TWI740101B - 降膽固醇之益生菌株的用途 - Google Patents
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Abstract
本發明提供一種降膽固醇之益生菌株及其組合物與用途,該益生菌係為胚芽乳桿菌TCI507,寄存編號為BCRC910806,該益生菌株或其代謝產物可有效減少膽固醇的含量、提升SCARB1基因、APOA1基因、及LDLR基因的表現量,以促進膽固醇代謝的功效、且可有效降低人體血液中總膽固醇、以及低密度脂蛋白的含量,因此可用於製備降低膽固醇之組合物的用途。
Description
本發明係關於一降膽固醇之益生菌株及其組合物與用途,尤其是一種胚芽乳桿菌TCI507或其代謝產物用於製備提升SCARB1基因、APOA1基因、及LDLR基因的表現量、以及降低人體血液中總膽固醇與低密度脂蛋白的含量之組合物的用途。
相關文獻統計顯示,全球約有三分之一人口死於心血管疾病(Cardiovascular disease,CVD),為全球死因排行中的第一名,心血管疾病又稱為循環系統疾病,係指人體內運送血液的器官和組織所產生之疾病,因此所有心臟及血管的病變都可稱為心血管疾病,如中風、心肌梗塞等。其中高膽固醇血症在心血管疾患者中有高達七成的盛行率,亦為造成冠狀動脈疾病、中風、與周邊血管疾病等血管粥狀硬化的主要危險因子。
然而,高膽固醇血症大多是沒有明顯的症狀,僅有頭暈、頸部僵硬與血壓升高等非典型症狀。當血管阻塞的程度越來越嚴重時,就會出現狹心症、心肌梗塞、中風、或間歇性跛行等嚴重的併發症。因此,為降低發生心血管疾病的風險,控制血液中膽固醇含量即成為首要的目標。
其中,目前為預防心血管疾病僅能透過改善生活、飲食、以及運動習慣,並避免如香菸等危險因子等方式改善。否則則須以藥物進行控制,其
中該些藥物主要為化學合成而得,且可分為兩大類:一是預防血栓、抗血小板凝結的藥物,二是控制高血壓、膽固醇、糖尿病的藥物,然而這兩類藥物常伴隨著較強的副作用。例如血管緊張素相關之拮抗藥可能會引起高血鉀,使肌肉出現無力或心跳過慢等症狀;或是強效降膽固醇藥物之Statins類藥物可能會使肝功能指數上升、或有頭痛、噁心、疲倦等副作用。且這些藥物必須長期服用以控制病情,然而長期服用藥物對於個體所造成的副作用更無法自根本改善個體身體健康。
綜合上面所述,因應心血管疾病的高發生率,且基於現代人生活水平提高且對於保健概念提高,研發一種能有效預防心血管疾病,並能有效控制血液中膽固醇含量,又能避免合成藥物對於人體產生之副作用之由天然植物之有效成分組成的組合物,著實有其必要性。
緣此,本發明之一目的在提供一種用於降膽固醇之益生菌株,其中該益生菌株係為一胚芽乳桿菌(Lactobacillus plantarum),寄存編號係BCRC910806。
本發明之又一目的在提供一種如前所述之益生菌株或其代謝產物用於製備降膽固醇之組合物的用途。
本發明之另一目的在提供一種用於降膽固醇之組合物,包含一有效量之如前所述之益生菌株或其代謝產物。
在本發明之一實施例中,該益生菌株係為具有活性或去活性的菌株。
在本發明之又一實施例中,該益生菌株或其代謝產物係提升清道夫受體B1(Scavenger Receptor Class B Member 1,SCARB1)基因、載脂蛋白A1(Apolipoprotein A1,APOA1)基因、及/或低密度脂蛋白受體(Low-density
lipoprotein receptor,LDLR)基因的表現量,以促進膽固醇的代謝;且該益生菌株之濃度至少為0.5%(w/w)。
在本發明之另一實施例中,該益生菌株或其代謝產物係降低人體血液中總膽固醇、及/或低密度脂蛋白的含量。
