TWI727159B - 蘭花葉片癒傷組織萃取物之應用 - Google Patents
蘭花葉片癒傷組織萃取物之應用 Download PDFInfo
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- TWI727159B TWI727159B TW107111783A TW107111783A TWI727159B TW I727159 B TWI727159 B TW I727159B TW 107111783 A TW107111783 A TW 107111783A TW 107111783 A TW107111783 A TW 107111783A TW I727159 B TWI727159 B TW I727159B
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Abstract
一種使用蘭花癒傷組織萃取物於製備一組合物之用途,其中該萃取物係一蘭花之葉片癒傷組織的極性溶劑萃取物,且該組合物係用於護膚,尤其是用於保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及/或促進傷口癒合。
Description
本發明係關於蘭花癒傷組織萃取物於護膚之應用,尤其係關於蘭花葉片之癒傷組織的萃取物於護膚之應用,包括使用該萃取物於保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及/或促進傷口癒合。
皮膚是人體最大的組織,也是抵抗外物侵入及外在環境刺激的第一道防線,具有保水、保暖及感覺等重要功能。當皮膚受到紫外線、輻射、環境中的微生物及懸浮粒子等的影響時,皆可能使皮膚的含水量降低並加速皮膚細胞老化,導致皮膚發生角質增厚、皮膚乾燥脫屑、產生細紋及斑點、皮膚鬆弛、及蛋白質的分解與流失等現象,甚至會損害皮膚細胞中DNA,而引發細胞毒性、造成皮膚細胞變異而導致皮膚病變。
膠原蛋白及透明質酸皆為增加皮膚含水量的重要物質,其中,膠原蛋白除具有保水功能以外,還可賦予皮膚韌性及彈性;皮膚真皮層中的彈力蛋白則具有支撐皮膚細胞結構的功能。因此,若可提升上述三者於皮膚中的含量,即可達到提升肌膚延展性及彈性、緊緻皮膚、以及撫平細紋的效果,並可改善皮膚凹陷及鬆弛的問題。
近年來,人們越來越重視皮膚保養的問題,且講究使用天然且安全的原料於皮膚保養上,因此,業界仍持續開發可透過天然且安全之原料的使用以有效保養皮膚的手段。本案發明人研究發現,蘭花癒傷組織萃取物可促進膠原蛋白、彈力蛋白、及透明質酸之合成,且具有幫助修復皮膚細胞中DNA的效果,故可用於保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及/或促進傷口癒合。
本發明之一目的,在於提供一種使用蘭花癒傷組織萃取物於製備一組合物之用途,其中該萃取物係一蘭花葉片之癒傷組織的極性溶劑萃取物,且該組合物係用於護膚。較佳地,該蘭花係白花蝴蝶蘭(Phalaenopsis amabilis)。較佳地,該極性溶劑係水、C1-C4醇類、或前述之組合。較佳地,該組合物係一保養品組合物、食品組合物、或醫藥組合物。
本發明之另一目的,在於提供一種護膚組合物,其係包含一有效量之蘭花癒傷組織萃取物,其中該萃取物係一蘭花葉片之癒傷組織的極性溶劑萃取物。較佳地,該極性溶劑係水、C1-C4醇類、或前述之組合。較佳地,該組合物係一保養品組合物、食品組合物、或醫藥組合物。
該根據本發明所提供之保養品組合物係可用於以下之至少一者:保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、及延緩皮膚老化。較佳地,該保養品組合物係以精華液、乳液或化妝水之形式提供。
該根據本發明所提供之食品組合物係可用於幫助提升皮膚膠原蛋白含量,尤其是幫助提升皮膚第一型膠原蛋白含量。較佳地,該食品組合物係以美容飲品之形式提供。
該根據本發明所提供之醫藥組合物係可用於以下之至少一者:修復皮膚組織、預防皮膚病變、及促進傷口癒合。該醫藥組合物係可呈一選自以下群組之劑型:經皮投予、口服及皮下注射。
