TW202038747A - 用於預防或治療幽門螺旋桿菌相關疾病之韓國泡菜 - Google Patents
用於預防或治療幽門螺旋桿菌相關疾病之韓國泡菜 Download PDFInfo
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Abstract
本申請案係關於含有乳酸菌之韓國泡菜,其可有效地用於預防及治療幽門螺旋桿菌相關疾病。
Description
本申請案係關於含有乳酸菌之韓國泡菜(kimchi),其可有效地用於預防及治療幽門螺旋桿菌(Helicobacter pylori
)相關疾病。
在1983年,澳大利亞微生物學家Marchall及Warren在患有B型胃炎之患者之胃黏膜生檢組織中首次發現彎曲細菌,且隨後培育細菌,且鑑別出其為與彎曲桿菌屬(Campylobacter
genus)相關之新穎物種。此後,已對幽門螺旋桿菌與上部胃腸道疾病(諸如胃炎、消化性潰瘍、及類似者)之間之相關性進行眾多研究。幽門螺旋桿菌為革蘭氏陰性芽孢桿菌,具有由薄膜包圍之鞭毛,釋放大量脲酶,且已知藉由糞口途徑擴散,且為在全世界吸引極大關注之病原體,因為世界衛生組織(WHO)在1994年鑑別出其為明確致癌物。
當前,由幽門螺旋桿菌引起之疾病之發病機制已知如下:隨著食物進入胃內腔之細菌藉由具有強運動性之鞭毛穿透胃黏液層,且寄居於上部上皮層及胃黏液層中之細胞之間的接合,且釋放大量脲酶以將黏液層變為鹼性,從而引起胃泌素胃酸分泌之異常過度刺激,且隨即導致發炎及潰瘍。另外,根據發現幽門螺旋桿菌為胃腺癌發病機制之重要危險因素的近期研究,世界衛生組織已劃定幽門螺旋桿菌為第I類致癌物。
特定言之,已報導包括南韓之開發中國家中常常出現幽門螺旋桿菌的感染。根據對無上胃腸道症狀之5,732人進行的國家流行病調查,幽門螺旋桿菌感染之總盛行率為46.6%,且16歲以上成人之感染率為69.4%,且當人在其40多歲老化時尤其高(78.5%)。另外,根據對患有消化性潰瘍之1,031個患者進行之研究,發現66%的胃潰瘍患者及79%的十二指腸潰瘍患者感染有幽門螺旋桿菌,且71%的患有胃及十二指腸潰瘍之患者據報導感染有幽門螺旋桿菌。
作為治療由幽門螺旋桿菌感染引起之以上疾病之方法,當前主要進行利用抗生素的化學療法。然而,雖然此方法的普遍使用可能在短期內有效,但此方法有一個問題,其涉及由於持續使用抗生素而出現抗生素抗藥性菌株之風險。另外,據報導持續使用抗細菌療法會導致各種副作用,諸如腹瀉、逆流性食道炎、抑制腸中正常菌落、出現抗藥性菌株、及類似者。
因此,需要開發適合的抗微生物材料或食品,其可抑制幽門螺旋桿菌,同時替換引起人體中上文所描述之副作用及抗藥性之治療劑。
同時,傳統醱酵食品韓國泡菜之經醱酵之乳酸菌為公認安全(Generally Recognized As Safe;GRAS)級細菌,其長期以來被人類與醱酵食品一起食用。由於乳酸菌之功能性及安全性之優點,因此關注聚焦於使用乳酸菌之功能性食品及藥物之開發。舉例而言,本案之申請人已報導產生減少量氣體之腸膜狀明串珠菌CJLM119菌株及使用其製備韓國泡菜之方法,以及胚芽乳酸桿菌CJLP133菌株及包含其之用於治療腸疾病及增強免疫之組成物(專利文件1至3)。然而,對藉由經醱酵之食品(諸如被經常食用之韓國泡菜)預防或治療由幽門螺旋桿菌感染引起之疾病之方法的研究不完整,且仍處於進展中。
先前技術文件
專利文件
(專利文件1)韓國專利第1,807,995號
(專利文件2)韓國專利第1,486,999號
(專利文件3)韓國專利第1,406,168號
技術問題
本申請案係關於含有作為活性成分對幽門螺旋桿菌具有抑制作用之乳酸菌之韓國泡菜,且係關於韓國泡菜及其萃取物用於預防及治療幽門螺旋桿菌相關疾病之用途。技術方案
為解決以上問題,本申請案之一個方面提供包括為韓國泡菜衍生之乳酸菌之腸膜狀明串珠菌(Leuconostoc mesenteroide
)CJLM119及胚芽乳酸桿菌(Lactobacillus plantarum
)CJLP133之韓國泡菜,且提供一種其製備方法。
本申請案之另一方面提供用於改善幽門螺旋桿菌相關疾病之組成物,該組成物包括韓國泡菜或其萃取物。
本申請案之再一方面提供一種用於預防、治療或改善幽門螺旋桿菌相關疾病之方法,該方法使用韓國泡菜或其萃取物。有利作用
根據本申請案之包括作為韓國泡菜衍生之乳酸菌之腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133之韓國泡菜或其萃取物具有針對幽門螺旋桿菌之抑制活性,且在長期投予後,可預防或治療由幽門螺旋桿菌引起之萎縮性胃炎及胃潰瘍或延遲其等之進展,且亦可有效地用於預防或治療由幽門螺旋桿菌引起之胃癌。
然而,本申請案之作用不限於以上提及之作用,且未提及之其他作用將由本發明所屬技術領域中具有通常知識者由以下描述明確理解。
在下文中,將詳細地描述本申請案。
1.包括腸膜狀明串珠菌 CJLM119 及胚芽乳酸桿菌 CJLP133 之韓國泡菜及其製備方法
