TW201907944A - 一種具抗老化、美白、抗過敏及細胞修護之龍眼花萃取物 - Google Patents
一種具抗老化、美白、抗過敏及細胞修護之龍眼花萃取物 Download PDFInfo
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- TW201907944A TW201907944A TW106124544A TW106124544A TW201907944A TW 201907944 A TW201907944 A TW 201907944A TW 106124544 A TW106124544 A TW 106124544A TW 106124544 A TW106124544 A TW 106124544A TW 201907944 A TW201907944 A TW 201907944A
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- flower extract
- longan flower
- longan
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Abstract
本發明係有關於一種具有一種具抗老化、美白、抗過敏及細胞修護之龍眼花萃取物，其係將一有效劑量之龍眼花萃取物投予至一皮膚細胞，清除DPPH．及ABTS．+自由基能力、還原力、螯合鐵離子及抑制脂質過氧化物效能；抑制酪胺酸酶活性；清除一氧化氮(NO)自由基、抑制脂氧合酶lipoxygenase-1(LOX-1)活性；抑制玻尿酸酶之能力，提高皮膚玻尿酸含量，促進傷口修復，減少皺紋產生；抑制基質金屬蛋白酶(MMPs)活性，降低膠原蛋白降解；促進細胞修復的能力及提高抗過敏之能力等機制作用。
Description
本發明係有關於一種龍眼花萃取之應用，尤其係指以龍眼花(DLFE)作為化妝品、保養品等皮膚外用品之添加物，以達到抗老化、抗氧化、抗發炎、抑制黑色素、抗皺、抗過敏及細胞修復等機制作用之目的。
現代的東方人對於白晳的膚色需求日益提高，並且對於健康也同樣的重視，因此如何提出一種健康的淡斑方式對於醫藥外用品產業實是一發展重點。傳統植物針對美容保養，多為一些補氣活血、調整體質的溫補藥物，亦有少許單、複方直接使用於皮膚美白、除痘、消炎等用途。但是對於植物萃取物中之有效成分之機制分析與其於醫藥外用品中之配方設計應用，仍是國內醫藥外用品產業需積極開發的重點。因此如能開發傳統植物成為低劑量、高效能之淡斑產品，將有助於提升醫藥外用品科技產業。
目前發現到的問題是，現有的美白產品裡面，多是藉由維生素C(Vitamin C)、對苯二酚(Hydroquinone)、杜鵑花酸(Acelaic acid)和熊果素(Arbutin)來達到淡斑的功效，雖然這些物質對於淡斑的確有顯著的功效，然而，有些淡斑劑之作用為直接破壞黑色素細胞，因此會造成細 胞的毒殺性。因此抑制黑色素生成與抗氧化成分之安全性與其作用濃度是值得重視的。本發明是萃取出天然植物龍眼花萃取物具有清除DPPH．及ABTS．+自由基能力、還原力、螯合鐵離子及抑制脂質過氧化物效能；抑制酪胺酸酶活性；清除一氧化氮(NO)自由基、抑制脂氧合酶lipoxygenase-1(LOX-1)活性。
