CN105228637A - 包含接骨木提取物和鼠李糖乳杆菌菌株的组合的组合物 - Google Patents
包含接骨木提取物和鼠李糖乳杆菌菌株的组合的组合物 Download PDFInfo
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- CN105228637A CN105228637A CN201480028624.2A CN201480028624A CN105228637A CN 105228637 A CN105228637 A CN 105228637A CN 201480028624 A CN201480028624 A CN 201480028624A CN 105228637 A CN105228637 A CN 105228637A
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Abstract
本发明涉及接骨木(黑接骨木)提取物和鼠李糖乳杆菌菌株的组合,旨在刺激免疫力和/或增强免疫防御和/或促进抗感染和/或抗炎免疫反应和/或帮助保持活力。
Description
技术领域
本发明涉及包含接骨木提取物和至少一种鼠李糖乳杆菌(Lactobacillusrhamosus)菌株的组合的组合物,旨在刺激免疫力和/或促进抗感染和/或抗炎反应。
背景技术
接骨木或黑接骨木(SambucusnigraL.)属于五福花科(Adoxaceae)。接骨木属(Sambucus)包含分布在世界温带地区的25个种。黑接骨木(S.nigrassp.nigra)广泛生长于欧洲(特别是丹麦、意大利和奥地利)、非洲北部地区和亚洲西部。加拿大接骨木(S.nigrassp.Canadensis)尽管是在加拿大进行的选择(sélection),但其源自北美洲东北部,并生长于俄勒冈州、宾夕法尼亚州和堪萨斯州。
接骨木的不同器官:树皮、根、茎、花、叶和果实传统地用于各种领域,如医药、食品工业以及工具和玩具的制造。目前最重要的工业产品涉及接骨木浆果提取物的产品,其用于营养食品市场和染料工业。
一些出版物报导了接骨木浆果提取物对细胞因子生成的作用(Barak等人,EuropeanCytokinenetwork,Vol12(2),290-296,2001)。所述作用已被接骨木提取物对与由流感病毒(A和/或B和/或C)引起的流感感染相联系的症状的减轻所特别地证明,以及被接骨木提取物对1型单纯疱疹病毒、呼吸道合胞病毒和流感病毒的抗病毒作用所特别地证明(Zakay-Rones等人,TheJournalofInternationalMedicalResearch,vol32,132-140,2004)。
大量的科学研究已经证明存在于发酵食品,特别是乳制品中的一些微生物对健康的有益作用。通常将这些微生物称为“益生菌”。根据目前普遍接受的定义,益生菌为:活的微生物,当适量食用它们时,其对宿主的健康具有有益作用(针对食物中,包括含有活乳酸菌的奶粉中的益生菌的健康和营养特性的评估的世界卫生组织报告,科尔多瓦,阿根廷,2001年十月1-4日)。
在专利申请WO96/20607、EP0794707、EP1283714和FR292912657中已经显示,特别地通过重新平衡肠道菌群,提高对感染的抵抗力,以及调节免疫应答,食用含有益生菌的食品能够对健康产生有利的作用。
用于人类饮食中的益生菌微生物通常是乳酸杆菌,主要属于乳酸杆菌属(Lactobacillus)和双歧杆菌属(Bifidobacterium),特别是副干酪乳杆菌(Lactobacillusparacasei)种。
乳酸杆菌属是革兰氏阳性菌的属,不运动,有多变的形状和尺寸,任选地厌氧。由于其大部分成员将乳糖和其他简单糖转化为乳酸而得名。在人类中,乳酸杆菌作为共生者是非常普遍的宿主,通常是有益的,或甚至是必要的。它们构成肠道菌群的重要组成元素。
鼠李糖乳杆菌是一种最初被视为干酪乳杆菌(Lactobacilluscasei)的亚种的细菌,但是遗传研究已经证明其是一个单独的种。通常,在胃和消化道中发现鼠李糖乳杆菌。这种细菌具有对肠道有益的特性,但也具有对免疫系统,特别是在打击肠道和泌尿道的病原体中有益的特性。
益生菌可以对食糜、菌群产生直接的作用,称为内腔作用(d'effetsluminaux),或者在肠细胞或Galt免疫活性细胞的水平,则称为表皮作用(d'effetspariétaux)。它们也可以具有与生态系统或局部免疫系统的修饰相联系的间接的作用。
一些益生菌具有粘附于消化道上皮的能力。这种特性可以构成生态优势,有助于与肠上皮细胞和局部免疫系统的密切相互联系的机会。研究表明,鼠李糖乳杆菌的粘附菌株可以长时间地定殖在一些受试者的空肠和/或直肠黏膜上(Alander等人,LettersinAppliedMicrobiology,24(5),vol361-364,1997)。
越来越多的工作证明多菌株的生物学作用,重要的是要注意这些作用似乎是菌株依赖的。
专利申请US2006/0233895描述了包含接骨木提取物的组合物,所述提取物可能与益生菌例如干酪乳杆菌组合。然而,这种组合物必须包含绒毛钩藤(UncariaTomentosa)、保德科(Paud'Arco)、黄芩(Scrutellariabaicalensis)和青蒿素的混合物。此外,这种组合物并非旨在刺激免疫力,而是用于治疗莱姆病,所述莱姆病是由细菌感染引起的,因此其与流感型病毒引起的感染是非常不同的。
