化學藥物對黑色素細胞及C57BL/6小鼠白化作用動物模式之研究

Abstract

Hydroquinone (HQ)為酚類化學藥物，為最早被發現之致白化藥物，廣泛應用於皮膚漂白，如黑斑(melasma)或美白產品，其相關衍生化合物 p-hydroxyanisole (MMEH)亦具有類似效果。研究黑色素生成(melanogensis)在色素代謝性疾病或美容相關領域，可針對具促進或抑制黑色素生成之物質或代謝路徑加以探討和研究。本實驗主要目的為研究去色素作用(depigmentation)之試驗模式探討，分別採用B16F10、A2058黑色素腫瘤細胞株與C57BL/6(B6)小鼠，進行比較去色素藥物之作用，以了解色素表現之差異性。在in vitro方面，以B16F10小鼠與A2058人類來源黑色素腫瘤細胞株，分別評估酚類化合物HQ與MMEH之致白化作用，並以酪胺酸酶之酵素活性(tyrosinase activity)及黑色素含量(melanin content)作為偵測指標，同時以 Kojic acid (KA)與Ascorbic acid (AA)兩種已知酪胺酸酶抑制藥物，作為陽性對照組並進行比較。結果顯示，HQ對於B16F10細胞Tyrosinase酵素活性在1mM抑制率約為27% (p <0.05)， MMEH於10mM 抑制率可達30% (p<0.05)，KA於1mM完全無抑制效果，但AA在1mM卻呈現增加現象 ; HQ對A2058細胞株於10mM抑制率約14%(p<0.05)，AA於1mM抑制率近40%(p<0.05)，MMEH與KA於10和1mM則無顯著差異，B16F10與A2058細胞對化學藥物去色素表現之差異，可能與細胞不而有所差異。Melanin含量方面，B16F10細胞株在HQ、MMEH與AA分別於1mM、10mM和1mM皆呈現抑制作用(p<0.05)，KA於1mM則無任何改變 ; 然而以上四種化合物對A2058細胞株皆無顯著差異。在in vivo方面，以HQ與MMEH 0.5%每週施打於小鼠背部皮下，可造成毛髮去色素作用， 且於不同時間點之MMEH可誘發毛髮白化區域逐漸增加，以此方式進行兩種中草藥Lai-1及Lai-2抑制毛髮白化之評估，但結果並不如理想，推測可能與藥劑之作用機制不同有關。

In pigment disorders and cosmetology, there are many experiments in researching whether the compounds could induce hypopigmentation or hyperpigmentation and knowing the pathways of pigment formation. Hydroquinone(HQ) is the first discovered phenoic chemical to cause depigmentation, and has been applied to treat hyperpigmentation in skin, such as melasma. There are many analogues of HQ have the same effect, such as p-hydroxyanisole(MMEH). The aim of this study was to find out the possible effects of melanogenesis, by using B16F10, A2058 melanoma cell line and C57BL/6(B6) mice. In vitro, for the study of B16F10 and A2058 cells were incubated with HQ and MMEH for 72 hours to evaluate the effect of melanin content and tyrosinase activity. In addition, the well-known tyrosinase inhibitors of kojic acid(KA) and ascorbic acid(AA) were also tested as positive control. Results revealed the inhibition ratio of B16F10 cells on tyrosinase activity at concenstrations of 1mM HQ and 10mM MMEH in treatment was 27% and 30%. However, KA had no effect, but AA increased tyrosinase activity in B16F10 cells. In A2058 cells, the inhibition ratio of 10mM HQ was 14%, 1mM AA was 40%, but MMEH and KA had no effect on tyrosinase activity. In melanin content assay, HQ, MMEH and AA revealed dose-depend inhibiton in B16F10 cells, but not in KA. The A2058 cells had no effect to those of four test chemicals. It was suggested that the sensitivity of chemical-induced depigmentation might be related to the species difference. In vivo study, female B6 mice, 4-weeks old, were subcutaneously injected 0.5% HQ and MMEH to evaluate the potential depigmentation. Results revealed that the change of depigment area was significantly increased in a time-related. However, no inhibition effect of Lai-1 and Lai-2 herbal complexes was found under this depigmentation model in mice, and suggested that the action of these herbs might differ to the chemical-caused depigmentation.