TWI698247B - 茶樹癒傷組織萃取物於護膚之用途 - Google Patents
茶樹癒傷組織萃取物於護膚之用途 Download PDFInfo
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- TWI698247B TWI698247B TW107107479A TW107107479A TWI698247B TW I698247 B TWI698247 B TW I698247B TW 107107479 A TW107107479 A TW 107107479A TW 107107479 A TW107107479 A TW 107107479A TW I698247 B TWI698247 B TW I698247B
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Abstract
一種使用茶樹(Camellia sinensis
)癒傷組織萃取物於製備一組合物之用途,其中該萃取物係一茶樹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,尤其是用於調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及/或促進傷口癒合。
Description
本發明係關於茶樹(Camellia sinensis)癒傷組織萃取物於護膚之應用,尤其係關於茶樹葉片之癒傷組織的萃取物於護膚之應用,包括使用前述萃取物於調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及/或促進傷口癒合。
膠原蛋白是動物體內最主要的結構性蛋白質,其主要分布於細胞外基質及結締組織中,能夠支持細胞組織結構,維持組織強韌度。尤其是皮膚真皮層,其構造90%是由膠原蛋白所組成。膠原蛋白可賦予皮膚韌性及彈性,並具有保水功能。已知,老化、紫外光照射及自由基的生成皆會破壞膠原蛋白間的結構,導致膠原蛋白的分解及流失,而膠原蛋白分解及流失會加速皮膚老化(例如皮膚鬆弛、皺紋等)。
醣基化反應係指葡萄糖附著到蛋白質的化學反應過程,此反應會產生高度醣化終產物(advanced glycation end product,AGEs)。堆積於皮膚細胞中的高度醣化終產物不僅容易引起蛋白質變性、使蛋白質失去彈性,因而導致皮膚皺紋產生、皮膚老化,亦會造成皮膚細胞DNA損傷、影響皮膚細胞DNA正常功能,甚至導致皮膚病變。
紫外線(UV)亦是造成皮膚老化及病變的主要因素之一。已知,日光中的紫外線有超過90%是UVA,其對於皮膚的穿透力極強,可深達真皮層而對皮膚造成傷害。經常暴露於UVA輻射下將使得細胞中的細胞基質降解酵素過度活化,造成膠原蛋白、彈性蛋白流失並加速細胞老化,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、皮膚失去彈性等現象。此外,UVA亦可能破壞細胞中的DNA、造成DNA受損,過多受損DNA的累積不僅造成細胞老化,也可能使細胞產生變異進而導致皮膚病變,甚至是皮膚癌。
業界目前仍積極開發透過天然且安全之原料的使用以有效解決上述問題的方法。本案發明人研究發現,茶樹癒傷組織萃取物具有降低紫外線對細胞所造成的傷害、抑制皮膚中膠原蛋白的流失、促進皮膚中膠原蛋白的分泌、及抑制皮膚細胞蛋白質之醣基化反應的效果,故可用於調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及/或促進傷口癒合。
本發明之一目的,在於提供一種使用茶樹癒傷組織萃取物於製備一組合物之用途,其中該萃取物係一茶樹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,該極性溶劑係水、C1-C4醇類、或前述之組合。較佳地,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。