专利申请WO2010/043696公开了包含黑接骨木提取物和副干酪乳杆菌、干酪乳杆菌、保加利亚乳杆菌(Lactobacillusbulgaricus)或嗜热链球菌(Streptococcusthermophilus)的菌株的组合的组合物,旨在刺激免疫力。在该申请中,IL-10和干扰素-伽马的生成证明了接骨木和副干酪乳杆菌之间的协同作用。然而,炎症反应的限制是未知的。其他细胞因子和CXCL10类型的趋化因子的数据将消除在这种炎症反应中过于重要的增加的风险。
发明内容
因此,本发明涉及包含接骨木提取物和至少一种鼠李糖乳杆菌菌株的组合。
有利地,这样的组合是协同组合。
本发明还涉及包含这样的组合的组合物,并且有利地涉及包含接骨木提取物和至少一种鼠李糖乳杆菌菌株的组合物。
本发明还涉及包含接骨木提取物和至少一种鼠李糖乳杆菌菌株以及维生素和/或矿物盐的组合物。
在一个特别的实施方案中,根据本发明的组合物可以通过包括以下步骤的方法制备:
-组合的制备;
-将组合加入至根据本发明的组合物中。
在这种情况下,因此,在将组合加入至组合物之前制备组合。然而,也可以通过以单独的方式将组合的每个组分加入至组合物制备根据本发明的组合物,也就是说不事先制备组合。
优选地,根据本发明的组合物旨在用于通过口服途径施用。
有利地,根据本发明的组合物为食品、食品补充剂、药物或OTC(非处方)产品。
有利地,根据本发明的接骨木提取物是从接骨木浆果和/或花中获得的,优选从接骨木浆果中获得。以有利的方式,根据本发明的接骨木提取物是水溶性提取物。
在本发明的范围内使用的接骨木提取物一方面可以以其花青素含量为特征,另一方面可以以大分子例如蛋白质(特别是凝集素)为特征。花青素是叶、花瓣和果实的天然色素,位于细胞的液泡中,可溶于水,在可见光谱中为橙红色至紫蓝色。
有利地,所述接骨木提取物包含花青素,以有利的方式包含花葵素(pelargonidines)和矢车菊素(cyanidines)家族,与提取物的干物质相比,其具有以重量计约0.5%至约25%,以有利的方式约3%至约25%,以更加有利的方式约8%至约16%的量。优选地,与提取物的干物质相比,花青素含量等于以重量计约12%。根据Wu等人描述的HPLC方法(J.Agric.FoodChem.52(26),7846-7856,2004),这些含量以矢车菊素-3-葡萄糖苷表示。
有利地,接骨木提取物包含以重量计2至10%的量的蛋白质,以相比于干物质表示(N×6.25根据Kjeldahl方法:Protéinesvégétales,Coord.B.Godon,CollectionSciencesettechniquesagro-alimentaires,TechniqueetDocumentationLavoisier,Paris,1985)。
在本发明的范围内,所述接骨木提取物是市售可得的,或者可以通过包括以下步骤的方法获得:
新鲜的或冷冻的接骨木浆果可以经历挤压以在过滤后获得果汁。其可以经历酶水解步骤(果胶酶或果胶甲酯酶和聚半乳糖醛酸酶的混合物)以在过滤前使其澄清(Girard和Fukumoto,Crit.Rev.FoodSci.Nutr.40(2),91-157,2000)。也可以对干燥且磨碎的浆果进行水性提取,然后通过过滤进行固/液分离。由此获得的提取物可以原样使用或浓缩、或干燥为粉末的形式使用。
在这个阶段,与提取物的干物质相比,花青素含量为以重量计0.5%至3%,有利地等于约1%(HPLC测定,以矢车菊素-3-葡萄糖苷表示)。
所述接骨木提取物可以富集特别是花青素和/或高分子量分子(蛋白质、多酚、多糖)。为此,本领域技术人员公知的几种方法是可行的:
-超滤:对预先稀释的果汁渗滤,然后在具有1至20kDa,优选3至10kDa的截留阈值的有机或无机膜上的过度浓缩(surconcentration)步骤(Girard和Fukumoto,Crit.Rev.FoodSci.Nutr.40(2),91-157,2000)。
-通过AmberliteXAD型吸收性聚合物树脂柱。
-用水-醇(水和C1至C4醇以所有比例的混合物)或水性溶液在酸性pH下提取(Bronnum-Hansen和Flink,Int.J.FoodSci.Tech.21(2),605-614,1986;Lee和Wrolstad,J.FoodSci.69(7),564-573,2004)。可以有利地直接在干燥且磨碎的浆果上进行这种提取,或在由新鲜浆果制备的果泥上进行这种提取。
由此获得的提取物可以加热浓缩(在不高于50℃的温度,并且在真空下)以增加白利糖度(Brix)程度并使其对微生物污染稳定。其也可以单独干燥或在载体(例如麦芽糊精、乳糖等)上干燥。
获得的经富集的果汁优选具有40至60°的白利糖度,与提取物的干物质相比以重量表示的3至25%的花青素和5至10%的蛋白质(Kjeldahl方法,N×6.25)。
有利地,根据本发明,组合物中存在的每单位剂量的接骨木干提取物的量为约10mg至约1g。更有利地,所述组合物包含每单位剂量约20mg至约200mg的接骨木干提取物,以更有利的方式每单位剂量约20至100mg的接骨木干提取物,以甚至更有利的方式每单位剂量40mg至80mg的接骨木干提取物,以另一个同样有利的方式每单位剂量20至60mg的接骨木干提取物。
在本发明的范围内,术语“单位剂量”表示单次施用的根据本发明的组合物的量。有利地,根据本发明的单位剂量可以对应于,例如100或125ml的酸奶、常规尺寸的胶囊或2g的片剂。因此,无论组合物的重量是多少,每单位剂量接骨木干提取物和/或鼠李糖乳杆菌菌株的量保持不变。
