行政院國家科學委員會專題研究計畫 成果報告
荖葉對皮膚黑色素細胞去色素化/過度色素化之研究
計畫類別： 個別型計畫
計畫編號： NSC91-2320-B-041-019-
執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 嘉南藥理科技大學藥學系
計畫主持人： 陳秋蘭
計畫參與人員： 賴慧芬 賴慧娟 黃文良
報告類型： 精簡報告
處理方式： 本計畫涉及專利或其他智慧財產權，2 年後可公開查詢
中 華 民 國 92 年 10 月 6 日
行政院國家科學委員會補助專題研究計畫 ■ 成 果 報 告 □期 中進度
報 告
荖葉對皮膚黑色素細胞去色素化/過度色素化之研究
計畫類別： ■ 個別型計畫 □ 整合型計畫 計畫編號：NSC 91－2320－B－041－019
執行期間：91 年 08 月 01 日至 92 年 07 月 31 日
計畫主持人：陳秋蘭 共同主持人：劉宗榮
計畫參與人員：賴慧芬、賴慧娟、黃文良
成果報告類型(依經費核定清單規定繳交)： ■ 精簡報告 □完整 報告
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
□出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
處理方式：除產學合作研究計畫、提升產業技術及人才培育研究 計畫、列管計畫及下列情形者外，得立即公開查詢 □涉及專利或其他智慧財產權，□一年□二年後可公開
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執行單位：嘉南藥理科技大學 藥學系
中 華 民 國 92 年 10 月 06 日
一、中文摘要
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、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，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 是 黑 色 素 形成過 程 中的一 個
速率決定酵素，因此我們先以 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 活，我性 。此 影的 響 外
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 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
（0.1~100µg/ml ）。同 時 ， 荖 葉 萃 取 液 在 1~100µg/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（100µg/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 及丁香油酚 在 低 劑 量 (1
µ
M) 時 可 降 低 黑 色 素 的 含 量，其程度與正對照組的維他命 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 化 作 素 色 去 的用
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。
關鍵詞：荖葉、黑色素、酪胺酸
Abstract
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, chavicol and hydroxychavicol.
Phenolic derivatives are known for depigmentation, such as hydroquinone and sometimes even induce postinflammatory hyperpigmentation. In this study, we hypothesizes that PBL extracts induces leukomelanosis through inhibition of melanin synthesis and/or melanocytotoxicity. We first screened the inhibition potential of PBL on tyrosinase activity in the presence of
L-Dopa as a substrate. We further determined the cytotoxicity of PBL extracts in mouse melanoma B16 cells by using tetrazolium salt and trypan blue exclusion assays. The effects of PBL extracts on melanin content in B16 cells were also measured by UV at OD405. The data showed that PBL extracts were cytotoxic to B16 cells in a dose-dependent manner (10~100
µg/ml) and inhibited mushroom
tyrosinase activityin vitro. These results
demonstrate that PBL extracts induced leukomelanosis may through the inhibition of melanin synthesis and melanocytotoxicity.Keywords: Piper betle leaf, melanin, tyrosinase I. Introduction
Betel quid (BQ) chewing is a common habit in some Asian countries, including Taiwan. BQ is generally composed of areca nut,
Piper betle leaf (PBL) or inflorescence,
lime and additives such as tobacco.However, the composition of BQ varies in different geographic locations.
Piper betle
inflorescence is used in the preparation of BQ in Taiwan and Papua New Guinea,whereas the leaf of Piper betle Linn. is used in almost all of the BQ chewing countries.
Chewing BQ has been associated with oral submucous fibrosis, leukoplakia, and oral squamous cell carcinoma (OSCC). In Taiwan, tobacco is not included in the preparation of BQ, however, epidemiological studies showed that BQ chewing is still the main cause of OSCC (1).
An unexpected disease related to the use of BQ component appeared in Taiwan recently. After the advertisement by a local newspaper that steamed betel leaves can serve as a facial bleaching remedy, this has gained popularity in some local female groups. In one report indicated that the bleaching effect occurred within 1 week in 8 of 15 patients (2). Among them, 4 claimed that significant bleaching occurred within 3 days after nightly use of steamed betel leaves before bedtime. The rapidity of this bleaching effect is faster than any of the commercially available bleaching agents (3).
However, many patients developed severe facial leukomelanosis after prolonged use of this home remedy. The underlying mechanism for this leukomelanosis has remained elusive.
PBL, the mature green leaves of Piper betle vines, has been used in the preparation
of BQ since ancient times (4). The Working Group of IARC concluded that the data are inadequate to allow an evaluation of the carcinogenicity of betel leaf to experimental animals (4). Besides having been used as a part of BQ together with areca nuts and slaked lime for centuries,Piper betle
leaves have been found to possess diverse biologic effects including antifungal, antiseptic, and antihelmintic effects (5).Piper betle leaves contain volatile oils,
nitrate, and small quantities of sugar, starch and tannin. The most important constituents of betel leaves may be the various chemicals in the essential oils, especially eugenol and hydroxychavicol (6,7). We hypothesize that the essential oils in betel leaves may be responsible for this leukomelanosis, perhaps through inhibition of melanin synthesis or melanocytotoxicity.Melanogenesis is the process of the production and subsequent distribution of
melanin by melanocytes within the skin and hair follicles (8,9). The copper- containing enzyme tyrosinase catalyzes the first two rate-limiting reactions, the oxidation of tyrosine into dopa and subsequently the corresponding
ortho-quinone (dopaquinone).