本發明之另一目的,在於提供一種護膚的組合物,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,且係包含一有效量之茶樹癒
傷組織萃取物。該萃取物係一茶樹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。
該根據本發明所提供之保養品組合物係可用於以下至少一者:調理皮膚、修補皮膚、改善膚質、及延緩皮膚老化。較佳地,該保養品組合物係以精華液之形式提供,且該精華液中之茶樹癒傷組織萃取物(以乾重計)之濃度為0.01至10重量%。較佳地,該保養品組合物係以化妝水之形式提供,且該化妝水中之茶樹癒傷組織萃取物(以乾重計)之濃度為0.01至5重量%。
該根據本發明所提供之食品組合物係可用於幫助維持皮膚膠原蛋白含量,較佳地,該食品組合物係以美容飲品之形式提供,且該美容飲品中之茶樹癒傷組織萃取物(以乾重計)之濃度係為1至1000ppm。
該根據本發明所提供之醫藥組合物係可用於以下至少一者:抗光損傷、預防皮膚病變、及促進傷口癒合。該醫藥組合物係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上述組合物。本發明方法可用於以下之至少一者:調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及促進傷口癒合。
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。
第1圖係茶樹癒傷組織萃取物與維生素C之抗氧化能力的結果比較圖,其中,橫軸係顯示茶樹癒傷組織萃取物之濃度,縱軸則係顯示維生素C之濃度;第2圖係顯示相較於控制組,其他各組之細胞存活率,其中,控制組係培養於不含茶樹癒傷組織萃取物之培養基中、且未經UVB照射之人類皮膚纖維母細胞,「UVB」組係培養於不含茶樹癒傷組織萃取物之培養基中、但經UVB照射之人類皮膚纖維母細胞,「UVB+萃取物(0.375)」組、「UVB+萃取物(0.75)」組、「UVB+萃取物(1.5)」組、「UVB+萃取物(3)」組、及「UVB+萃取物(6)」組則分別係培養於含有0.375、0.75、1.5、3及6毫克/毫升之茶樹癒傷組織萃取物之培養基中、且經UVB照射之人類皮膚纖維母細胞;第3圖係顯示相較於控制組,其他各組細胞中粒線體的表現情形,其中,控制組係培養於不含茶樹癒傷組織萃取物之培養基中、且未經UVA照射之人類皮膚纖維母細胞,「UVA」組係培養於不含茶樹癒傷組織萃取物之培養基中、但經UVA照射之人類皮膚纖維母細胞,「UVA+萃取物」組則係培養於含有5毫克/毫升之茶樹癒傷組織萃取物之培養基中、且經UVA照射之人類皮膚纖維母細胞;第4圖係顯示分別以不同濃度之茶樹癒傷組織萃取物處理後之高度醣化終產物生成量;第5圖係顯示相較於控制組,「萃取物」組之膠原蛋白分泌量,其中,控制組係培養於不含茶樹癒傷組織萃取物之培養基中的人類皮膚纖維母細胞,「萃取物」組則係培養於含有10毫克/毫升之茶樹癒傷組織萃取物之培養基中的人類皮膚纖維母細胞;以及
第6圖係顯示相較控制組,其他各組細胞之MMP2及TIMP1相對基因表現量,其中,控制組係培養於不含茶樹癒傷組織萃取物之培養基中的人類皮膚纖維母細胞,「萃取物(0.125)」組及「萃取物(0.25)」組則係分別培養於含有0.125及0.25毫克/毫升之茶樹癒傷組織萃取物之培養基中的人類皮膚纖維母細胞。
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「茶樹癒傷組織」係指茶樹之葉片的癒傷組織。
本案發明人研究發現,茶樹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抗紫外光傷害、抑制蛋白質之醣基化反應、促進膠原蛋白分泌、及減緩膠原蛋白分解與流失。
因此,本發明係關於使用茶樹癒傷組織萃取物以護膚之應用,此包括:提供一包含茶樹癒傷組織萃取物的組合物、使用茶樹癒傷組織萃取物於製造一組合物之用途、以及對一有需要之個體投予一含有有效量茶樹癒傷組織萃取物之組合物,以調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及/或促進傷口癒合的方法。其中,該茶樹癒傷組織尤其是指茶樹之葉片的癒傷組織。