大部分文獻皆指出龍眼花具有抗氧化及抗發炎之功效，但較少有抑制黑色素生合成及DNA修復之活性的相關研究，亦未有其美白及抗老化的功能發現，而市售產品所含之美白成分例如：維生素C(Vitamin C)、對苯二酚(Hydroquinone)、杜鵑花酸(Acelaic acid)和熊果素(Arbutin)，雖具有顯著功效，但是有些美白劑之作用為直接破壞黑色素細胞，因此會造成細胞的毒殺性。
發明人即是鑑於上述現有美白、抗老化之產品組合物於實際實施使用時仍具有多處缺失，本於孜孜不倦之精神，藉由其豐富專業知識及多年之實務經驗加以改善，並據此研創出本發明。
龍眼花含有大量的多酚類及黃酮類化合物，具有很好的抗氧化能力，且具有降血脂及抑制脂質合成之功效。然而相關文獻討論卻不多見，，因此本發明即以龍眼花萃取物進行一系列抗氧化、DNA修復、抑制黑色素生合成及抗發炎活性評估。
本發明主要目的為提供一種龍眼花萃取物之應用，其係指以龍眼花(Dimocarpus longan flower,DLF)水萃及乙醇萃取物作為具 有清除自由基能力及抑制酪胺酸酶活性之化妝品添加物；藉此，龍眼花萃取物可作為皮膚外用品以用於預防或緩和一個體的皮膚老化現象，達到抗老化、抗氧化、抗發炎、抑制黑色素、抗皺、抗過敏及細胞修護等機制作用。
為了達到上述實施目的，本發明一種龍眼花萃取物之應用，其係將一有效劑量之龍眼花萃取物投至一皮膚細胞，具清除自由基、抑制脂質過氧化物效能以及抑制脂氧合酶lipoxygenase-1(LOX-1)活性，可提高抗氧化、抑制黑色素生成、抗發炎及細胞修復。
較佳者，該龍眼花萃取物包含一水萃DLFW及一乙醇萃取物DLFE。
較佳者，該龍眼花萃取物係具有清除或減少自由基以防止細胞老化之能力。
較佳者，該自由基係為DPPH(2,2-diphenyl-1-picrylhydrazyl)自由基或ABTS(2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid)自由基。
較佳者，該龍眼花萃取物係作為美白或抗氧化之化妝材料組成物、食品添加物或醫藥組成物。
較佳者，該龍眼花萃取物具抑制UV照射引起之皮膚發炎反應或皮膚消炎劑。
較佳者，該龍眼花萃取物係作為抗發炎之化妝材料組成物或醫藥組成物。
較佳者，該龍眼花萃取物具有調節p53及NER修復系統之相關因子來修復由UV照射引起的DNA之損傷。
較佳者，該龍眼花萃取物具有抑制細胞外蘑菇酪胺酸酶之活性，進而降低黑色素生成。
較佳者，該龍眼花萃取物可抑制NO及LOX-1的活性達到抑制發炎反應。
較佳者，該龍眼花萃取物具有抑制玻尿酸酶之能力，提高皮膚玻尿酸含量，促進傷口修復，減少皺紋產生。
較佳者，該龍眼花萃取物及具有抑制基質金屬蛋白酶(MMPs)活性，降低膠原蛋白降解。
較佳者，該龍眼花萃取物具有促進細胞修復的能力。
較佳者，該龍眼花萃取物提高抗過敏之能力。
圖1係本發明細胞毒理性測試圖。
圖2係本發明清除自由基能力測試圖。
圖3係本發明美白效能測試圖。
圖4係本發明抗發炎試驗圖。
圖5係本發明DNA保護試驗圖。
圖6係本發明DNA保護對照圖。
圖7係本發明抗皺試驗圖。
圖8係本發明抑制玻尿酸酶能力試驗圖。
圖9係本發明細胞之傷口癒合能力試驗圖。
圖10係本發明UVB細胞受損試驗圖。
圖11係本發明抗敏能力試驗圖。
本發明之目的及其結構功能上的優點，將依據以下圖面所示之結構，配合具體實施例予以說明，俾使審查委員能對本發明有更深入且具體之瞭解。
