TWI601827B - 美白功效物質篩查方法 - Google Patents
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Description
本發明是關於一種具有美白功效之物質之篩查方法,具體而言,本發明是關於一種藉由利用作為長壽基因之FoxO3(叉頭框O3)(Fork head box O3)基因來被編碼之蛋白質FoxO3a(叉頭框)O3A(Fork head box O3A),來確認測試物質是否活化FoxO3a或促進FoxO3a之表現,從而確認測試物質是否具有美白功效之方法。
黑色素為決定人之膚色之色素,藉由此種黑色素之量和分佈而決定膚色。形成黑色素之細胞於位於皮膚表皮底之稱為黑色素形成細胞(Melanocyte)之細胞中形成,並藉由皮膚之新陳代謝作用而移動至角質表面後剝離。黑色素形成細胞之數量大致相同,此與膚色無關。只是由於黑色素生成量和種類、分佈不同,導致出現不同之膚色。於皮膚中,酪氨酸(Tyrosine)藉由稱為酪氨酸酶(Tyrosinase)之人體酶而轉換成多巴(DOPA),藉由持續之一系列氧化過程,最終生成作為黑褐色之聚合物之黑色素。
黑色素於生物體內無法分解,當角質形成細胞自表皮
脫落時,黑色素與角質形成細胞一同自皮膚脫落,藉此能夠去除黑色素。此種黑色素過度生成之情形下,將誘發黃褐斑或雀斑、斑點等色素沈澱症,於美容問題上帶來不好之結果,目前於運動等戶外活動時希望阻擋因紫外線導致之黑色素色素沈澱之需求越來越多。對此,用於防止生成過量黑色素之美白劑之開發需求越來越大。
藉此,尋找此種藉由阻礙黑色素之生成而具有美白功效之物質成為重要之課題,需要一種藉由客觀性標準而對測試物質之美白功效進行確認及判斷之方法,而非僅憑感官評價或根據肉眼之評價。
對此,本發明人著眼於如何藉由借助作為長壽基因之FoxO3(叉頭框O3)(Fork head box O3)基因來被編碼之蛋白質FoxO3a(叉頭框O3A)(Fork head box O3A)來確認是否具有美白功效,而完成了本發明。
藉此,本發明之目的在於提供一種藉由FoxO3a(叉頭框O3A)(Fork head box O3A)來篩查具有美白功效之物質之方法。
為了達到上述目的,本發明提供一種包括確認是否活化FoxO3a(Fork head box O3A)或促進FoxO3a(Fork head box O3A)之表現之具有美白功效之測試物質之篩查方法。
根據本發明之FoxO3a(Fork head box O3A)為借助稱為長壽基因之FoxO3來被編碼之蛋白質,作為位於胰島素訊息傳遞路徑(IIS,Insulin/IGF-like signaling)之轉錄因子,它是對錳超氧化物歧化酶(Mn-SOD)、過氧化氫酶(Catalase)等酶之表現發揮作用之蛋白質。當FoxO3a被活化時,藉由生物體內之防禦機制之活化洞而實現抗老化功效。上述FoxO3a之活化可藉由FoxO3a朝細胞核之移動而判斷。即,存在於細胞質之FoxO3a移動至細胞核,藉此開始進行酶之表現。
本發明人確認了根據FoxO3a之活性度之細胞核移動程度和黑色素生成減少程度呈正比之結果。藉此,藉由對被測定出針對皮膚細胞具有美白功效之候補物質進行處理,確認是否可活化FoxO3a或促進FoxO3a表現,藉此能夠簡單而客觀地確認上述物質是否為藉由減少黑色素之生成而表現出美白功效之物質。
藉此,本發明提供一種美白功效物質篩查方法,其特徵在於,作為對具有美白功效之物質進行篩查之方法,確認待測試物質是否活化用於編碼FoxO3a(Fork head box O3A)蛋白質之基因或促進上述基因之表現。
於本發明之實施例中,對於上述測試物質是否活化FoxO3a進行確認,可包含:a)將測試物質向皮膚細胞進行處理之步驟;以及b)觀察FoxO3a於上述a)步驟中經測試物質處理之皮膚細胞中之移動之步驟。
上述篩查方法更可包含:c)於FoxO3a蛋白質在上述皮膚細胞中向細胞核移動之情形下,判定為美白功效物質之步驟,其由於可將FoxO3a朝細胞核之移動,看作為叉頭框O3A蛋
白質編碼基因被活化。又,藉由上述篩查方法被判定之美白功效物質,可藉由FoxO3a而開始抗氧化酶之表現,藉此,能夠將其判定為表現出抗老化和抗氧化之功效,同時更具有美白功效之物質。
優選地,於上述a)步驟中,根據測試物質之種類,以1nM至100nM之濃度進行處理。如果以小於1nM之濃度進行處理,那麼將無法達到測試物質之功效濃度;而如果以大於100nM之濃度進行處理,那麼將由於過度之功效,導致細胞動態平衡性出現異常,反而會減少核移動及美白功效。
