TWI690323B - 泡葉藻萃取物用於調節基因群組表現之用途 - Google Patents
泡葉藻萃取物用於調節基因群組表現之用途 Download PDFInfo
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Abstract
本發明提供一種泡葉藻萃取物用於製備調節一基因群組表現之組合物之用途，其中，該基因群組包含一選自由轉麩胺酸醯胺基酶(TGM1)、角質蛋白14(KRT14)、絲聚蛋白(FLG)、水通道蛋白3(AQP3)、β葡萄糖腦苷酯酶(GBA)、玻尿酸合成酶3(HAS3)、及其任意組合所組成群組之A群基因，及一選自由玻尿酸合成酶2(HAS2)、基質金屬蛋白酶2(MMP2)、賴胺醯氧化酶(LOX)、及其任意組合所組成群組之B群基因。該泡葉藻萃取物透過調節上述基因群組表現而提升皮膚含水量與彈性及對紫外線的抵抗力。
Description
本發明係關於一種泡葉藻萃取物之用途，特別係關於一種泡葉藻萃取物用於製備調節特定基因群組表現之組合物之用途。
皮膚是人體隔絕外界環境中的傷害，例如紫外線、病原體、摩擦力等，與防止水分流失的第一道防線。皮膚由外向內依序包含表皮層、主要由結締組織構成的真皮層、及皮下組織。表皮層是皮膚的最外層並且不斷更新。表皮層與真皮層間存在持續分裂的細胞(如纖維母細胞、角質細胞、黑色素細胞)，該些細胞的活動對紫外線非常敏感。真皮層含有膠原蛋白(collagen)、彈力蛋白(elastin)、及玻尿酸(hyaluronic acid)，其賦予肌膚彈性和支撐力量。隨著年齡增長，皮膚會出現皺紋、細紋、鬆弛、凹陷、毛孔粗大等老化現象。這些皮膚老化現象的形成與諸多因素有關，例如暴露於高量的紫外線(主要是紫外線A)會使膠原蛋白或彈力蛋白受損，真皮層內的膠原蛋白、彈力蛋白、及玻尿酸等分子的含量隨年齡增加而減少，這些因素皆會降低皮膚飽滿度與彈性。
市面上用以改善前述皮膚老化現象的方法包含使用防曬品以減少紫外線所致皮膚老化，直接注射膠原蛋白或玻尿酸至真皮層，及以口服方式補充膠原蛋白或玻尿酸等。然而，防曬品含有的化學物質可能引發光敏性，以致該些化學物質與紫外線的結合對皮膚產生不良影響，例如皮疹或更嚴重的曬傷。此外，注射至皮膚的膠原蛋白或玻尿酸易隨時間被體內酵素分解，導致必須定期施打該些物質，成本高昂。以口服方式補充膠原蛋白或玻尿酸會造成該些大分子在腸胃道中被消化為小分子的胺基酸或單醣，儘管身體能利用這些胺 基酸或單醣合成蛋白質或多醣，但不必然形成膠原蛋白或玻尿酸，因此補充膠原蛋白或玻尿酸的實質效果有限。
有鑒於此，開發一種成分天然又能有效保護皮膚免於紫外線傷害及延緩皮膚老化的新穎組成物，實有其必要。
緣此，本發明之一目的在提供一種泡葉藻(Ascophyllum nodosum)萃取物用於製備促進一基因群組表現之組合物之用途，其中該泡葉藻萃取物係以一溶劑萃取一泡葉藻而獲得，且其中該基因群組包含一選自由轉麩胺酸醯胺基酶(transglutaminase 1，TGM1)、角質蛋白14(keratin 14，KRT14)、絲聚蛋白(filaggrin，FLG)、水通道蛋白3(aquaporin 3，AQP3)、β葡萄糖腦苷酯酶(glucosylceramidase beta，GBA)、玻尿酸合成酶3(hyaluronan synthase 3，HAS3)、及其任意組合所組成群組之A群基因，及一選自由玻尿酸合成酶2(hyaluronan synthase 2，HAS2)、基質金屬蛋白酶2(matrix metalloproteinase 2，MMP2)、賴胺醯氧化酶(lysyl oxidase，LOX)、及其任意組合所組成群組之B群基因。
在本發明之一實施例中，該泡葉藻萃取物促進轉麩胺酸醯胺基酶、角質蛋白14、絲聚蛋白、水通道蛋白3、β葡萄糖腦苷酯酶、玻尿酸合成酶3、玻尿酸合成酶2、賴胺醯氧化酶、或其任意組合之基因表現。
在本發明之一實施例中，該泡葉藻萃取物抑制基質金屬蛋白酶2之基因表現。
在本發明之一實施例中，該溶劑為水、醇類、或醇水混合物，該溶劑與該泡葉藻之液固比為5~20：1~5，且該萃取係在50℃~100℃進行。
在本發明之一實施例中，該泡葉藻萃取物係為一泡葉藻之水萃取物，其濃度為至少1mg/mL。
