Abstract
Melanin pigmentation in human skin is a major defense mechanism against ultraviolet light of the sun, but abnormal pigmentation such as freckles, liver spot could be a serious aesthetic problem. Nearly all studies are mainly concentrated on searching for the materials that have inhibitory activities on tyrosinase. In this work, to isolate phenolic compounds from Gastrodia elata, we purified the extract through solvent fractionation, column chromatography, and recrystallization. They were identified as 4-hydroxybenzyl alcohol 1, bis(4-hydroxyphenyl)methane 2, gastrodin (4-${\beta}$-D-glucopyranosyloxybenzyl alcohol) 3 on the base of spectroscopic evidences. In order to investigate their depigmentation effect, inhibitory activity against mushroom tyrosinase and inhibitory activity of melanin synthesis in B16 melanoma cells were evaluated in vitro. We have found that 4-hydroxybenzyl alcohol 1 and gastrodin (4- ${\beta}$-D-glucopyranosyloxybenzyl alcohol) 3 have no tyrosinase inhibitory activity, but inhibit the melanin synthesis in B16 melanoma cells. Tyrosinase inhibitory activities of bis(4-hydroxyphenyl)methane 2 (IC$\_$50/ = 400 $\mu\textrm{g}$/mL) and butanol fraction (IC$\_$50/ = 46 $\mu\textrm{g}$/mL) were lower/higher than that of arbutin (IC$\_$50/ = 114 $\mu\textrm{g}$/mL), but inhibitory activities of melanin synthesis in B16 melanoma cells were much higher than that of arbutin. Especially, tyrosinase inhibitory activities of isolated phenolic fraction (IC$\_$50/ = 2.37 $\mu\textrm{g}$/mL) from butanol fraction was very higher than that of arbutin (IC$\_$50/ = 114 $\mu\textrm{g}$/mL). Therefore, these results suggest that isolated phenolic compounds from Gastrodia elata have inhibitory activity against mushroom tyrosinase and inhibitory activity of melanin synthesis in 816 melanoma cells in vitro.
본 연구에서는 천마(Gastrodia elata)추출물의 분획과 butanol 분획층의 연속적인 silicagel column chromatography를 통하여 유효성 분인 4-hydroxybenzyl alcohol 1. bis(4-hydroxyphenyl) methane 2. gastrodin(4-$\beta$-D-glucopyranosyloxybenzyl alcohol) 3을 분리하였다. 4-Hydroxybenzyl alcohol 1과 gastrodin(4-$\beta$-D-glucopyranosyloxybenzyl alcohol) 3은 tyrosinase에 대한 저해작용은 없으나 B16 melanoma 세포의 melanin 새성을 억제한다는 것을 발견하였다. Bis(4-hydroxyphenyl)methane 2 ($IC_{50}$/ = 400 $\mu\textrm{g}$/mL)은 arbutin ($IC_{50}$/ = 400 $\mu\textrm{g}$/mL) 보다 약 $\frac{1}{4}$의 tyrosinase 활성 저해작용을 나타내었지만 B16 melanoma 세포의 melanin 생성 억제는 오히려 arbutin 보다 높게 나왔다. 또한 butanol 분획층 ($IC_{50}$/ = 46$\mu\textrm{g}$/mL)의 tyrosinase에 대한 활성 저해 작용이 arbutin ($IC_{50}$/ = 114 $\mu\textrm{g}$/mL)보다 높은 억제 작용을 나타내었고 B16 melanoma 세포의 melanin 생성 억제도 arbutin 보다 높게 나왔다. 특히 butanol 분획층으로부터 분리된 페놀성 혼합물 ($IC_{50}$/ = 2.37 $\mu\textrm{g}$/mL)은 arbutin 보다 약 50배 가까운 매운 높은 tyrosinase 활성 억제 작용을 나타내었다. 이러한 결과로부터 천마추출물로부터 분리된 유효성분들이 tyrosinase에 대한 저해작용 뿐만 아니라 B16 melanoma 세포의 melanin 생성을 억제하는 것을 알 수 있었다.