特别适用于本发明的范围内的鼠李糖乳杆菌菌株为鼠李糖乳杆菌GG(ATCC53103)菌株。这种鼠李糖乳杆菌菌株为1983年在健康人的肠道中分离的菌株。该菌株是专利US4839281的主题。已证明鼠李糖乳杆菌GG菌株对于预防在成人和儿童中不同类型的腹泻是有益的(Guandalini等人,J.Pediatr.Gastroenterol.Nutr.30(1),54-60,2000;Osterlund等人,Br.J.Cancer97,1028-1034,2007)。也已报导鼠李糖乳杆菌GG菌株是儿童呼吸道感染风险降低的原因(Hojsak等人,Clin.Nutr.29(3),312-316,2010)。
鼠李糖乳杆菌对于免疫系统也是重要的援助手段,特别是在对抗肠道和尿道的致病因子中。鼠李糖乳杆菌将自身附着于肠道的粘膜,在那里它促进帮助消化的有益菌生长。鼠李糖乳杆菌是帮助消灭并阻止有害菌在肠内生长的细菌。
有利地,所述组合物将包含每单位剂量1.107至1.1011UFC,优选每单位剂量1.108至1.109UFC,以甚至更优选的方式每单位剂量5.108UFC的鼠李糖乳杆菌。
在本发明的一个特别的实施方案中,每单位剂量组合物包含,
-10mg至1g,优选20mg至200mg,有利地20mg至100mg,更优选40mg至80mg,和以另一个同样有利的方式20mg至60mg的接骨木干提取物。
-每单位剂量1.107至1.1011,优选1.108至1.109UFC的鼠李糖乳杆菌。
在本发明的一个特别的实施方案中,每单位剂量组合物包含,
-2mg至200mg,优选2mg至100mg,更优选2mg至20mg,和以另一个同样有利的方式20mg至50mg的接骨木花青素。
-2mg至100mg,优选10mg至20mg的接骨木蛋白质;和
-每单位剂量1.107至1.1011,优选1.108至1.109UFC的鼠李糖乳杆菌。
以一个有利的方式,然后观察协同效应。
根据本发明的接骨木提取物和鼠李糖乳杆菌菌株的组合已显示出良好的诱导和/或刺激非特异性免疫应答的能力,由此表明其在食品和/或药物领域的重要性。
根据本发明的组合物可以有利地以通常用于食品和/或药物领域的用于口服或舌下途径施用的全部剂型出现。
在食品的情况中,其可以特别地为鲜乳制品、发酵乳制品、酸奶、发酵乳、婴儿乳、粉末、锭剂、蔬菜汁、饮料及其混合物,有利地选自鲜乳制品、水果和/或蔬菜汁或水果罐头(compotesdefruits)。
以有利的方式,所述水果选自苹果、橙子、红果、草莓、桃、杏、李子、覆盆子、黑莓、红醋栗、柠檬、葡萄柚、香蕉、菠萝、猕猴桃、梨、樱桃、椰子、西番莲果、芒果、无花果、大黄、甜瓜、奇异果、荔枝、葡萄干、越桔或其混合物。
在本发明的意义之内,“食品补充剂”是指食品,其目的是使普通饮食完整,其构成营养物或其他具有单独的或组合的营养或生理学作用的物质的浓缩源。
有利地,所述食品补充剂可以包含维生素和/或矿物质和/或微量元素。
在这些维生素中,可以特别地列举维生素A、C、D和E,以及B族维生素,尤其是,B1、B2、B3、B5、B6、B8、B9和B12。
维生素A(视黄醇或类胡萝卜素)、维生素E和维生素C是3种主要的抗氧化维生素。维生素A独特地存在于动物源性食品中。然而,某些植物含有维生素A原,也就是身体能将其转化为维生素A的物质。它对于视力,以及支气管、肠或皮肤的生长是必不可少的。维生素A也参与免疫机制。抗氧化物质通过减弱自由基的损伤、修复细胞遭受的损伤、清除废物、阻止氧化现象来对抗自由基的作用。当自由基在体内过量时,它们可以促进细胞衰老。这些抗氧化维生素具有控制来源于氧代谢的自由基的产生的能力,并且自由基的过度产生是很多种疾病的原因,尤其是在心血管水平、免疫、癌症、皮肤、加速老化、神经退化性疾病等。特别地,维生素C是众所周知的抗氧化剂,可溶于水。人类依赖于维生素C的外部来源,以满足其维生素C的需求。因此,抗坏血酸、抗坏血酸钠或其混合物是维生素C的来源。维生素C对于血管和肌肉的合成是必需的。其促进食物中的铁的吸收,并干预一些激素机制。其也在有毒物质的清除中发挥作用。维生素C缺乏可以降低对感染的抵抗力。维生素E也是众所周知的抗氧化剂。维生素E可以与维生素C协同作用,以保护重要的细胞功能免受一般氧化剂的作用。醋酸α-生育酚、醋酸三甲基生育酚和/或维生素E琥珀酸盐是维生素E的来源。维生素E对于身体的细胞具有特别重要的保护作用。其在生育机制中发挥重要作用,并参与红细胞的合成。
维生素D被称为脂溶性维生素,尽管其首先是一种在光线中UVB射线的作用下在人体内由胆固醇衍生物合成的激素。其以2种形式存在,D2(钙化醇)或D3(胆钙化醇)。这两种分子是9,10-开环甾类化合物。维生素D参与肠对钙和磷的吸收,以及肾脏对它们的重吸收,它是真正的激素。另一方面,其影响超过200个基因,直到最近阐明其在多种疾病(关节炎、与牛皮癣相关的皮肤病、糖尿病、某些癌症和甚至痴呆)中出乎意料的重要性。
B族维生素形成一组水溶性维生素,其在细胞代谢中发挥重要作用,在其中可以发现:
-维生素B1或硫胺素,其是复合丙酮酸脱羧酶的辅助因子;
-维生素B2或核黄素,其是黄素腺嘌呤二核苷酸的前体;
-维生素B3(PP)或烟酰胺,其是烟酰胺腺嘌呤二核苷酸的前体;
-维生素B5或泛酸,其是辅酶A的前体和组成部分;
-维生素B6或吡哆醇,其是与氨基酸和蛋白质代谢相联系的反应的辅酶;
-维生素B8(H)或生物素,其是脂肪酸、碳水化合物和氨基酸代谢反应的辅酶;
