TW202106321A - 沙棘萃取物用於改善下呼吸道狀態的組合物的用途 - Google Patents
沙棘萃取物用於改善下呼吸道狀態的組合物的用途 Download PDFInfo
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- TW202106321A TW202106321A TW109127839A TW109127839A TW202106321A TW 202106321 A TW202106321 A TW 202106321A TW 109127839 A TW109127839 A TW 109127839A TW 109127839 A TW109127839 A TW 109127839A TW 202106321 A TW202106321 A TW 202106321A
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- sea buckthorn
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- respiratory tract
- buckthorn extract
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Abstract
一種沙棘萃取物的用途,其用於製備一組合物。此組合物具有下列一種或多種改善下呼吸道狀態的功能:修復肺損傷、改善肺功能及抑制下呼吸道發炎相關基因的表現量。
Description
本發明是關於沙棘相關產品,特別是關於沙棘萃取物用於製備改善下呼吸道狀態的組合物的用途。
自有機及天然的飲食概念興起後,生技公司及食品業者積極投入關於天然植物的相關產品之研發。為使植物相關產品對身體健康助益有科學驗證的基礎,植物的活性成分分析及功效評估成為產品開發的重點項目。
沙棘(Hippophae rhamnoides
)又稱海沙棘,其是胡頹子科沙棘屬的植物。沙棘是一種原產于亞洲和歐洲的帶有棘刺的耐寒落葉灌木,其果實呈現橘黃色。沙棘可防風固沙,並且沙棘果實具有很高的藥用價值。因此,沙棘亦成為生技公司及食品業者積極研究開發的對象之一。
有鑑於此,本發明提供一種沙棘萃取物的用途,其是用於製備改善下呼吸道狀態的組合物。其中,沙棘萃取物具有下列一種或多種能力:修復肺損傷、改善肺功能及抑制下呼吸道發炎相關基因的表現量,以致沙棘萃取物能改善下呼吸道狀態。
在一實施例中,一種沙棘萃取物的用途,其是用於製備修復肺損傷的組合物的用途。
在一些實施例中,沙棘萃取物用以促進肺上皮細胞之創傷修復力,以致修復肺損傷。
在一實施例中,一種沙棘萃取物的用途,其是用於製備改善肺功能的組合物的用途。
在一些實施例中,改善肺功能是指提升下列肺功能指標中的至少一種:用力肺活量及用力呼氣中斷流速。
在一些實施例中,沙棘萃取物用以去痰及平喘,以致改善肺功能。
在一實施例中,一種沙棘萃取物的用途,其是用於製備抑制下呼吸道發炎相關基因的表現量的組合物的用途。其中,下呼吸道發炎相關基因包括下列至少一者:白介素5受體α(interleukin-5 receptor alpha;IL5RA)基因、粘膜相關淋巴組織淋巴瘤易位基因1(Mucosa associated lymphoid tissue lymphoma translocation gene 1;MALT1 gene)及瘤壞死因子受體超家族成員14(TNF superfamily member 14;TNFSF14)基因。
在一些實施例中,前述的組合物為醫藥品。
在一些實施例中,前述的組合物為食品產品或食品添加物。
在一些實施例中,前述的組合物為化妝品或保養品。
關於本文中所使用之濃度符號「wt%」通常是指重量百分濃度,而濃度符號「vol%」通常是指體積百分濃度。
在一些實施例中,由沙棘(Hippophae rhamnoides