首先，本發明人由龍眼花分離製得一水萃DLFW、一乙醇萃取物DLFE，，上述材料之水萃DLFW及乙醇萃取物DLFE具有降低因H2O2刺激而引起的HaCaT細胞內氧化壓力，提高細胞內glutathione(GSH)含量，及提高抗氧化酵素superoxide dismutase(SOD)、catalase和glutathione peroxidase(GPx)的表現量。
龍眼花萃取物(水萃DLFW、乙醇萃取物DLFE)具抑制細胞內酪胺酸酶(tyrosinase)活性及降低黑色素生成量。以免疫螢光試驗及西方墨點法試驗，龍眼花萃取物(水萃DLFW、乙醇萃取物DLFE)作用於細胞後，證實具有抑制黑色素生合成途經中接受器MC1R(melanocortin-lreceptor)和MITF(microphthalmia-associated transcription factor)之表現，並影響酪胺酸酶、TRP-2(tyrosinase-related proteins-2)和TRP-1(tyrosinase-relatedproteins-1)的表現，進而抑制黑色素生成。
在抗發炎能力試驗中，龍眼花萃取物具抑制細胞內發炎 因子(TNF-α、IL-1β及IL-6)的含量，且具有降低發炎相關路徑的p38、IL-6、IL-1β和TNF-α蛋白質表現。動物試驗SKH-1小鼠經UVB照射後以免疫組織化學染色法immunohistochemistry(IHC)及西方墨點法試驗證實龍眼花萃取物具有降低發炎相關路徑的p38、IL-6、IL-1β和TNF-α蛋白質表現。
在DNA修復及保護能力試驗中，龍眼花萃取物具有調節nucleotideexcision repair(NER)修復系統之相關因子來修復由UV照射引起的DNA之損傷，以及調節p53及NER修復系統之相關因子來修復由UV照射引起之DNA損傷。
藉由下述具體實施例，可進一步證明本發明可實際應用之範圍，但不意欲以任何形式限制本發明之範圍。
實驗一：細胞毒理性測試
鑑於現有之美白劑或皮膚黑色素抑制劑之作用為會造成細胞毒性，因此本發明人於取得此龍眼花萃取物後，先進行此化合物之細胞毒殺測試實驗，利用100、250和500μg/ml濃度之龍眼花萃取物作用於HaCaT細胞24小時之後，利用MTT試驗測定其存活度。其結果如圖1所示，龍眼花之水萃DLFW及乙醇萃取物DLFE在細胞中不具毒性，與市售常用成分ascorbic acid(AA)相比來的安全。
實驗二：清除自由基能力測試
本實驗探討龍眼花之水萃DLFW、乙醇萃取物DLFE清除自由基的功效，利用DPPH．和ABTS+．兩種自由基測試其清除能力，其結果如圖2所示， 乙醇萃取物DLFE在清除DPPH自由基試驗中約在45μg/ml時就達到50%的清除率，且乙醇萃取物DLFE清除兩種自由基的能力更優於水萃DLFW，其結果顯示龍眼花萃取物對於兩種自由基有很好的抑制率。
實驗三：美白效能測試
酪胺酸酶為黑色素生合成途徑中重要的酵素，將龍眼花之水萃DLFW及乙醇萃取物DLFE以不同濃度100、250和500μg/ml進行細胞外蘑菇酪胺酸酶活性試驗評估。其結果如圖3所示，經過龍眼花之水萃DLFW及乙醇萃取物DLFE作用後，抑制細胞外蘑菇酪胺酸酶活性隨濃度增加而提高，且乙醇萃取物DLFE(EC50為410μg/ml)的抑制率優於水萃DLFW(EC50為624μg/ml)，證實龍眼花萃取物具有抑制細胞外蘑菇酪胺酸酶之活性。
實驗四：抗發炎試驗
