CN103635102A - 皮肤病学活性酵母提取物 - Google Patents
皮肤病学活性酵母提取物 Download PDFInfo
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- CN103635102A CN103635102A CN201280031922.8A CN201280031922A CN103635102A CN 103635102 A CN103635102 A CN 103635102A CN 201280031922 A CN201280031922 A CN 201280031922A CN 103635102 A CN103635102 A CN 103635102A
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- acid
- extract
- carry out
- yeast
- yeast extract
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Abstract
本发明涉及一种用于制备皮肤病学活性酵母提取物的方法,所述方法包括以下步骤:提供酵母细胞的预培养物;将所述细胞在1.8-4的pH下培养至少15分钟;收获所述细胞并将所述细胞裂解。本发明还涉及由此制备的酵母提取物以及包含所述酵母提取物的产品。
Description
技术领域
本发明涉及一种用于制备皮肤病学活性酵母提取物的方法,所述方法包括以下步骤:提供酵母细胞的预培养物;将所述细胞在1.8-4的pH下培养至少15分钟;收获所述细胞并将所述细胞裂解。本发明还涉及由此制备的酵母提取物以及包含所述酵母提取物的产品。
背景技术
皮肤是人体最大的器官。在其许多的功能中,例如热控制和用作感觉器官,其屏障功能,即防止皮肤干燥从而最终防止整个身体的干燥,当然是最重要的。同时,皮肤充当针对外部物质穿透和吸收的保护设施。这种屏障功能由最外层的表皮提供,其形成针对环境的实际保护覆盖物。表皮的厚度为总厚度的约十分之一,其还是皮肤最薄的层。表皮是层状组织,其中最外层的角质层(角化层)是屏障功能最重要的部分。
化妆品皮肤护理主要理解为增强或恢复皮肤针对环境因素(如污垢、化学品、微生物)和针对内源物质(如水、天然脂肪、电解质)的丧失的屏障的天然功能。如果这种功能被破坏,则存在增加的毒性或致敏性物质的吸收或者微生物攻击,以及相应的毒性或致敏性皮肤反应。
皮肤护理的目的还在于弥补每日盥洗引起的脂肪和水的损失。当再生的天然能力不足时这尤为重要。此外,皮肤护理产品应当提供针对环境因素特别是日光和风的保护,并且应当延缓皮肤老化。
用于疲劳特别是老化的皮肤的护理产品本身是已知的。它们包含例如类视黄醇(维生素A酸和/或其衍生物)或者维生素A和/或其衍生物。然而,它们对结构损伤的作用在程度上是有限的。而且,在产品开发中,充分稳定活性物质以防止氧化破坏具有相当的难度。
而且,使用包含维生素A酸的产品通常导致严重的红斑皮肤刺激。疲劳的皮肤还伴有超重的趋势和/或伴有通常相关的所谓的脂肪团。最近消费者的身体意识明确地增加。除了清理和护理应用,还越来越多地考虑用于增加体形的步骤。脂肪团是一种广泛存在的现象,在这种情况下处于中心位置。观察到的脂肪团是由于皮下脂肪层的增加、结缔组织的弱化以及血液和淋巴系统中灌注条件的降低。因此,原因是结缔组织部分的组成型弱化,以及由超重导致的增大脂肪细胞区室的同时发展、不平衡的营养和缺乏运动。脂肪团的发展还可以归因于毛细血管壁通透性的增加,这使得水渗入结缔组织。