本發明以水、醇類、或醇水混合物為溶劑所萃取得之泡葉藻萃取物能調節TGM1、KRT14、FLG、AQP3、GBA、HAS3、HAS2、MMP2、及LOX等基因之表現，最終導致提升皮膚含水量與彈性及對紫外線的抵抗力。該泡葉藻萃取物可用於製備一具有護膚功效的組合物，例如食品、飲品、營養補充劑、及醫藥品組合物，且該組合物可具有粉末、顆粒、溶液、膠體、或膏體之劑型，藉由口服、塗抹於皮膚等方式給予一個體。
以下將配合圖式進一步說明本發明的實施方式，下述所列舉的實施例係用以闡明本發明之發明特點及應用，而非以限定本發明之範圍，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可做些許更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。
圖1A顯示人類表皮角質細胞在有或無泡葉藻萃取物處理的情況下，其TGM1、KRT14、FLG、AQP3、GBA、及HAS3基因的相對表現量。
圖1B顯示人類表皮角質細胞在有或無泡葉藻萃取物處理的情況下，其MMP2、LOX、及HAS2基因的相對表現量。
圖2顯示泡葉藻萃取物提升人類皮膚纖維母細胞對紫外線A照射的抵抗力。
本發明提供一種泡葉藻萃取物用於製備調節一基因群組表現之組合物之用途，其中該基因群組包含一選自由TGM1、KRT14、FLG、AQP3、GBA、HAS3、及其任意組合所組成群組之A群基因，及一選自由HAS2、MMP2、LOX、及其任意組合所組成群組之B群基因。該泡葉藻萃取物係以一溶劑萃取一泡葉藻的植株而獲得，其中，該溶劑為水、醇類、或醇水混合物，該溶劑與該泡葉藻之液固比為5~20：1~5，且該萃取係在50℃~100℃進行。以下實施例進一步說明該泡葉藻萃取物對前述基因群組的調控作用及其提升皮膚纖維母細胞對紫外線抵抗力的效果，以證明該基因調控作用對維護皮膚健康的重要性。
本文中所使用數值為近似值，所有實驗數據皆表示在20%的範圍內，較佳為在10%的範圍內，最佳為在5%的範圍內。
自Thermo Fisher Scientific公司購買含Earle’s平衡鹽溶液之Eagle’s最低基本培養基(Eagle’s minimum essential medium，簡稱MEM培養基)，角質細胞SFM培養基(Keratinocyte-SFM(1X))，胎牛血清(fetal bovine serum，簡稱FBS)，非必需胺基酸，碳酸氫鈉，丙酮酸鈉，磷酸緩衝鹽溶液(phosphate buffered saline， 簡稱PBS溶液)。自Amersco公司購買3-(4,5-二甲基-2-噻唑基)-2,5-二苯基四氮唑溴化物(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，簡稱MTT)。自ECHO Chemical公司購買二甲基亞碸(dimethyl sulfoxide，簡稱DMSO)。
以下實施例使用初代人類表皮角質細胞(human epidermal karatinocyte)HPEK-50(CELLnTEC，瑞士)及人類皮膚纖維母細胞(human skin fibroblast)CCD-966SK(BCRC 60153)進行實驗。HPEK-50在37℃、5%二氧化碳的條件下培養於SFM培養基。CCD-966SK細胞在37℃、5%二氧化碳的條件下培養於添加10% FBS、0.1mM非必需胺基酸、1.5g/L碳酸氫鈉、1mM丙酮酸鈉之MEM培養基，以下稱細胞培養基。
細胞中特定基因的表現量係利用定量聚合酶鏈鎖反應(quantitative polymerase chain reaction，簡稱qPCR)技術測定，其步驟簡述如下。依據廠商使用說明，以核糖核酸萃取套組(RNA Extraction Kit；Geneaid)自細胞分離出RNA後，於37℃下以反轉錄酶SuperScript® III Reverse Transcriptase(Invitrogen)將RNA反轉錄為互補去氧核醣核酸(cDNA)。其後，利用qPCR套組(KAPA CYBR FAST qPCR Kit(2X)；KAPA Biosystems)以及目標基因與作為內部對照之甘油醛3-磷酸脫氫酶(Glyceraldehyde 3-phosphate dehydrogenase，GAPDH)基因之引子對(表1)在PCR反應儀(Applied Biosystems StepOnePlusTM Real-Time PCR Systems；Thermo Fisher Scientific)對該cDNA進行PCR，以取得各基因的解鏈曲線(melting curve)及循環閾值(CT)。