本申請案提供包括腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133之韓國泡菜及其製備方法。
腸膜狀明串珠菌CJLM119為以寄存編號KCTC 13043BP寄存在韓國生物科學及生物技術研究院(Korea Research Institute of Bioscience and Biotechnology)基因庫之微生物菌株,且韓國專利第1,807,995號中具體揭示了此菌株之分離方法及鑑別。另外,胚芽乳酸桿菌CJLP133為以寄存編號KCTC 11403BP寄存在韓國生物科學及生物技術研究院基因庫之微生物菌株,且韓國專利第1,486,999號中具體揭示了此菌株之分離方法及鑑別。
在本申請案中,「韓國泡菜」意欲包括但不限於藉由對蔬菜加鹽及調味,且隨後使其醱酵而製備之食品。在本申請案之一個實施方式中,蔬菜可為大白菜(Kimchi cabbage)、白蘿蔔、大蔥、芥菜、黃瓜、及類似者。特定言之,蔬菜可為大白菜,且任何其他蔬菜可用作本申請案之標的之韓國泡菜之原料。在本申請案之一個實施方式中,蔬菜可為但不限於具有高含量番茄紅素之蔬菜。在本申請案之另一個實施方式中,可以每100 g蔬菜0.05至5 mg,諸如0.1至3 mg、0.2至2 mg、0.3至1 mg、0.5至0.8 mg之含量包括番茄紅素,但含量不限於此。
具有高含量番茄紅素之蔬菜可為大白菜,且具體言之,使用TheHan1ho之大白菜品種之大白菜(2015年12月15日公開之品種申請案公開第2015-688號)。當具有高含量番茄紅素之蔬菜用腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133醱酵時,防止番茄紅素含量降低,且因此可維持番茄紅素之癌症預防功能。
在本申請案之一個實施方式中,調味料藉由混合諸如紅辣椒粉、大蒜、薑、鹹魚、等等之成分來製備,且除此等之外,針對組成調味料之成分,在必要時視個人偏好、醱酵方法、醱酵時間段、等等而定,可視需要添加或排除任何其他成分。
在本申請案之一個實施方式中,本申請案之韓國泡菜可根據製備韓國泡菜之習知方法,藉由使鹽漬蔬菜(例如鹽漬大白菜)與調味料混合,且隨後使其醱酵的方法來製備。作為蔬菜,使用具有高含量番茄紅素之蔬菜可能更合適。然而,本申請案不限於此,且可使用習知蔬菜(但不限於其等)來製備本申請案之韓國泡菜。
在本申請案之另一個實施方式中,本申請案之韓國泡菜可進一步包括對於食品而言可接受之已知添加劑。作為添加劑,本申請案之韓國泡菜中可包括各種天然香料,諸如李子香料、檸檬香料、鳳梨香料、草本香料、或類似者;各種天然色素,諸如天然果汁、葉綠酸、類黃酮、或類似者;增甜組分,諸如果糖、蜂蜜、糖醇或糖;或酸化劑,諸如檸檬酸或檸檬酸鈉,但不限於其等。
在本申請案之一個實施方式中,按韓國泡菜之總重量計,本申請案之韓國泡菜可包括0.01至5重量%、特定言之0.02至3重量%、更特定言之0.05至2.0重量%腸膜狀明串珠菌CJLM119。
在本申請案之另一個實施方式中,按韓國泡菜之總重量計,本申請案之韓國泡菜可包括0.01至5重量%、特定言之0.02至3重量%、更特定言之0.05至2.0重量%胚芽乳酸桿菌CJLP133。
在本申請案之一個實施方式中,本申請案之韓國泡菜可藉由將0.01至5重量%、特定言之0.02至3重量%、更特定言之0.05至2.0重量%腸膜狀明串珠菌CJLM119及0.01至5重量%、特定言之0.02至3重量%、更特定言之0.05至2.0重量%胚芽乳酸桿菌CJLP133添加至鹽漬蔬菜,例如具有高含量番茄紅素之鹽漬蔬菜中,且隨後在特定溫度下進行醱酵特定時間段來製備。
醱酵期間之溫度不受特定限制,但可在例如1℃至30℃、特定言之3℃至15℃、或5℃至10℃下進行醱酵。
醱酵時間段不受特定限制,但可例如進行醱酵1至365天、特定言之5至180天、10至90天、15至60天或20至40天。
在本申請案之一個實施方式中,可使用腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株自身,但亦可使用包括此等菌株之培養物。在本申請案之一個實施方式中,腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株可以活細胞、死細胞、或其等之混合物的形式使用。
在本申請案中,術語「培養物」意謂接種至培養基中之後由腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株之培養產生之物質。舉例而言,培養物可包括藉由在培養基中培養腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株所獲得之培養物自身(亦即,培養物可包括腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株、此等菌株之培養基或代謝產物)、藉由過濾或離心培養物以移除菌株所獲得之濾液(例如,離心上清液)、或類似者。另外,本申請案之培養物可包括但不限於藉由使培養物乾燥(例如,冷凍乾燥)及粉末化所獲得者。