在这种背景下,对施用于皮肤而使得所述不利效果的缓解或至少延缓的物质的需求日益增加,即特别是对皮肤具有致密和强化效果的物质。在消费者不明了遗传工程技术的背景下,亟需根据严格的标尺呗被视为纯生物的相应物质,特别是可以无需遗传工程技术和/或相应有机体而制备的物质。
WO2010087503描述了耶氏酵母属(Yarrowia)用于制备琥珀酸盐的生物技术用途,但是并没有教导耶氏酵母属提取物用于皮肤病学活性剂的用途。
本发明要解决的问题是提供一种新的皮肤病学活性剂,其在皮肤上的施用对抗上述效果,并且提供制备所述物质的方法。此外,本发明要解决的问题之一是开发皮肤病学活性剂,其根据严格的标准是纯生物学的,即特别是不使用遗传工程技术或遗传修饰的有机体制备的。本发明要解决的另一问题是开发一种可以从可再生原料制备的皮肤病学活性剂。
这些和其他问题通过本申请的目的得到解决,特别是所附的独立权利要求的目的,其中从属权利要求为实施方案。
发明内容
在本发明的第一方面,通过用于制备皮肤病学活性酵母提取物的方法来解决问题,所述方法包括以下步骤:a)提供酵母细胞的预培养物;c)将所述细胞在1.8-4的pH下培养至少15分钟;d)收获所述细胞;以及e)将所述细胞裂解。
在第一方面的第一实施方案中,所述方法还包括步骤b)将所述细胞在34-39℃的温度和pH>5下培养至少1小时。
在第一方面的第二实施方案中,其还代表第一实施方案的实施方案,步骤e)利用基于水的裂解剂进行。
在第一方面的第三实施方案中,其还代表第一和第二实施方案的实施方案,首先进行步骤b),然后进行步骤c)。
在第一方面的第四实施方案中,其还代表第一至第三实施方案的实施方案,步骤c)在34-39℃的温度下进行。
在第一方面的第五实施方案中,其还代表第一至第四实施方案的实施方案,步骤b)进行3-5小时。
在第一方面的第六实施方案中,其还代表第一至第五实施方案的实施方案,步骤c)进行45-75分钟。
在第一方面的第七实施方案中,其还代表第一至第六实施方案的实施方案,步骤b)和c)在36-38℃的温度下进行。
在第一方面的第八实施方案中,其还代表第一至第七实施方案的实施方案,步骤c)在1.9-2.2的pH下进行。
在第一方面的第九实施方案中,其还代表第一至第八实施方案的实施方案,酵母细胞是来自包括以下的属的酵母细胞:耶氏酵母属、酵母属(Saccharomyces)和毕赤酵母属(Pichia),并且优选耶氏酵母属。
在第一方面的第十实施方案中,其还代表第一至第九实施方案的实施方案,步骤b)在36-38℃的温度下进行3-5小时,步骤c)在36-38℃的温度和1.9-2.2的pH下进行45-75分钟,所用的酵母细胞为耶氏酵母属的酵母细胞。
在本发明的第二方面,通过第一方面或第一方面的实施方案之一制备的酵母提取物解决问题。
在本发明的第三方面,通过皮肤病学活性剂解决问题,所述皮肤病学活性剂包含耶氏酵母属的酵母细胞的酵母提取物或者本发明的第二方面的酵母提取物。
在本发明的第三方面的第一实施方案中,所述皮肤病学活性剂具有皮肤和/或组织致密作用。
在本发明的第四方面,通过包含第二方面的酵母提取物的食品补充剂解决问题。