本發明之又一目的,在於提供一種護膚的方法,其係包含對一有需要之個體投予一有效量之蘭花癒傷組織萃取物。本發明方法可用於以下之至少一者:保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及促進傷口癒合。於根據本發明之方法中,所採用之蘭花癒傷組織萃取物係可以上述之組合物的形式投予至該有需要之個體。
本發明之詳細技術內容及部分具體實施態樣,將描述於以下內容中,以供本發明所屬領域具通常知識者據以明瞭本發明之特徵。
圖1至圖4係顯示本發明蘭花癒傷組織萃取物於提升皮膚膠原蛋白、彈力蛋白與透明質酸含量、及提升皮膚抗氧化能力的效果,其中,圖1係顯示COL1A1基因及COL1A2基因之相對表現量(倍)(*表示p<0.05、**表示p<0.01),圖2係顯示ELN基因之相對表現量(倍)(**表示p<0.01),圖3係顯示HAS3基因之相對表現量(倍),圖4則顯示CAT基因及SOD2基因之相對表現量(倍)(***表示p<0.001),且其中,控制組係以不含蘭花癒傷組織萃取物之培養基培養24小時,「萃取物」組則以含有蘭花癒傷組織萃取物之培養基培養24小時;以及
圖5至圖7係顯示本發明蘭花癒傷組織萃取物於幫助修復皮膚細胞中受損DNA的效果,其中,圖5係顯示MSH2基因、MLH1基因及MSH6基因(前述三者係屬於鹼基錯誤配對之修復基因)之相對表現量(倍)(**表示p<0.01、***表示p<0.001),圖6係顯示ERCC1基因(屬於核苷酸切除之修復基因)之相對表現量(倍)(**表示p<0.01),圖7則顯示UNG基因、OGG1基因及APE1基因(前述三者係屬於鹼基切除之修復基因)之相對表現量(倍)(**表示p<0.01、***表示p<0.001)。
以下將描述根據本發明之部分具體實施態樣;惟,在不背離本發明精神下,本發明尚可以多種不同形式之態樣來實踐,不應將本發明保護範圍解釋為限於說明書所陳述者。此外,除非文中有另外說明,於本說明書中(尤其是在後述專利申請範圍中)所使用之「一」、「該」及類似用語應理解為包含單數及複數形式;所謂“個體”是指人類或非人的哺乳動物。另,於本發明中,除非特別說明,否則所稱「蘭花癒傷組織」係指蘭花之葉片的癒傷組織。
本案發明人研究發現,蘭花植株之葉片在受傷後所產生之癒傷組織具有類似哺乳類全能幹細胞的特性,該癒傷組織萃取物可有效提升皮膚細胞之抗氧化能力、幫助皮膚細胞之DNA修復、促進膠原蛋白合成、促進彈力蛋白合成、及促進透明質酸合成。
因此,本發明係關於使用蘭花癒傷組織萃取物於護膚之應用,此包括:提供一包含蘭花癒傷組織萃取物的組合物、使用蘭花癒傷組織萃取物於製造一組合物之用途、以及使用該包含蘭花癒傷組織萃取物之組合物的方法,該組合物係用於護膚,尤其係用以保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮
膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及/或促進傷口癒合。其中,該蘭花癒傷組織尤其是指蘭花之葉片的癒傷組織。
根據本發明所採用之蘭花癒傷組織萃取物,係可透過以極性溶劑萃取蘭花之葉片的癒傷組織而提供,其中,該極性溶劑可以為水、醇(例如C1-C4醇類)、或其組合。此外,萃取溶劑的用量並無特殊限制,通常係採用可以分散原料之用量。舉例言之,可於萃取步驟中採用萃取溶劑:蘭花癒傷組織=100-300:1的重量比用量。視需要地,可於超音波震盪操作下進行萃取,以提升萃取效果。較佳地,可以在進行萃取之前,先進行一乾燥處理及/或一破碎處理。
於本發明之部分實施態樣中,係於進行萃取之前,先冷凍乾燥蘭花癒傷組織。舉例言之,可以100:1(水:經冷凍乾燥之蘭花癒傷組織)之重量比混合水與經冷凍乾燥之蘭花癒傷組織,並於70℃下對所得到之混合物進行超音波震盪歷時45分鐘以完成萃取。其中,該蘭花癒傷組織可透過包含如下處理之操作而提供:I.以6%次氯酸鈉溶液沖洗蘭花植株,其後以滅菌水沖洗蘭花植株,視需要地,可重複前述沖洗操作;II.在蘭花植株之葉片的表面上切出傷口,以誘導產生癒傷組織(歷時1至3個月);III.在25℃、50至60%濕度下,以MS培養基(Murashige and Skoog Basal Medium)培養步驟II所得之癒傷組織(歷時1至1.5個月)。
根據本發明所採用之蘭花癒傷組織萃取物,可以萃取所得之萃取原液直接使用,或視需要對該萃取原液進行例如過濾、滅菌、濃縮、稀釋等一
