TWI746917B - 代代花萃取物用於減緩皮膚老化及減少細胞中脂肪含量的用途 - Google Patents
代代花萃取物用於減緩皮膚老化及減少細胞中脂肪含量的用途 Download PDFInfo
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Abstract
本發明提供一種代代花萃取物用於減緩皮膚老化及減少細胞中脂肪含量的用途,能提升其清除糖化終產物之能力、抑制脂肪堆積、及促進脂肪分解,以減緩皮膚老化並達到皮膚美化的效果,以及減少細胞中脂肪的含量並達到減脂的效果。其中該代代花萃取物係代代花以水、醇、或醇水混合物之溶劑所萃取而得。
Description
本發明係關於一代代花萃取物之用途,尤其是一種代代花萃取物用於減緩皮膚老化及減少細胞中脂肪含量的用途。
糖化終產物(Advanced Glycation End Products,AGEs)是一群高度氧化的化合物,因此被認為是一種糖毒素(Glycotoxin),研究顯示糖化終產物會與細胞表面的接受器結合,而改變其結構與功能,並促使細胞的氧化壓力與發炎反應增加,更與糖尿病、動脈硬化及腎臟慢性疾病等的形成關係密不可分,其中對於還沒有產生糖尿病等慢性疾病的人來說,糖化作用會導致皮膚的膠原蛋白劣化,破壞膠原蛋白的纖維與彈力纖維,導致皮膚出現皺紋、鬆弛甚至蠟黃暗沉等現象。除了人體正常代謝過程中會產生糖化終產物外,許多加工食品中也會含有糖化終產物,研究也已指出避免從食物攝取糖化終產物,能有助於延緩慢性疾病與老化的進展。
隨著飲食習慣改變及生活品質改善,台灣的肥胖盛行率高達百分之二十左右,換算成數字,約有480萬人有肥胖的困擾,而其中就有20萬人以上已達病態型肥胖的標準而須手術治療。肥胖會引起許多相關的合併症,如糖尿病、高血脂、睡眠呼吸中止、狹心症、退化性關節炎、尿酸過高,甚至某些癌症等,進而可能造成死亡,因此病態性肥胖病患的平均壽命比起正常體重者少了許多。目前治療肥胖最有效的方法為手術治療,另外合法藥物(目前只
有羅氏鮮)、運動、熱量控制、及低卡代餐等亦被證實是有效的方式。然而,除了手術治療之外,大部份的病患使用其他方法減肥,大多在減重治療結束後便會失去戒心而復胖,如此一瘦一胖的現象(溜溜球效應)而對身體的傷害更大。
綜上所述,因應現代人因生活及飲食習慣改變所面臨的肥胖及因肥胖造成之整體健康問題,且基於現代人生活水平提高且對於保健概念提高,研發一種能有效增加抗糖化活性、及減少身體脂肪含量之有效成分組成的醫藥組合物,著實有其必要性。
緣此,本發明之一目的在提供一種代代花萃取物用於製備提升細胞抗糖化活性之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物。
本發明之又一目的在提供一種代代花萃取物用於製備抑制細胞脂肪堆積之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物。
本發明之另一目的在提供一種代代花萃取物用於製備促進細胞脂肪分解之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物。
在本發明之一實施例中,其中該代代花係代代花乾料,且該溶劑與該代代花乾料之重量比為5-20:1-5;且該萃取溫度係50℃-100℃。
在本發明之又一實施例中,其中該代代花萃取物係具有皮膚抗老化及美肌之功效,且該代代花萃取物的濃度為至少0.6mg/mL。
在本發明之又一實施例中,其中該代代花萃取物的濃度為至少0.0625mg/mL,且該代代花萃取物係具有減少細胞脂肪之功效。
本發明以水、醇類、或醇水混合物為溶劑所萃取得之代代花萃取物,能有效提升其清除糖化終產物之能力、有效抑制脂肪堆積、及有效促進脂肪分解,以減緩皮膚老化並達到皮膚美化的效果,以及減少細胞中脂肪的含量並達到減脂的效果。因此本發明之代代花萃取物可用於製備減緩皮膚老化及減少細胞中脂肪含量之組合物的用途,該組合物該組合物是一醫藥品、一食品或一保養品,可藉由口服、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,並非用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係為本發明之代代花萃取物之抗糖化活性之效果的長條圖。
圖2係為本發明之代代花萃取物於抑制脂肪堆積之效果的長條圖。*** p<0.001。
圖3係為本發明之代代花萃取物於促進脂肪分解之效果的長條圖。* p<0.05。
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。