在第三方面的第二实施方案中,所述皮肤病学活性剂包含第三方面或其实施方案之一或者第四方面的食品补充剂,其中所述酵母提取物中蛋白质的比例为0.5-50mg/l。
在本发明的第五方面,通过一种用于美容处理皮肤和/或毛发的方法解决问题,所述方法包括局部施用第二方面的酵母提取物或者第三方面或第三方面的实施方案之一的皮肤病学活性剂。
在本发明的第六方面,通过用于制备医药产品的本发明的第二方面的酵母提取物解决问题。
在本发明的第七方面,通过用于制备针对肥胖、糖尿病、动脉粥样硬化、炎性疾病或心血管疾病或者用于伤口处理的医药产品的本发明的第二方面的酵母提取物解决问题。
在本发明的第八方面,通过使用用于体外刺激角质形成细胞、成纤维细胞和/或脂肪细胞的增殖的本发明第二方面的酵母提取物解决问题。
本发明基于本发明人出人意料的发现,即使用替代性酵母菌株和条件允许制备相对于常规产品更具有皮肤病学活性的产品。特别地,本发明人发现,基于耶氏酵母属的菌株酵母提取物令人惊讶地被证实特别有效。而且,本发明人发现特别地暴露于低pH环境的通常酵母细胞以及耶氏酵母属的菌株的酵母细胞适合于从所述细胞制备皮肤病学活性剂。此外,本发明人发现,此类物质的皮肤病学效力可以通过通过组合所述酵母细胞暴露于低pH环境与暴露于温度应激来令人惊讶地进一步改进,优选以温度应激然后pH应激的顺序。最后,本发明人发现,在皮肤模型上施用所述酵母提取物令人惊讶地调节wnt信号转导途径以及这些细胞的细胞外基质,并且以有利于皮肤生理的方式影响它们。
不希望被理论束缚,本发明人认为,耶氏酵母属的酵母细胞释放皮肤病学活性提取物,因为它们具有特别地与疏水、高脂肪底物良好地相互作用的细胞膜蛋白,和/或是细胞包含一种或多种目前为止尚未鉴定对皮肤细胞的代谢具有积极影响的的因子。
本发明包括耶氏酵母属细胞的两种提取物,其以每种可能的方式进行应激,特别是通过温度、pH和氧化应激。本发明还包括通过极端pH、优选低pH应激的任何属的酵母细胞。在优选的实施方案中,它们是酵母属、毕赤酵母属和耶氏酵母属的细胞,并且在更优选的实施方案中,它们是耶氏酵母属的细胞,特别是解脂耶氏酵母(Yarrowia lipolytica)菌株的细胞。
现有技术中已知,酵母细胞生长于酸性环境中,即pH低于7。然而,对酵母细胞也存在与酸性培养基相关的限制,并且从某些pH、应激反应发展开始。在优选的实施方案中,本文所用的术语“pH应激”表示将酵母细胞暴露于低于对其生长最优的pH。在更优选的实施方案中,这种pH为1.8-4的范围内。在甚至更优选的实施方案中,pH为1.9-2.2的范围内。本领域技术人员了解可以实现pH应激的方法和方案,在最简单的情况下,通过加入酸来降低营养培养基的pH,或者通过离心收获细胞然后重悬浮于相应的更酸性的培养基中。pH应激,特别是本发明第一方面的步骤c)的形式中,以优选的顺序,持续至少20、30、45、60、75、90、120或180分钟,或者以优选的顺序,持续20-180、30-90、45-75或50-70分钟。在这个时间内,以优选,pH为低于5、4.5、4、3.5、3、2.5或2.2,或者以优选的顺序,为1.8-5、1.8-4、1.9-3、1.9-2.5、1.9-2.2、1.9-2.2、2-2.8、2-2.2的范围内,并且最优选2。
在优选的实施方案中,本文所用的术语细胞的“裂解”表示适合于破坏细胞的方法,从而其膜变得可透过并且通常包含于细胞中的因子可以从所述细胞释放。本领域技术人员了解裂解的方法,例如使用超声或合适的可商购设备或者悬浮在低盐缓冲液或蒸馏水中。