在本申請案之一個實施方式中,培養菌株可在10℃至30℃之溫度下進行6至48小時、特定言之在20℃至30℃之溫度下進行12至36小時或18至30小時。但本申請案不限於此。
作為培養基,可使用乳酸菌之任何已知培養基,但不限於其等。培養基可包括碳源及氮源。碳源可包括蔗糖、葡萄糖、果糖、及類似者之一或多者,且氮源可包括但不限於酵母萃取物、蛋白腖、牛肉萃取物、麥芽萃取物、玉米浸液、檸檬酸銨、硫酸銨、氯化銨、磷酸銨、碳酸銨、硝酸銨、及類似者之一或多者。另外,培養基可進一步包括吐溫80、檸檬酸鈉、磷酸鉀、乙酸鈉、硫酸錳、硫酸鎂、及蒸餾水之一或多者。2. 用於改善幽門螺旋桿菌相關疾病之組成物 ,其 包括本申請案之韓國泡菜或其萃取物
本申請案提供用於改善幽門螺旋桿菌相關疾病之組成物,該組成物包括包含腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133的韓國泡菜或其萃取物。
本申請案之韓國泡菜及其製備方法與「1. 包括腸膜狀明串珠菌 CJLM119 及胚芽乳酸桿菌 CJLP133 之韓國泡菜及其製備方法
」中提及之韓國泡菜及其製備方法相同,且因此省略其描述。
在本申請案中,幽門螺旋桿菌相關疾病可包括但不限於胃炎、胃潰瘍、胃癌、及類似者。
在本申請案中,「韓國泡菜萃取物」係指藉由任何方法自韓國泡菜(其為原料)萃取之材料,且意欲包括但不限於由此萃取之所有萃取物、自其所獲得之濃縮物、濃縮物之乾燥產物及粉末。
當藉由萃取韓國泡菜(其為原料)獲得萃取物時,可使用習知萃取方法,諸如溶劑萃取、超音波萃取、過濾及回流萃取,但不限於其等。特定言之,可使用溶劑萃取或回流萃取,但本申請案不限於其等。萃取程序可重複若干次,隨後萃取物可進一步經歷諸如濃縮或冷凍乾燥之步驟。特定言之,高濃度韓國泡菜萃取物粉末可藉由在減壓下濃縮所得韓國泡菜萃取物以獲得濃縮物,且冷凍乾燥濃縮物,且使用研磨機來製備。
在本申請案之一個實施方式中,韓國泡菜萃取物意欲包括但不限於藉由用水、有機溶劑或其等之混合溶劑萃取韓國泡菜所獲得之萃取物、該萃取物之濃縮物或粉末。有機溶劑可為選自由以下組成之群之任何者:醇,尤其C1
-C4
低碳數醇,諸如甲醇、乙醇、丙醇或丁醇、己烷(正己烷)、醚、丙三醇、丙二醇、丁二醇、乙酸乙酯、乙酸甲酯、二氯甲烷、氯仿、苯及其等之混合溶劑。特定言之,萃取溶劑可為選自由以下組成之群之任何者:水、C1
-C4
醇以及C1
-C4
醇及水之混合溶劑。舉例而言,萃取溶劑可為甲醇水溶液或乙醇水溶液。甲醇或乙醇之水溶液可為10%(v/v)至99%(v/v)水溶液、特定言之20%(v/v)至70%(v/v)水溶液、且更特定言之30%(v/v)至60%(v/v)水溶液,但本申請案不限於其等。
在本申請案之一個實施方式中,萃取可在5℃至100℃之溫度下、特定言之在20℃至95℃之溫度下、且更特定言之在50℃至90℃之溫度下進行,但本申請案不限於其等。另外,萃取可進行1至20小時、特定言之2至16小時、且更特定言之3至12小時,但本申請案不限於其等。
在本申請案之一個實施方式中,萃取可進行一次至八次、特定言之一次至六次、且更特定言之一次至五次,但本申請案不限於其等。萃取物可為自各萃取獲得之單一萃取物或自各萃取獲得之萃取物之混合萃取物,特定言之自各萃取獲得之萃取物之混合萃取物。
在本申請案中,術語「改善」係指但不限於至少減小參數,諸如與藉由投予本申請案之韓國泡菜或其萃取物來進行的治療相關之症狀的程度之所有作用。本申請案之幽門螺旋桿菌相關疾病之改善可為腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133自身之作用,且可為由蔬菜被菌株醱酵引起之作用,但機制不限於其等。
在本申請案中,該組成物可為健康功能性食品。
術語「健康功能性食品」為與用於特殊健康用途之食品(food for special health use,FoSHU)相同的術語,且係指除了養營素供應之外,經處理以有效地發揮身體調節功能,因此具有高醫藥及醫療作用之食品。本文中,術語「功能(性)」意謂涉及控制用於人體之結構及功能之營養素或獲得於衛生用途之有益作用(諸如在生理功能中)。
健康食品組成物可使用本發明所屬技術領域中習用之方法製備。本發明所屬技術領域中習用之材料及成分可用於製備健康食品組成物。另外,可將健康食品組成物製備成被視為食品之任何調配物,但不限於其等。健康食品組成物可被製備成各種形式之調配物,且比起一般藥物具有以下優勢:健康食品組成物無在長期攝取藥物時可能出現的副作用,而此係因為其係基於食品材料。此外,健康食品組成物具有高便攜性,使得其可以以用於促進對幽門螺旋桿菌相關疾病之症狀之改善作用的助劑的形式攝取。
健康食品係指與一般食品相比具有活性健康維持或促進作用之食品,且健康補充食品係指用於健康補充目的之食品。視具體情況,互換地使用術語功能性食品、健康食品及健康補充食品。