使用Excel軟體進行統計分析。數據以平均值±標準差(SD)表示,個此之間的差異以學生t檢驗(student's t-test)分析。
代代(Citrus aurantium)為芸香科(Rutaceae)柑橘屬(Citrus)之常綠灌木植物,又稱酸橙、青橙、玳玳、苦橙。源於地中海,於古希臘時代,已被古希臘人用作芳香療法的殺菌劑,和植物療法的鎮靜劑。並在17世紀,於法國、義大利一帶,開始利用苦橙來提煉精油,以作生產香水用。代代喜溫暖濕潤氣候且不耐寒,花白色或帶紫紅色,果實呈略扁球形,直徑約7-9公分,成熟時黃色,外具顯著皺紋與凹點。代代已知具有理氣解鬱、減緩腹脹痛及嘔吐、促進血液循環、破氣行痰等功效,也因代代的花香氣濃郁,聞之令人忘倦,而可用於鎮定心情、解除緊張不安。
如本文中所使用的,用語「代代花萃取物」意為代代之花朵與溶劑以1-5:5-20(w/w)比例經一特定時間與溫度萃取而得。
依據本發明,醫藥品可利用熟習此技藝者所詳知的技術而被製造成一適合於非經腸道地(parenterally)或局部地(topically)投藥的劑型,這包括,但不限於:注射品(injection)[例如,無菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]、無菌的粉末(sterile powder)、外部製劑(external preparation)以及類似之物。
依據本發明,醫藥品可進一步包含有一被廣泛地使用於藥物製造技術之醫藥上可接受的載劑(pharmaceutically acceptable carrier)。例如,該醫藥上可接受的載劑可包含一或多種選自於下列的試劑:溶劑(solvent)、緩衝液(buffer)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、崩解劑(disintegrating agent)、分散劑(dispersing agent)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤濕劑(wetting agent)、潤滑劑(lubricant)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以
及類似之物。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。
依據本發明,該醫藥上可接受的載劑包含有一選自於由下列所構成之群組中的溶劑:水、生理鹽水(normal saline)、磷酸鹽緩衝生理鹽水(phosphate buffered saline,PBS)、含有醇的水性溶液(aqueous solution containing alcohol)以及它們的組合。
依據本發明,該醫藥品可以一選自於由下列所構成之群組中的非經腸道途徑(parenteral routes)來投藥:皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)以及病灶內注射(intralesional injection)。
依據本發明,醫藥品可利用熟習此技藝者所詳知的技術而被製造成一適合於局部地施用於皮膚上的外部製劑(external preparation),這包括,但不限於:乳劑(emulsion)、凝膠(gel)、軟膏(ointment)、乳霜(cream)、貼片(patch)、擦劑(liniment)、粉末(powder)、氣溶膠(aerosol)、噴霧(spray)、乳液(lotion)、乳漿(serum)、糊劑(paste)、泡沫(foam)、滴劑(drop)、懸浮液(suspension)、油膏(salve)以及繃帶(bandage)。
依據本發明,該外部製劑是藉由將本發明的醫藥品與一為熟習此項技藝者所詳知的基底(base)相混合而被製備。
依據本發明,該基底可包含有一或多種選自於下列的添加劑(additives):水、醇(alcohols)、甘醇(glycol)、碳氫化合物(hydrocarbons)[諸如石油膠(petroleum,jelly)以及白凡士林(white petrolatum,)]、蠟(wax)[諸如石蠟(paraffin)以及黃蠟(yellow wax)]、保存劑(preserving agents)、抗氧化劑(antioxidants)、界面活性劑(surfactants)、吸收增強劑(absorption enhancers)、安定劑(stabilizing agents)、膠凝劑(gelling agents)[諸如卡波普®974P