酵母细胞的理解优选在pH应激处理后利用基于水的裂解剂进行。在优选的实施方案中,本文所用的这一术语表示裂解剂主要包含水作为溶剂。在下文的另一实施方案中,应当理解溶剂是除了醇以外的物质。在另一优选实施方案中,溶剂是醇,特别优选丙二醇。在另一优选实施方案中,酵母提取物可以是总裂解物或溶剂提取物。
本发明人令人惊讶地发现,温度应激与pH应激的组合对酵母提取物的皮肤病学作用具有特别有利的效果。在特别优选的实施方案中,本文所用的术语“温度应激”表示将酵母细胞在高于其最优生长温度的温度下温育。在更优选的实施方案中,这一温度高于33、34、35、36、37、38或39℃,或者在34-42、34-39、35-38或36-38℃的范围内。在特别优选的实施方案中,所述温度为37℃。在特别优选的实施方案中,用温度应激处理后直接用pH应激下的培养基处理酵母细胞,即不给酵母细胞任何在温度应激后恢复的机会。换言之,本发明第一方面的步骤b)后立即进行步骤c),例如通过向步骤b)中的培养基中添加酸。在特别优选的实施方案中,保持步骤c)中产生应激的温度,即在步骤c)中,将酵母细胞暴露于温度和pH应激的组合。在另一实施方案中,步骤b)和c)可以为任何次序。在另一优选的实施方案中,在仍然进行步骤b)的时候进行步骤c),任选地步骤b)和c)同时开始。
在优选的实施方案中,步骤b)在37℃的温度下进行3.5-4.5小时,步骤c)在1.9-2.1的pH和36-38℃的温度下进行45-75分钟,并且所用的酵母细胞为耶氏酵母属的酵母细胞。在一优选的实施方案中,步骤b)在35-39℃的温度下进行2-6小时,步骤c)在1.9-2.1的pH和36-38℃的温度下进行45-75分钟,并且所用的酵母细胞为解脂耶氏酵母属的酵母细胞。
温度应激步骤b)的持续时间必须是这样的:强制使得细胞调节至对其不利的条件,特别是通过改变的蛋白表达,例如改进对升高温度的抗性的因子的表达。在特别优选的实施方案中,根据本发明的第一方面,以优选的顺序,步骤b)进行至少0.25、0.5、0.75、1、1.5、2、3或4小时,或者进行2-10、3-6、3-5、3.5-4.5小时。
本申请中所述的酵母提取物可以加工成皮肤病学活性护理制剂,还同义且互换地称为皮肤病学物质。在一优选的实施方案中,本文所用的术语“皮肤病学物质”表示用于非医疗处理皮肤的物质。在一优选的实施方案中,酵母细胞为包括以下属的组的酵母细胞:耶氏酵母属、酵母属、克鲁维酵母属(Kluyveromyces)、有孢圆酵母属(Torulaspora)、裂殖酵母属(Schizosaccharomyces)、德巴利酵母属(Debaromyces)、假丝酵母属(Candida)、毕赤酵母属、曲霉属(Aspergillus)和青霉属(Penicillium),甚至更优选来自包括耶氏酵母属、酵母属和毕赤酵母属的组。在特别优选的实施方案中,酵母细胞是解脂耶氏酵母菌株的酵母细胞。
本发明的护理制剂相对于护理制剂的总重量包含0.001wt%-20wt%、优选0.01wt%-5wt%、特别优选0.05wt%-3wt%甚至更优选2-3wt%的提取物。