本發明之胚芽乳桿菌TCI507的活性菌株、去活性菌株、或其代謝產物在體外能可有效減少膽固醇的含量,顯示其可用於人體降膽固醇的潛力;而在細胞實驗中發現本發明之胚芽乳桿菌TCI507或其代謝產物可有效提升SCARB1基因、APOA1基因、及LDLR基因的表現量,顯示其可促進膽固醇代謝的功效;在人體試驗中更發現本發明之胚芽乳桿菌TCI507或其代謝產物可有效降低人體血液中總膽固醇、以及低密度脂蛋白的含量,顯示其具有人體降膽固醇的功效;且因高膽固醇體質容易引起高血壓、動脈硬化、心肌梗塞等心血管疾病,因此本發明之胚芽乳桿菌TCI507或其代謝產物可應用於心血管保健之用途。故,本發明之胚芽乳桿菌TCI507或其代謝產物可用於製備降低膽固醇之組合物的用途,該組合物係為一食品、一飲品、一營養補充劑、或一醫藥品,且該組合物係粉末、顆粒、溶液、或膠體之劑型,並可藉由口服等方式給予一個體。
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係為本發明之一實施例的胚芽乳桿菌TCI507降低總膽固醇含量之長條圖。
圖2係為本發明之一實施例的胚芽乳桿菌TCI507提升SCARB1基因、APOA1基因、及LDLR基因表現量之長條圖。*p值<0.05;**p值<0.01;***p值<0.001。
圖3係為本發明之一實施例的胚芽乳桿菌TCI507降低人體血液中總膽固醇含量之長條圖。*p值<0.05。
圖4係為本發明之一實施例的胚芽乳桿菌TCI507降低人體血液中低密度脂蛋白含量之長條圖。**p值<0.01。
本發明之胚芽乳桿菌(Lactobacillus plantarum)是一種可降膽固醇以促進心血管保健的益生菌株(Probiotic bacteria)。本發明係一新穎之胚芽乳桿菌,本文命名為TCI507,於民國106年12月21日寄存於食品工業發展研究所,寄存編號BCRC910806,本發明經由體外實驗證實本發明之胚芽乳酸桿菌TCI507或其代謝產物可有效減少膽固醇的含量;經由細胞實驗證實本發明之胚芽乳酸桿菌TCI507或其代謝產物可有效提升SCARB1基因、APOA1基因、及LDLR基因的表現量,以促進膽固醇代謝的功效;經由人體試驗證實本發明之胚芽乳酸桿菌TCI507或其代謝產物可有效降低人體血液中總膽固醇、以及低密度脂蛋白的含量。顯示本發明之胚芽乳酸桿菌TCI507或其代謝產物可用於製備降膽固醇的心血管保健之組合物的用途,且該組合物係一醫藥品、或一食品,可藉由口服等方式給予一個體。
使用Excel軟體進行統計分析。數據以平均值±標準差(SD)表示,個組之間的差異以學生t檢驗(student's t-test)分析。
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。
益生菌株(probiotic或probiotic bacteria)係為一微生物,其菌體、混合菌株、萃取物或代謝產物對於宿主本身係具有正面影響,通常源自於人體內、有益於腸道健康的活菌,亦可指外來補充、對身體可能有益的某些微生物,其中該益生菌株的代謝產物係為該益生菌株之分泌物,包含培養該菌的培養液。
依據本發明,有關細菌培養的操作程序與參數條件等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。
如本文中所使用的,用語「代謝產物」意為培養細菌時,經該細菌代謝後所分泌至細菌培養液中之物質,包含培養該菌的培養液。
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,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)。