最終，使用2-△△CT方法測定目標基因的相對表現量。所謂相對表現量定義為實驗組之一目標基因相對於控制組之同一基因的RNA表現量倍數變化。該方法以GAPDH基因的循環閾值作為內部對照之參考基因的循環閾值，按照以下公式計算倍數變化：△CT=實驗組或控制組的目標基因的CT-內部對照的CT △△CT=實驗組的△CT-控制組的△CT 倍數變化=2-△△Ct平均值
細胞存活率或增生率係以MTT分析測定。簡言之，將MTT溶液(4mg/ml MTT溶於PBS溶液)依15μl/孔添加至96孔盤中的細胞，於37℃反應4小時。移除反應液後，將DMSO依50μl/孔添加至細胞並震盪反應10分鐘以溶解所生成的甲
(formazan)結晶。最終，使用ELISA讀盤機(enzyme-linked immunosorbent assay reader；BioTek)測量該細胞混合物在570nm的吸光值(O.D.570)。
統計上顯著差異係以Excel軟體之學生t檢定判定。
首先，將泡葉藻洗淨，並且以例如切割、研磨等方式將其處理為適當大小，再以水、醇類、或醇水混合物為溶劑對該經處理的泡葉藻進行萃取。 該溶劑較佳為水，且該溶劑與泡葉藻之液固比5~20：1~5。萃取溫度為介於50℃~100℃，較佳為80℃~95℃。本實施例中萃取時間為0.5~3小時。
經上述萃取步驟所得泡葉藻萃取物冷卻至室溫後，可以400目(mesh)之濾網過濾，以移除殘餘固體物。該過濾後的泡葉藻萃取物可進一步在45℃~70℃進行減壓濃縮而獲得一濃縮產物。為獲得固態的泡葉藻萃取物，可將前述經減壓濃縮的泡葉藻萃取物以噴霧乾燥方式去除溶劑，因此獲得泡葉藻萃取物粉末。
為探討泡葉藻萃取物對皮膚細胞中基因表現的調節作用，本實施例以qPCR技術測定初代人類表皮角質細胞HPEK-50經泡葉藻之水萃取物處理後的基因表現變化。首先，將1.5×105個HPEK-50細胞接種於含有2mL細胞培養基的6孔盤的各孔，置於37℃下培養。其次，該些細胞以2mL含1mg/mL泡葉藻水萃取物之SFM培養基處理(三重複試驗)，此係作為實驗組。同時，另設置一組以不含泡葉藻水萃取物之SFM培養基處理的HPEK-50細胞作為控制組。經過6或24小時後，收集該些細胞以進行qPCR。
上述HPEK-50細胞中TGM1、KRT14、FLG、AQP3、GBA、HAS3、HAS2、MMP2、及LOX等基因的相對表現量如圖1A及圖1B所示。依據圖1A，相對於控制組，施予濃度1mg/mL的泡葉藻萃取物6小時或24小時會明顯提升HPEK-50細胞中TGM1、KRT14、FLG、AQP3、GBA、及HAS3的基因表現。依據圖1B，相對於控制組，施予濃度1mg/mL的泡葉藻萃取物24小時會顯著提升LOX及HAS3的基因表現及抑制MMP2的基因表現。由於TGM1、KRT14、FLG、AQP3、GBA、及HAS3基因之表現上調與增加皮膚屏障及含水量相關，LOX及HAS3基因之表現上調與MMP2基因之表現下調與提升皮膚彈性相關，前述實驗結果說明泡葉藻萃取物有助於提高皮膚保濕度及彈性。
為檢驗泡葉藻萃取物是否影響皮膚對紫外線照射的抵抗力，利用細胞存活分析(MTT分析)評估經過紫外線A照射的人類皮膚纖維母細胞 CCD-966SK在泡葉藻萃取物處理後的細胞存活率。簡言之，將5×103個CCD-966SK細胞接種於含有200μL細胞培養基的96孔盤的各孔，在37℃下培養24小時後，移除細胞培養基，並添加200μL含1mg/mL泡葉藻水萃取物之細胞培養基至細胞以作為實驗組，其於37℃下再培養24小時。其後，使細胞在紫外線照射箱(Vilber)中接受12J/cm2紫外線A(波長315-400nm)照射1小時，此輻射劑量會造成半數細胞死亡。同時，另設置一組經紫外線A照射但以不含泡葉藻水萃取物之細胞培養基處理的細胞以作為負控制組，及設置一組未予紫外線A照射且僅以不含泡葉藻水萃取物之細胞培養基處理的細胞以作為空白對照組。最終，進行MTT分析及依下列公式計算各組細胞之細胞增生率：細胞增生率=各組的O.D.570/空白對照組的O.D.570×100%