)的果實所獲得的沙棘萃取物可具有下列至少一種能力:修復肺損傷、改善肺功能及抑制下呼吸道發炎相關基因的表現量,以致沙棘萃取物能改善下呼吸道狀態。因此,沙棘萃取物可用於製備具有下列至少一種能力的組合物:修復肺損傷、改善肺功能及抑制下呼吸道發炎相關基因的表現量。
在一些實施例中,一種沙棘萃取物的用途,其是用於製備修復肺損傷的組合物的用途。在一些實施例中,沙棘萃取物是藉由促進肺上皮細胞之創傷修復力而修復肺損傷。
在一實施例中,一種沙棘萃取物的用途,其是用於製備改善肺功能的組合物的用途。在一些實施例中,改善肺功能是指提升下列肺功能指標中的至少一種:用力肺活量及用力呼氣中斷流速。在一些實施例中,沙棘萃取物是藉由去痰及平喘而改善肺功能。
在一實施例中,一種沙棘萃取物的用途,其是用於製備抑制下呼吸道發炎相關基因的表現量的組合物的用途。其中,下呼吸道發炎相關基因包括下列至少一者:白介素5受體α(Interleukin-5 receptor alpha;IL5RA)基因、粘膜相關淋巴組織淋巴瘤易位基因1(Mucosa associated lymphoid tissue lymphoma translocation gene 1;MALT1 gene)及瘤壞死因子受體超家族成員14(TNF superfamily member 14;TNFSF14)基因。
在一些實施例中,前述的組合物可為醫藥品。換言之,此醫藥品包含有效含量的沙棘萃取物。其中,此醫藥品可利用熟習此技藝者所詳知的技術而被製造成適合於經腸道地、非經腸道地(parenterally)、口服地、或局部地(topically)投藥劑型。
在一些實施例中,經腸道或口服的投藥劑型可為,但不限於,錠劑(tablet)、片劑(troche)、口含錠(lozenge)、丸劑(pill)、膠囊(capsule)、分散性粉末(dispersible powder)或細顆粒(granule)、溶液、懸浮液(suspension)、乳劑(emulsion)、糖漿(syrup)、酏劑(elixir)、濃漿(slurry)或類似之物。在一些實施例中,非經腸道地或局部地投藥劑型可為,但不限於,注射品(injection)、無菌的粉末(sterile powder)、外部製劑(external preparation)或類似之物。在一些實施例中,注射品的投藥方式可為皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)或病灶內注射(intralesional injection)。
在一些實施例中,含有沙棘萃取物的醫藥品可更包含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)。在一些實施例中,醫藥上可接受的載劑可為下列載劑中一種或多種: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、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。關於選用之載劑的種類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。其中,作為醫藥上可接受的載劑的溶劑可為水、生理鹽水(normal saline)、磷酸鹽緩衝液(phosphate buffered saline,PBS)或含有醇的水性溶液(alcohol containing aqueous solution )。
在一些實施例中,前述的組合物為食用組合物。換言之,食用組合物包含特定含量的沙棘萃取物。其中,食用組合物的型態可為粉末、顆粒、溶液、膠體或膏體。
在一些實施例中,含有沙棘萃取物的食用組合物可為食品產品或食品添加物(food additive)。