在本申請案中,本申請案之組成物可進一步包括生理學上可接受之載劑。載劑之類型不受特定限制,且可使用但不限於任何載劑,只要其等在本發明所屬技術領域中常用即可。此外,本申請案之組成物可包括常用於食品組成物中以改善氣味、味道、視覺外觀、及類似者之其他成分。舉例而言,本申請案之組成物可包括維生素A、維生素C、維生素D、維生素E、維生素B1、維生素B2、維生素B6、維生素B12、菸鹼酸、生物素、葉酸、泛酸、及類似者。另外,本申請案之組成物可包括礦物質,諸如鋅(Zn)、鐵(Fe)、鈣(Ca)、鉻(Cr)、鎂(Mg)、錳(Mn)、銅(Cu)、及類似者;及胺基酸,諸如離胺酸、色胺酸、半胱胺酸、纈胺酸、及類似者。
另外,本申請案之組成物可包括食品添加劑,諸如防腐劑、滅菌劑、抗氧化劑、著色劑、固色劑、漂白劑、調味料、甜味劑、調味劑、膨潤劑、增強劑、乳化劑、增稠劑、塗層、泡沫抑制劑、溶劑、改善劑等。可根據食品類型選擇添加劑,且以適當量使用。該添加劑之比例不重要,但通常選擇每100重量份本申請案之組成物0.01至5重量份之範圍內之該添加劑之比例。
可視使用目的而適當地決定本申請案之組成物中所含之韓國泡菜或其萃取物之含量。在本申請案之一個實施方式中,按組成物之總重量計,本申請案之組成物中所含之韓國泡菜或其萃取物之含量可為但不限於0.0001至20.0重量%,例如且具體言之0.001至1.0重量%。當韓國泡菜或其萃取物之含量在以上範圍內時,有可能用低成本量對幽門螺旋桿菌相關疾病之症狀獲得改善作用。然而,用於健康及衛生或健康控制之長期攝取之韓國泡菜或其萃取物之量可低於以上範圍。因為韓國泡菜或其萃取物不具有任何安全問題,其可以比以上範圍更多的量使用。
除了本申請案之韓國泡菜或其萃取物之外,本申請案之組成物可進一步包括製造食品中通常所添加之成分,且例如,組成物可包括蛋白質、碳水化合物、脂肪、營養素、調味品、調味劑、及類似者。該等碳水化合物之實例為單糖,諸如葡萄糖、果糖、及類似者;雙糖,諸如麥芽糖、蔗糖、寡糖、及類似者;及習知糖,諸如多糖,諸如糊精、環糊精、及糖醇,諸如木糖醇、山梨醇、及赤藻糖醇。作為調味劑,可使用天然調味劑,諸如索馬甜(Thaumatin)及甜菊(Stevia)萃取物、及合成調味劑。舉例而言,當將本申請案之健康食品組成物製備為飲料時,除了本申請案之韓國泡菜萃取物外,可進一步包括檸檬酸、高果糖玉米糖漿、糖、葡萄糖、乙酸、蘋果酸、果汁、天然萃取物、及類似者。
組成物之類型不受特別限制。可將本申請案之韓國泡菜或其萃取物添加至其中的食品之實例為肉、香腸、麵包、巧克力、糖果、點心、糖果甜點、批薩、拉麵、其他麵條、口香糖、包括冰淇淋之乳製品、各種湯、飲品、茶、飲料、酒精類飲品、及維生素錯合物,且可包括所有通常意義上的健康食品。
因為本申請案之韓國泡菜或其萃取物包括腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133,且此兩種乳酸菌具有高度抑制幽門螺旋桿菌之活性之特徵,所以包括本申請案之韓國泡菜或其萃取物之組成物可有效地用於預防或治療由幽門螺旋桿菌引起之相關疾病。
3.用於預防、治療或改善幽門螺旋桿菌相關疾病之方法 , 該方法使用本申請案之韓國泡菜或其萃取物
本申請案提供一種用於預防、治療或改善幽門螺旋桿菌相關疾病之方法,其包含以下步驟:向患有或有可能罹患幽門螺旋桿菌相關疾病之個體投予本申請案之韓國泡菜或其萃取物。
本申請案提供本申請案之韓國泡菜或其萃取物用於預防、治療或改善幽門螺旋桿菌相關疾病之用途。
本申請案提供本申請案之韓國泡菜或其萃取物用於製備供預防、治療或改善幽門螺旋桿菌相關疾病之用的組成物之用途。
本申請案之韓國泡菜或其萃取物及其製備方法與「1. 包括腸膜狀明串珠菌 CJLM119 及胚芽乳酸桿菌 CJLP133 之韓國泡菜及其製備方法
」及「2. 用於改善幽門螺旋桿菌相關疾病之組成物 ,其 包括本申請案之韓國泡菜或其萃取物
」中提及者相同,且因此省略其描述。
在本申請案中,術語「預防」意欲包括藉由投予本申請案之韓國泡菜或其萃取物抑制或延緩幽門螺旋桿菌相關疾病之症狀之所有作用。
另外,在本申請案中,術語「治療」意欲包括藉由投予本申請案之韓國泡菜或其萃取物改善或有益地改變幽門螺旋桿菌相關疾病之症狀之所有作用。
另外,在本申請案中,術語「投予」係指以適當方式將預定物質引入個體中。
另外,在本申請案中,術語「個體」係指所有動物,諸如大鼠、小鼠及家畜,包括人類。其具體實例可為哺乳動物,包括人類。作為另一實例,個體可為患有或有可能罹患幽門螺旋桿菌相關疾病者。
在本申請案之方法中,本申請案之韓國泡菜或其萃取物之投藥途徑及投藥模式不受特別限制,且只要包含本申請案之韓國泡菜或其萃取物之組成物可達至所需目標位置,則可遵循任何投藥途徑及任何投藥模式。特定言之,韓國泡菜或其萃取物可藉由各種途徑,包括經口或非經腸途徑投予,但經口投予為常見的。
在本申請案之一個實施方式中,新近研究作為用於幽門螺旋桿菌感染之膳食干預之食品組成物的癌症預防韓國泡菜之組成物。在本申請案之另一實施方式中,假設小鼠模型中之用癌症預防性韓國泡菜進行之膳食干預可典型地預防、改善或治療幽門螺旋桿菌相關疾病,諸如胃炎、胃潰瘍、胃癌、及類似者。癌症預防性韓國泡菜可藉由在習知韓國泡菜製備方法中添加具有高含量番茄紅素之植物、腸膜狀明串珠菌CJLM119菌株及胚芽乳酸桿菌CJLP133菌株來製備,但本申請案不限於其等。在本申請案之一個實施方式中,根據本申請案之韓國泡菜或其萃取物展示針對幽門螺旋桿菌相關疾病,尤其胃炎、胃潰瘍、及胃癌之極佳防治及治療作用。