根據本發明所採用之茶樹癒傷組織萃取物,係可透過以極性溶劑萃取茶樹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,只要可以使原料均勻分散即可。舉例言之,可於萃取步驟中採用萃取溶劑:茶樹癒傷組織=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。
於本發明之部分實施態樣中,係於進行萃取之前,先冷凍乾燥茶樹癒傷組織。舉例言之,可以100:1(水:經冷凍乾燥之茶樹癒傷組織)之重量比混合水與經冷凍乾燥之茶樹癒傷組織,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。
該茶樹癒傷組織可透過包含如下處理之操作提供:I.以2%次氯酸鈉溶液沖洗茶樹植株,其後以滅菌水沖洗茶樹植株,視需要地,可重複前述沖洗操作;II.在茶樹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1/2MS培養基(1/2 x 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茶樹癒傷組織萃取物,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,透過例如濃縮乾固、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:調理皮膚、修補皮膚、改善膚質、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。
當施用根據本發明所提供之保養品組合物於皮膚表面以調理皮膚、修補皮膚、改善膚質、及/或延緩皮膚老化時,可視產品型態而含有不同濃度之茶樹癒傷組織萃取物。舉例言之,當該保養品組合物以精華液之形式提供,該精華液中之茶樹癒傷組織萃取物(以乾重計)之濃度可以為0.01至10重量%,較佳為1重量%。當該保養品組合物以化妝水之形式,該化妝水中之茶樹癒傷組織萃取物(以乾重計)之濃度則可以為0.01至5重量%,較佳為0.1重量%。
根據本發明所提供之食品組合物係用於幫助維持皮膚膠原蛋白含量。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吞食或飲用的形式,例如健康食品、美容飲品等,但不以此為限。當該食品組合物以美容飲品之形式提供,該美容飲品中之茶樹癒傷組織萃取物(以乾重計)之濃度可以為1至1000ppm,較佳為500ppm。
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,茶樹癒傷組織萃取物)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、健康況狀、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組合物,其中該組合物係含有一有效量之茶樹癒傷組織萃取物。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
實施例1:茶樹癒傷組織萃取物的製備
對購自國光花市之茶樹(Camellia sinensis)植株施以如下處理,以提供茶樹癒傷組織:I.以2%次氯酸鈉溶液沖洗茶樹植株,其後以滅菌水沖洗茶樹植株,視需要地,可重複前述沖洗操作;II.在茶樹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1/2MS培養基(1/2 x Murashige and Skoog Basal