在一些實施例中,含有沙棘萃取物的食品產品可為飲料(beverages)、發酵食品(fermented foods)、烘培產品(bakery products)、健康食品(health foods)或膳食補充品(dietary supplements)等。在一些實施例中,含有沙棘萃取物的食品產品可更包括一佐劑。舉例來說,佐劑可為麥芽糖糊精(Maltodextrin)、蘋果酸、蔗糖素、檸檬酸、水果香料、蜂蜜香料、甜菊糖苷或其組合等。關於選用之載劑的種類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。
在一些實施例中,含有沙棘萃取物的食品添加物可為調味料、甜味料、香料、pH值調整劑、乳化劑、著色料或穩定劑等。
在一些實施例中,前述的組合物為化妝品或保養品。換言之,化妝品或保養品包含特定含量的沙棘萃取物。
在一些實施例中,含有沙棘萃取物的化妝品或保養品可為下列任一種型態:化妝水、凝膠、凍膜、泥膜、乳液、乳霜、唇膏、粉底、粉餅、蜜粉、卸妝油、卸妝乳、洗面乳、沐浴乳、洗髮精、護髮乳、防曬乳、護手霜、指甲油、香水、精華液及面膜。
在一些實施例中,含有沙棘萃取物的化妝品或保養品可視需要更包含外用品可接受成分。在一些實施例中,外用品可接受成分可例如為乳化劑、滲透促進劑、軟化劑、溶劑、賦型劑、抗氧化劑、或其組合。
在一些實施例中,沙棘萃取物可由合適的萃取溶劑與沙棘果實進行一萃取程序所得。
舉例來說,在萃取程序中,以水於50℃-100℃下萃取沙棘果實30分鐘至90分鐘適當時間以得到初萃原液。其中,進行萃取的沙棘果實可為完整果實,或為經物理前處理而分離成碎片、顆粒或粉末等型態之果實。採用之物理前處理可包括下列至少一種:切碎、剪碎、搗碎、及研磨。在一些實施例中,在萃取程序中,初萃原液可進一步進行過濾以去除雜質而得到濾液。在一些實施例中,在萃取程序中,濾液可進一步進行濃縮以得到濃縮液。其中,濃縮方式可採用減壓濃縮。在一些實施例中,在萃取程序中,濃縮液可進一步乾燥成粉末。其中,乾燥方式可採用噴霧乾燥。
也就是說,可依實際需求以萃取程序所得的初萃原液、濾液、濃縮液或粉末作為沙棘萃取物。
在一些實施例中,沙棘萃取物亦可為市售之沙棘果實的原汁、濃縮原汁或原汁的乾燥粉末。
例一:維生素C定量
秤取10mg的維生素C(L-Ascorbic Acid Sodium Salt,純度≧98%,購自SIGMA-ALORICH)置於定量瓶中,然後以純水(H2
O)溶解並定量至10mL,以得到初始溶液(即含1000ppm的維生素C)。然後,依據下表一配置10ppm、25 ppm、50 ppm、100ppm及200ppm的標準溶液。
表一
標準溶液(ppm) | 10 | 25 | 50 | 100 | 200 |
初始溶液(mL) | 0.1 | 0.25 | 0.5 | 1.0 | 2.0 |
純水(mL) | 9.9 | 9.75 | 9.5 | 9.0 | 8.0 |
總量(mL) | 10 | 10 | 10 | 10 | 10 |
秤取1.0g的沙棘果汁粉(其含有100%沙棘萃取物)(購自Puredia)置於定量瓶中,然後以純水(H2
O)溶解並定量至50mL,以得到待測溶液。
接著,將待測溶液及標準溶液分別注入高效液相層析儀(HPLC)(機型:Hitachi Chromaster HPLC system,廠牌:Hitachi, Tokyo, Japan)以進行液相層析並就待測溶液與標準溶液所得波峰之滯留時間及吸收圖譜比較鑑別之。其中,HPLC裝設Ascentis Express C18層析管柱(廠牌:Supelco, 內徑2.1mm,長100mm,粒徑2μm)。HPLC的設定值中,移動相0.1wt%草酸水溶液、流速為每分鐘流0.2毫升(0.2mL/min),進樣體積為2μL,進樣次數為3重複,偵測波長為245nm,及管柱溫度設定為30℃。
然後,依據下式(1)計算維生素C的含量。
維生素C的含量(ppm)=C×V÷M (1)
其中,C為由標準曲線求得待測溶液中維生素C的濃度(ppm)。V為待測溶液最後定量的體積,即50mL。M為取樣分析的檢體的重量,即1g。
於此,維生素C的滯留時間為1.8 min(分鐘)。並且,測得沙棘果汁粉中具有9334.03ppm的維生素C。
例二:肺損傷修復試驗
於此,實驗將會分為實驗組以及控制組(未添加沙棘萃取物)二組進行,各組分別進行三重複試驗。