本发明的护理制剂可以包含至少一种选自包括以下的额外组分:软化剂、乳化剂和表面活性剂、增稠剂/粘度控制剂/稳定剂、UV光保护滤剂、抗氧化剂和维生素、助水溶剂(或多元醇)、固体材料和填充剂、成膜剂、珠光(nacreous)添加剂、除臭剂和止汗活性物质、驱虫剂、自晒黑剂(self-tanningagent)、防腐剂、调节剂、香料、着色剂、生物源活性物质、护理添加剂、加脂剂(overfatting agent)和溶剂。
作为软化剂,可以使用所有的化妆品油,特别是具有2-44个碳原子的直链和/或支链的单和/或二羧酸与具有1-22个碳原子的直链和/或支链的饱和或不饱和醇的单或二酯。也可以使用具有2-36个碳原子的脂肪族双官能醇与具有1-22个碳原子的单官能脂肪族羧酸的酯化产物。此外,长链芳基酸酯是合适的,例如苯甲酸的酯,如具有1-22个碳原子的直链或支链的饱和或不饱和醇的苯甲酸酯,或者苯甲酸异硬脂基酯或苯甲酸辛基十二烷基酯。可以适合作为软化剂和油组分的其他单酯有例如具有12-22个碳原子的脂肪酸的甲酯和异丙酯,例如月桂酸甲酯、硬脂酸甲酯、油酸甲酯、芥酸甲酯、棕榈酸异丙酯、肉豆蔻酸异丙酯、硬脂酸异丙酯、油酸异丙酯。其他合适的单酯有例如硬脂酸正丁酯、月桂酸正己基酯、油酸正癸基酯、硬脂酸异辛基酯、棕榈酸异壬基酯、异壬酸异壬基酯、棕榈酸2-乙基己基酯、月桂酸2-乙基己基酯、硬脂酸2-己基癸基酯、棕榈酸2-辛基十二烷基酯、油酸油酯、芥酸油酯、油酸芥酯以及可获得自工艺脂肪醇部分和工艺脂肪族羧酸混合物的酯,例如具有12-22个碳原子的不饱和脂肪醇与具有12-22个碳原子的饱和和不饱和脂肪酸如可以获得自动物和植物脂肪的脂肪酸的酯。然而,天然存在的单酯或蜡酯混合物如存在于西蒙得木油或鲸油中的酯也是合适的。合适的二羧酸酯有例如己二酸二正丁酯、癸二酸二正丁酯、己二酸二(2-乙基己基)酯、琥珀酸二(2-己基癸基)酯、D-isotridecylacelaat。合适的二醇酯有例如二油酸乙二醇酯、乙二醇-二异十三烷基酯、丙二醇-二-(2-乙基己酸)酯、丁二醇-二异硬脂酸酯、丁二醇-二辛酸酯/癸酸酯和新戊二醇二辛酸酯。可以用作软化剂的其他脂肪酸酯有例如C12-15烷基苯甲酸酯、二辛酰基碳酸酯、二乙基己基碳酸酯。更长链的甘油三酯(即具有三个酸分子,其中至少一个是更长链的甘油的三酯)也可以用作软化剂和油组分。例如,可以提及脂肪酸甘油三酯,例如天然植物油,如橄榄油、向日葵油、大豆油、花生油、菜籽油、杏仁油、芝麻油、鳄梨油、蓖麻油、可可脂、棕榈油以及椰子油或棕榈仁油的液体部分;和动物油,如鲨鱼肝油、鳕鱼肝油、鲸油、牛油和乳脂;蜡,如蜂蜡、巴西棕榈蜡、鲸腊、羊毛脂和牛脚油、牛油的液体部分或辛酸-癸酸混合物的合成甘油三酯、来自工艺油酸的甘油三酯、异硬脂酸的甘油三酯或者来自棕榈酸-油酸混合物的甘油三酯可以用作软化剂和油组分。此外,可以使用烃,特别是液体石蜡和异石蜡。可用的烃的实例有石蜡油、异十六烷、聚癸烯、凡士林、paraffinum perliquidum、角鲨烷、地蜡。此外,也可以使用直链或支链脂肪醇如油醇或辛基十二烷醇,以及脂肪醇醚如二辛基醚。合适的硅油和蜡有例如聚二甲基硅氧烷、环甲基硅氧烷以及芳基-或烷基-或烷氧基-取代的聚甲基硅氧烷或环甲基硅氧烷。