本發明提供一種胚芽乳桿菌(Lactobacillus plantarum)TCI507或其代謝產物用於製備降膽固醇之組合物的用途,本發明胚芽乳桿菌代謝產物係取該胚芽乳桿菌TCI507之培養液所獲得。該胚芽乳桿菌TCI507或其代謝產物可用於提升SCARB1基因、APOA1基因、及/或LDLR基因的表現量,以促進膽固醇的代謝;亦可降低人體血液中總膽固醇、及/或低密度脂蛋白的含量。
同時,本發明用於降膽固醇之組合物,亦可包含一有效量之胚芽乳桿菌TCI507或其代謝產物,以及一醫藥上可接受之載體,該組合物係一醫藥品、或一食品。
以下將詳細說明本發明之胚芽乳桿菌TCI507或其代謝產物於試管實驗中降低膽固醇含量之功效測試;其提升肝臟細胞中SCARB1基因、APOA1基因、及LDLR基因表現量之功效測試;以及其降低人體血液中總膽固醇、以
及低密度脂蛋白的含量之功效測試,證實本發明之胚芽乳桿菌TCI507或其代謝產物具有促進膽固醇的代謝及降低人體膽固醇之功效,而能用於製備降膽固醇之組合物。
在本發明之一實施例為測試本發明之胚芽乳桿菌(Lactobacillus plantarum)TCI507降膽固醇的功效,於試管進行體外降低膽固醇含量之實驗。首先,將1%之胚芽乳桿菌TCI507的菌種培養在含有5000ppm膽固醇之MRS(de Man,Rogosa and Sharpe,本實施例係使用BD DifcoTM Lactobacilli MRS Broth,編號為DF0881175)培養液的試管中,於37℃培養24小時後,將菌液以5000rpm離心10分鐘後,蒐集5mL之上清液並使用膽固醇定量試劑套組(Cholesterol Quantitation Kit,購於西格瑪奧瑞奇,美國,編號為MAK043)進行膽固醇總含量檢測。其中,以MRS作為空白控制組;以已知具有降膽固醇功效之胚芽乳桿菌LP1及LP2二菌株作為控制組;且該培養液係取55g之MRS溶於1L純水中再於121℃下滅菌15分鐘所製成。
本發明之胚芽乳桿菌TCI507降膽固醇的實驗測試結果如圖1所示。經本發明之胚芽乳桿菌TCI507作用後,可大幅降低87%的膽固醇含量,且相較於二正控制組之胚芽乳桿菌株,降低更高比例的膽固醇。此結果顯示本發明之胚芽乳桿菌TCI507可有效降低膽固醇含量,可用於人體降膽固醇的心血管保健用途。
本發明之一實施例為測試本發明之胚芽乳桿菌TCI507調控膽固醇相關基因表現量的功效,使用人類肝臟上皮細胞HepG2(購自美國典型培養
物保藏中心ATCC,編號ATCC® HB-8065TM)進行實驗。其中,該細胞係培養於含有10%之胎牛血清(Fetal Bovine Serum,購自Gibco,美國)、1%青黴素-鏈黴素(購自Gibco,美國)之DMEM(Dulbecco's Modified Eagle Medium,購自Gibco,美國,12100-046)細胞培養液中。
首先,將0.5x105個HepG2細胞培養於6孔培養盤之每孔中,於37℃培養16小時,接著將細胞分成以下五組:(1)僅加入前述細胞培養液之空白控制組、(2)僅加入前述細菌培養液(即空培液)之空白控制組、(3)加入1% LP1代謝產物之正控制組、(3)加入1% LP2代謝產物之正控制組、(4)加入1%本發明胚芽乳桿菌TCI507代謝產物之實驗組,並將該些組別之細胞分別於37℃下作用24小時後,測試各組HepG2細胞中膽固醇代謝相關基因的表現量。接著,將HepG2細胞以細胞裂解液(購自Geanaid公司,臺灣,編號RBD300)回收細胞後,使用RNA萃取試劑套組(購自Geneaid公司,臺灣,編號RBD300)分別收集該五組細胞內之RNA,接著利用SuperScript® III反轉錄酶(購自Invitrogene公司,美國,編號18080-051)以2000ng之萃取RNA為模板並以引子產生mRNA反轉錄之相應cDNA產物,接著利用ABI StepOnePlusTM Real-Time PCR system(Thermo Fisher Scientific公司,美國),以及KAPA SYBR FAST(購自Sigma公司,美國,編號38220000000)將該五組反轉錄後產物分別以表1之組合引子進行定量即時聚合酶連鎖反應(Quantitative real-time polymerase chain reaction,qPCR)試驗,條件為95℃反應1分鐘預熱後,再進行以下95℃反應15秒、58℃反應15秒、72℃反應30秒之總共40個迴圈的程序。