(carbopol®974P)、微結晶纖維素(microcrystalline cellulose)以及羧基甲基纖維素(carboxymethylcellulose)]、活性劑(active agents)、保濕劑(humectants)、氣味吸收劑(odor absorbers)、香料(fragrances)、pH調整劑(pH adjusting agents)、螯合劑(chelating agents)、乳化劑(emulsifiers)、閉塞劑(occlusive agents)、軟化劑(emollients)、增稠劑(thickeners)、助溶劑(solubilizing agents)、滲透增強劑(penetration enhancers)、抗刺激劑(anti-irritants)、著色劑(colorants)以及推進劑(propellants)等。有關這些添加劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。
依據本發明,保養品可進一步包含有一被廣泛地使用於保養品製造技術之可接受的佐劑(acceptable adjuvant)。例如,該可接受的佐劑可包含有一或多種選自於下列的試劑:溶劑、膠凝劑、活性劑、防腐劑、抗氧化劑、遮蔽劑(screening agent)、螯合劑、界面活性劑、染色試劑(coloring agent)、增稠劑(thickening agent)、填料(filler)、香料以及氣味吸收劑。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。
依據本發明,保養品可利用熟習此技藝者所詳知的技術而被製造成一適合於護膚(skincare)或化妝(makeup)的形式,這包括,但不限於:水性溶液(aqueous solution)、水-醇溶液(aqueous-alcohol solution)或油性溶液(oily solution)、呈水包油型(oil-in-water type)、油包水型(water-in-oil type)或複合型之乳劑、凝膠、軟膏、乳霜、面膜(mask)、貼片、貼布(pack)、擦劑、粉末、氣溶膠、噴霧、乳液、乳漿、糊劑、泡沫、分散液、滴劑、慕斯(mousse)、防曬油(sunblock)、化妝水(tonic water)、粉底(foundation)、卸妝產品(makeup remover products)、肥皂(soap)以及其他身體清潔產品(body cleansing products)等。
依據本發明,保養品亦可與一或多種選自於下列之已知活性的外用劑(external use agents)一起合併使用:美白劑(whitening agents)[諸如維生素A
酸(tretinoin)、兒茶素(catechin)、麴酸、熊果苷以及維生素C]、保濕劑、抗發炎劑(anti-inflammatory agents)、殺菌劑(bactericides)、紫外線吸收劑(ultraviolet absorbers)、植物萃取物(plant extracts)[諸如蘆薈萃取物(aloe extract)]、皮膚營養劑(skin nutrients)、麻醉劑(anesthetics)、抗痘劑(anti-acne agents)、止癢劑(antipruritics)、止痛劑(analgesics)、抗皮膚炎劑(antidermatitis agents)、抗過角化劑(antihyperkeratolytic agents)、抗乾皮膚劑(anti-dry skin agents)、抗汗劑(antipsoriatic agents)、抗老化劑(antiaging agents)、抗皺劑(antiwrinkle agents)、抗皮脂溢出劑(antiseborrheic agents)、傷口治療劑(wound-healing agents)、皮質類固醇(corticosteroids)以及激素(hormones)。有關這些外用劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。
依據本發明,食品產品可被當作食品添加物(food additive),藉由習知方法於原料製備時添加,或是於食品的製作過程中添加,而與任一種可食性材料配製成供人類與非人類動物攝食的食品產品。
依據本發明,食品產品的種類包括但不限於:飲料(beverages)、發酵食品(fermented foods)、烘培產品(bakery products)、健康食品(health foods)以及膳食補充品(dietary supplements)。