可以考虑的其他油有例如基于具有6-18、优选8-10个碳原子的脂肪醇的Guerbet醇、直链C6-C22脂肪酸与直链C6-C22脂肪醇的酯、支链C6-C13羧酸与直链C6-C22脂肪醇的酯、直链C6-C22脂肪酸与支链C8-C18醇特别是2-乙基己醇或异壬醇的酯、支链C6-C13羧酸与支链醇特别是2-乙基己醇或异壬醇的酯、直链和/或支链脂肪酸与多元醇(如,丙二醇、二聚二醇或三聚二醇)和/或Guerbet醇的酯、基于C6-C10脂肪酸的甘油三酯、基于C6-C18脂肪酸的液态甘油单酯/甘油二酯/甘油三酯混合物、C6-C22脂肪醇和/或Guerbet醇与芳香族羧酸特别是苯甲酸的酯、植物油、支链伯醇、取代的环己烷、直链C6-C22脂肪醇碳酸酯、Guerbet碳酸酯、苯甲酸与直链和/或支链C6-C22醇的酯(如FinsolvTM TN)、二烷基醚、环氧化脂肪酸酯与多元醇的开环产物、硅油和/或脂肪族或环烷烃。
非离子、阴离子、阳离子或两亲表面活性剂可以用作乳化剂或表面活性剂。以下组中的至少一种的化合物可以用作非离子化乳化剂或表面活性剂:2-100mol环氧乙烷和/或0-5mol环氧丙烷在具有8-22个碳原子的直链脂肪醇上、在具有12-22个碳原子的脂肪酸上以及在烷基中具有8-15个碳原子的烷基酚上的加成产物,1-100mol环氧乙烷在甘油上的加成产物的C12/18脂肪酸单和二酯,具有6-22个碳原子的饱和和不饱和脂肪酸的甘油单和二酯以及山梨聚糖单和二酯及其环氧乙烷加成产物,烷基残基中具有8-22个碳原子的烷基单和寡糖苷及其环氧乙烷加成产物,2-200mol环氧乙烷在蓖麻油和/或硬化蓖麻油上的加成产物,基于直链、支链、不饱和或饱和C6-C22脂肪酸、蓖麻油酸和12-羟基硬脂酸的部分酯,以及甘油,聚甘油,季戊四醇,二季戊四醇,糖醇(如,山梨醇),烷基糖苷(如,甲基糖苷、丁基糖苷、月桂基糖苷)和聚糖苷(如,纤维素),单、二和三烷基磷酸酯以及单、二和/或三PEG-烷基磷酸酯及其盐,聚硅氧烷-聚醚共聚物(二甲基硅油共聚醇(dimethicone copolyol)),例如PEG/PPG-20/6二甲基硅油、PEG/PPG-20/20二甲基硅油、双-PEG/PPG-20/20二甲基硅油、PEG-12或PEG-14二甲基硅油、PEG/PPG-14/4或4/12或20/20或18/18或17/18或15/15,例如聚硅氧烷-聚烷基聚醚共聚物或相应的衍生物,例如月桂基或十六烷基二甲基硅油共聚醇,特别是十六烷基PEG/PPG-10/1二甲基硅油(ABIL EM 90(Evonik Goldschmidt GmbH)),根据DE 11 65 574的季戊四醇、脂肪酸、柠檬酸和脂肪醇的混合酯,和/或具有6-22个碳原子的脂肪酸、甲基葡萄糖和多元醇的混合酯,例如甘油或聚甘油,柠檬酸酯,如甘油硬脂酸柠檬酸酯、甘油油酸柠檬酸酯和二月桂基柠檬酸酯。
阴离子乳化剂或表面活性剂可以包含水溶性阴离子基团如羧酸根、硫酸根、磺酸根或磷酸根基团以及亲脂性残基。大量的皮肤相容的阴离子表面活性剂为本领域技术人员已知并且是可商购的。这些可以是碱、铵或烷醇铵盐形式的烷基硫酸酯或者碱或铵盐形式的烷基磷酸酯、烷基醚硫酸酯、烷基醚羧酸酯、酰基肌氨酸酯以及磺基琥珀酸酯和酰基谷氨酸酯。
还可以添加阳离子乳化剂和表面活性剂。为此,可以特别地使用季铵化合物,特别是带有至少一个具有8-22个碳原子的直链和/或支链、饱和或不饱和的烷基链的季铵化合物,烷基三甲基卤化铵,如十六烷基三甲基氯化铵或溴化铵或者二十二烷基三甲基氯化铵,以及二烷基二甲基卤化铵,如二硬脂基二甲基氯化铵。
此外,也可以使用单烷基酰氨基quat,如棕榈酰氨基丙基-三甲基氯化铵或者相应的二烷基酰氨基quat。
此外,可以使用具有良好的生物可降解性的季酯化合物,例如基于单、二或三乙醇胺的季铵化脂肪酸酯。此外,烷基胍盐还可以作为阳离子乳化剂添加。