-维生素B9或叶酸和维生素B12或氰钴胺素,其与S-腺苷甲硫氨酸是辅助因子。
B族维生素经常共同发挥作用以利于身体的健康。它们增强代谢,帮助保持健康的皮肤和肌肉的健康,改善免疫系统,改善神经系统并延缓阿尔茨海默症的认知障碍,促进细胞的生长及分裂,它们对抗压力过大引起的症状。
有利地,所述矿物质选自铁、锌、硒、铬、锰、钼及其混合物。矿物盐是来源于岩石的物质,其进入生物体的组成中,并存在于动物性和植物性饲料中。它们以离子形式出现,主要的矿物元素是众所周知的,例如钙、铁、镁、磷、钾、钠、硫,以及微量元素例如铝、砷、硼、氯、铬、钴、铜、氟、碘、锰、钼、镍、铅、硅、硒、钒、锌。
铁是对于身体的正确运作必不可少的矿物盐之一。它对于构成包含在红细胞中的血红蛋白,构成包含在肌肉中的肌红蛋白,以及对于构成身体运作必不可少的众多酶均具有基础性作用。缺铁常见于世界各地,其导致贫血,伴有身体和智力能力的降低,对感染的抵抗力的降低。
锌作为直接参与防御氧化剂的酶的辅助因子,发挥抗氧化的作用。在根据本发明的组合物中可以单独使用或以混合物使用氧化锌、葡萄糖酸锌、柠檬酸锌、乙酸锌、氯化锌、乳酸锌或硫酸锌。有利地其为葡萄糖酸锌。
硒作为酶(特别是谷胱甘肽过氧化物酶,其直接参与防御氧化剂)的辅助因子也发挥抗氧化的作用。
铬是一种参与碳水化合物代谢的微量元素,其促进胰岛素的作用。其也参与脂质代谢和胆固醇代谢,其有助于降低其浓度。铬也在核酸代谢中发挥其作用。
锰是一种对于维生素B1的功效必不可少的微量元素。其也干预某些金属蛋白,例如超氧化物歧化酶。其用作许多酶的辅助因子。其也是用于合成参与对抗氧化应激的酶的必要金属,这预防自由基造成的损害。
钼作为某些酶(特别是黄嘌呤氧化酶)的构成涉及众多层面。它在骨骼生长和牙齿结构中发挥作用。其对铁的代谢具有有利的作用。钼还参与身体的解毒,并且其参与尿酸的合成。
可以根据本发明添加任选的另外的活性成分。例如,辅酶Q10和/或人参提取物。辅酶Q10或泛醌是类似于维生素的物质,其对于人体运作是至关重要的。辅酶Q10天然存在于所有人体细胞中,并确保在线粒体中身体能量的产生。大约95%的身体能量需求是依靠辅酶Q10转化的。该物质不能被任何其他物质代替。如果通过膳食补充剂增加辅酶Q10的摄入,运动能力得到加强。脂质代谢更加有效,最大耗氧量和运动持续时间也如是。辅酶Q10水平的降低与衰老有关,但也与压力有关。
人参是药用植物,其在亚洲享有盛誉。人参首先是神经、身体和智力系统的刺激物,并且增强身体抵抗力。其是刺激剂、血管舒缩剂,其对身体具有基本的作用,其使得对各种压力具有更好的抵抗力。
可以通过口服途径或任何其它药物施用途径施用根据本发明的组合物。
可以配制根据本发明的组合物用于施用给哺乳动物,包括人。制备这些组合物,以便能够通过口服、舌下、皮下、肌内、静脉内、经皮、局部或直肠途径施用。在这种情况下,可以以单位施用形式,与常规药物载体混合,将活性成分施用于动物或人类。
在食品补充剂或药物的情况下,可以设想下列剂型:胶囊剂、吞服片剂、咀嚼片剂、泡腾片、锭剂、丸剂、粉末剂、颗粒剂、口服溶液或混悬液,以及舌下和颊内施用形式、皮下、局部、肌内、静脉内、鼻内或眼内和直肠施用形式。优选的剂型为胶囊剂。
当以片剂形式制备固体组合物时,将活性成分与药物载体混合,所述药物载体例如明胶、淀粉、乳糖、硬脂酸镁、滑石、阿拉伯胶、二氧化硅或类似物。可以用蔗糖或其它合适的材料将片剂包衣,或者作为代替可以处理它们以使得它们具有延长的或延迟的活性,并且它们以连续的方式释放预先确定的活性成分的量。
通过将活性成分与稀释剂混合(任选步骤),并通过将得到的混合物倒入软或硬胶囊中获得胶囊剂。
糖浆剂或酏剂形式的制剂可以包含活性成分,以及甜味剂、矫味剂和合适的着色剂。
在水中可分散的粉末剂或颗粒剂可以包含与悬浮剂、以及矫味剂或甜味剂混合的活性成分。
对于直肠施用,采用栓剂,用在直肠温度熔化的粘合剂制备所述栓剂,所述粘合剂例如可可脂或聚乙二醇。
对于肠胃外(静脉内、肌内等)、鼻内或眼内施用,采用水性混悬剂、等渗盐水溶液或无菌和可注射的溶液,其含有分散剂和/或药理学上相容的润湿剂。
也可以将活性成分可能地与一种或更多种附加的载体配制为微囊的形式。
有利地,根据本发明的组合物旨在用于通过口服途径施用。
本发明的另一个目的是根据本发明的组合物,其用作药物。
本发明的另一个目的是根据本发明的组合物,其用于刺激免疫力和/或增强免疫防御和/或促进抗感染和/或抗炎反应和/或保持活力。
可以理解,与保持活力相关的用途是建立在要求维生素B2、B5、B6、B12、C和铁之上的。这些是欧洲食品安全局(EFSA)认可的对这些营养物的健康声明(allégations)。它们基于科学数据,所述数据通过碳水化合物、脂质或蛋白质的使用测量代谢产生的能量值。这也影响作用于代谢的激素例如甾体激素的合成。
因此,该用途得到下列参考文献的支持:
EFSAJournal2009;7(9):1215
Scientificopiniononthesubstantiationofhealthclaimsrelatedtoironandformationofredbloodcellsandhaemoglobin(ID249,ID1589),functionoftheimmunesystem(ID252,ID259),cognitivefunction(ID253)andcelldivision(ID368)pursuanttoArticle13(1)ofregulation(EC)n°1924/2006.