或多個步驟,以提升萃取液之使用便利性。舉例言之,透過例如濃縮乾燥、噴霧乾燥、或冷凍乾燥等操作,可由萃取液得到方便攜帶或方便儲存之濃縮液或粉末。
根據本發明所提供之組合物係一保養品組合物、食品組合物、或醫藥組合物。其中,根據本發明所提供之保養品組合物係可用於以下之至少一者:保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、及延緩皮膚老化。根據本發明所提供之保養品組合物可呈任何合宜的形式,並無特殊的限制。舉例言之,該保養品組合物可呈供直接外用之乳液、乳霜、凝膠(例如水凝膠)、或溶液(例如精華液、化妝水)等形式,但不以此為限。
根據本發明所提供之食品組合物係用於幫助提升皮膚膠原蛋白含量,尤其是幫助提升皮膚第一型膠原蛋白含量。根據本發明所提供之食品組合物可呈任何合宜的形式,並無特殊的限制。舉例言之,可將本發明之食品組合物製備成可供食用或飲用的形式,例如健康食品、美容飲品等,但不以此為限。
視需要地,可於根據本發明所提供之保養品組合物、食品組合物、或醫藥組合物中另含有合宜用量之添加劑,例如可提高該食品組合物或醫藥組合物於服用時的口適感及視覺感受之調味劑、調色劑、著色劑等,以及可改善該保養品組合物、食品組合物、或醫藥組合物的穩定性及儲存性之緩衝劑、保存劑、防腐劑、抗菌劑、抗真菌劑等。
根據本發明所提供之醫藥組合物係可呈任何合宜的型式,並無特殊限制,端視所欲之用途而呈對應之合宜劑型。舉例言之,但不以此為限,該醫藥組合物可以口服或非經口服(例如:經皮投予、皮下注射)之投藥方式施用至
有需要之個體上。其中,視使用形式及用途而定,可選用合宜之載劑以提供該醫藥組合物,其中,該載劑包括賦形劑、稀釋劑、輔助劑、安定劑、吸收延遲劑、崩散劑、增溶劑、乳化劑、抗氧化劑、黏合劑、結合劑、增黏劑、分散劑、懸浮化劑、潤滑劑、吸濕劑等。
以適於口服之劑型為例,於根據本發明所提供之醫藥組合物中係可含有任何不會不利影響活性成分(即,蘭花癒傷組織萃取物)之所欲效益的醫藥上可接受之載劑,例如:水、食鹽水、葡萄糖(dextrose)、甘油、乙醇或其類似物、纖維素、澱粉、糖膨潤土(sugar bentonite)、及前述之組合。可利用任何合宜之方法,將該醫藥組合物以適於口服投藥的劑型提供,例如:錠劑(例如糖衣錠)、丸劑、膠囊劑、顆粒劑、散劑、流浸膏劑、溶液劑、糖漿劑、懸液劑、酊劑等。
以適於經皮投予之劑型為例,根據本發明所提供之醫藥組合物可呈供直接外用之貼布、乳液、乳霜、凝膠(例如水凝膠)、膏狀物(例如分散膏、軟膏)、噴霧劑、或溶液(例如懸浮液)等形式,但不以此為限。
至於適於皮下注射之針劑或點滴劑型,則可於該醫藥組合物中含有一或多種例如等張溶液、鹽類緩衝液(如磷酸鹽緩衝液或檸檬酸鹽緩衝液)、增溶劑、乳化劑、5%糖溶液、以及其他載劑等成分。或者,將該醫藥組合物製備成一注射前固體,以可溶於其他溶液或懸浮液中之劑型、或可乳化之劑型提供該注射前固體,並於投予至有需要之個體之前,將該注射前固體溶於其他溶液或懸浮液中或將其乳化,提供所欲之注射劑。
根據本發明之應用所提供之組合物係可以一日一次、一日多次、或數日一次等不同頻率施用,端視投予個體之需求、年齡、體重、健康狀況、及施用目的而異。
本發明亦提供一種護膚的方法,其係包含對一有需要之個體投予一有效量之蘭花癒傷組織萃取物。於根據本發明之方法中,所採用之蘭花癒傷組織萃取物係可以一組合物的形式投予至該有需要之個體。有關該組合物之投予態樣、投予途徑、投予形式、施用頻率、以及相關之應用,均如上述之說明。
茲以下列實施例進一步例示說明本發明。其中該等實施例僅提供作為說明,而非用以限制本發明之保護範圍。本發明保護範圍係如後附申請專利範圍所示。
實施例
[製備實施例]
A.蘭花癒傷組織萃取物之製備
對購自台灣國光花市232蘭園之白花蝴蝶蘭(Phalaenopsis amabilis)植株施以如下處理,以提供蘭花癒傷組織:I.以6%次氯酸鈉溶液沖洗蘭花植株,其後以滅菌水沖洗蘭花植株,視需要地,可重複前述沖洗操作;II.在蘭花植株之葉片的表面上切出傷口,以誘導產生癒傷組織(歷時1至3個月);III.在25℃、50至60%濕度下,以MS培養基(Murashige and Skoog Basal Medium;購自SIGMA公司;產品編號:M5519)培養步驟II所得之癒傷組織(歷時1至1.5個月)。
