|摘要: ||隨著美白化粧品原料多樣化，消費者希望買到具有幼藺吨峖w全性的產品，但由於檢測美白化粧品幼蘆瘍擗爾桲蝖]in vivo）耗時，因此本研究目的是藉由B16黑色素瘤細胞（melanoma）黑色素形成機轉來建立一個快速評估美白化粧品幼蘆熔茩M模式。本研究將探討引發黑色素形成的主要因素包括紫外光的照射、細胞內H2O2及黑色素細胞（melanocyte）中黑色素顆粒體(melanosome)胞器內pH值變化，相關研究顯示UV照射造成黑色素生成增加是藉由調整黑色素細胞內pH值，而促進黑色素細胞成熟化，增加黑色素的釋放及酪胺酸酶( tyrosinase )活性增加的現象。
本研究利用氯化銨（ammonium chloride）來模擬UV照射後黑色素顆粒體內的環境，因為氯化銨可以提高黑色素細胞內胞器pH值，促進黑色素顆粒體成熟化，而刺激黑色素形成的影響。利用此影響來探討桑枝、桑椹果實、乾燥桑椹、桑白皮、桑葉之萃取物、H2組織胺受體阻斷劑（cimetidine、famotidine）等材料的美白幼纂A評估的項目包含：（一）利用MTT assay來測細胞存活率；（二）對B-16 黑色素瘤細胞上清液中黑色素釋放的抑制率（三）對細胞溶解物中酪胺酸酶活性的影響（四）用螢光顯微鏡及流式細胞儀來觀察黑色素細胞內pH值變化。
With the variety of whitening agents for cosmetic formulation, consumers hope to buy the products with efficiency and safety. However, in vivo test the efficiency of cosmetics is time-consuming, the aim of this study is to establish a cellular model that can evaluate the efficiency of the cosmetics fast by the mechanism of melanogenesis in melanoma. This study will discuss the primary factor of induce melanogenesis, which include ultraviolet irradiation, intracellular H2O2 and differences in the pH environment of the melanosome .
The published lecture indicate that the ultraviolet irradiation increase the eumelanin production by regulating melanosomal pH to facilitate maturation of melanosomes in mouse melanoma which increases the melanin release and the tyrosinase activity.
This research use ammonium chloride to simulate the environment of melanosomal after ultraviolet irradiation. Due to ammonium chloride moves into acidic organelles that raises the pH of the organelle in melanosomes, it facilitate maturation of melanosomes and stimulate melanogenesis in mouse melanoma. We use this model to test the efficiency of whitening of extracts which include mulberry's branch, mulberry's fruits, unripe mulberry's fruits, mulberry white roots, mulberry's leaves, cimetidine and famotidine.
The evaluated items are: (1) the viability of sample-treated cells was determined by cell viability assay (2) The percentage inhibition of released melanin in B-16 melanoma cell supernatant, (3) The percentage inhibition of tyrosinase activity in cell lysate (4) melanosomal pH level was measured by flow cytometry and fluorescence microscope.
The results indicated: (1) 10mM ammonium chloride could increase 21% released melanin and 3mM ammonium chloride had a dose-dependent increase in tyrosinase activity, at this concentration tyrosinase activities were 4-5 times that of untreated cells. (2) all of the various mulberry's extracts (1mg/ml), cimetidine (2mM), famotidine (0.2mM) had no cytotoxicity. (3) The percentage inhibition of released melanin in B-16 melanoma cell supernatant for mulberry's branch, mulberry's leaves, mulberry's fruits, unripe mulberry's fruits, mulberry white roots (1mg/ml) were 74.2%, 64.1%, 65.8%, 57.9%, 54.2%. (4) The percentage inhibition of tyrosinase activity in cell lysate for mulberry's branch, unripe mulberry's fruits, mulberry's fruits, mulberry's leaves, mulberry white roots (1mg/ml), cimetidine (2mM) and famotidine (0.2mM) were 74.1%, 69.6%, 63.7%, 61.2%, 59.0%, 65.9%, 69.7%. (5)The percentage inhibition of tyrosinase zymorgraphy activity for mulberry's fruits, mulberry's branch, mulberry white roots, mulberry's leaves , unripe mulberry's fruits (1mg/ml) , cimetidine (100uM) were 60.7% , 45.2% , 42.5% , 37.9%, 35.94% , 86% .
(6) mulberry's fruits ,mulberry's branch, mulberry white roots, cimetidine could reduced melanosomal pH.
Our results demonstrate that this model could quickly test sample efficiency of whitening, This study indicates the mechanism of melanogenesis in melanoma of mulberry's extracts and H2 blocker maybe reduce melanosomal pH. This cell model can be applied to screen whitening agents for cosmetic formulation and basis of new product development in the future.