首先,以每孔1.5×105
個細胞的細胞數,將人肺(支氣管)上皮細胞株BEAS-2b(購自美國典型培養物保存中心(ATCC),編號CRL-9609)接種於含有支氣管上皮細胞基礎培養基(Bronchial epithelial cell basal Medium,BEME,品牌:Lonza)的24孔培養盤的各孔中,並置於37°C下培養過夜以形成細胞單層(cell monolayer)。
之後,使用200μL的吸管尖(pipette tip)於細胞單層創造傷口,然後移除培養基並以PBS(phosphate buffered saline,酸鹽緩衝液,品牌:Gibco)潤洗。然後,添加0.5mL無血清培養基至各孔中,並以顯微鏡(廠牌:ZEISS)觀察及拍攝各孔中的細胞的照片(以下稱0小時照片)。於此所使用的無血清培養基為Dulbecco的改良Eagle培養基(Dulbecco's Modified Eagle Medium,DMEM,品牌:Gibco)。
接著,將沙棘溶液添加至作為實驗組的各孔中並移除等體積的無血清培養基,使實驗組的無血清培養基含有0.25mg/ml的沙棘果汁粉。作為對照組的各孔則不做任何處理。其中,沙棘溶液為將2.23μL沙棘果汁添加至0.5mL無血清培養基中所形成的溶液,而沙棘果汁(0.056mg/μL)以沙棘果汁粉(其含有100%沙棘萃取物)(購自Puredia)以純水配置成糖度(Degrees Brix)為5.6的溶液。
接著,將各組的細胞於37℃下培養16小時。於16小時培養後,以顯微鏡(廠牌:ZEISS)觀察及拍攝各孔中的細胞的照片(以下稱16小時照片)。然後,使用Image J軟體分析0小時照片與16小時照片,以得到各組的傷口癒合百分比。各組的傷口癒合百分比為各組在16小時的傷口區域的總細胞面積相對於0小時的傷口區域的總面積的百分比。因實驗係進行三重複,故將各組的三重複實驗之結果平均以得平均值,然後將控制組的平均值視為100%相對癒合百分比來將實驗組的平均值換算成相對癒合百分比(%),如圖1所示。在圖1中,「*」代表在控制組比較下其p值小於0.05。
參照圖 1,相較於控制組,沙棘萃取物之處理增加肺上皮細胞所修復傷口區域的面積達約36%。此結果說明沙棘萃取物能促進肺上皮細胞之創傷修復力,因此具有修復肺損傷的功效,進而能有效阻隔肺部細胞纖維化的可能性。
例三:肺功能檢測
令7位受試者(其年齡介於30歲至45歲且具有菸癮、常接觸二手菸、騎車通勤、肺部慢性發炎或呼吸道過敏等習慣)每日食用一包0.5g的沙棘果汁粉(其含有100%沙棘萃取物)(購自Puredia),連續食用4週。於食用前(即第0週)及食用4週後(即第4週),以Mir Spirobank New®功肺功能檢測儀(廠牌:義大利Mir)搭配渦流氣探頭與吹嘴檢測各受試者的用力肺活量(Forced vital capacity,FVC)以及用力呼氣中斷流速(Forced Expiratory Flow in the middle 1/2 of an expiration, FEF25-75),如圖2及圖3所示。其中,FVC為測定呼吸道阻力的指標。FEF25-75為測定氣道阻塞的敏感指標,且其代表FVC中間50%用力吐氣氣流速度的平均。在圖2及圖3中,各長條是以7位受試者的對應量測結果的平均值繪示,並且第0週與第4週之間的統計學顯著差異是藉由學生t-試驗來統計分析。在圖2中,「*」代表在與第0週比較下其p值小於0.05。
並且,請各受試者於食用前(即第0週)及食用4週後(即第4週)進行肺功能問卷(如下表2所示)填寫回饋並分析之,以評估肺功能改善之狀況,如圖4及圖5所示。其中,在肺功能問卷中,評估項目A、B、E、F、G、H為生痰情況評估項目,評估項目C、D為易喘情況評估項目,評估項目I則依據受試回覆之症況列入生痰情況評估項目和易喘情況評估項目中之一或不列入評估。在圖4中,呼吸道生痰的嚴重情況是以7位受試者對生痰情況評估項目的評定分數之平均值呈現。在圖5中,容易喘的嚴重情況是7位受試者對易喘情況評估項目的評定分數之平均值呈現。