以下列步驟處理上述蘭花癒傷組織,以製備蘭花癒傷組織萃取物:(1)蘭花癒傷組織置於-22℃下進行冷凍乾燥,歷時12小時,接著粉碎前述經乾燥之蘭花癒傷組織,以提供蘭花癒傷組織粉末;(2)將水與步驟(1)之蘭花癒傷組織粉末以100:1(水:蘭花癒傷組織粉末)的重量比混合,在70℃下,對所獲得的混合物進行超音波震盪,歷時45分鐘;(3)以濾膜過濾步驟(2)所得之混合物,以提供一濾液;(4)加熱由步驟(3)所獲得之濾液至95℃,並維持20分鐘,以進行滅菌;(5)待步驟(4)之濾液冷卻後,將其分裝並冷藏保存以供後續實驗使用;以及(6)對步驟(5)之濾液進行冷凍乾燥,以提供一乾燥物(即,本發明之蘭花癒傷組織萃取物)。
B.細胞處理
於MEM培養基(Minimum essential medium,購自Gibco公司;產品編號:61100-061)中培養人類皮膚纖維母細胞(CCD-966SK;購自ATCC)歷時24小時,接著,將細胞分成二組,並進行以下處理:
1.控制組:將細胞置於MEM培養基中培養24小時(即,將細胞培養於不含蘭花癒傷組織萃取物的培養液中)。
2.「萃取物」組:將細胞置於含有1重量%獲得自[製備實施例A]的蘭花癒傷組織萃取物(以乾重計)的MEM培養基中培養24小時。
C.cDNA之製備
以RNA萃取套組(RNA extraction kit,購自GENEmark公司)對經[製備實施例B]處理之各組細胞進行RNA萃取,再以反轉錄酶將各組RNA反轉錄為cDNA,供後續之基因表現量檢測使用。
實施例1:蘭花癒傷組織萃取物於提升皮膚膠原蛋白、彈力蛋白與透明質酸含量的效果
研究已知,皮膚細胞的COL1A1、COL1A2、ELN、HAS3等基因的表現量提升係有助於促進膠原蛋白、彈力蛋白、及透明質酸的合成,可參見例如:Modulation of collagen synthesis and its gene expression in human skin fibroblasts by tocotrienol-rich fraction.Arch Med Sci.7,5:889-895(2011)、The complexity if elastic fiber biogenesis in the skin-a perspective to the clinical heterogeneity of cutis laxa.Exp Dermatol.22(2):88-92(2013)、Hyaluronan Synthase 3 Regulates Hyaluronan Synthesis in Cultured Human Keratinocytes.The Journal of Investigative Dermatology.118:43-48(2002)、及An overview of the Beneficial Effects of Hydrolysed Collagen as a Nutraceutical on Skin Properties:Scientific Background and Clinical Studies.The Open Nutraceuticals Journal.8:29-42(2015),該等文獻之全文併於此處以供參考。為確認本發明蘭花癒傷組織萃取物可以提升皮膚之膠原蛋白的含量、彈力蛋白含量、及透明質酸含量,使用ABI Step One Plus儀器及KAPA SYBR FAST qPCR套組對[製備實施例C]所提供之各組cDNA進行qPCR(quantitative polymerase chain reaction),以檢測各組細胞之COL1A1、COL1A2、ELN、HAS3等基因的表現量。接著,以學生t檢驗(Student t-test)進行統計分析,並以控制組的結果為基準(即,將控制組的基因表現設定為1倍),計算「萃取物」組的基因相對表現量。結果示於圖1至3。
由圖1至3可知,相較於控制組,萃取物組的COL1A1、COL1A2、ELN、及HAS3基因表現量皆明顯提升。前述結果顯示,本發明蘭花癒傷組織萃取物確實可以提升OL1A1、COL1A2、ELN、HAS3等基因的表現量,故可有效促進膠原蛋白、彈力蛋白及透明質酸的合成與分泌,從而達到保濕、提升皮膚柔潤度、提升皮膚飽滿度、提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、及/或促進傷口癒合的效果。