Medium;購自SIGMA公司;產品編號:M5524)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茶樹癒傷組織,以製備茶樹癒傷組織萃取物:(1)茶樹癒傷組織置於-22℃下進行冷凍乾燥,歷時12小時,接著粉碎前述經乾燥之茶樹癒傷組織,以提供茶樹癒傷組織粉末;(2)將水與步驟(1)之茶樹癒傷組織粉末以100:1(水:茶樹癒傷組織粉末)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(即,後續實施例所使用之茶樹癒傷組織萃取物原液)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)之萃取原液進行冷凍乾燥,以提供一乾燥物(即,本發明之茶葉癒傷組織萃取物)。
實施例2:茶樹癒傷組織萃取物之抗氧化功效
取實施例1步驟(6)所提供之茶樹癒傷組織萃取物乾燥物,分成七組,並以逆滲透(RO)水作為溶劑,分別配製成濃度為0.83、1.00、1.25、1.67、2.50、4.00、及5.00毫克/毫升之茶樹癒傷組織萃取物溶液後,再對各組溶液進行以下處理:I.將2.5毫升的茶樹癒傷組織萃取物溶液或標準品與2.5毫升之0.2M磷酸鹽緩衝液(pH為6.6)及2.5毫升之1%鐵氰化鉀(potassium ferricyanide,PFC)混和,並置於50℃下作用20分鐘,以提供一混和液;
II.待步驟I之混和液冷卻後,於其中加入2.5毫升之三氯乙酸(trichloroacetic acid,TCA)溶液混和均勻,再以3000g進行離心,歷時10分鐘;以及III.取3毫升離心後之上清液,於其中加入3毫升之蒸餾水及1.2毫升之0.1%氯化鐵溶液混和均勻,並置於室溫下作用10分鐘,以提供一溶液供後續於700nm下測量吸光值。
取購自SIGMA公司之維生素C,分成七組,以水作為溶劑,分別配製成濃度為0、20、40、60、80、100、及120毫克/毫升之維生素C溶液後,再對各組溶液進行上述步驟I至III之處理。
接著,以分光光度計測量步驟I至III所提供之各組溶液於波長700nm時的吸光值。其中,吸光值越高表示還原力越強,抗氧化能力越好。最後,以茶樹癒傷組織萃取物之吸光值為基準,計算具有相同吸光值之維生素C的濃度,結果示於第1圖。
如第1圖所示,當茶樹癒傷組織萃取物之濃度為1毫克/毫升時,其抗氧化能力相當於濃度為30微克/毫升之維生素C。前述結果顯示,茶樹癒傷組織萃取物具有優異的抗氧化能力。
實施例3:茶樹癒傷組織萃取物於抗光損傷的效果
(3-1)MTT檢測
於MEM培養基(Minimum essential medium,購自Gibco;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,其後,將細胞分為七組,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,將細胞培養於不含茶樹癒傷組織萃取物的培養基中)。
2.「UVB」組:將細胞置於MEM培養基中培養24小時(即,將細胞培養於不含茶樹癒傷組織萃取物的培養基中),再以UVB照射細胞(15焦耳/平方公分),歷時1小時。
3.「UVB+萃取物(0.375)」組、「UVB+萃取物(0.75)」組、「UVB+萃取物(1.5)」組、UVB+萃取物(3)」組、及「UVB+萃取物(6)」組:分別將細胞置於含有0.375、0.75、1.5、3、及6毫克/毫升由實施例1步驟(6)獲得之茶樹癒傷組織萃取物乾燥物的MEM培養基中培養24小時,接著以UVB照射細胞(15焦耳/平方公分),歷時1小時。
其後,以MTT檢定法檢測各組細胞之存活率。結果示於第2圖。由第2圖可知,相較於控制組,UVB組之細胞存活率明顯較低。然而,經茶樹癒傷組織萃取物處理之組別(包括「UVB+萃取物(0.375)」組、「UVB+萃取物(0.75)」組、「UVB+萃取物(1.5)」組、「UVB+萃取物(3)」組、及「UVB+萃取物(6)」組)的細胞存活率皆顯著回升,且茶樹癒傷組織萃取物之使用濃度係與細胞存活率成正比。
(3-2)螢光染色觀察
於MEM培養基中培養人類皮膚纖維母細胞(CCD-966SK;購自ATCC)歷時24小時,其後,將細胞分為三組,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,將細胞培養於不含茶樹癒傷組織萃取物的培養基中)。