圖2顯示上述CCD-966SK細胞在不同處理後的細胞存活率。依據圖2，負控制組相比空白對照組有顯著降低的細胞存活率，顯示紫外線A照射會造成皮膚纖維母細胞大量死亡。然而，泡葉藻水萃取物之處理卻使細胞存活率明顯回升，說明泡葉藻萃取物能提升皮膚對紫外線的抵抗力。此結果與實施例2中所述泡葉藻萃取物會促進增加皮膚屏障的基因表現相符。
綜上所述，本發明以水、醇類、或醇水混合物為溶劑所萃取得之泡葉藻萃取物能調節TGM1、KRT14、FLG、AQP3、GBA、HAS3、HAS2、MMP2、及LOX等基因之表現，因而導致提升皮膚含水量與彈性及對紫外線的抵抗力。該泡葉藻萃取物可用於製備一具有護膚功效的食品、飲品、營養補充劑、或醫藥品組合物，且該組合物可具有粉末、顆粒、溶液、膠體、或膏體之劑型，藉由口服、塗抹於皮膚等方式給予一個體。
<110> 大江生醫股份有限公司
<120> 泡葉藻萃取物用於調節基因群組表現之用途
<130> 106B0185-I1
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Claims (5)
- 一種泡葉藻萃取物用於製備促進一基因群組表現之醫藥組合物之用途，其中該泡葉藻萃取物係以水萃取一泡葉藻而獲得且具有至少1mg/mL之濃度，且其中該基因群組包含一選自由轉麩胺酸醯胺基酶(TGM1)、角質蛋白14(KRT14)、絲聚蛋白(FLG)、水通道蛋白3(AQP3)、β葡萄糖腦苷酯酶(GBA)、玻尿酸合成酶3(HAS3)、及其任意組合所組成群組之A群基因，及一選自由玻尿酸合成酶2(HAS2)、賴胺醯氧化酶(LOX)、及其任意組合所組成群組之B群基因。
- 如申請專利範圍第1項所述之用途，其中該泡葉藻萃取物促進轉麩胺酸醯胺基酶、角質蛋白14、絲聚蛋白、水通道蛋白3、β葡萄糖腦苷酯酶、玻尿酸合成酶3、玻尿酸合成酶2、及賴胺醯氧化酶之基因表現。
- 如申請專利範圍第1項所述之用途，其中該水與該泡葉藻之液固比為5~20：1~5。
- 如申請專利範圍第1項所述之用途，其中該萃取係在50℃~100℃進行。
- 如申請專利範圍第1項所述之用途，其中該醫藥組合物具有粉末、顆粒、溶液、膠體、或膏體之劑型。
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| US201762503763P | 2017-05-09 | 2017-05-09 | |
| US62/503,763 | 2017-05-09 |
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| KR (1) | KR20180123640A (zh) |
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| US5508033A (en) * | 1989-12-06 | 1996-04-16 | Societe D'engrais Composes Mineraux Et Amendments | Utilization of algae extract for the preparation of pharmaceutical, cosmetic, food or agricultural compositions |
| JP2000136124A (ja) * | 1998-10-30 | 2000-05-16 | Pias Arise Kk | 皮膚外用剤 |
| EP2968107B1 (en) * | 2013-03-15 | 2019-05-08 | Mary Kay, Inc. | Cosmetic compositions and uses thereof |
