말오줌나무 추출물의 미백활성 검증

Whitening Activity of Sambucus Sieboldiana Var. Pendula (Nakai) Extract

Abstract

본 연구는 말오줌나무 70% 에탄올 추출물의 항산화 효과와 미백 효과를 검증하였다. 말오줌나무 추출물의 전자공여능 측정실험은 $1,000{\mu}g/ml$에서 86.21%의 효과를 나타내었고, ABTS 소거능을 측정한 결과 $1,000{\mu}g/ml$ 농도에서 97.9%의 소거능을 보였다. Tyrosinase 저해활성 측정은 $1,000{\mu}g/ml$ 농도에서 37%의 효과를 보였다. 말오줌나무 추출물의 세포 생존율을 melanoma cell (B16F10)에서 확인하기 위해 MTT assay를 진행하였는데 세포 생존율을 측정한 결과, $100{\mu}g/ml$ 농도에서 90% 이상의 생존율을 보였다. 말오줌나무 추출물의 MITF, TRP-1, TRP-2 및 tyrosinase의 단백질 발현 효과를 25, 50, $100{\mu}g/ml$ 농도에서 측정하였으며 양성 대조군으로 ${\beta}-actin$을 사용하였다. 그 결과, MITF, TRP-1, TRP-2 및 tyrosinase의 각각 $100{\mu}g/ml$ 농도에서 34.5%, 45.6%, 58.4%, 79.6%의 단백질 발현 억제 효과를 보였다. MITF, TRP-1, TRP-2 및 tyrosinase의 mRNA발현을 RT-PCR로 25, 50, $100{\mu}g/ml$ 농도에서 측정하였고, 양성 대조군으로 GAPDH를 사용하였다. 그 결과, 말오줌나무 추출물의 $100{\mu}g/ml$ 농도에서 각각 85.4%, 67.5%, 85.2%, 67.1%의 mRNA 발현이 감소함을 확인 할 수 있었다. 따라서, 말오줌나무 추출물이 미백 소재로서의 가능성을 확인할 수 있었다.

This study evaluated the anti-oxidant and whitening effects of a 70% ethanol extract of the Sambucus sieboldiana var. pendula (Nakai) (SS). At $1,000{\mu}g/ml$ concentration, the electron donating ability of this SS extract was found to be 86.21% and the ABTS+ radical scavenging ability was 97.9%. In terms of whitening activity, the tyrosinase inhibitory effect of the extract was 37%, also at $1,000{\mu}g/ml$ concentration. To explore the extractefftoxicity to B16F10 melanoma cells, a 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide assay was performed. Results showed 90% or more cells remained viable at $100{\mu}g/ml$ concentration. A Western blot of the SS extract was used to measure microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), tyrosinase relate protein-2 (TRP-2), and the tyrosinase protein expression inhibitory effect at 25, 50, $100{\mu}g/ml$ concentrations; ${\beta}-actin$ was used as a positive control. Consequently, the MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect were seen to decrease by 34.5%, 45.6%, 58.4%, and 79.6%, respectively, at $100{\mu}g/ml$ concentration. These were also then measured by reverse transcription-polymerase chain reaction at 25, 50, $100{\mu}g/ml$ concentrations with GAPDH as a positive control. As a result, the SS extract was seen to decrease MITF, TRP-1, TRP-2, and the tyrosinase protein expression inhibitory effect by 85.4%, 67.5%, 85.2%, 67.1%, respectively at the $100{\mu}g/ml$ concentration. We therefore confirmed the possibility of Sambucus sieboldiana var. pendula (Nakai) extract as a whitening material.

Fig. 1. Electron donating ability of extract from Sambucus sieboldiana var. pendula (Nakai).

Fig. 2. ABTS⁺ radical scavenging activity of Sambucus sieboldiana var. pendula (Nakai) extracts.

Fig. 3. Inhibition rate of Sambucus sieboldiana var. pendula (Nakai) extracts on tyrosinase.

Fig. 4. Cell viability of extracts from Sambucus sieboldiana var. pendula (Nakai) on melanoma cell (B16F10).

Fig. 5. MITF protein expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cell (B16F10).

Fig. 6. TRP-1 protein expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cell (B16F10).

Fig. 7. TRP-2 protein expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cell (B16F10).

Fig. 8. Tyrosinase protein expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cell (B16F10).

Fig. 9. MITF mRNA expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cells (B16F10).

Fig. 10. TRP-1 mRNA expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cells (B16F10).

Fig. 11. TRP-2 mRNA expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cells (B16F10).

Fig. 12. Tyrosinase mRNA expression rate of Sambucus sieboldiana var. pendula (Nakai) extracts on melanoma cells (B16F10).

Table 1. Sequence of the primers used for RT-PCR