HuskissonE.,MagginiS.,RufM.,JournalofInternationalMedicalResearch2007,25:277-289.
Theroleofvitaminsandmineralsinenergymetabolismandwell-being.
InstituteofMedicine(1998),NationalAcademypress,Washington,DC(592pages).DietaryreferenceintakesforThiamine,Riboflavin,Niacin,VitaminB6,Folate,VitaminB12,Pantothenicacid,Biotin,andCholine.
Areportofthestandingcommitteeonthescientificevaluationofdietaryreferenceintakesanditspaneloffolate,otherBvitamins,andcholineandsubcommitteeonupperreferencelevelsofnutrientsfoodandnutritionboardinstituteofmedicine.
本发明的另一个目的是一种组合物,其用于治疗和/或预防呼吸道和/或肠道感染。
本发明的另一个目的是一种组合物,其用于治疗和/或预防尤其由1型单纯疱疹病毒、呼吸道合胞病毒和流感病毒(A和/或B和/或C)和副流感病毒感染引起的症状,有利地为尤其由流感病毒(A和/或B和/或C)引起的流感样状态的症状。
根据本发明的组合物特别适合于治疗和/或预防由流感病毒(正粘病毒(Orthomyxovirus),流感A、B或C)感染引起的流感样状态的症状。这些症状包括感冒、鼻炎、咳嗽、鼻粘膜炎症(鼻涕、鼻塞、打喷嚏)、咽喉痛(不同的表现:刺痛、吞咽困难)、发烧、头痛、疼痛、疲劳。
这些“冬季疾病”与我们免疫防御的减弱相关。
本发明的另一个目的是组合物用于制备食品、食品补充剂或药物的用途。
本发明的另一个目的是组合物用于制备药物的用途,所述药物旨在用于治疗和/或预防呼吸道和/或肠道感染。
本发明的另一个目的是组合物用于制备药物的用途,所述药物旨在用于治疗和/或用于预防由1型单纯疱疹病毒、呼吸道合胞病毒和流感病毒(A和/或B和/或C)以及副流感病毒感染的症状,有利地为尤其由流感病毒(A和/或B和/或C)引起的流感样状态的症状。
本发明的另一个目的是组合物用于制备药物的用途,所述药物用于刺激免疫力和/或促进抗感染和/或抗炎反应和/或帮助保持活力。
最后,本发明的另一个目的是提供一种加强免疫防御的方法,所述方法在于通过口服途径施用基于接骨木提取物和至少一种鼠李糖乳杆菌菌株的组合的组合物。
根据本发明的组合物适合于所有年龄的人。其特别适合于非常年幼的儿童。在本发明的意义之内,“非常年幼的儿童”是指6个月至3岁的儿童。根据本发明的组合物相当特别地适合于“青少年”,其中在本发明的意义之内“青少年”是指3岁至17岁的儿童。根据本发明的组合物也特别适合于成年人,其中在本发明的意义之内“成年人”是指18岁至60岁的人。不过根据本发明的组合物也相当特别地适合于“老年人”,其中在本发明的意义之内“老年人”是指至少60岁的人。
可以通过下面的非限制性实施例更好地理解本发明,所述实施例构成根据本发明的组合物的特别的实施方案。
具体实施方式
实施例1:在本发明范围内使用的接骨木提取物的制备
从经富集花青素的接骨木浆果果汁中获得接骨木提取物,并在麦芽糊精上干燥。通过根据如下方法的膜过滤方法进行富集:用284升水稀释26升澄清的接骨木果汁(1体积果汁对应11体积水)。搅拌由此获得的310升液体并加热至45℃,然后通过向每体积果汁中加入7体积水(即181L)在第一个5kDa膜上进行渗滤(diafiltrés)。然后,通过在5kDa膜(5.8m2单位表面的5/10kDa中试膜(membranepilote))上的超滤浓缩渗析渗透物。在不超过50℃的温度在真空下热浓缩超滤截留物,然后在麦芽糊精载体上雾化,以使得麦芽糊精的量低于或等于30%(p/p)。
由此获得的提取物的特征在于:
-花青素含量:与提取物的干物质相比,以重量计10-14%(以矢车菊素-3-葡萄糖苷表示,根据Wu等人,J.Agric.FoodChem.52(26),7846-7856,2004描述的HPLC方法)。
-蛋白质含量:与提取物的干物质相比,以重量计5-7%(N×6.25根据Kjeldahl方法)。
实施例2:胶囊剂形式的食品补充剂,这种组合物特别适合于成年人。
-冻干益生菌株,鼠李糖乳杆菌:35mg,即5.108UFC;
-根据实施例1的接骨木提取物(黑接骨木):50mg;
-维生素A:400μg;
-维生素B1:550μg;
-维生素B2:700μg;
-维生素B5:3mg;
-维生素B6:700μg;
-维生素B8:25μg;
-维生素B9:100μg;
-维生素B12:0.625μg;
-维生素C:40mg;
-维生素D:2.5μg;
-维生素E:6mg;
-铁:2.5mg;
-锌:2.5mg;
-硒:15μg;
-铬:12.5μg;
-锰:500μg;
-钼:12.5μg。
实施例3:小袋形式的食品补充剂,这种组合物特别适合于非常年幼的儿童。
-冻干益生菌株,鼠李糖乳杆菌:35mg,即5.108UFC;
-根据实施例1的接骨木提取物(黑接骨木):25mg;
-维生素C:18mg;
-维生素B6:600μg;
-维生素E:2.4mg;
-锌:2.5mg;
-硒:15μg;
实施例4:在来自外周血的单核细胞中鼠李糖乳杆菌菌株与接骨木
提取物的联合免疫调节剂作用的体外研究。
材料和方法
从健康受试者分离来自外周血的单核细胞,重新悬浮并以5×105细胞/孔的比率分配在24孔板中。在5%CO2下于37℃孵育这些细胞18h。或者在100μg/ml接骨木提取物存在下,即用5.105鼠李糖乳杆菌,或者在100μg/ml接骨木提取物和5.105鼠李糖乳杆菌存在下,用10μg/ml脂多糖刺激这些细胞。回收培养物上清液以通过流式细胞术分析不同的细胞因子,于4℃将它们以1600rpm离心10分钟,并保存在-20℃直至分析。
通过基于流式细胞术原理的Luminex技术进行细胞因子的分析。该Luminex技术基于ELISA原理,其用按照精确的比例包含两种荧光染料的微珠作为载体在96孔微孔板上分析,所述荧光染料赋予它们识别它们的颜色代码(不同的荧光)。细胞仪(Bio-Plex200)的光学系统由两个激光器组成:红色激光器(λ=635nm)激发每个微珠中定义它的染料混合物,因而确认待分析的细胞因子。第二个绿色激光器(λ=532nm)激发附着于特定检测抗体的报告荧光染料(fluorochromerapporteur),以量化细胞因子。由装备有数据采集和分析软件(Bio-PlexManagerversion4.1)的计算机管理系统。解冻后,上清液为测试纯的,并在培养基中稀释至1/20,使用Milliplex试剂盒(Millipore,参考HCYTOMAG-60K-29)。其包含用于分析下列细胞因子的特定珠、检测抗体和标准物:IL-1β、IL-1RA、IL-2、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12p70、IL-13、IL-15、IL-17A、CCL2、CCL3、CCL4、CCL5、CCL22、CXCL8、CXCL10、TNFα、IFNγ、GM-CSF。
每种细胞因子的浓度以pg/ml表示,其是获得自6个供体的值的平均值。每种细胞因子的检测阈值是3.2pg/ml。对于在检测阈值以下的样品,归为对应于检测阈值×1/2(即1.6pg/ml)的任意值。统计分析通过ANOVA比较每种细胞因子浓度的log10。
结果
每种刺激条件获得的细胞因子的浓度汇集在表1和2中。对于在细胞因子水平具有协同作用,以下是必要的:
-通过同时用益生菌和接骨木提取物刺激来自外周血的单核细胞而实验性地获得的细胞因子的浓度,大于单独地用接骨木和单独地用益生菌所观察到的细胞因子的浓度之和;
-6个供体用组合获得的细胞因子的中值浓度显著大于用接骨木获得的和用益生菌获得的细胞因子中值浓度。
表1总结了与益生菌鼠李糖乳杆菌组合的接骨木提取物的活性结果,以及每种活性成分对来自人类外周血的单核细胞生成细胞因子的贡献。
表1.