| ES2441469B2 (es) * | 2013-04-12 | 2014-07-04 | Universidade De Santiago De Compostela | Extracto antioxidante a partir de macroalgas pardas y procedimiento de obtención |
| CN107693406A (zh) * | 2017-11-17 | 2018-02-16 | 皮光明 | 美白紧致植物精华化妆品及其制备方法 |
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2018
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- 2018-05-07 US US15/972,708 patent/US20180325803A1/en not_active Abandoned
- 2018-05-09 KR KR1020180052921A patent/KR20180123640A/ko not_active Application Discontinuation
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- 2018-05-09 CN CN201810445513.1A patent/CN108852919A/zh active Pending
Non-Patent Citations (4)
| Title |
|---|
| "FucowhiteTM Whitening the skin"，2019年9月23日取自網址: http://www.lessonia.com/vars/fichiers/Fiches-anglais/cosmetic-ingredients/marine-actives/Fucowhite.pdf，2016年11月9日 * |
| "FucowhiteTM Whitening the skin"，2019年9月23日取自網址: http://www.lessonia.com/vars/fichiers/Fiches-anglais/cosmetic-ingredients/marine-actives/Fucowhite.pdf，2016年11月9日。 |
| "RESPONSE OF KERATINOCYTES TO UV RADIATION AND ITS MODULATION BY HYALURONAN,MASARYK UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF EXPERIMENTAL BIOLOGY,2013"，2019年9月23日取自網址: https://is.muni.cz/th/npyzf/Moravcova_2013-Ph.D..pdf * |
| "RESPONSE OF KERATINOCYTES TO UV RADIATION AND ITS MODULATION BY HYALURONAN,MASARYK UNIVERSITY FACULTY OF SCIENCE DEPARTMENT OF EXPERIMENTAL BIOLOGY,2013"，2019年9月23日取自網址: https://is.muni.cz/th/npyzf/Moravcova_2013-Ph.D..pdf。 |
Also Published As
| Publication number | Publication date |
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| TW201900194A (zh) | 2019-01-01 |
| CN113730446A (zh) | 2021-12-03 |
| US20180325803A1 (en) | 2018-11-15 |
| CN108852919A (zh) | 2018-11-23 |
| KR20180123640A (ko) | 2018-11-19 |
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