TWI736855B - 西番蓮花萃取物促進皮膚細胞粒線體活性及抗老化基因表現之用途 - Google Patents
西番蓮花萃取物促進皮膚細胞粒線體活性及抗老化基因表現之用途 Download PDFInfo
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Abstract
本發明提供一種西番蓮花萃取物用於製備促進皮膚細胞粒線體活性或抗老化基因表現之組合物之用途。該西番蓮花萃取物不僅有助於增加細胞能量產生，而且提高與粒線體更新、細胞壓力反應、及端粒延長相關之多種抗老化基因之表現，例如PINK1、ATG1、ATG8、SIRT1、FOXO3、TERT及TERC，因此能維持皮膚細胞的活力及皮膚的年輕狀態。
Description
本發明係關於一種西番蓮花萃取物的保健用途，特別係關於一種西番蓮花萃取物用於製備促進皮膚細胞粒線體活性或抗老化基因表現之組合物之用途。
隨著人口平均年齡的延長，擁有健康而高品質的老年生活成為大眾關注的焦點之一，關於老化的科學研究也越發受重視。老化可大致定義為生理機能隨時間而衰退，其與許多流行疾病相關，例如心血管疾病、癌症、代謝疾病、神經退化疾病等。老化的過程複雜而受到諸多因素影響，包括飲食、運動、心理狀態、及遺傳因子。在分子或細胞層次，可能的內部機制包括染色體末端的端粒(telomere)縮短、基因突變、基因表現失調、蛋白質恆定喪失、粒線體功能下降等。此外，細胞周圍環境中由物理或化學因素產生的自由基(如活性氧物質)會損害細胞的染色體去氧核醣核酸(DNA)、蛋白質、及脂質生物膜，因而破壞細胞的機能。
鑒於上述造成老化的因素複雜，延緩身體乃至於各種細胞老化可以從多方面著手，包括改變飲食與運動習慣、保持心情愉快、減少暴露於誘導自由基形成的環境因子(如紫外光)、及加強細胞保護機制的運作(例如促進抗老化基因之表現)。其中，促進細胞保護機制的方法正處於發展階段，究竟何種手段能加強細胞保護機制尚在研究當中。因此，開發一種能促進細胞保護機制的新穎組合物，為減緩細胞機能衰退與維持細胞健康狀態提供新方向，實有其必要。
緣此，本發明之一目的在提供一種西番蓮(Passiflora spp.)花萃取物用於製備促進皮膚細胞粒線體活性之組合物之用途，其中該西番蓮花萃取物係以一溶劑萃取一西番蓮花而獲得。
在本發明之一實施例中，該西番蓮花萃取物係以水、醇類、或醇水混合物為溶劑而萃取。
在本發明之一實施例中，該西番蓮花萃取物抑制一聚二磷酸腺苷核糖聚合酶(亦稱聚ADP核糖聚合酶，poly(ADP-ribose)polymerase，PARP)之基因表現，且該聚二磷酸腺苷核糖聚合酶包含聚二磷酸腺苷核糖聚合酶1(PARP1)及聚二磷酸腺苷核糖聚合酶2(PARP2)。已知PARP與粒線體機能失調相關，故西番蓮花萃取物可能但不限於藉由對PARP基因表現的抑制作用達到提升粒線體活性之效果。
本發明之另一目的在提供一種西番蓮花萃取物用於製備促進皮膚細胞抗老化基因表現之組合物之用途，其中該西番蓮花萃取物係以一溶劑萃取一西番蓮花而獲得。
在本發明之一實施例中，該抗老化基因編碼一自噬相關蛋白(autophagy-related protein，ATG)，包含自噬相關蛋白1(ATG1)及自噬相關蛋白8(ATG8)。
在本發明之一實施例中，該抗老化基因編碼一磷酸酶及張力蛋白同源物誘導激酶1(phosphatase and tensin homolog(PTEN)-induced putative kinase 1，PINK1)。
在本發明之一實施例中，該抗老化基因編碼一沉默信息調節因子2同源蛋白1(silent mating type information regulation 2 homolog 1或sirtuin 1，SIRT1)或一叉頭框蛋白O3(forkhead box class O 3，FOXO3)。