NO為主要參與發炎反應的介質之一，而LOX-1是最常見引起發炎反應的酵素，藉由此試驗評估龍眼花之水萃DLFW及乙醇萃取物DLFE抗發炎效果。其結果如圖4所示，龍眼花之水萃DLFW及乙醇萃取物DLFE，有不錯的清除NO自由基的能力及抑制LOX-1活性，證實龍眼花之水萃DLFW及乙醇萃取物DLFE能經由抑制NO及LOX-1的活性來達到抑制發炎反應的活性，具有抗發炎之效果。
實驗五：DNA保護試驗
紫外線曝曬或生理代謝，均會增加氧化壓力造成DNA的損傷。本試驗以pUC119 DNA以H2O2(1mM)誘導及以UVB(20mJ/cm2)照射，引發DNA 損傷，評估龍眼花之水萃DLFW及乙醇萃取物DLFE是否具有保護DNA避免因氧化壓力及UVB照射所造成的DNA損傷。請參照圖5，pUC119 DNA原為超螺旋體結構(Supercoiled form,SC-form)，經H2O2(1mM)誘導及UVB(20mJ/cm2)照射後會造成DNA斷裂，形成(Linear form,L-form)和(Open form,O-form)之DNA片段。續參照圖6，當加入不同濃度之乙醇萃取物DLFE(100、250和500μg/ml)再以H2O2誘導及UVB照射後，結果顯示乙醇萃取物DLFE能有效保護DNA，且隨濃度的增加保護效果越佳，證實龍眼花有保護DNA免於因氧化壓力及UVB照射所造成的損傷。
實驗六：抗皺試驗
膠原蛋白主要存在於皮膚組織中真皮層的結締組織中，是人體內含量最高的蛋白質，有很強的伸展力且能支撐真皮層整體結構，膠原蛋白也是細胞外基質的主要組成成分，保持皮膚彈性並具有修復之能力，而膠原蛋白會隨著老化及環境等因素而流失，使皮膚產生皺紋及骨質疏鬆等問題。其中基質金屬蛋白酶(Matrix metalloproteinases，MMPs)為膠原蛋白分解酵素，MMP-2及MMP-9主要是分解第II型膠原蛋白。為調節光老化重要因子，纖維母細胞受UV曝曬會引起基質金屬蛋白酶大量表現，進而降解細胞外基質(ECM)使真皮組織的膠原蛋白降解。
本試驗將龍眼花之乙醇萃取物DLFE(100、250和500μg/ml)以不同濃度作用於Hs68細胞中培養72小時後，測定膠原蛋白含量。藉此試驗評估該龍眼花之乙醇萃取物DLFE是否具有抑制MMPs之 活性。其結果如圖7所示，經龍眼花之乙醇萃取物DLFE作用後，與Control相比，膠原蛋白含量隨著濃度提高而增加，因此可推知龍眼花之乙醇萃取物DLFE具有抑制MMP-9及MMP-2之活性，並可以有效的增加纖維母細胞生成膠原蛋白之含量。
實驗七：抑制玻尿酸酶能力試驗
玻尿酸存在於人體皮膚組織中的真皮層及結締組織中，減少皺紋的產生，並具有促進傷口修復之功效，而玻尿酸酶是一種會使玻尿酸降解的酵素，使玻尿酸的結構瓦解，喪失原有功能。藉由此試驗評估龍眼花之乙醇萃取物DLFE抑制玻尿酸酶之能力。
將龍眼花之乙醇萃取物DLFE(500μg/ml)及標準品表沒食子兒茶素(Epigallocatechin gallate,EGCG)，分別加入玻尿酸酶(3000unit/mL)作用18小時，結果如圖8所示，經過龍眼花之乙醇萃取物DLFE作用後的位置和Control相比顏色明顯深了許多，顯示DLFE可以有效防止玻尿酸被玻尿酸酶分解，經定量分析出龍眼花之乙醇萃取物DLFE對玻尿酸酶活性之抑制率為82%。結果證實，龍眼花之乙醇萃取物DLFE具有抑制玻尿酸酶活性之能力。
實驗八：細胞之傷口癒合能力試驗