表2
請依據您近1週的感受來判斷程度 | ||||||
呼吸道狀況 | 無 | 些微 | 普通 | 經常 | 持續有 | |
A. | 最近呼吸道過敏的情況如何? | 1 | 2 | 3 | 4 | 5 |
B. | 最近有呼吸道生痰的情況嗎? | 1 | 2 | 3 | 4 | 5 |
C. | 最近覺得呼吸不順 | 1 | 2 | 3 | 4 | 5 |
D. | 最近覺得肺活量小(容易喘) | 1 | 2 | 3 | 4 | 5 |
E. | 最近您每日陣發性咳嗽次數多嗎? | 1 | 2 | 3 | 4 | 5 |
F. | 最近咳嗽會讓您聲音嘶啞嗎? | 1 | 2 | 3 | 4 | 5 |
G. | 最近咳嗽會影像您的睡眠嗎? | 1 | 2 | 3 | 4 | 5 |
H. | 最近咳嗽會讓您胸痛或肚子痛嗎? | 1 | 2 | 3 | 4 | 5 |
I. | 其他症狀: | 1 | 2 | 3 | 4 | 5 |
此外,於食用前(即第0週)及食用4週後(即第4週),還會蒐集各受試者的血液,以供後續實驗使用。
參照圖2,相較於第0週,食用沙棘萃取物4週後,受試者的用力肺活量顯著提升0.14L(3.4%),換言之,沙棘萃取物能提升肺活量。參照圖3,相較於第0週,食用沙棘萃取物4週後,受試者的用力呼氣中斷流速提升2.02%,換言之,沙棘萃取物能使受試者呼吸更加暢通。參照圖4及圖5,食用沙棘萃取物4週後,受試者呼吸道生痰及容易喘的情況皆有改善。此結果說明沙棘萃取物具有改善肺功能的功效。
例四:抗下呼吸道發炎試驗
於此,以例三蒐集到的各受試者的第0週及第4週血液進行下呼吸道發炎相關基因(包括IL5RA基因、MALT1基因及TNFSF14基因)的表現量的檢測。其中,IL5RA基因所表現的IL5RA會與多配體聚糖結合蛋白(Syndecan binding protein,SDCBP)相互作用活化與氣管發炎相關之細胞激素IL5。MALT1基因所表現的MALT1蛋白質(Mucosa associated lymphoid tissue lymphoma translocation protein 1;MALT1 protein)為淋巴細胞抗原受體(如,B細胞抗原受體(B cell receptor,BCR)及/或T細胞抗原受體(T cell receptor, TCR))至核因子活化B細胞κ輕鏈增強子(Nuclear factor kappa-light-chain-enhancer of activated B cells,NF-κB)活化過程中重要的訊息傳遞分子。
首先,將蒐集到的血液移至離心管中,並以300g之轉速離心15分鐘。於離心後,移除上層血漿,取2mL之膚色血球層(即,buffy coat)置於另一離心管中,再加入2mL之PBS均勻混和,以提供經稀釋的buffy coat。將經稀釋的buffy coat緩慢加入至內有3mL之Ficoll-Plague Plus溶液(購自Sigma)的另一離心管中,然後以400g之轉速離心40分鐘。於離心後,移除上層液體後,取出約2mL-3mL之中間層(即,單核細胞層)置於新的離心管中。將離心管中的單核細胞以PBS清洗3至5次後,再以300g之轉速離心10分鐘。於離心後,移除上清液,而所獲得之沉澱物即為周邊血單核球細胞(peripheral blood mononuclear cell,PBMC)。
獲得之PBMC以細胞裂解液(購自Geneaid公司)破細胞,以得到細胞裂解液。再以RNA萃取試劑套組(購自Geneaid公司)萃取細胞裂解液內的RNA。接著,取2000奈克(ng)之萃取到的RNA為模板,並利用SuperScript®III反轉錄酶(購自Invitrogene公司)將進行反轉錄作用,以產生相應的cDNA。後續,以cDNA作為模版並使用用來擴增標的基因的引子對(如下表3所示),在ABI StepOnePlusTM Real-Time PCR system(ABI StepOne Plus即時PCR系統,購自Thermo Fisher Scientific公司)中,利用KAPA SYBR FAST套組(購自Sigma公司)進行定量即時反轉錄聚合酶連鎖反應 (quantitative real-time reverse transcription polymerase chain reaction),以定量IL5RA基因、MALT1基因與TNFSF14基因之mRNA表現量,進而推斷各基因編碼的蛋白質的表現量。