用以定量SCARB1基因、APOA-1基因、LDLR基因、ABCA1基因、及CETP基因之mRNA表現量,其中定量數值係取由閾值循環數(Ct),而目標基因的mRNA相對量係推導自方程式2-△Ct,其中△Ct=Ct目標基因-Ct ACTB (β-肌動蛋白,beta-actin),再利用Excel軟體進行非成對單尾student t-test以決定變異係數與是否在統計上具有顯著差異(*p值<0.05;**p值<0.01;***p值<0.001)。
SCARB1(Scavenger receptor class B member 1)基因編碼的蛋白質SRB1(Scavenger receptor class B type 1)是高密度脂蛋白(High-density lipoprotein,HDL)的細胞膜上受體。高密度脂蛋白會收集血液中的膽固醇,並將其沉積於肝臟待最終被代謝掉,在此過程中SR-B1扮演了關鍵的角色。因此,SCARB1基因表現量上升與降低血液中膽固醇相關。
APOA1(Apolipoprotein A1)基因編碼的蛋白質APOA-1為血液中高密度脂蛋白中主要的蛋白質成分,其經水解加工後,會執行促進膽固醇從組織流出至肝臟排泄的功能。APOA-I附著於細胞膜並促進膽固醇和磷脂從細胞內部向外移動,並與APOA-I結合形成HDL。APOA-I亦會引發膽固醇酯化反應,將膽固醇轉化為可以完全整合到HDL中並通過血液運輸的形式。
ABCA1(ATP binding cassette subfamily A member 1)基因編碼的蛋白質ABCA1主要功能為將膽固醇從細胞內運送到胞外,為APOA-1的受體。因此,APOA1基因及ABCA1基因的表現量上升與降低血液中膽固醇相關。
LDLR(Low density lipoprotein receptor)基因編碼的蛋白質LDLR為低密度脂蛋白(Low-density lipoprotein,LDL)的受體,LDLR位於不同種類細胞的表面,會與在血液中的低密度脂蛋白結合並將其運輸到細胞中,低密度脂蛋白進入細胞後,會被分解並釋放出膽固醇。LDLR在調節血液中膽固醇含量扮演關鍵的角色,其在肝臟細胞數量特別多,其中肝臟是負責從體內去除過量膽固醇的器官。肝臟細胞表面的LDLR數量決定了從血液中去除膽固醇的速度。因此,LDLR基因的表現量上升與降低血液中膽固醇相關。
CETP(Cholesteryl ester transfer protein)基因編碼的蛋白質CETP為一種血漿蛋白,主要功能為協助膽固醇由高密度脂蛋白轉移至低密度脂蛋白,此基因的突變會導致高密度脂蛋白血症,以及動脈粥様硬化。因此,CETP基因的表現量上升與降低血液中膽固醇相關。
本發明之胚芽乳桿菌TCI507提升肝臟細胞中SCARB1基因、APOA1基因、及LDLR基因表現量之結果如圖2所示。經本發明之胚芽乳桿菌TCI507作用後,相較於空培液組,能顯著提高SCARB1基因、APOA1基因、及LDLR基因的表現量,且皆較已知具有降低膽固醇功效的LP1及LP2能更加地提升該三基因的表現量;而CETP基因及ABCA1基因則差異不大。此結果顯示本發明之胚芽乳桿菌TCI507能有效提升SCARB1基因、APOA1基因、及LDLR基因的表現量,能促進膽固醇的代謝,並可用於人體降膽固醇的心血管保健用途。