本發明提供一種代代花萃取物用於製備減緩皮膚老化及減少細胞中脂肪含量之組合物的用途,本發明之代代花萃取物係以一溶劑萃取一代代花乾料所獲得,該溶劑為水、醇、或醇水混合物,其可用於減緩皮膚老化、以及減少細胞中脂肪的含量。
同時,本發明用於製備減緩皮膚老化及減少細胞中脂肪含量之組合物,亦可包含一有效量之代代花萃取物及一醫藥上可接受之載體,該組合物係一醫藥品、一食品或一保養品。
以下將詳細說明本發明代代花萃取物之詳細萃取方法,與該代代花萃取物於抗糖化活性之測試、於抑制脂肪累積能力之測試、以及促進脂肪分解能力之測試,以證實該代代花萃取物對於減緩皮膚老化並達到皮膚美化的效果,以及減少細胞中脂肪的含量並達到減脂的效果。
在本發明一實施例中,將代代花乾料與水、醇、或醇水混合物之萃取溶劑,萃取溶劑較佳為水,以5-20:1-5之液固比混合,均質後在溶劑中以50-100℃進行萃取0.5-3小時。萃取後冷卻至室溫,將該粗萃取物經由400mesh之濾網過濾以獲得濾液。最後,將該濾液於45-70℃進行減壓濃縮,得到本發明之代代花萃取物。
本實施例係為了測試本發明之代代花萃取物的抗糖化活性,因此以抑制D-果糖(D-fructose)使膠原蛋白(Collagen)產生糖化的效率,進行糖化活性之定量。首先,分別取0.2mL之水作為控制組,或取0.2mL濃度為0.6mg/mL之本發明代代花萃取物,加入0.2mL之含有0.06%之NaN3的60mg/mL之膠原蛋白溶液(以200mM之磷酸鈉緩衝液配置,pH7.4)中,並與0.2mL的1.5M之D-果糖(以200mM之磷酸鈉緩衝液配置,pH7.4)溶液均勻混合,取出0.1mL混合溶液作為原點產物,並將剩餘的混合溶液於50℃下反應24小時後,取出0.1mL作為終點產物,再分別測量原點產物與終點產物於激發波長360nm、放射波長460nm的螢光數值。最後,以下列公式計算清除糖化終產物(Advanced glycation end-products,AGEs)能力之效率,以代表其抗糖化作用的活性,其中糖化終產物生成量越少表示抗糖化活性越高。
測試本發明之代代花萃取物抗糖化活性功效之結果如圖1所示,抑制非酵素褐變,避免體內功能性蛋白質變性。經本發明之代代花萃取物處理後,相較於控制組減少了約39%之糖化終產物的生成量,此結果顯示本發明之代代花萃取物能有效提升其清除糖化終產物之能力,而有助於延緩慢性疾病與老化的進展,且研究顯示糖化作用會導致皮膚的膠原蛋白劣化,破壞膠原蛋白的纖維與彈力纖維,導致皮膚出現皺紋、鬆弛甚至蠟黃暗沉等現象,因此本發明之代代花萃取物亦能有效減緩皮膚老化,達到皮膚美化的效果。
本實施例以小鼠骨髓基質細胞(bone marrow stromal cell)進行本發明之代代花萃取物於抑制脂肪堆積之功效測試。該小鼠骨髓基質細胞係購自美國典型培養物保藏中心(美國ATCC®),編號CRL-2749TM。該細胞於分化前係培養於前脂肪細胞擴增培養液(Pre-adipocyte Expansion Medium),其中包含90%之MEMAM(Minimum Essential Medium Alpha Medium,購自Gibco,美國,12100-046)細胞培養液、10%之胎牛血清(Fetal Bovine Serum,購自Gibco,美國),且加入0.1%之青黴素/鏈黴素(Penicillin-streptomycin,購自Gibco,美國);並使用分化培養液(Differentiation Medium)使小鼠骨髓基質細胞進行分化,其中包含90%之MEMAM細胞培養液、20%之胎牛血清,且加入0.1%之青黴素/鏈黴素。細胞中的脂質以油紅O染色試劑(購自Sigma,美國),其中以100%之異丙醇配置3mg/mL的油紅O儲備溶液,並將儲備溶液以ddH2O配置成60%的作用溶液。
為證實本發明之代代花萃取物具有抑制脂肪堆積的功效,首先將小鼠骨髓基質細胞分化成脂肪細胞,將8x104個小鼠骨髓基質細胞培養於含有0.5μL上述前脂肪細胞擴增培養液之24孔培養盤中,於37℃下培養7天,且期間每3天更換一次新鮮之上述分化培養液,7天後以顯微鏡觀察脂滴的形成,確保細胞已完全分化,接著將細胞分成(1)加入0.0625mg/mL本發明之代代花萃取物之實驗組及(2)僅含細胞培養液之控制組,並於37℃培養7-10天,且同樣每3天更換一次新鮮之分化培養液。