在本發明之一實施例中，該抗老化基因係編碼一端粒酶反轉錄酶(telomerase reverse transcriptase，TERT)、一端粒酶核醣核酸組分(telomerase RNA component，TERC)、或其組合。因此，該西番蓮花萃取物能促進皮膚細胞內端粒酶之合成。
在本發明之一實施例中，用於促進皮膚細胞粒線體活性或抗老化基因表現之西番蓮花萃取物之濃度為0.125mg/mL至0.25mg/mL。
本發明基於體外細胞實驗及基因表現分析結果，揭示西番蓮花萃取物提升皮膚細胞粒線體活性以及多種抗老化基因之表現。鑒於粒線體活性之維持對細胞活力極為重要，且前述抗老化基因涉及老化粒線體更新、細胞壓力反應(即協助逆境中細胞減少損傷的多種分子機制)、及端粒延長等有利細胞回春之事件，西番蓮花萃取物具備維持皮膚細胞活力及個體皮膚年輕狀態之潛力。因此，本發明提供西番蓮花萃取物用於製備促進皮膚細胞粒線體活性或抗老化基因表現之組合物之用途。該組合物可為粉末狀、顆粒狀、液狀、膠狀或膏狀，且可製成食品、飲品、藥品、試劑或營養補充劑，藉由口服、皮膚塗抹等方式給予一個體。
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1顯示人類皮膚纖維母細胞在有或無西番蓮花萃取物處理24小時後，細胞中JC-1聚集體的相對量(%)。
圖2顯示人類皮膚纖維母細胞在有或無西番蓮花萃取物處理48小時後，相對於控制組細胞的PARP1與PARP2基因的相對表現量。
圖3顯示人類皮膚纖維母細胞在有或無西番蓮花萃取物處理48小時後，相對於控制組細胞的PINK1、ATG1，ATG8、SIRT1、FOXO3、TERT、及TERC基因的相對表現量。
本發明提供一種西番蓮花萃取物用於製備促進皮膚細胞粒線體活性或抗老化基因表現之組合物之用途。本發明之西番蓮花萃取物係以一溶劑萃取一西番蓮花而獲得，其中，該溶劑可為水、醇類、或醇水混合物，較佳為水。下列實施例顯示該西番蓮花萃取物之處理能提升人類皮膚細胞之粒線體活性，並且抑制與粒線體機能失調有關之多種聚ADP核糖聚合酶(PARP；例如PARP1及PARP2)之基因表現。此外，該西番蓮花萃取物促進人類皮膚細胞中抗
老化基因之表現。前述抗老化基因表現之產物可為核糖核酸(RNA)或蛋白質，包括但不限於：磷酸酶及張力蛋白同源物誘導激酶1(PINK1)、多種自噬相關蛋白(ATG；例如ATG1及ATG8)、沉默信息調節因子2同源蛋白1(SIRT1)、叉頭框蛋白O3(FOXO3)、及參與端粒酶反應的端粒酶反轉錄酶(TERT)及端粒酶RNA組分(TERC)。
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。
本文中所謂「粒線體活性」指粒線體運作的效力，包括能量產生、物質代謝、活性氧物質清除效率等。粒線體活性之評估可依據本發明所屬技術領域所習知之生化、分子生物及細胞學技術而完成，例如本文所述粒線體膜電位之測定。
本文中所謂「抗老化基因」泛指其存在與生物體長壽相關或其RNA或蛋白質產物之作用可維持細胞正常功能的基因。該抗老化基因所編碼蛋白質之作用包括促使粒線體正常運作、協助其他蛋白質摺疊、促進端粒酶合成、及調控參與養分代謝或細胞壓力反應之蛋白質的基因表現等。
含有Earle’s平衡鹽溶液(Earle’s balanced salt solution，EBSS)之Eagle’s最低基本培養基(Gibco Eagle’s minimum essential medium，MEM)、胎牛血清(Gibco fetal bovine serum，FBS)、丙酮酸鈉、青黴素/鏈黴素(penicillin/streptomycin)、及磷酸緩衝鹽溶液(Gibco phosphate buffered saline，PBS)係購自Thermo Fischer Scientific公司。