本發明之一實施例為試驗本發明之胚芽乳桿菌TCI507於降低人體低密度膽固醇之功效,首先募集6位受試者,每日服用一顆內含5 x 109
CFU/cap之本發明胚芽乳桿菌TCI507的膠囊,並分別於服用前、服後兩週後、及服用四週後,測試受試者血液中總膽固醇、以及低密度膽固醇之變化,其中係委外給檢驗單位進行各受試者血液樣品中總膽固醇及低密度膽固醇的含量。
本發明之胚芽乳桿菌TCI507降低人體血液總膽固醇含量的結果如圖3所示;降低人體血液中低密度脂蛋白含量之結果如圖4所示。服用前受試者血液中的總膽固醇含量約為210mg/dL,低密度脂蛋白含量約為148mg/dL;服用本發明之胚芽乳桿菌TCI507兩週後,能使受試者血液中總膽固含量降低至約195mg/dL,低密度脂蛋白則降低至約137mg/dL;服用本發明之胚芽乳桿菌TCI507四週後,能使受試者血液中總膽固含量顯著地降低至約194mg/dL,且相較於服用前降低了7.2%,而服用四週後低密度脂蛋白則顯著地降低至約131mg/dL,相較於服用前降低了11%。此些結果顯示,本發明之胚芽乳桿菌TCI507可有效降低人體血液中總膽固醇、以及低密度脂蛋白的含量,可用於人體降膽固醇的心血管保健用途。
綜上所述,本發明之胚芽乳桿菌TCI507的活性菌株、去活性菌株、或其代謝產物在體外能可有效減少膽固醇的含量,顯示其可用於人體降膽固醇的潛力;而在細胞實驗中發現本發明之胚芽乳桿菌TCI507或其代謝產物可有效提升SCARB1基因、APOA1基因、及LDLR基因的表現量,顯示其可促進膽固醇代謝的功效;在人體試驗中更發現本發明之胚芽乳桿菌TCI507或其代謝產物可有效降低人體血液中總膽固醇、以及低密度脂蛋白的含量,顯示其具有人體降膽固醇的功效;且因高膽固醇體質容易引起高血壓、動脈硬化、心肌梗塞等心血管疾病,因此本發明之胚芽乳桿菌TCI507或其代謝產物可應用於心血管保健之用途。故,本發明之胚芽乳桿菌TCI507或其代謝產物可用於製備降低膽固醇之組合物的用途,該組合物係為一食品、一飲品、一營養補充劑、或一醫藥品,且該組合物係粉末、顆粒、溶液、或膠體之劑型,並可藉由口服等方式給予一個體。
食品工業發展研究所(台灣);民國106年12月13日;編號BCRC910806。
<110> 大江生醫股份有限公司
<120> 降膽固醇之益生菌株及其組合物與用途
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<170> PatentIn version 3.5
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Claims (4)
- 一種益生菌株的代謝產物用於製備降膽固醇之組合物的用途,其中該益生菌株係為一胚芽乳桿菌(Lactobacillus plantarum),寄存編號係BCRC910806,且該益生菌株的代謝產物係提升一基因群的表現量,以促進膽固醇的代謝,其中該基因群係由以下基因所組成:清道夫受體B1(Scavenger Receptor Class B Member 1,SCARB1)基因、載脂蛋白A1(Apolipoprotein A1,APOA1)基因、以及低密度脂蛋白受體(Low-density lipoprotein receptor,LDLR)基因。
- 如申請專利範圍第1項所述之用途,其中該益生菌株係為具有活性或去活性的菌株。
- 如申請專利範圍第1項所述之用途,其中該益生菌株之濃度至少為0.5%(w/w)。
- 如申請專利範圍第1項所述之用途,其中該益生菌株的代謝產物係降低人體血液中總膽固醇、及/或低密度脂蛋白的含量。
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