2.「UVA」組:將細胞置於MEM培養基中培養24小時(即,將細胞培養於不含茶樹癒傷組織萃取物的培養基中),再以UVA照射細胞(15焦耳/平方公分),歷時100分鐘。
3.「UVA+萃取物」組:將細胞置於含有5毫克/毫升由實施例1獲得步驟(6)之茶樹癒傷組織萃取物乾燥物的MEM培養基中培養24小時,接著以UVA照射細胞(15焦耳/平方公分),歷時100分鐘。
其後,以Rhodamine 123(購自Thermo公司)及Hoechest 33342(購自Thermo公司)對各組細胞進行螢光染色(其中,Hoechest 33342為細胞核染劑,Rhodamine 123為粒線體染劑),並以螢光顯微鏡觀察各組細胞之細胞核(藍色螢光)及粒線體(紅色螢光)。結果示於第3圖。
由第3圖可知,相較於控制組,「UVA」組之細胞中的粒線體數量明顯較少。然而,「UVA+萃取物」組細胞中的粒線體數量則明顯回復,甚至與控制組相當。
(3-1)及(3-2)之結果顯示,茶樹癒傷組織萃取物可有效降低UVA及UVB對細胞的傷害,有效協助人類皮膚纖維母細胞中粒線體的防護及修復紫外光所造成的損害,且在試驗濃度範圍內對正常人類皮膚纖維母細胞不具毒性。
實施例4:茶樹癒傷組織萃取物於抑制蛋白質醣基化反應的效果
為了解本發明茶樹癒傷組織萃取物是否可抑制皮膚細胞中蛋白質之醣基化反應,係進行以下之高度醣化終產物生成測試。
(I)以200mM的磷酸鈉緩衝液(pH為7.4)作為溶劑,分別配置濃度為60毫克/毫升之胎牛血清白蛋白溶液(含有0.06%疊氮化鈉)及濃度為1.5M之果糖溶液;(II)取實施例1步驟(5)所提供之茶樹癒傷組織萃取物原液進行減壓濃縮,
分別提供濃縮10倍及100倍之茶樹癒傷組織萃取濃縮液;(III)於實施例1步驟(5)所提供之茶樹癒傷組織萃取物原液、及步驟(II)所提供之10倍濃縮液、100倍濃縮液各取0.25毫升,分別加入0.25毫升步驟(I)所提供之胎牛血清白蛋白溶液(含有0.06%疊氮化鈉)及0.25毫升步驟(I)所提供之果糖溶液,混合均勻後置於50℃下反應24小時,以提供「原液」組、「10倍濃縮液」組、及「100倍濃縮液」組之反應溶液;(IV)取步驟(III)所提供之各組反應溶液(各組取0.1毫升),並以螢光光譜儀測量在激發光波長360nm、放射光波長460nm下的螢光值(即,各實驗組0小時之螢光值);以及(V)另取步驟(III)所提供之各組反應溶液(各組取0.45毫升),置於50℃下繼續培養24小時後(此為反應終點),於各組取0.1毫升之溶液並測量在激發光波長360nm、放射光波長460nm下的螢光值(即,各實驗組24小時之螢光值)。
(i)以200mM的磷酸鈉緩衝液(pH為7.4)作為溶劑,配置一濃度為3mM的胺基胍(aminoguanidine,AG)溶液;(ii)取0.25毫升步驟(i)之AG溶液,分別加入0.25毫升步驟(I)所提供之胎牛血清白蛋白溶液(含有0.06%疊氮化鈉)及0.25毫升步驟(I)所提供之果糖溶液,混合均勻後置於50℃下反應24小時,以提供控制組之反應溶液;(iii)取0.1毫升步驟(ii)所提供之反應溶液,並以螢光光譜儀測量在激發光波長360nm、放射光波長460nm下的螢光值(即,控制組0小時之螢光值);
以及(iv)另取0.45毫升步驟(ii)所提供之各組反應溶液,置於50℃下繼續培養24小時後(此為反應終點),取其中0.1毫升之溶液並測量在激發光波長360nm、放射光波長460nm下的螢光值(即,控制組24小時之螢光值)。
其後,經由下式計算各實驗組之相對高度醣化終產物生成量(%)。結果示於第4圖。
由第4圖可知,茶樹癒傷組織萃取物可有效抑制高度醣化終產物的生成,且茶樹癒傷組織萃取物濃縮液的效果較佳。前述結果顯示茶樹癒傷組織萃取物可有效抑制蛋白質之醣基化反應,故可用於抑制皮膚細胞之蛋白質的醣基化反應,達到延緩皮膚老化及預防皮膚病變的效果。
實施例5:茶樹癒傷組織萃取物於幫助維持皮膚膠原蛋白含量的效果
(5-1)促進膠原蛋白分泌