以下實施例使用購自食品工業發展研究所生物資源保存及研究中心(Bioresource Collection and Research Center，BCRC)之人類皮膚纖維母細胞(human skin fibroblast)CCD-966SK(BCRC 60153)。CCD-966SK細胞在37℃、5%二氧化碳的條件下培養於MEM培養基(含有EBSS)，該培養基並添加10%胎牛血
清、1mM丙酮酸鈉、0.01M非必需胺基酸、及1%青黴素-鏈黴素，以下稱MEM細胞培養基。
本文中所謂西番蓮花萃取物係以一溶劑對一西番蓮屬(Passiflora spp.)植物的花(簡稱西番蓮花，例如百香果(Passiflora edulis)、毛西番蓮(Passiflora foetida)的花)進行萃取而獲得。該溶劑可為水、醇類、或醇水混合物，較佳為水。在一實施例中，進行萃取前先洗淨及乾燥西番蓮花，使用均質機將其粗碎。其後，以攪拌方式均勻混合該溶劑與該西番蓮花均質物0.5至3小時即可製得西番蓮花萃取物；該溶劑與該西番蓮花均質物的重量比範圍為20：1至2：1，較佳為5：1；萃取溫度為40℃至75℃。以下實施例所用西番蓮花萃取物係購自陝西嘉禾生物科技股份有限公司，其依類似前述步驟製備而得。
為評估細胞的粒線體活性，利用流式細胞儀(flow cytometer；Beckman Coulter Life Sciences)及粒線體膜電位檢測套組(BDTM MitoScreen)測定粒線體膜電位之改變，其步驟簡述如下。依據廠商使用說明，以PBS溶液潤洗待測細胞，再將細胞收集至1.5mL微量離心管及進行離心(400 xg，5分鐘)。經移除上清液，以PBS溶液再懸浮細胞並且再次離心(400 xg，5分鐘)。將100μL JC-1(5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide)操作溶液與沉澱之細胞在暗處均勻混合15分鐘以進行螢光標記，再以清洗溶液及離心步驟(400 xg，5分鐘)清洗細胞2次。最終，將細胞再懸浮於含2% FBS之PBS溶液，使用流式細胞儀偵測指示粒線體膜電位的JC-1聚集體的相對量(激發波長為約490nm，偵測波長為約595nm)。JC-1聚集體的量越多表示粒線體內膜的電位差越大，被視為粒線體活性越高。
基於定量聚合酶鏈鎖反應(quantitative polymerase chain reaction，簡稱qPCR)測定細胞中有關粒腺體活性與抗老化之基因表現，其步驟簡述如下。依據廠商使用說明，利用RNA萃取套組(RNA Extraction Kit；Geneaid)自細胞分離出RNA，於37℃下以反轉錄酶SuperScript® III Reverse Transcriptase(Invitrogen)將2000ng RNA反轉錄為cDNA。其次，利用qPCR套組(KAPA CYBRFAST qPCR Kit(2X)；KAPA Biosystems)以及下表1所列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)基因之引子對在PCR反應儀(Step One Plus Real-Time PCR system；Applied Biosystems)對前述cDNA進行qpCR，以取得解鏈曲線(melting curve)。
最終，使用2-△△CT方法測定目標基因的相對表現量。該方法以GAPDH基因的循環閾值(CT)作為內部對照之參考基因的循環閾值，按照以下公式計算相對倍數變化：△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
統計分析係使用Excel軟體中的STDEV函數計算各基因相對表現量的標準差，並以單尾學生T檢驗(TTEST)計算統計上差異。
