells were seeded at 1 × 10⁴ cells/well and cultured for 48 h. Cells were treated with SFN (A) or SFN+ PF (B) in the absence or presence of α-MSH for 24 h. Melanin contents were measured at 490 nm. Arbutin was used as a melanogenesis control. The results were expressed as the mean ± SD from the three independent experiments (*p < 0.05).

Figure 2. Effect of sulforaphane, PF-3758309 or sulforaphane+ PF-3758309 on NO production. RAW 264.7 cells (1 × 10⁴) were seeded into 24-well plates and pre-treated with SFN, PF, SFN+PF, ASA for 30 min and stimulated with or without LPS (1μg/ml) for 18 h. Cells were then treated with SFN (A), PF (B) or SFN+PF (C). The culture media was collected and subjected to Griess assay for measurement of NO generated. The results were expressed as the mean ± SD from the three independent experiments (*p < 0.05).

Table 1. EPA Dermal Classification System

Table 2. Skin Safety Evaluation Using Patch Test

Table 3. Statistical Results of Skin Brightness (V)

Table 4. The MI of Skin Pigmentation