實施例2:蘭花癒傷組織萃取物於提升皮膚抗氧化能力的效果
研究已知,皮膚細胞的CAT及SOD2基因的表現量提升係有助於皮膚細胞抗氧化能力的提升,可參見例如:Anti-oxidant gene expression imbalance,aging and Down syndrome.Life Sciences.76:1407-1426(2005),該文獻之全文併於此處以供參考。為確認本發明蘭花癒傷組織萃取物的抗氧化力,使用ABI Step One Plus儀器及KAPA SYBR FAST qPCR套組對[製備實施例C]所提供之各組cDNA進行qPCR,以檢測各組細胞之CAT及SOD2等抗氧化基因的表現量。接著,以學生t檢驗(Student t-test)進行統計分析,並以控制組的結果為基準(即,將控制組的基因表現設定為1倍),計算「萃取物」組的基因相對表現量。結果示於圖4。
由圖4可知,相較於控制組,萃取物組的CAT及SOD2基因表現量皆明顯提升。前述結果顯示,本發明蘭花癒傷組織萃取物確實可以提升抗氧化基因的表現量,故可有效提升皮膚細胞抵抗過氧化物質傷害的能力,從而達到延緩皮膚老化、修復皮膚組織、及/或預防皮膚病變的效果。
實施例3:蘭花癒傷組織萃取物於幫助修復皮膚細胞中受損DNA的效果
研究已知,生物體中的鹼基錯誤配對修復(mismatch repair,MMR)、核苷酸切除修復(nucleotide excision repair,NER)、及鹼基切除修復(base excision repair,BER)等DNA修復機制可避免因DNA突變或斷裂所造成的傷害,若可提升皮膚細胞之MMR相關基因、NER相關基因、及BER相關基因的表現量,則可有助於皮膚細胞中受損DNA的修復,可參見例如:DNA repair mechanisms in dividing and non-dividing cells.DNA repair(Amst).12(8):620-636(2013),該文獻之全文併於此處以供參考。為確認本發明蘭花癒傷組織萃取物可以幫助修復皮膚細胞中受損的DNA,使用ABI Step One Plus儀器及KAPA SYBR FAST qPCR套組對[製備實施例C]所提供之各組cDNA進行qPCR,以檢測各組細胞中MMR相關基因(包括MSH2、MLH1及MSH6)、NER相關基因(包括ERCC1)、及BER相關基因(包括UNG、OGG1及APE1)的表現量。接著,以學生t檢驗(Student t-test)進行統計分析,並以控制組的結果為基準(即,將控制組的基因表現設定為1倍),計算「萃取物」組的基因相對表現量。結果分別示於圖5至7。
由圖5至7可知,相較於控制組,萃取物組的MMR相關基因(包括MSH2、MLH1及MSH6)、NER相關基因(包括ERCC1)、及BER相關基因(包括UNG、OGG1及APE1)的表現量皆明顯提升。前述結果顯示,本發明蘭花癒傷組織萃取物確實可以提升DNA修復相關基因的表現量,故可有效修復皮膚細胞中受損DNA,從而達到延緩皮膚老化、修復皮膚組織、及/或預防皮膚病變之效果。
如上述實施例所示,本發明蘭花癒傷組織萃取物確實可促進皮膚中膠原蛋白、彈力蛋白、與透明質酸的合成、提升皮膚細胞抗氧化能力、及幫助修復皮膚細胞中受損DNA,故可用於保濕、提升皮膚柔潤度、提升皮膚飽滿度、
提升皮膚彈性、緊緻皮膚、減少皮膚紋理、延緩皮膚老化、幫助提升皮膚膠原蛋白含量、修復皮膚組織、預防皮膚病變、及/或促進傷口癒合。
Claims (5)
- 一種使用蘭花癒傷組織萃取物於製備一組合物之用途,其中該萃取物係白花蝴蝶蘭(Phalaenopsis amabilis)之葉片癒傷組織的極性溶劑萃取物,且該組合物係用於提升以下基因之至少一者的表現:MSH2、MLH1、MSH6、ERCC1、UNG、OGG1及APE1。
- 如請求項1之用途,其中該極性溶劑係選自以下群組:水、C1-C4醇類、及前述之組合。
- 如請求項1或2中任一項之用途,其中該組合物係一保養品組合物、食品組合物、或醫藥組合物。
- 如請求項3之用途,其中該保養品組合物係以精華液、乳液或化妝水之形式提供。
- 如請求項3之用途,其中該食品組合物係以美容飲品之形式提供。
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