Quinones are chemically reactive compounds that are potentially harmful, but in melanocytes the normal process of melanogenesis is not usually associated with significant toxicity due to the compartmentation of the reaction within membrane-limited organelles (melanosomes) and because of the rapid cyclization of the intermediate quinone. Such formed melanin has many biological functions including the scavenge of oxidative free radicals (10,11).
To date, research on the regulation of melanogenesis has focused on factors which affect tyrosinase, the rate-limiting enzyme in the melanogenic pathway, by searching for chemicals which competitively inhibit tyrosinase function.
Various dermatologic disorders result in the accumulation of excessive levels of epidermal pigmentation. However, a global market demand has developed recently for skin-lightening agents as vanity cosmeceutical products, because lighter skin color is preferred by some dark-skinned individuals in many countries and races (12).
Unfortunately, several purportedly active agents (e.g. arbutin and kojic acid, among others) have not been demonstrated yet to be clinically efficacious when critically analyzed in carefully controlled studies. The U.S.
FDA-approved pharmaceutical products containing 2-4% hydroquinone (HQ) are moderately efficacious, but HQ is considered to be cytotoxic to melanocytes and potentially mutagenic to mammalian cells (12,13).
Desir able skin-whitening agents inhibit the synthesis of melanin in melanosomes by acting specifically to reduce the synthesis or activity of tyrosinase, exhibit low cytotoxicity, and are non-mutagenic.
II. Material and Methods
1. Piper betle leaf extract (PBLE) preparation
PBL will be purchased in a shop in
Tainan. PBL will be minced and warmed with hot water for 3 hours. When it is cold,the filtrate is then frozen immediately and lyophilized.
2. Cell culture and treatment
The pigmented human melanoma cell line RPMI 7951 is cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and appropriate amounts of antibiotics and fungizone. The test agents are added to the cell cultures for various times.
3. Cytotoxicity
The cytotoxicity of PBL or HC is determined by assay for the reduction of tetrazolium-based compound MTT.
Melanocytes are plated at a density of 104 cells/well into 96-well tissue culture plates.
Cells are treated with the indicated concentrations of PBL or HC for various times, the medium was then removed and 0.5 mg/ml MTT in medium is added to each well.
Following a 2 h-incubation period the medium is removed, and 100
µl DMSO is added to
each well. The viable cells can be calculated from the A570 values determined with a microtiter plate reader. PBLE- or HC-induced cytotoxicity is also determined by counting the living cell exclusion of trypan blue dye.4. Melanin content assay
Cells are collected by trypsin/EDTA after treatment, and are counted with trypan blue exclusion method. The colors of cell pellets are evaluated visually, and pellets of 106 cells were solubilized in boiling 0.1N NaOH for 10 min. Spectrophotometric analysis of melanin content is performed at 400 nm absorbance (14,15).
5. Tyrosinase assay
Cells pellets are lysed in 0.1 M sodium phosphate buffer (pH 6.8) containing 1%
Triton X-100, 1 mM phenylmethylsuphonyl fluoride (PMSF), 10
µ
g/ml aprotinin and 10µ
g/ml leupeptin. The radiometric determination of tyrosinase activity is performed as previously described. In brief, 0.09ml of each cell extract (20µg protein
content) is incubated for 60 min at 370C with 0.01 ml sodium phosphate buffer containing 1µCi of
L-[ring-3,5-3H]tyrosine, 5 µg of L-dopa and 1% Triton X-100. One milliliter of activated charcoal (10% w/v) in 0.1 M citric acid is then added and specimens arecentrifuged for 10 min at 2000g at 40C. The supernatants are applied to 0.2 ml columns of Dowex-50 (Bio-Rad Laboratories, CA, USA), equilibrated in 0.1 M citric acid, washed with 0.5 ml of 0.1 M citric acid, and the effluents are counted by scintillation spectrometry for the formation of 3H2O (14).