以傷口癒合(wound healing)試驗評估DLFE是否具有促進細胞修復之能力。首先以龍眼花萃取物DLFE(500μg/ml)作用於HaCaT細胞4小時後，抽乾培養液，以UVB(50mJ/cm2)照射，再加入DLFE作用，培養4小時後，以顯微鏡觀察細胞移動之變化。
結果如圖9所示，Control及單純照射UVB的細胞沒有太大的移動變化，尤其是單純照射UVB的細胞，從0至48小時幾乎沒有變化。經龍眼花萃取物DLFE作用後之細胞在12小時後有明顯的向中間移動的趨勢，到了48小時已經沒有縫隙。結果再次證實，龍眼花萃取物DLFE具有促進細胞修復的能力。
實驗九：UVB細胞受損試驗
彗星試驗是以膠體電泳並以EtBr進行細胞核之染色，偵測單一細胞中DNA經UVB照射後之損傷程度。以DLFE(500μg/ml)作用於HaCaT細胞4小時後，抽乾培養液，以UVB(50mJ/cm2)照射，再加入龍眼花萃取物DLFE作用，培養4小時後，以顯微鏡觀察細胞之損傷程度。
結果如圖10所示，單純受UVB照射之細胞，DNA受損且有明顯的拖尾現象，1000顆細胞中，約有50%顆細胞之DNA有受損之情形。經過DLFE作用過後之細胞，在高濃度500μg/ml作用下，1000顆細胞中，約有8%顆細胞之DNA有受損之情形。藉此判斷DLFE具有保護DNA免於UVB照射所造成的傷害。
CPD為DNA受UVB照射後所產生的光產物，結果如圖10所示，經UVB(50mJ/cm2)照射後之細胞，CPD的螢光表現量增加，而經過DLFE(500μg/ml)作用後，明顯降低了UVB光產物的生成，再次證實DLFE具有保護DNA並降低因UVB照射所造成的DNA損傷。
實驗十：抗敏能力試驗
請參照圖11以卡西霉素A23187或抗原誘導之β-葡萄糖醛酸酶去顆粒試 驗：以卡西霉素A23187和抗原誘導RBL-2H3肥大細胞去顆粒的程度，是由β-葡萄糖醛酸酶釋放試驗，經由下列方式改良的方法來測定：接種RBL-2H3肥大細胞(2×104cells/well)在96孔微量盤和RBL-2H3肥大細胞(3×104cells/well)在48孔微量盤中，分別供給卡西霉素A23187誘導實驗和抗原誘導實驗所用，在細胞中加入各種濃度的樣品，靜置20小時；使用迪皮質醇(10nM)作為正對照組。在抗原誘導實驗中，細胞首先以抗-二硝基苯酚IgE單克隆抗體(anti-dinitrophenol IgE,anti-DNP IgE)(5μg/mL)被動高敏至少2小時，將細胞以預熱的台羅德氏緩衝液(135mM氯化鈉，5mM氯化鉀，1.8mM氯化鈣，1.0mM氯化鎂，5.6mM葡萄糖，20mM 4-羥乙基呱嗪乙磺酸，pH 7.4)徹底沖洗後，各別以台羅德氏緩衝液配製的鈣離子載體A23187(卡西霉素，1μM)或抗原二硝基苯-胎牛血清共軛物(100ng/mL)刺激一小時。為測量β-葡萄糖醛酸酶釋放的總量，將未受刺激的細胞以0.5%的Triton X-100溶液裂解，或者不進行處理使細胞自發性的釋放β-葡萄糖醛酸酶；分裝上清液(50μL)與等量的1μM聚氮-乙醯葡萄糖胺(抗原)配製於0.1M檸檬酸鹽緩衝液(pH 4.5)中，用作釋放β-葡萄糖醛酸酶的基底。在37℃下培養1小時後，加入100μL的終止緩衝液(0.1M Na2/NaHCO3,pH 10.0)猝滅反應。將微量盤分析儀(Multiskan Ascent,Thermo Scientific)設定在405nm波長，測量吸光度；β-葡萄糖醛酸酶釋放的抑制百分比是實驗組除以對照值(未處理細胞)的百分比來計算；對二甲基亞碸最大的耐受劑量為0.5%，且所有實驗重複三次。