仅在经测试的6个供体中的2个中观察到响应于接骨木的IL-1β增加。益生菌在5/6的供体中触发IL-1β的生成。接骨木和益生菌的组合增加IL-1β的生成,但是没有达到统计学显著。
经测试的所有供体响应于接骨木生成IL-1RA(IL-1受体的拮抗剂),表明接骨木的抗炎活性。经测试的所有供体响应于鼠李糖乳杆菌生成IL-1RA。6个供体用接骨木益生菌组合获得的IL-1RA的浓度显著大于接骨木和益生菌。此外,用接骨木益生菌组合获得的IL-1RA的浓度大于通过益生菌获得的和通过接骨木获得的之和。因此,对于IL-1RA的生成2种活性成分具有协同作用。
仅在经测试的6个供体中的单独一个中观察到响应于接骨木的IL-2增加。6个供体中的三个通过中等程度的IL-2生成响应于益生菌。2种活性成分的组合对IL-2的生成没有影响。
没有一个经测试的供体通过IL-5的生成响应于接骨木。以同样的方式,没有一个经测试的供体通过IL-5的生成响应于益生菌。2种活性成分的组合对IL-5的生成没有影响。
经测试的全部供体响应于接骨木生成IL-6。6个供体中的五个响应于益生菌,应当注意的是,无响应者具有最高的IL-6基础率(tauxbasal)。对于响应者,相比于接骨木益生菌是更强大的诱导剂。2种活性成分的组合引起IL-6的协同生成。
表2显示与益生菌鼠李糖乳杆菌组合的接骨木提取物的活性结果的示例,以及每种活性成分对于获得自6个供体的人类外周血的单核细胞生成的IL-6的贡献。
表2
组别 | 供体1 | 供体2 | 供体3 | 供体4 | 供体5 | 供体6 |
对照 | 1.6 | 1.6 | 7 | 1.6 | 69 | 4 |
Prob | 148 | 301 | 968 | 362 | 51 | 1148 |
接骨木 | 17 | 27 | 44 | 12 | 405 | 17 |
Prob+接骨木 | 1258 | 1426 | 5398 | 1721 | 6081 | 5369 |
Prob:益生菌,鼠李糖乳杆菌
没有一个经测试的供体通过IL-9的生成响应于接骨木。类似地,没有一个经测试的供体通过IL-9的生成响应于益生菌。2种活性成分的组合对IL-9的生成没有影响。
经测试的6个供体中的四个响应于接骨木生成IL-10。经测试的所有供体响应于益生菌生成IL-10。6个供体用接骨木+益生菌获得的IL-10的浓度显著大于用接骨木获得的和用益生菌获得的IL-10的浓度。此外,用接骨木益生菌组合获得的IL-10的浓度大于通过益生菌获得的和通过接骨木获得的之和,因此,对于IL-10的生成2种活性成分具有协同作用。
没有一个经测试的供体通过IL-12p70的生成响应于接骨木。另一方面,所有供体响应于益生菌生成IL-12p70。接骨木和益生菌的组合没有增加(potentialisé)IL-12p70的生成。
没有一个经测试的供体通过IL-15的生成响应于接骨木。类似地,没有一个经测试的供体通过IL-15的生成响应于益生菌。2种活性成分的组合对IL-15的生成没有影响。
没有一个经测试的供体通过IL-17A的生成响应于接骨木。对于对益生菌的响应同样是如此:在任何供体中其不诱导IL-17A的生成。2种活性成分的组合对IL-17A的生成没有影响。
经测试的所有供体通过CCL2的生成响应于接骨木。6个供体中的五个响应于益生菌,应当注意的是,无响应者具有最高的CCL2基础率,相比于接骨木益生菌触发更强的响应。2种活性成分的组合刺激CCL2的高分泌,其显著大于接骨木但不大于益生菌。
经测试的全部供体通过CCL3的生成响应于接骨木。6个供体中的五个响应于益生菌,应当注意的是,无响应者具有最高的CCL3基础率,相比于接骨木益生菌触发更强的响应。6个供体用接骨木+益生菌获得的CCL3的浓度显著大于用接骨木获得的和用益生菌获得的CCL3的浓度。此外,用接骨木益生菌组合获得的CCL3的浓度大于通过益生菌获得的和通过接骨木获得的之和,因此,对于CCL3的生成2种活性成分具有协同作用。
经测试的全部供体通过CCL4的生成响应于接骨木。除了一个具有高CCL4基础率的供体,经测试的所有供体响应于益生菌生成CCL4,相比于接骨木益生菌触发更强的响应。6个供体用接骨木+益生菌获得的CCL4的浓度显著大于用接骨木获得的和用益生菌获得的CCL4的浓度。此外,用接骨木益生菌组合获得的CCL4的浓度大于通过益生菌获得的和通过接骨木获得的之和,因此,对于CCL4的生成2种活性成分具有协同作用。
没有一个经测试的供体通过CCL5的生成响应于接骨木。6个供体中的单独一个响应于益生菌生成CCL5。接骨木和益生菌的组合对CCL5没有作用。
经测试的6个供体中仅2个响应于接骨木生成CCL22。经测试的全部供体响应于益生菌生成CCL22。2种活性成分的组合对CCL22没有协同作用。
经测试的全部供体响应于接骨木生成CXCL8。6个供体中的五个响应于益生菌,应当注意的是,无响应者具有最高的CXCL8基础率,相比于接骨木益生菌触发更强的响应。2种活性成分的组合引起CXCL8的协同生成。
没有一个经测试的供体响应于接骨木生成CXCL10,反之,接骨木显著抑制CXCL10的组成性生成(productionconstitutive)。6个供体中的五个响应于益生菌。2种活性成分的组合显示接骨木显著抑制由益生菌诱导的生成。
经测试的全部供体响应于接骨木生成TNFα。经测试的全部供体响应于益生菌生成TNFα。相比于接骨木触发的,这种生成更加强烈。接骨木和益生菌的组合对TNFα的生成没有影响。
没有一个经测试的供体响应于接骨木生成IFNγ,另一方面,经测试的全部供体响应于益生菌生成IFNγ。接骨木和益生菌的组合对IFNγ的生成没有影响。
没有一个经测试的供体响应于接骨木生成GM-CSF。另一方面,6个供体中的5个响应于益生菌生成GM-CSF。6个供体用接骨木+益生菌获得的GM-CSF的浓度显著大于用接骨木获得的和用益生菌获得的GM-CSF的浓度。此外,用接骨木益生菌组合获得的GM-CSF的浓度大于通过益生菌获得的和通过接骨木获得的之和,因此,对于GM-CSF的生成2种活性成分具有协同作用。
刺激T细胞增殖的细胞因子IL-2、IL-5和IL-9生成的缺乏,表明所述组合应该不会激活T淋巴细胞的克隆扩增。