上述皮膚細胞能夠是角質形成細胞、成纖維細胞或黑色素形成細胞。
又,於本發明之一實施例中,於確認上述測試物質是否促進FoxO3a之表現時,可藉由西方墨點法(Western blot)、酶聯免疫吸附試驗(ELISA)或逆轉錄聚合酶鏈反應(RT-PCR)來執行。
藉由上述確認結果,於測試物質促進FoxO3a表現之情形下,可判定為美白功效物質。又,藉由上述篩查方法被判定之美白功效物質,基於作為長壽基因之FoxO3之特性,可根據FoxO3a來開始表現抗氧化酶,藉此,可判定為除於本發明中發現之具有美白功效外,更同時表現出抗老化及抗氧化效果之物質。
根據本發明之美白功效物質篩查方法,實際黑色素形成之減少和叉頭框O3A之活性化程度呈正比,藉此,藉由客觀
性標準,可確認測試物質是否具有美白功效,於不依賴臨床實驗或感官評估等方法之前提下,可迅速簡便而又正確地判斷出美白功效物質。又,藉由上述篩查方法被判定之美白功效物質可藉由叉頭框O3A開始抗氧化酶之表現,藉此,可篩查出同時表現出抗老化和抗氧化效果以及美白功效之物質。
圖1a為於分別向MNT-1細胞(NT)、MNT-1細胞轉染(transfection)野生型FoxO3a質粒(plasmid)之情形(WT)以及於轉染核定位序列中存在突變之核定位序列(NLS)突變FoxO3a質粒之情形(NLS)之細胞沈澱之溶解液之顏色比較圖。
圖1b為於分別向MNT-1細胞(NT)、MNT-1細胞轉染野生型FoxO3a質粒之情形(WT)以及於轉染核定位序列中存在突變之核定位序列(NLS)突變FoxO3a質粒之情形(NLS)之溶解液之西方墨點法結果圖。
圖2為表示向MNT-1細胞進行維生素C、N-乙醯半胱氨酸(Acetylcysteine)、水溶性維生素E處理之情形下之黑色素量和酪氨酸酶活性之圖。
圖3為於向MNT-1細胞進行維生素C、N-乙醯半胱氨酸、水溶性維生素E處理之情形下觀察了FoxO3a之核移動之螢光顯微鏡圖。
以下,將藉由實施例及試驗例對本發明之內容進行更
具體之說明。該些實施例是為了幫助理解本發明之內容而提出,該些實施例及試驗例並非用於限定本發明之保護範圍,可執行本領域通常之公知之變形、置換及插入等,與此相關之內容亦包含於本發明之範圍。
[試驗例1]FoxO3a活性化與黑色素生成減少實驗
將1×106個MNT-1細胞(Human melanoma cell line,Lonza,SWISS)放入MEM培養基(10% DMEM),藉由野生型(WT)FoxO3a質粒(Plasmid 1787:HA-FOXO3a WT,Addgene公司)、核定位序列(NSL)突變FoxO3a質粒(nuclear localization sequence mutant plasmid;Plasmid 8361:FLAG-FOXO3a TM,Addegene公司),使用Lipofectamin 2000(Inveitrogen),分別向MNT-1細胞進行轉染,於37℃溫度條件下培養了48小時。將上述MNT-1細胞以500μl之LIPA緩衝液(25mM三羥甲基氨基甲烷鹽酸鹽(Tris-HCl),pH 7.4,0.1%十二烷基硫酸鈉(SDS),0.1%聚乙二醇辛基苯基醚(Triton X-100),1%脫氧膽酸鈉(sodium deoxycholate),150mM氯化鈉(NaCl),1mM乙二胺四乙酸(EDTA),1mM正釩酸鈉(Na3VO4),1mM苯甲基磺醯氟(PMSF),10mg/mL抑肽酶(aprotinin),5mg/mL亮抑酶肽(leupeptin))進行溶解(lysis)後,將細胞溶解質進行離心分離(Centrifuge 5415R,Eppendorf產品,德國),分離出所得沈澱並觀察顏色,使用200μl之1N氫氧化鈉,溶解上述沈澱中之內容物,所獲得之溶解液之顏色如圖1a所示。作為對照組,使用了未轉染質粒之MNT-1細胞。
裝入上述溶解液20μg以西方墨點法對叉頭框O3A之蛋白質表現量進行了確認,作為細胞質成分之標記蛋白質使用了Gapdh並且作為核成分之標記蛋白質使用了Lamin B,結果如圖1b所示。
於圖1a之結果中,於轉染野生型FoxO3a質粒之情形下,由於FoxO3a之過表現,可確認黑色素生成顯著減少,而於轉染核定位序列存在突變之核定位序列突變FoxO3a質粒之情形下,由於FoxO3a不向細胞核移動,藉此,可確認未表現出黑色素生成之減少。又,於圖1b之結果中,於轉染野生型FoxO3a質粒之情形下,可確認由FoxO3a被活化而向細胞核移動。藉此,於未發生FoxO3a之活化時,不會對黑色素生成產生影響,可確認FoxO3a向細胞核之移動能夠減少黑色素。即,可得出FoxO3a之活化為於皮膚細胞中有效妨礙黑色素生成之重要因子之結論。