粒線體是供給細胞能量的胞器，並且參與細胞的氧化還原恆定及養分代謝，其正常運作對維持細胞的活力與增殖能力至關重要。為探討西番蓮花萃取物對皮膚細胞粒線體功能的影響，本實施例以流式細胞儀評估人類皮膚纖維母細胞CCD-966SK經西番蓮花萃取物處理後，其粒線體活性的變化。首先，將CCD-966SK細胞依1×105個細胞/孔接種於6孔盤，各孔含有2mL MEM細胞培養基。在37℃培養細胞24小時後，移除該細胞培養基，並以1mL含0.125mg/mL西番蓮花萃取物之MEM細胞培養基處理細胞(實驗組)，或者僅用MEM細胞培養基處理細胞以作為控制組。各組細胞於37℃培養24小時後用於粒線體活性分析，以螢光染劑JC-1指示粒線體膜電位及活性。
圖1顯示前述各組CCD-966SK細胞中JC-1聚集體的相對量(%)，其值越高表示細胞的平均粒線體活性越高；***表示相比控制組為p<0.001。依據圖1，相比控制組細胞，施予0.125%西番蓮花萃取物顯著提高CCD-966SK細胞的JC-1聚集體相對量約40%，說明西番蓮花萃取物能提升皮膚細胞粒線體活性，使皮膚細胞有足夠活力執行特定生理功能或增殖以汰換老舊細胞。因此，西番蓮花萃取物有利於維持人類個體皮膚的年輕狀態。
為探討西番蓮花萃取物對皮膚細胞抗老化力的影響，利用qPCR測定人類皮膚纖維母細胞CCD-966SK經西番蓮花萃取物處理後，其聚ADP核糖聚合酶1及2(PARP1及PARP2)、磷酸酶及張力蛋白同源物誘導激酶1(PINK1)、自嗜相關蛋白1及8(ATG1及ATG8)、沉默信息調節因子2同源蛋白1(SIRT1)、叉頭框蛋白O3(FOXO3)、端粒酶反轉錄酶(TERT)、及端粒酶RNA組分(TERC)之基因表現變化。簡言之，將CCD-966SK細胞依1.5×105個細胞/孔接種於6孔盤，各孔含有2mL MEM細胞培養基。在37℃培養細胞24小時後，移除該細胞培養基，並以1mL含0.125mg/mL或0.25mg/mL西番蓮花萃取物之MEM細胞培養基處理細
胞(實驗組)，或者僅以MEM細胞培養基處理細胞以作為控制組。前述細胞於37℃培養48小時後用於qPCR分析。
圖2顯示CCD-966SK細胞以0.125mg/mL西番蓮花萃取物處理48小時後相對於控制組細胞的PARP1與PARP2基因的相對表現量。依據圖2，西番蓮花萃取物之處理會抑制CCD-966SK細胞中多種聚ADP核糖聚合酶(PARP)之基因表現。鑒於先前研究指出該種酵素活性之抑制可改善粒線體活性，此結果暗示西番蓮花萃取物能增加粒線體活性，進而改善細胞的能量產生及對抗老化。
圖3顯示CCD-966SK細胞以0.25mg/mL西番蓮花萃取物處理48小時後相對於控制組細胞的PINK1、ATG1，ATG8、SIRT1、FOXO3、TERT、及TERC基因的相對表現量；*、**、及***分別表示相比控制組為p<0.05、p<0.01、及p<0.001。依據圖3，相比控制組，對CCD-966SK細胞施予西番蓮花萃取物顯著提高與老化粒線體更新相關之PINK1、ATG1、及ATG8基因之表現，並且促進與細胞壓力反應相關之SIRT1及FOXO3基因之表現。此外，西番蓮花萃取物亦提升參與端粒酶反應以延長端粒之TERT及TERC基因之表現。此結果說明西番蓮花萃取物提高皮膚細胞中多種抗老化基因之產物，因此有助於維持皮膚細胞的活力及延緩肌膚老化。
綜上所述，西番蓮花萃取物能提升皮膚細胞之粒線體活性以及多種抗老化基因之表現，進而維持皮膚細胞的活力及個體皮膚的年輕狀態。因此，本發明提供西番蓮花萃取物用於製備促進皮膚細胞粒線體活性或抗老化基因表現之組合物之用途。該組合物可為粉末狀、顆粒狀、液狀、膠狀或膏狀，且可製成食品、飲品、營養補充劑、或醫藥品，藉由口服、皮膚塗抹等方式給予一個體。
<110> 大江生醫股份有限公司