其中,ABI StepOne Plus即時PCR系統的設定條件為95°C反應1 秒,60°C反應20秒,總共40個迴圈,並使用2-ΔCt方法進行基因定量。IL5RA基因對應的引子對為IL5RA-F與IL5RA-R。MALT1基因對應的引子對為MALT1-F與MALT1-R。TNFSF14基因對應的引子對為TNFSF14-F與TNFSF14-R。
表3
引子名稱 | 序列編號 | 序列 |
IL5RA-F | SEQ ID NO:1 | TGAAGTAAAAATACACAATACAAGGAATGG |
IL5RA-R | SEQ ID NO:2 | CACTGCTGCTCTCACTTGAACAT |
MALT1-F | SEQ ID NO:3 | ATGTGTGATGCCTACGTTACTGATTT |
MALT1-R | SEQ ID NO:4 | TATAGAGGCAATGCTTGGGAAGAT |
TNFSF14-F | SEQ ID NO:5 | GGCACACACCACGAAGTTTACTC |
TNFSF14-R | SEQ ID NO:6 | CCGACATCACCATCCTTCCT |
於此,將7位受試者對應基因的定量結果取平均以得到平均值,然後透過將第0週的平均值視為1(相對表現量)來將第4週的平均值換算成相對表現量(%),如圖6所示。並且,第0週的量測結果與第4週的量測結果之間的統計學顯著差異是藉由學生t-試驗來統計分析。在圖6中,「***」代表在與第0週比較下其p值小於0.001。
參照圖6,相比第0週,食用沙棘萃取物4週後,呼吸道及肺部發炎基因(即MALT1基因、TNSF14F基因與IL5RA基因)的表現量皆顯著降低。此結果說明沙棘萃取物具有減緩肺部發炎情況的功效。
綜上所述,根據本發明任一實施例的沙棘萃取物的用途,其用於可製備改善下呼吸道狀態的組合物。換言之,前述之組合物具有下列一種或多種功能:修復肺損傷、改善肺功能及抑制下呼吸道發炎相關基因的表現量,以致能改善下呼吸道狀態。
無
圖1是繪示控制組與實驗組的傷口區域的相對癒合百分比之長條圖。
圖2是繪示沙棘萃取物食用前與後的用力肺活量的變化之長條圖。
圖3是繪示沙棘萃取物食用前與後的用力呼氣中斷流速的變化之長條圖。
圖4是繪示沙棘萃取物食用前與後的呼吸道生痰情況的變化之長條圖。
圖5是繪示沙棘萃取物食用前與後的易喘情況的變化之長條圖。
圖6是繪示沙棘萃取物食用前與後的呼吸道及肺部發炎基因的表現量的變化之長條圖。
Claims (9)
- 一種沙棘萃取物用於製備修復肺損傷的組合物的用途。
- 如請求項1所述之用途,其中該沙棘萃取物用以促進肺上皮細胞之創傷修復力。
- 一種沙棘萃取物用於製備改善肺功能的組合物的用途。
- 如請求項3所述之用途,其中改善該肺功能是指提升下列肺功能指標中的至少一種:用力肺活量及用力呼氣中斷流速。
- 如請求項3所述之用途,其中該沙棘萃取物用以去痰及平喘。
- 一種沙棘萃取物用於製備抑制下呼吸道發炎相關基因的表現量的組合物的用途,其中該下呼吸道發炎相關基因包括下列至少一者:白介素5受體α基因、粘膜相關淋巴組織淋巴瘤易位基因1及瘤壞死因子受體超家族成員14基因。
- 如請求項1至6中任一項所述之用途,其中該組合物為醫藥品。
- 如請求項1至6中任一項所述之用途,其中該組合物為食品產品或食品添加物。
- 如請求項1至6中任一項所述之用途,其中該組合物為化妝品或保養品。
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