温和的,即特别皮肤相容的表面活性剂的典型实例为脂肪醇-聚乙二醇醚硫酸盐、甘油一硫酸酯盐、磺基琥珀酸单烷基酯和/或磺基琥珀酸二烷基酯、脂肪酸羟乙基磺酸盐、脂肪酸肌氨酸盐、脂肪酸氨基乙磺酸盐、脂肪酸谷氨酸盐、醚羧酸、烷基寡聚葡糖苷、脂肪酸葡糖酰胺、烷基酰胺甜菜碱和/或蛋白脂肪酸缩合物,后者例如基于小麦蛋白。
此外,可以使用两亲表面活性剂,例如甜菜碱、两亲乙酸盐或两亲丙酸盐,例如以下物质如N-烷基-N,N-二甲基甘氨酸铵,如椰子烷基二甲基甘氨酸铵,N-酰基氨基丙基-N,N-二甲基甘氨酸铵,如椰子酰基氨基丙基二甲基甘氨酸铵,以及在每种情况下在烷基或酰基中具有8-18个碳原子的2-烷基-3-羧基甲基-3-羟基乙基咪唑啉和椰子酰基氨基乙基羟基乙基羧基甲基甘氨酸盐。
在两亲表面活性剂中,可以使用这样的表面活性化合物:除了分子中的C8/18烷基或酰基,其包含至少一个游离氨基和至少一个-COOH-或-SO3H-基团,并且能够形成内盐。合适的两亲表面活性剂的实例为N-烷基甘氨酸、N-烷基丙酸、N-烷基氨基丁酸、N-烷基亚氨基二丙酸、N-羟基乙基-N-烷基酰氨基丙基甘氨酸、N-烷基牛磺酸、N-烷基肌氨酸、2-烷基氨基丙酸和烷基氨基乙酸,在每种情况下,在烷基中具有约8-18碳原子。两亲表面活性剂的其他实例为N-椰子烷基氨基丙酸盐、椰子酰基氨基乙基氨基丙酸盐和C12/18-酰基肌氨酸。
合适的增稠剂例如多糖,特别是黄原胶、瓜尔胶-瓜尔胶、琼脂-琼脂、藻酸盐和纤基乙酸钠、羧甲基纤维素和羟乙基纤维素,还有脂肪酸的较高分子量的聚乙二醇单酯和二酯、聚丙烯酸酯(例如Carbopole TM或Synthalene TM)、聚丙烯酰胺、聚乙烯醇和聚乙烯吡咯烷酮,表面活性剂如乙氧基化脂肪酸甘油酯、脂肪酸与多元醇的酯如季戊四醇或三羟甲基丙烷、具有较窄分布的同系物的脂肪醇乙氧基化物或烷基低聚葡糖苷以及电解质如常见的盐和氯化铵。
本领域技术人员已知的所有增稠试剂均可以考虑作为增稠油相的增稠剂。特别应当提到蜡,例如氢化蓖麻蜡、蜂蜡或微蜡。此外,还可以使用无机增稠试剂,例如二氧化硅、氧化铝或层状硅酸盐(例如锂蒙脱石、Laponite、滑石粉)。这些无机油相增稠剂可以是疏水修饰的。对于增稠/稳定的油包水乳剂,特别地可以使用Aerosil、层状硅酸盐和/或脂肪酸的金属盐如硬脂酸锌。
例如,对于水性表面活性剂系统,作为粘度控制剂可以包含NaCl,低分子量的非离子表面活性剂如椰油酰胺DEA/MEA和月桂醇聚醚-3,或者聚合的高分子量的缔合的高度乙氧基化的脂肪衍生物如PEG-200氢化棕榈酸甘油酯。
作为UV光保护滤剂,可以例如使用能够吸收紫外线并以长波辐射的形式如热重发射所吸收的能量的有机物质。UVB滤剂可以是油溶性或水溶性的。以下物质可以作为油溶性UVB光保护滤剂的实例:3-亚苄基樟脑及其衍生物,例如3-(4-甲基亚苄基)樟脑,4-氨基苯甲酸衍生物,例如4-(二甲基氨基)苯甲酸-2-乙基己基酯、4-(二甲基氨基)苯甲酸-2-乙基己基酯和4-(二甲基氨基)-苯甲酸戊基酯,肉桂酸酯,例如4-甲氧基肉桂酸-2-乙基己基酯、4-甲氧基肉桂酸异戊基酯、2-氰基-3-苯基-肉桂酸-2-乙基己基酯(氰双苯丙烯酸辛酯),水杨酸酯,例如水杨酸-2-乙基己基酯、水杨酸-4-异丙基苄基酯、水杨酸三甲环己酯,二苯甲酮衍生物,例如2-羟基-4-甲氧基二苯甲酮、2-羟基-4-甲氧基-4′-甲基二苯甲酮、2,2′-二羟基-4-甲氧基二苯甲酮,亚苄基丙二酸酯,例如4-甲氧基亚苄基丙二酸二-2-乙基己基酯,三嗪衍生物,例如2,4,6-三苯氨基-(p-碳-2′-乙基-1′-己基氧基)-1,3,5-三嗪和辛基三嗪酮,丙烷-1,3-二酮,例如1-(4-叔丁基苯基)-3-(4′-甲氧基苯基)丙烷-1,3-二酮。
