在1997年初，台灣皮膚科門診發現，婦女為求美白而使用蒸過的荖葉來敷臉，結果臉部出現非常嚴重的黑白斑症之案例，而荖葉所造成的這種獨特現象之機轉究竟如何還是一個未知數。荖葉是檳榔塊的組成之一，內含有許多酚類的物質，包括像丁香油酚（eugenol）、hydroxychavicol（HC）及HC-diacetate (HC-dA) 等化合物。已知有一些酚類化合物，像是對苯二酚（hydroquinone；HQ），具有去色素化的效果，而有些則會誘發發炎後的過度色素化的作用。究竟荖葉萃取液或其所含的酚類化合物是否會造成去色素化或過度色素化的作用，則是一個值得探討的一個課題。
酪氨酸酶（tyrosinase）是黑色素形成過程中的一個速率決定酵素，因此首先我們以L-dopa當作受質來篩選荖葉萃取液或其中的酚類成份在體外對酪氨酸酶活性的影響。此外，我們也利用MTT的方法以及錐蟲藍排除法來偵測荖葉萃取液或其中的酚類成份對小鼠的黑色素瘤B16細胞是否具細胞毒殺作用。最後，我們利用小鼠黑色素瘤B16細胞來探討荖葉萃取液或其酚類成份在細胞內對黑色素含量的影響，以黑色素在波長415nm呈現最大吸收的特性來加以偵測。
從我們的實驗結果得知：荖葉萃取液對於酪氨酸酶的活性具有抑制的效果，但此抑制的效果並不具有劑量的相關性（0.1~100g/ml）。同時，荖葉萃取液在1~100g/ml的劑量下，是具有細胞的毒性作用且呈現劑量相關性。此外，這些試劑對於黑色素的產生並不具有明顯抑制的效果，反而在高劑量（100g/ml）的處理之下，具有過度色素化的作用。但是，荖葉中的酚類化合物HC、HC-dA及eugenol在低劑量 (1M) 時可降低黑色素的含量，其程度與正對照組的Vit.C作用相當。因此，由我們的實驗結果可知，荖葉中的酚類化合物具有去色素化作用的濳力。同時，使用蒸過的荖葉來敷臉造成黑白斑症的原因，可能是因為荖葉對黑色素細胞產生毒殺作用（白斑症）和過度色素化的作用（黑斑症）所致。 In 1997, dermatologists documented a kind of severe facial leukomelanosis to the use of facial dressing with steamed Piper betle leaf (PBL) as a bleaching agent in Taiwan. The underlying mechanisms for this leukomelanosis have yet to be resolved. PBL is a component of areca quid and it contains many phenolic ingredients including eugenol, hydroxychavicol (HC), and HC-diacetate (HC-dA). Phenolic derivatives (such as hydroquinone) are known for depigmentation but sometimes induce postinflammatory hyperpigmentation. Therefore, we wound to test whether PBL extracts or its phenolic compounds induce depigmentation and/or hyperpigmentation on melanogenesis procesis.
Tyrosinase is a rate-limiting enzyme in melanogenesis process. Thus, we first screened the inhibit potential of PBL extracts or its phenolic compounds on tyrosinase activity in the presence of L-dopa a substrate. We further determined the cytotoxicity of PBL extracts in mouse melanoma B16 cells by using tetrazolium salt (MTT) and trypan blue exclusion assays. The effects of PBL extracts on melanin content in B16 cells were also measured by UV at OD415.
The data showed that PBL extracts can inhibit mushroom tyrosinase activity in vitro and in a dose-independent manner (0.1~100g/ml). We also found PBL extracts were cytotoxicity to mouse melanoma cells in a dose-dependent manner (1~100g/ml). Finally, PBL extracts can not significant inhibit melanin content, but induce hyperpigmentation at high dose (100g/ml). However, low dose (1M) of HC, HC-dA and eugenol can decrease melanin content as well as Vit.C (postive control). These results demonstrate that the phenolic compounds of PBL have the potential to act as a hypopigmenting agents. We also demonstrate that the use of facial dressing with steamed PBL can induced leukomelanosis is mediated through the melanocytotoxicity (leukosis) and hyperpigmentation (melanosis).