IFNγ和IL-12p70的生成,典型的1型应答,归因于鼠李糖乳杆菌,而IL-4的适度生成,典型的2型应答,与接骨木提取物有关。通过IFNγ/IL-4的比例评估应答的类型:对于大于100的比例为1型应答,当该比例在0.01至100之间时为0型,如果比例小于0.01则为2型。对于鼠李糖乳杆菌该比例为600,对于接骨木提取物其为3,对于接骨木+鼠李糖乳杆菌组合其为119,因此其为1型。因此,益生菌诱导的强烈1型应答被由接骨木触发的IL-4的生成充分地抵消。
通过接骨木和鼠李糖乳杆菌组合的IL-1β、IL-6、CCL2、CCL3、CCL4、CCL5、CCL22、CXCL8、TNFα和GM-CSF的生成表明在炎症位点有细胞的募集和活化的炎症活动。鉴于所涉及的趋化因子(CCL2、CCL3、CCL4、CCL5、CCL22、CXCL8),优选将吸引具有先天免疫的细胞,粒细胞和单核/巨噬细胞类型,以及淋巴细胞。这种促炎性活性将通过抗炎细胞因子IL-1RA生成调节,IL-1RA将自身连接于与IL-1相同的膜受体,防止其向细胞发送其信号。此外,接骨木对趋化因子CXCL10(其吸引活化的淋巴细胞、NK细胞以及嗜酸性粒细胞)的组成性和诱导生成的抑制作用,以同样的意义进行。因此,益生菌的促炎性活性似乎被接骨木的抗炎作用所抵消。
总结
在测试条件下,本发明人显示接骨木提取物和鼠李糖乳杆菌的组合在IL-1RA、IL-6、IL-10、CCL3、CCL4、CXCL8、GM-CSF的诱导方面起到协同作用。该组合诱导IL-1β、IL-1RA、IL-4、IL-6、IL-10、IL-12p70、CCL2、CCL3、CCL4、CCL5、CCL22、CXCL8、IFNγ、TNFα和GM-CSF的生成。这种模式表明通过在炎症位点募集单核细胞、粒细胞和淋巴细胞,接骨木+鼠李糖乳杆菌的组合刺激免疫防御。然后,1型模式将促进细胞型应答的发展,特别地伴有单核细胞/巨噬细胞和嗜中性粒细胞的活化,但没有淋巴组织增生作用。炎症反应将受制(contenue)于IL-1RA和IL-10的生成,并且受制于特别地与接骨木相联系的CXCL10生成的抑制。
Claims (8)
1.包含接骨木提取物和至少一种鼠李糖乳杆菌Lactobacillusrhamnosus菌株的组合的组合物,其特征在于,所述接骨木提取物包含与提取物的干物质相比以重量计0.5至25%的花青素含量,以及与提取物的干物质相比以重量计2至10%的蛋白质含量。
2.根据权利要求1所述的组合物,其特征在于,其包含1.107至1.1011UFC/单位剂量的鼠李糖乳杆菌的量。
3.根据权利要求1或2中任一项所述的组合物,其特征在于,其为食品、食品补充剂或药物。
4.根据权利要求1至3中任一项所述的组合物,其特征在于,其为选自胶囊剂、吞服片剂、咀嚼片剂、泡腾片剂、锭剂、丸剂、粉剂、颗粒剂、口服溶液或混悬剂、和舌下和颊内施用形式的剂型的食品补充剂。
5.根据权利要求1至4中任一项所述的组合物,其特征在于,其包含每单位剂量20mg至200mg的接骨木干提取物,优选每单位剂量20mg至100mg的接骨木干提取物,以及每单位剂量1.107至1.1011UFC的鼠李糖乳杆菌,优选每单位剂量1.108至1.109UFC的鼠李糖乳杆菌。
6.根据权利要求1至5中任一项所述的组合物,其特征在于,其进一步包含维生素和/或矿物盐。
7.根据权利要求1至6中任一项所述的组合物,其用于刺激免疫力和/或增强免疫防御和/或促进抗感染和/或抗炎反应和/或帮助保持活力。
8.根据权利要求1至6中任一项所述的组合物,其用于治疗和/或预防尤其由流感病毒感染引起的流感样状态的症状。
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PCT/EP2014/057082 WO2014166960A1 (fr) | 2013-04-09 | 2014-04-08 | Composition comprenant une association d'un extrait de sureau et d'une souche de lactobacillus rhamnosus |
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KR101938865B1 (ko) * | 2016-11-03 | 2019-01-16 | 주식회사 쎌바이오텍 | 골 질환, 비만 및 지질 관련 대사성 질환의 예방 또는 치료용 조성물 |
GB2562260A (en) * | 2017-05-10 | 2018-11-14 | Sis Science In Sport Ltd | Compositions |
CN112574917B (zh) * | 2020-12-16 | 2022-08-09 | 无锡特殊食品与营养健康研究院有限公司 | 一株缓解高尿酸血症的鼠李糖乳杆菌ccfm1131 |
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TR201902055T4 (tr) | 2019-03-21 |
RU2015147874A (ru) | 2017-05-16 |
EP2983685B1 (fr) | 2018-11-21 |
BR112015025733A2 (pt) | 2017-07-18 |
MX2015014266A (es) | 2016-03-01 |
CY1121491T1 (el) | 2020-05-29 |
TN2015000442A1 (fr) | 2017-01-03 |
US20160082055A1 (en) | 2016-03-24 |
ZA201508222B (en) | 2017-01-25 |
FR3004109B1 (fr) | 2016-01-01 |
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JP2016516757A (ja) | 2016-06-09 |
RU2668126C2 (ru) | 2018-09-26 |
FR3004109A1 (fr) | 2014-10-10 |
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