接著,使用油紅O將細胞內的脂質染色,以評估本發明之代代花萃取物是否確實能減少脂肪堆積,首先將培養基輕輕地取出,並以1mL之磷酸鹽緩衝溶液(Phosphate buffered saline,PBS)清洗細胞兩次,再加入1mL之10%甲醛(購自Echo chemical,台灣,Cat.TG1794-4-0000-72NI)並於室溫下反應30分鐘以固定細胞,接著移除甲醛後以1mL之PBS輕輕地清洗細胞兩次,接著於每孔細胞內加入1mL之60%異丙醇(購自Echo chemical,台灣,PH-3101)反應1分鐘後,移除異丙醇後加入1mL之油紅O作用溶液,於室溫下反應1小時,接著移除油紅O溶液並迅速地以1mL之60%異丙醇進行脫色5秒鐘,再使用顯微鏡拍照並進行量化。接著,加入100%異丙醇於染色之細胞中,並置於振盪器上反應10分鐘以溶解油滴,接著取100μL至96孔培養盤中,以測量ELISA讀數器讀取各組之OD510nm讀值,以量化油紅O。再利用Excel軟體進行student t-test以決定兩個樣本群體之間是否在統計上具有顯著差異(*p值<0.05;**p值<0.01;***p值<0.001)。
測試本發明之代代花萃取物抑制脂肪堆積之結果如圖2所示。經本發明之代代花萃取物處理後,相較於控制組減少了約44%之細胞脂肪堆積,此結果顯示本發明之代代花萃取物能有效抑制脂肪堆積,並減少細胞中脂肪的含量。
本實施例以上述小鼠骨髓基質細胞進行本發明之代代花萃取物於促進脂肪分解功效的測試。首先以實施例3之實驗步驟將小鼠骨髓基質細胞分化成脂肪細胞,同樣於開始培養7天後以顯微鏡觀察脂滴的形成,確保細胞已完全分化,接著將細胞分成(1)加入0.0625mg/mL本發明之代代花萃取物之實驗組及(2)僅含細胞培養液之控制組,並於37℃培養7-10天,且同樣每3天更換一次新鮮之分化培養液。
接著使用甘油檢測試劑盒(購於Cayman Chemical,美國)檢測細胞外甘油之數值,其中儲存在細胞中之三酸甘油脂被分解之後,會產生游離之甘油及脂肪酸,因此藉由測量細胞外的甘油含量,可以推知細胞中脂肪分解之數值。本試劑盒係利用測量會產生明亮之紫色產物的偶聯酵素反應系統之產物含量,來定量細胞外甘油的數值,並以標準品建立標準曲線以推算出樣品之細胞外甘油的數值。其中收集每孔之細胞培養上清液,並將25μL之細胞培養上清液轉移到新的96孔培養盤中,再於每孔中加入100μL重構的游離甘油測定試劑(Free Glycerol Assay Reagent)在室溫下反應15分鐘,接著讀取OD540nm處之數值。並利用Excel軟體進行student t-test以決定兩個樣本群體之間是否在統計上具有顯著差異(*p值<0.05;**p值<0.01;***p值<0.001)。
測試本發明之代代花萃取物促進脂肪分解之結果如圖2所示。經本發明之代代花萃取物處理後,相較於控制組增加了約7%之脂肪分解,此結果顯示本發明之代代花萃取物能有效促進脂肪分解,並減少細胞中脂肪的含量。
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代代花萃取物,能有效提升其清除糖化終產物之能力、有效抑制脂肪堆積、及有效促進脂肪分解,以減緩皮膚老化並達到皮膚美化的效果,以及減少細胞中脂肪的含量並達到減脂的效果。因此本發明之代代花萃取物可用於製備減緩皮
膚老化及減少細胞中脂肪含量之組合物的用途,該組合物該組合物是一醫藥品、一食品或一保養品,可藉由口服、皮膚塗抹等方式給予一個體。
Claims (8)
- 一種代代花萃取物用於製備提升細胞抗糖化活性之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物,其中該代代花萃取物的濃度為至少0.6mg/mL。
- 如申請專利範圍第1項所述之用途,其中該代代花萃取物係具有皮膚抗老化及美肌之功效。
- 一種代代花萃取物用於製備抑制細胞脂肪堆積之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物,其中該代代花萃取物的濃度為至少0.0625mg/mL。
- 一種代代花萃取物用於製備促進細胞脂肪分解之組合物的用途,其中該代代花萃取物係以一溶劑萃取一代代花所獲得,該溶劑為水、醇、或醇水混合物,其中該代代花萃取物的濃度為至少0.0625mg/mL。
- 如申請專利範圍第3項或第4項所述之用途,其中該代代花萃取物係具有減少細胞脂肪之功效。
- 如申請專利範圍第1項、第3項或第4項所述之用途,其中該代代花係一代代花乾料,且該溶劑與該代代花乾料之重量比為5-20:1-5。
- 如申請專利範圍第1項、第3項或第4項所述之用途,其中該萃取溫度係50℃-100℃。
- 如申請專利範圍第1項、第3項或第4項所述之用途,其中該組合物是一醫藥品。
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