III. Results & Conclusions
Tyrosinase assay
Absorbance at 492 nm
0.0 0.2 0.4 0.6 0.8
control HWE (µg/ml) OE-n-Hexane (µg/ml) OE-CH2CL2 (µg/ml) OE-Ethylacetate (µg/ml) OE-pure-n-Hexane (µg/ml) OE-pure-CH2CL2(µg/ml)
Conc of drug
control 10 1000 0.1 10 0.1 10 0.1 10 0.1 100 0.1 10
* * *
* * *
** * *
* ** *
***
Tyrosinase assay
Conc of drug
Absorbance at 492 nm
0.0 0.2 0.4 0.6 0.8
control Hydroquinone (µM) VitC(µM) VitE (µM) Hrydoxychavicol (µM) HC-dA(µM) Oil (µg/ml) Eugenol (µM)
control 0.1 100 1 100 0.1 100 0.1100 0.1 100 0.1 100 0.1 100
* * ** * * ** * **** ** ** *
PS : HWE(Hot water extract) ; OE : organic extract
Cytotoxicity of B16-F0 cells by MTT assay
Conc of drug
Absorbance at 550 nm
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
control Hydroquinone(µM) VitC(µM) VitE(µM) Eugenol(µM) Hydroxychavicol(µM)
HC-dA(µM)
control 1 100 1 100 1 100 1 100 1 100 1 100 n=3
*
*
* *
*
*
* *
Cytotoxicity of B16-F0 cells by MTT assay
Absorbance at 550 nm
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
2.0 control
Waterf raction(µg/ml) n-Hexane(µg/ml) CH2CL2(µg/ml) Ethylacetate(µg/ml) oil(µg/ml) pure-n-Hexane(µγ/ml) pure-CH2CL2(µg/ml)
Conc of drug
control 1 100 1 100 1 100 1 100 1 100 1 100 1 100
*
*
*
*
*
*
*
*
**
*
**
* * *
*
n=3
The data showed that PBL extracts were cytotoxic to B16 cells in a dose-dependent manner (10~100
µ
g/ml) and inhibited mushroom tyrosinase activityin vitro.
These results demonstrate that PBL extracts induced leukomelanosis may through the inhibition of melanin synthesis and melanocytotoxicity.
IV. References
1. Ko, Y.C., Huang, Y.L., Lee, C.H., Chen, M.J., Lin, L.M. and Tsai, C.C. (1995) Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan. J.
Oral Pathol. Med., 24, 450-453.
2. Liao, Y.L., Chiang, Y.C., Tsai, T.F., Lee, R.F., Chan, Y.C. and Hsiao, C.H. (1999) Contact leukomelanosis induced by the leaves of Piper betle L. (Piperaceae): a clinical and histopathologic survey. J. Am. Acad. Dermatol., 40, 583-589.
3. Kameyama, K., Sakai, C., Kondoh, S., Yonemoto, K., Nishiyama, S., Tagawa, M., T., M., Ohnuma, T., Quigley, J., Dorsky, A., Bucks, D. and Blanock, K. (1996) Inhibitory effect of magnesoum L-ascorbyl-2-phosphate (VC-PMG) on melanogenesis in vitro and in vivo. J. Am.
Acad. Dermatol., 34, 29-33.
4. IARC (1985) Betel-quid and areca-nut chewing, IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Vol.
37. IARC, Lyon, pp. 137-202.
5. Evans, P.H., Bowers, W.B. and Funk, E.J. (1984) Identification of fungicidal and nematocidal components in the leaves of Piper betle (Piperaceae). J. Agric. Food Chem., 32, 1254-1256.
6. Ueda, E. and Sasaki, T. (1951) Chemical studies of Formosan plants. I. Chemical constituents of the leaves of Piper betle L. J. Pharm. Soc.
Japan, 71, 559-560.
7. Ganguly, P. and Choudhury, M. (1975) Phytochemical studies on Bangla variety of betel leaf (Piper betle Linn.). Indian Agric., 19, 199-200.
8. Hearing, V.J. (1987) Monophenol monooxygenase (tyrosinase): purification, properties, and reactions catalyzed. Methods Enzymol., 142, 154-165.
9. Spritz, R.A. and Hearing, V.J. (1994) Genetic-disorders of pigmentation. Adv. Hum.
Genet., 22, 1-45.
10. Riley, P.A., Cooksey, C.J., Johnson, C.I., Land, E.J., Latter, A.M. and Ramsden, C.A. (1997) Melanogenesis-targeted anti-melanoma pro-drug development: effect of side-chain variations on the cytotoxicity of tyrosinase-generated ortho-quinones in a model screening system.
Eur. J. Cancer, 33, 135-143.
11. Prota, G. (1993) Regulatory mechanisms of melanogenesis: beyond the tyrosinase concept. J.
Invest. Dermatol., 100, 156S-161S.
12. Dooley, T.P. (1997) Topical skin depigmentation agents: current products and discovery of novel inhibitors of melanogenesis. J. Dermatol. Treat, 8, 275-279.
13. Curto, E.V., Kwong, C., Hermersdorfer, H., Glatt, H., Santis, C., Virador, V., Hearing, V.J.
and Dooley, T.P. (1999) Inhibitors of mammalian melanocyte tyrosinase: in vitro comparisons of alkyl esters of gentisic acid with other putative inhibitors. Biochem. Pharmacol., 57, 663-672.
14. Funasaka, Y., Chakraborty, A.K., Komoto, M., Ohashi, A. and Ichihashi, M. (1999) The depigmenting effect of α-tocopheryl ferulate on human melanoma cells. Br. J. Dermatol., 141, 20-29.
15. Oka, M., Ichihashi, M. and Chakraborty, A.K.
(1996) Enhanced expression of protein kinase C subspecies during stimulation of melanogenesis in B16 mouse melanoma cells. J. Invest.
Dermatol., 106, 377-378.