為了解本發明茶樹癒傷組織萃取物於促進膠原蛋白分泌之效果,係於MEM培養基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48小時(即,將細胞培養於不含茶樹癒傷組織萃取物的培養基中)。
2.「萃取物」組:將細胞置於含有10毫克/毫升由實施例1步驟(6)獲得之茶樹癒傷組織萃取物乾燥物的MEM培養基中培養48小時。
接著,以SircolTM Soluble Collagen Assay kit(購自Biocolor)及酵素免疫分析測讀儀(ELISA reader;購自BioTek)檢測上述二組之培養液中的膠原蛋白含量。最後,以學生t檢驗(Student t-test)進行統計分析,並以控制組作為基準(即,將控制組的膠原蛋白含量設定為100%)計算「萃取物」組的相對膠原蛋白含量。結果示於第5圖。
由第5圖可知,相較於控制組,「萃取物」組的膠原蛋白含量係提升28.29%。此結果顯示,茶樹癒傷組織萃取物可有效促進細胞之膠原蛋白分泌,具有調理皮膚、修補皮膚、改善膚質、延緩皮膚老化及/或促進傷口癒合之效果。
(5-2)減緩膠原蛋白分解及流失
已知MMP2的基因表現量下降及TIMP1的基因表現量上升皆代表膠原蛋白的分解被抑制。為了解本發明茶樹癒傷組織萃取物是否可減緩皮膚之膠原蛋白分解及流失,係於MEM培養基中培養人類皮膚纖維母細胞歷時24小時,接著,將細胞分成三組,並進行以下處理:
1.控制組:將細胞置於MEM培養基中培養6小時(即,將細胞培養於不含茶樹癒傷組織萃取物的培養基中)。
2.「萃取物(0.125)」組:將細胞置於含有0.125毫克/毫升由實施例1步驟(6)獲得之茶樹癒傷組織萃取物乾燥物的MEM培養基中培養6小時。
3.「萃取物(0.25)」組:將細胞置於含有0.25毫克/毫升由實施例1步驟(6)獲得之茶樹癒傷組織萃取物乾燥物的MEM培養基中培養6小時。
進行qRT-PCR,以檢測各組細胞中MMP2及TIMP1的基因表現量。最後,以學生t檢驗(Student t-test)進行統計分析,並以控制組作為基準(即,將控制組的基因表現設定為1倍)計算其餘各組的相對基因表現量。結果示於第6圖。
由第6圖可知,相較於控制組,經茶樹癒傷組織萃取物處理的組別(包括「萃取物(0.125)」組及「萃取物(0.25)」組),的MMP2基因表現量係明顯下降,且TIMP1基因表現量明顯提升。前述結果顯示,茶樹癒傷組織萃取物可有效抑制膠原蛋白分解,故可用於減緩膠原蛋白分解及/或流失,達到調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、及/或促進傷口癒合之效果。
如上述實施例所示,本發明茶樹癒傷組織萃取物確實可抑制皮膚細胞蛋白質之醣基化反應、促進皮膚細胞分泌膠原蛋白、抑制膠原蛋白分解、減緩膠原蛋白流失,並可提升皮膚細胞於紫外光照射下的存活率,故具有調理皮膚、修補皮膚、改善膚質、延緩皮膚老化、幫助維持皮膚膠原蛋白含量、抗光損傷、預防皮膚病變、及/或促進傷口癒合等護膚功效。
Claims (6)
- 一種使用茶樹(Camellia sinensis)癒傷組織萃取物於製備一用於抗光損傷、預防皮膚病變、及/或促進傷口癒合之組合物的用途,其中該萃取物係一茶樹葉片之癒傷組織的極性溶劑萃取物,該癒傷組織係在25℃、50至60%濕度下、以1/2MS培養基培養1至1.5個月而得,且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2之用途,其中該保養品組合物係以精華液之形式提供,且該精華液中之茶樹癒傷組織萃取物(以乾重計)之濃度係為0.01至10重量%。
- 如請求項2之用途,其中該保養品組合物係以化妝水之形式提供,且該化妝水中之茶樹癒傷組織萃取物(以乾重計)之濃度係為0.01至5重量%。
- 如請求項2之用途,其中該食品組合物係以美容飲品之形式提供,且該美容飲品中之茶樹癒傷組織萃取物(以乾重計)之濃度係為1至1000ppm。
- 如請求項2之用途,其中該醫藥組合物係呈一選自以下群組之劑型:經皮投予、口服、及皮下注射。
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