<120> 西番蓮花萃取物用於促進皮膚細胞粒線體活性及抗老化基因表現之用途
<130> 107B0594-I1
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Claims (5)
- 一種西番蓮花萃取物用於製備促進皮膚細胞粒線體活性之組合物之用途，其中該西番蓮花萃取物係以一溶劑萃取一西番蓮花而獲得，其中該西番蓮花萃取物之濃度為0.125mg/mL至0.25mg/mL。
- 如申請專利範圍第1項所述之用途，其中該西番蓮花萃取物抑制一聚二磷酸腺苷核糖聚合酶(PARP)之基因表現，且該聚二磷酸腺苷核糖聚合酶包含聚二磷酸腺苷核糖聚合酶1及聚二磷酸腺苷核糖聚合酶2。
- 如申請專利範圍第1項所述之用途，其中該西番蓮花萃取物提高磷酸酶及張力蛋白同源物誘導激酶1(PINK1)、自嗜相關蛋白1(ATG1)、或自嗜相關蛋白8基因之表現。
- 一種西番蓮花萃取物用於製備一透過延長端粒促進皮膚細胞抗老化基因表現之組合物之用途，其中該西番蓮花萃取物係以一溶劑萃取一西番蓮花而獲得，且其中該抗老化基因為端粒酶反轉錄酶(TERT)、或端粒酶核醣核酸組分(TERC)之蛋白質，其中該西番蓮花萃取物之濃度為0.125mg/mL至0.25mg/mL。
- 如申請專利範圍第1項或第4項所述之用途，其中該溶劑為水、醇類、或醇水混合物。
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| TW108107295A TWI736855B (zh) | 2019-03-05 | 2019-03-05 | 西番蓮花萃取物促進皮膚細胞粒線體活性及抗老化基因表現之用途 |
| CN201910342301.5A CN111658682A (zh) | 2019-03-05 | 2019-04-25 | 西番莲花萃取物促进皮肤细胞线粒体活性及抗老化基因表现的用途 |
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| TW108107295A TWI736855B (zh) | 2019-03-05 | 2019-03-05 | 西番蓮花萃取物促進皮膚細胞粒線體活性及抗老化基因表現之用途 |
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| TW202033207A TW202033207A (zh) | 2020-09-16 |
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| JP2002332224A (ja) * | 2001-03-06 | 2002-11-22 | Kose Corp | 老化防止用皮膚外用剤および老化防止用皮膚外用剤組成物 |
| FR2931070B1 (fr) * | 2008-05-13 | 2010-07-30 | Nuxe Lab | Association d'extraits de passiflore et d'anchuse utilisable en cosmetique |
| FR2974298B1 (fr) * | 2011-04-20 | 2013-11-29 | Nuxe Lab | Complexe d'extraits vegetaux pour la protection de la peau |
| WO2016176771A1 (en) * | 2015-05-05 | 2016-11-10 | Idunn Technologies | Anti-aging composition comprising a plant extract |
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