根據本申請案之一個實施方式,與屬於相同物種之其他菌株相比,用於製備本申請案之韓國泡菜之乳酸菌腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株具有針對幽門螺旋桿菌之顯著抑制活性。
根據本申請案之另一實施方式,包括腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株之韓國泡菜展示在預定濃度下針對幽門螺旋桿菌之抑制作用(參見圖1)。
根據本申請案之另一實施方式,本申請案之韓國泡菜可顯著抑制正常胃細胞中血管生長因子及發炎性因子之表現,且恢復細胞之緊密接合(參見圖2至4)。
根據本申請案之另一實施方式,本申請案之韓國泡菜可顯著抑制胃癌細胞之生長,誘發胃癌細胞之凋亡(參見圖5及6),且抑制經幽門螺旋桿菌感染之動物模型中胃癌之形成(參見圖10)。
因此,本申請案之包括腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株之韓國泡菜呈現針對幽門螺旋桿菌之顯著抑制活性,且因此可有效地用於預防、治療或改善可由幽門螺旋桿菌引起之各種疾病,諸如胃炎、胃潰瘍、及胃癌。
在下文中,將藉由實施例詳細描述本申請案。
然而,以下實施例僅特定說明本申請案,且本申請案之內容不受以下實施例限制。
首先,將描述本申請案之韓國泡菜之製備方法、測試方法及統計資料。統計分析用GraphPad Prism(GraphPad Software, 拉荷雅, CA, USA)及SPSS軟體(12.0版;SPSS Inc., 芝加哥, IL, USA)進行。各組之間之統計顯著性藉由Mann-Whitney U測試測定。統計顯著性在p < 0.05下為可接受的。實施例 1 :分離針對幽門螺旋桿菌細菌具有高生長抑制活性之菌株
<1-1>菌株分離及鑑別
腸膜狀明串珠菌CJLM119(其為衍生自韓國泡菜之乳酸菌且用於製備本申請案之韓國泡菜)為以寄存編號KCTC 13043BP寄存在韓國生物科學及生物技術研究院基因庫之微生物菌株,且在韓國專利第10-1807995號中具體揭示了此菌株之分離方法及鑑別。另外,胚芽乳酸桿菌CJLP133(其為衍生自韓國泡菜之乳酸菌且用於製備本申請案之韓國泡菜)為以寄存編號KCTC 11403BP寄存在韓國生物科學及生物技術研究院基因庫之微生物菌株,且在韓國專利第10-1486999號中具體揭示了此菌株之分離方法及鑑別。因此,省略其詳細描述。
在本申請案中,根據習知製備方法使用腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株製備韓國泡菜。
<1-2>選擇針對幽門螺旋桿菌細菌具有高抑制活性之菌株
量測在實施例<1-1>中分離及鑑別之腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株以及用作對照組之腸膜狀明串珠菌KCTC3100/KCTC3722及胚芽乳酸桿菌KCTC3108/DSM17853的幽門螺旋桿菌抑制活性。
結果,與用作對照組及屬於相同物種之其他菌株相比,腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株展示針對幽門螺旋桿菌之顯著抑制活性。實施例 2 :製備韓國泡菜
如下製備標準韓國泡菜(在下文中,有時稱為『s韓國泡菜』或『SK』)(其為對照組)以及根據本申請案之癌症預防性韓國泡菜(在下文中,有時稱為『Cp韓國泡菜』或『CPK』)。
<2-1>製備標準韓國泡菜(s韓國泡菜)
基於韓國泡菜世界學會(World Institute of Kimchi)之標準化韓國泡菜配方,藉由混合鹽漬大白菜與調味料(包括紅辣椒粉、大蒜、薑、鮆醬、白蘿蔔泥、大蔥、糯米糊、及糖)且隨後在低溫下進行醱酵來製備標準韓國泡菜。
<2-2> 製備癌症預防性韓國泡菜(Cp韓國泡菜)
癌症預防性韓國泡菜主要由具有高含量番茄紅素之大白菜製成。購買具有高含量的普通大白菜中不存在之番茄紅素之大白菜品種「TheHan1ho」(2015年12月15日公開的品種申請案公開第2015-688號)之種子。大白菜透過與大白菜農民契作獲得,且在根據韓國泡菜製備方法加鹽之後,收穫的大白菜用於癌症預防性韓國泡菜。癌症預防性韓國泡菜以與製備標準韓國泡菜相同之方式製備,不同之處在於:使用具有高含量番茄紅素之大白菜作為所用大白菜,且添加兩種類型之衍生自韓國泡菜之乳酸菌,即腸膜狀明串珠菌CJLM119及胚芽乳酸桿菌CJLP133菌株,其等在實施例 <1-1>中分離及鑑別。
<2-3>製備韓國泡菜萃取物之濃縮物