樣品誘導之β-葡萄糖醛酸酶直接去顆粒試驗：在RBL-2H3肥大細胞中，由樣品引發的β-葡萄糖醛酸酶的釋放量是以改良過的β-葡萄糖醛酸酶釋放實驗來決定；簡單地說，將RBL-2H3肥大細胞(4×104cells/well)接種於48孔微量盤中，加入樣品後靜置10小時。將台羅德氏緩衝液加入5.6mM葡萄糖、2mg/mL牛血清白蛋白(BSA)和2mM谷氨酸以製備樣品和細胞所用。50μL的上清液轉移至96孔微量盤，依照前述方式測量β-葡萄糖醛酸酶的釋放量，卡西霉素A23187(1μM)做為正對照組，所有實驗進行重複三次以上。
綜上所述，本發明證實龍眼花之水萃DLFW及乙醇萃取物DLFE具有清除活性氧和抑制黑色素生成，並具有皮膚表皮細胞之DNA保護、修復能力以及具有抑制發炎反應的產生，具發展成為美白、抗氧化及抗發炎的保養品或保健食品添加劑的潛力，並確能藉由上述所揭露之實施例，達到所預期之使用功效，且本發明亦未曾公開於申請前，誠已完全符合專利法之規定與要求。爰依法提出發明專利之申請，懇請惠予審查，並賜准專利，則實感德便。
惟，上述所揭之圖示及說明，僅為本發明之較佳實施例，非為限定本發明之保護範圍；大凡熟悉該項技藝之人士，其所依本發明之特徵範疇，所作之其它等效變化或修飾，皆應視為不脫離本發明之設計範疇。
Claims (14)
- 一種具抗老化、美白、抗過敏及細胞修護之龍眼花萃取物，係將一有效劑量之龍眼花萃取物投至一皮膚細胞，具清除自由基、抑制脂質過氧化物效能以及抑制脂氧合酶lipoxygenase-1(LOX-1)活性，可提高抗氧化、抑制黑色素生成、抗發炎及細胞修復。
- 如請求項1所述之應用，其中該龍眼花萃取物包含一水萃DLFW及一乙醇萃取物DLFE。
- 如請求項1所述之應用，其中該龍眼花萃取物係具有清除或減少自由基以防止細胞老化之能力。
- 如請求項1所述之應用，其中該自由基係為DPPH(2,2-diphenyl-1-picrylhydrazyl)自由基或ABTS(2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic acid)自由基。
- 如請求項1所述之應用，其中該龍眼花萃取物係作為美白或抗氧化之化妝材料組成物、食品添加物或醫藥組成物。
- 如請求項1所述之應用，其中該龍眼花萃取物具抑制UV照射引起之皮膚發炎反應或皮膚消炎劑。
- 如請求項1所述之應用，其中該龍眼花萃取物係作為抗發炎之化妝材料組成物或醫藥組成物。
- 如請求項1所述之應用，其中該龍眼花萃取物具有調節p53及NER修復系統之相關因子來修復由UV照射引起的DNA之損傷。
- 如請求項1所述之應用，其中該龍眼花萃取物具有抑制細胞外蘑菇酪胺酸酶之活性，進而降低黑色素生成。
- 如請求項1所述之應用，其中該龍眼花萃取物可抑制NO及LOX-1的活性達到抑制發炎反應。
- 如請求項1所述之應用，其中該龍眼花萃取物具有抑制玻尿酸酶之能力，提高皮膚玻尿酸含量，促進傷口修復，減少皺紋產生。
- 如請求項1所述之應用，其中該龍眼花萃取物及具有抑制基質金屬蛋白酶(MMPs)活性，降低膠原蛋白降解。
- 如請求項1所述之應用，其中該龍眼花萃取物具有促進細胞修復的能力。
- 如請求項1所述之應用，其中該龍眼花萃取物提高抗過敏之能力。
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