[試驗例2]根據美白物質處理之黑色素生成減少實驗
向1×106個MNT-1細胞(Human melanoma cell line,Lonza,SWISS)將公知之美白物質維生素C、N-乙醯半胱氨酸(N-acetyl cysteine;NAC)、水溶性維生素E(Trolox)分別以0.1nM、10nM、100nM、1μM、10μM、100μM之濃度進行處理後,於37℃溫度條件下培養了48小時。將上述培養之細胞以500μl之LIPA緩衝液進行溶解(lysis)後,將上述細胞溶解質中之500μl進行離心分離(Centrifuge 5415R,Eppendorf產品,德國),分離出所得沈澱並觀察顏色,使用200μl之1N氫氧化鈉,溶解上述沈澱中之內容物。於對黑色素具有特異性之490nM中對上述溶解液進行了吸光度測定,藉由BCA protein assay
kit(Pierce)測定了蛋白質總量。以(吸光度)/(蛋白質總量)對上述測定值進行校正,以黑色素/μg蛋白質表示黑色素之量。當於各個美白物質實驗組中之非處理組(0nM)之黑色素量視為1時,實驗組之黑色素量之相對值如圖2所示。
[試驗例3]根據美白物質處理之酪氨酸酶活性抑制評估
向上述試驗例2中之100μl細胞溶解質中,放入100μl之10mM二羥基苯丙氨酸(dihydroxyphenylalanine),於37℃條件下反應1小時,測定490nM上之吸光度,求出因酪氨酸酶之作用而生成之多巴色素(dopachrome)。當非處理組(0nM)之OD490值視為1時,藉由計算各實驗組之吸光度之相對值,藉此可求出酪氨酸酶活性抑制能,如圖2所示。
[試驗例4]根據美白物質處理之FoxO3a之細胞核移動觀察
向1×106個MNT-1細胞(Human melanoma cell line,Lonza,SWISS)將公知之美白物質維生素C、N-乙醯半胱氨酸(N-acetyl cysteine;NAC)、水溶性維生素E(Trolox)分別以100nM、100μM之濃度進行處理後,於37℃溫度條件下培養了48小時。作為對照組,使用了非處理組。將培養之細胞分別以苯基吲哚(DAPI)(核染色劑-blue)、FoxO3a IF antibody(red)(Cell Signaling Technology)進行染色,藉由螢光顯微鏡(EVOSfl digital fluorescence microscope,Advanced Microscopy Group)進行觀察,其結果如圖3所示。
於圖3之結果中,可觀察出維生素C、N-乙醯半胱氨酸(N-acetyl cysteine;NAC)、水溶性維生素E(Trolox)處理時
之FoxO3a之核移動,於各個情形下,未能觀察出100μM中之核移動,相反,於100nM中能夠被明顯觀察出。
根據上述試驗例2至試驗例4之結果,於觀察出FoxO3a之核移動之物質濃度下表現出美白功效,對FoxO3a之活性化起到媒介作用之物質就為同時具有抗老化、抗氧化功效及美白功效之抗老化-美白物質。藉此,根據本發明之篩查方法,能夠客觀準確地篩查出測試物質之抗老化、抗氧化效果和美白效果。
Claims (6)
- 一種美白功效物質的活體外篩查方法,其作為對美白功效物質進行篩查之方法,其特徵在於,確認待測試物質是否活化FoxO3a(叉頭框O3A)或促進FoxO3a的表現,其中上述確認包含:a)將上述待測試物質向皮膚細胞進行處理之步驟,其中上述皮膚細胞為黑色素形成細胞;及b)觀察FoxO3a於上述a)步驟中經上述待測試物質處理之上述皮膚細胞中之移動之步驟。
- 如申請專利範圍第1項所述的美白功效物質的活體外篩查方法,其更包括:c)於FoxO3a在上述皮膚細胞中向細胞核移動之情形下,判定上述待測試物質為美白功效物質之步驟。
- 如申請專利範圍第1項所述的美白功效物質的活體外篩查方法,其中上述待測試物質以1nM至100nM的濃度向皮膚細胞進行處理。
- 如申請專利範圍第1至3項中任一項所述的美白功效物質的活體外篩查方法,其中藉由西方墨點法、酶聯免疫吸附試驗或逆轉錄聚合酶鏈反應確認上述FoxO3a的活化或上述FoxO3a表現的促進。
- 如申請專利範圍第4項所述的美白功效物質的活體外篩查方法,其中於上述待測試物質促進FoxO3a的表現之情形下,判定為美白功效物質。
- 如申請專利範圍第5項所述的美白功效物質的活體外篩查方法,其中上述待測試物質同時具有美白功效及抗老化功效。
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