將在實施例<2-1>及<2-2>中製備之韓國泡菜冷凍乾燥,且隨後碾磨成粉末。向粉末中添加20倍重量之甲醇粉末,且隨後用攪拌器進行萃取12小時。所萃取上清液經由濾紙過濾,且再次將甲醇添加至殘留沈澱物中以進行萃取12小時。萃取物在約40℃之溫度下利用濃縮器進行熱濃縮以製備濃縮物,且濃縮物在4℃下儲存以用於進一步實驗。實施例 3 :測定針對幽門螺旋桿菌之生長抑制作用
<3-1>培養幽門螺旋桿菌菌株
幽門螺旋桿菌菌株ATCC 43504(美國菌種保存中心(American Type Culture Collection),其為cagA+及vacA s1-m1型的菌株)在微好氣性條件(BD GasPaK EZ Gas Generating Systems, BD Biosciences)下在37℃下在具有5%綿羊血之BBL胰酪酶大豆(TS)洋菜培養基(TSAII;BD Biosciences, Franklin Lakes, NJ)中培養3天。幽門螺旋桿菌菌株在TS培養基中收集,以3000 × g離心5分鐘,且以109
個群落形成單位(CFU)/mL之最終濃度再懸浮於培養基中。在所有實驗中,使用在TS洋菜培養基中生長72小時之培養物。
<3-2>測定針對幽門螺旋桿菌之生長抑制活性
為測定針對幽門螺旋桿菌之本申請案之韓國泡菜之抑制作用,將實施例<3-1>之以幽門螺旋桿菌菌株接種之培養基用不同濃度的實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理及培養,且量測抑制區之大小。
結果,發現到本申請案之韓國泡菜萃取物之濃縮物於500 mg/ml或更高之濃度對幽門螺旋桿菌細菌具有抑制作用(圖1)。實施例 4 : 測定 對 正常胃細胞系 ( RGM1 ; 正常胃黏膜細胞 ) 中幽門螺旋桿菌相關疾病之作用
<4-1>測定血管新生因子之抑制活性
為測定本申請案之韓國泡菜對血管生長因子之作用,將正常胃細胞系RGM1細胞用50 MOI幽門螺旋桿菌處理,且隨後用5 mg/mL之濃度的在實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理6小時。處理後,移除培養基組分,且用PBS(Dulbecco)溶液洗滌細胞兩次。隨後,將RiboEX(500 μl,GeneAll, 首爾,南韓)添加至在4℃下培育10分鐘之板中以進行RT-PCR。將RiboEX收集,且置放於1.5 ml管中,且緩慢添加100 μl氯仿及混合。在冰上培育10分鐘之後,以10,000 g離心樣本30分鐘。將上清液用200 μl異丙醇萃取且混合,且在4℃下培育混合物1小時。以13,000 g離心30分鐘之後,將沈澱物用70%(vol/vol)乙醇洗滌。完全蒸發乙醇,且隨後將沉澱丸粒溶解於100 μl經DEPC(Invitrogen Life Technologies, 卡爾斯巴德,加州)處理的水中。隨後,進行PCR以測定相關基因之變化。
結果,發現到為藉由用幽門螺旋桿菌處理而增加之血管新生因子之指標的VEGF及bFGF之表現在癌症預防性韓國泡菜處理組(CPK)中降低(圖2)。
<4-2>比較抗炎作用
為確認本申請案之韓國泡菜之抗炎作用,將RGM1細胞用50 MOI幽門螺旋桿菌處理,且隨後用5 mg/mL之濃度的在實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理。隨後,根據實施例<4-1>中之RT-PCR方法測定相關基因之變化。
結果,發現到為藉由用幽門螺旋桿菌處理而增加之發炎之指標的COX-2及iNOS之表現在癌症預防性韓國泡菜處理組(CPK)中降低(圖3)。
<4-3>比較細胞接合作用
為測定本申請案之韓國泡菜對細胞之緊密接合之指標之作用,將RGM1細胞用100 MOI幽門螺旋桿菌處理,且隨後用5 mg/mL及10 g/mL之濃度的在實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理6小時。此時,將由於細胞增殖而匯合之單細胞層之上半部分用0.5 ml冷D-PBS(-)洗滌三次,且用0.5 ml冷HBSS填充,並轉移至含有1.5 ml冷HBSS之新槽孔中。在使槽孔靜置30分鐘之後,量測上層與下層之間的跨上皮電阻(TERR%)。
結果,發現到在幽門螺旋桿菌感染模型中,細胞之緊密接合在用高濃度的本申請案之癌症預防性韓國泡菜處理時恢復(圖4)。實施例 5 : 測定胃癌細胞系 ( MKN28 ; 胃癌細胞 ) 中幽門螺旋桿菌相關疾病之作用
<5-1>測定對胃癌細胞系之生長抑制作用
為測定本申請案之韓國泡菜對胃癌細胞系之生長抑制作用,將MKN28細胞用分別為10、20及50 mg/ml之濃度之標準韓國泡菜及癌症預防性韓國泡菜之實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理6小時。隨後,比較各處理組之細胞活力。
結果,發現到與標準韓國泡菜相比,本申請案之癌症預防性韓國泡菜可以濃度依賴性方式更顯著抑制胃癌細胞系之生長(圖5)。
<5-2>測定對胃癌細胞系之凋亡誘發作用
為測定具有無凋亡之特徵之癌細胞中癌症預防性韓國泡菜處理之凋亡誘發作用,各細胞用藉由適當濃度的標準韓國泡菜及癌症預防性韓國泡菜之實施例<2-3>中之方法製備之韓國泡菜萃取物之濃縮物處理48小時,且隨後用含有1% FBS之1× PBS回收。將AnnexinV & Dead Cell Reagent以1:1分配至以不同濃度回收之各細胞,且隨後將細胞在室溫下在黑暗條件下反應20分鐘,且量測及分析凋亡誘發細胞之比率。
結果,發現到在用本申請案之癌症預防性韓國泡菜處理MKN28胃癌細胞系時,凋亡在70%或更多細胞中誘發(圖6)。
<5-3>測定胃癌細胞系之凋亡調節因子
為測定本申請案之韓國泡菜對胃癌細胞系之凋亡調節因子之作用,將MKN28胃癌細胞系用20 mg/ml濃度的實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理,且隨後移除培養基組分,且用PBS(Dulbecco)溶液洗滌細胞兩次。將細胞用含有1 mM PMSF(苯甲基磺醯氟,Sigma Aldrich, 聖路易, MO)之冰冷細胞溶胞緩衝液(Cell Signaling Technology, 丹佛, MA)溶胞。反應20分鐘之後,以10,000 × g離心各樣本10分鐘。隨後,獲得上清液。將溶胞產物之蛋白質由十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳(SDS-PAGE)分離,轉移至聚偏二氟乙烯(PVDF)膜,與初級抗體反應,洗滌,且隨後與和過氧化酶結合的二級抗體反應,再次洗滌,且隨後凋亡調節因子之表現之變化藉由ECL系統(GE Healthcare, 白金漢郡, UK)測定。
結果,發現到與在標準韓國泡菜處理組(SK)中者相比,涉及凋亡之經切割凋亡蛋白酶3、經切割凋亡蛋白酶8、及經切割PARP之表現在本申請案之癌症預防性韓國泡菜-處理組(CPK)中顯著提高(圖7)。
<5-4>測定胃癌細胞系之細胞週期相關因子
為測定本申請案之韓國泡菜對胃癌細胞系之細胞週期相關因子之作用,將MKN28胃癌細胞系用20 mg/ml濃度的實施例<2-3>中製備之韓國泡菜萃取物之濃縮物處理,且隨後移除培養基組分,且用PBS(Dulbecco)溶液洗滌細胞兩次。將細胞用含有1 mM PMSF(Sigma Aldrich, 聖路易, MO)之冰冷細胞溶胞緩衝液(Cell Signaling Technology, 丹佛, MA)溶胞。反應20分鐘之後,以10,000 × g離心各樣本10分鐘。隨後,獲得上清液。將溶胞產物之蛋白質由SDS-PAGE分離,轉移至PVDF膜,與初級抗體反應,洗滌,且隨後與和過氧化酶結合的二級抗體反應,再次洗滌,且隨後細胞週期相關蛋白質之表現之變化藉由ECL系統(GE Healthcare, 白金漢郡, UK)測定。
結果,與在標準韓國泡菜處理組(SK)中者相比,涉及細胞週期之週期蛋白D1、CDK4、CDK2、週期蛋白E、及週期蛋白A之表現在本申請案之癌症預防性韓國泡菜-處理組(CPK)中顯著減少。由此發現,胃癌細胞系之凋亡之機制係歸因於細胞週期調節(圖8)。實施例 6 : 測定在感染有幽門螺旋桿菌之動物之長期模型中對幽門螺旋桿菌相關疾病之作用
為測定在感染有幽門螺旋桿菌之動物之長期模型中本申請案之韓國泡菜對病理性參數之抑制作用,使五週齡雌性C57BL/6小鼠(Charles River Japan)可自由飲食滅菌的市售丸粒飼料(Biogenomics,南韓)及無菌水,且將其圈養於溫度24℃有空調房間中。適應1週之後,注射20 mg/kg泮托拉唑(pantoprazole)三次以藉由降低胃酸性促進幽門螺旋桿菌菌落化。且隨後將各動物使用胃插管針用相等體積(0.3 mL)之乾淨TS培養液用幽門螺旋桿菌之懸浮液(含有109
CFU/mL)胃內接種。給予對照組乾淨TS培養液之注射,且於一週內共四次給予所有實驗組幽門螺旋桿菌之注射。餵食小鼠含有7.5% NaCl之基於AIN76之特殊丸粒飼料4週以引起胃炎。
隨後,將幽門螺旋桿菌陽性小鼠隨機劃分成四組(n = 10)。投予含有7.5% NaCl之丸粒飲食AIN76 24週以在所有經感染動物中促進幽門螺旋桿菌誘導之致癌過程。對照組及實驗組分別展示於圖9中。分別對第1組:對照組,第2組:幽門螺旋桿菌感染組,第3組:幽門螺旋桿菌感染及癌症預防性韓國泡菜低濃度飼料組,第4組:幽門螺旋桿菌感染及癌症預防性韓國泡菜高濃度飼料組,第5組:幽門螺旋桿菌感染及標準韓國泡菜飼料組進行實驗。
結果,大體損傷指數在對照組G2中顯著提高,而在癌症預防性韓國泡菜處理組G3及G4中顯著降低(p<0.01)。另外,相當大尺寸的胃癌在對照組G2中形成,而胃癌之形成在癌症預防性韓國泡菜處理組G3及G4中減少(圖10及11)。
儘管上文已描述本申請案之例示性實施方式,但本申請案之範圍不限於如上所述之特定實施方式,本發明所屬技術領域中具有通常知識者可在本申請案之申請專利範圍中所述之範圍內改變本申請案。
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[圖1]為展示本申請案之韓國泡菜對幽門螺旋桿菌細菌之抑制作用的圖。
[圖2]為展示本申請案之韓國泡菜對各種血管生長因子之表現之作用的電泳圖。
[圖3]為展示本申請案之韓國泡菜對各種發炎性因子之表現之作用的電泳圖。
[圖4]為展示本申請案之韓國泡菜對幽門螺旋桿菌感染模型中之細胞之緊密接合之作用的圖。
[圖5]為展示本申請案之韓國泡菜對胃癌細胞系之生長之作用的圖。
[圖6]為展示本申請案之韓國泡菜對胃癌細胞系之凋亡之作用的圖。
[圖7]為展示本申請案之韓國泡菜對胃癌細胞系之各種凋亡因子之表現之作用的電泳圖。
[圖8]為展示本申請案之韓國泡菜對胃癌細胞系之細胞週期相關因子之表現之作用的電泳圖。
[圖9]展示測定本申請案之韓國泡菜對幽門螺旋桿菌相關疾病之作用之動物實驗的設計。
[圖10]為展示本申請案之韓國泡菜對幽門螺旋桿菌感染動物模型中之大體損傷指數評分之作用的圖。
[圖11]為展示本申請案之韓國泡菜對幽門螺旋桿菌感染動物模型之胃癌之發展之作用的照片。
Claims (12)
- 一種韓國泡菜,其係藉由對蔬菜加鹽及調味,且隨後用乳酸菌使其醱酵來製備,其中該乳酸菌包含腸膜狀明串珠菌(Leuconostoc mesenteroides )CJLM119(寄存編號:KCTC 11403BP)及胚芽乳酸桿菌(Lactobacillus plantarum )CJLP133(寄存編號:KCTC 13043BP)。
- 如請求項1之韓國泡菜,其中該等蔬菜選自由以下組成之群:大白菜(Kimchi cabbage)、白蘿蔔、大蔥、芥菜、黃瓜、及其等之組合。
- 如請求項2之韓國泡菜,其中該等蔬菜為大白菜。
- 如請求項3之韓國泡菜,其中該等大白菜為具有高番茄紅素含量者。
- 如請求項4之韓國泡菜,其中以每100 g該等大白菜0.05至5 mg之量含有該番茄紅素。
- 如請求項1之韓國泡菜,其中按該韓國泡菜之總重量計,以0.01至5重量%含有該等腸膜狀明串珠菌CJLM119,且按該韓國泡菜之總重量計,以0.01至5重量%含有該胚芽乳酸桿菌CJLP133。
- 一種用於改善幽門螺旋桿菌相關疾病之組成物,該組成物包含如請求項1至6中任一項之韓國泡菜或其萃取物。
- 如請求項7之組成物,其中該韓國泡菜萃取物為藉由用水、有機溶劑或其等之混合溶劑萃取韓國泡菜所獲得之萃取物。
- 如請求項8之組成物,其中該有機溶劑選自由以下組成之群:醇、己烷、醚、丙三醇、丙二醇、丁二醇、乙酸乙酯、乙酸甲酯、二氯甲烷、氯仿、苯及其等之混合溶劑。
- 如請求項8之組成物,其中該混合溶劑選自由以下組成之群:水、C1 -C4 醇以及該C1 -C4 醇及水之混合溶劑。
- 如請求項10之組成物,其中該混合溶劑為甲醇水溶液或乙醇水溶液。
- 如請求項7之組成物,其中該韓國泡菜萃取物選自由以下組成之群:萃取物、該萃取物之濃縮物、該濃縮物之乾燥產物及粉末。 酪酪酪酪
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CN115094001B (zh) * | 2022-06-24 | 2023-09-26 | 上海利康精准医疗技术有限公司 | 肠膜明串珠菌及其在制备抑制幽门螺杆菌的药物中的应用 |
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2018
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- 2019-12-27 EP EP19905385.1A patent/EP3903589A4/en active Pending
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US20220095636A1 (en) | 2022-03-31 |
EP3903589A2 (en) | 2021-11-03 |
TWI734322B (zh) | 2021-07-21 |
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JP7196314B2 (ja) | 2022-12-26 |
MY197563A (en) | 2023-06-23 |
CA3125104A1 (en) | 2020-07-02 |
AU2019416609A1 (en) | 2021-07-22 |
JP2022516116A (ja) | 2022-02-24 |
KR102651296B1 (ko) | 2024-03-26 |
AU2019416609B2 (en) | 2022-12-08 |
WO2020139020A3 (ko) | 2021-04-22 |
EP3903589A4 (en) | 2022-08-31 |
WO2020139020A2 (ko) | 2020-07-02 |
CN113412060A (zh) | 2021-09-17 |
KR20200081878A (ko) | 2020-07-08 |
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