以下物质可以作为水溶性UVB光保护滤剂:2-苯基苯并咪唑-5-磺酸及其碱金属、碱土金属、铵、烷基铵、链烷醇铵和葡糖铵(glucammonium)盐,二苯甲酮的磺酸衍生物,例如2-羟基-4-甲氧基二苯甲酮-5-磺酸及其盐,3-亚苄基樟脑的磺酸衍生物,例如4-(2-氧代-3-亚冰片基甲基)苯磺酸和2-甲基-5-(2-氧代-3-亚冰片基)磺酸及其盐。
特别地可以考虑苯甲酰基甲烷的衍生物作为典型的UVA光保护滤剂,例如1-(4′-叔丁基苯基)-3-(4′-甲氧基苯基)丙烷-1,3-二酮或1-苯基-3-(4′-异丙基苯基)丙烷-1,3-二酮。当然,UV-A和UV-B滤剂还可以在混合物中使用。
除了上述可溶性物质,为了这个目的,还可以考虑不溶性色素,即细碎的金属氧化物或盐,例如二氧化钛、氧化锌、氧化铁、氧化铝、二氧化铈、氧化锆、硅酸盐(滑石)、硫酸钡和硬脂酸锌。颗粒应当具有少于100nm的平均直径,例如5-50nm,特别是15-30nm。它们可以呈球形,但是还可以使用呈椭圆形或者以一些其他方式偏离球形的颗粒。一种相对新的类别的光保护滤剂是微粒化的有机色素,例如具有<200nm的粒径的2,2′-亚甲基-双-{6-(2H-苯并三唑-2-基)-4-(1,1,3,3-四甲基丁基)酚},其可以例如获得为50%水性分散液。
其他合适的UV光保护滤剂可以在 122,543(1996)中P.Finkel的综述中找到。除了上述两组主要UV光保护滤剂,还可以使用抗氧化剂类型的第二防晒剂,其中断在UV辐射穿透皮肤时触发的光化学反应链。
可以使用的抗氧化剂和维生素例如超氧化物歧化酶、生育酚(维生素E)、生育酚山梨酸酯、生育酚乙酸酯、生育酚的其他酯、二丁基羟基甲苯和抗坏血酸(维生素C)和它们的盐以及它们的衍生物(例如磷酸抗坏血酸酯镁盐、磷酸抗坏血酸酯钠盐、山梨酸抗坏血酸酯)、脂肪酸的抗坏血酸酯、丁基化羟基苯甲酸及其盐、过氧化物如过氧化氢、过硼酸盐、硫基乙醇酸盐、过硫酸盐、6-羟基-2,5,7,8-四甲基苯丙二氢吡喃-2-羧酸(TROLOX.RTM)、鞣酸及其烷基酯、尿酸及其盐和烷基酯、山梨酸及其盐、硫辛酸(lipoic acid)、阿魏酸、胺(例如N,N-二乙基羟基胺、氨基-胍)、巯基化合物(例如谷胱甘肽)、二羟基富马酸及其盐、甘氨酸吡酮酸盐、精氨酸吡酮酸盐、去甲二氢愈创木酸、生物类黄酮、姜黄素、赖氨酸、L-甲硫氨酸、脯氨酸、超氧化物歧化酶、水飞蓟素、茶提取物、柚皮/核提取物、黑素、迷迭香提取物、硫辛酸(thioctic acid)、白藜芦醇、氧化白藜芦醇等。
例如乙醇、异丙基醇或多元醇可以用作助水溶剂以改善流动行为和应用特征。这里可以考虑的多元醇可以具有2-15个碳原子以及至少2个羟基。典型实例包括:甘油亚烷基二醇,例如乙二醇、二乙二醇、丙二醇、丁二醇、己二醇以及具有100-1000道尔顿的平均分子量的聚乙二醇,具有1.5-10的内在缩合度的技术低聚甘油混合物,例如具有40-50wt%的二甘油含量的技术二甘油混合物,羟甲基化合物,特别是例如三羟甲基乙烷、三羟甲基丙烷、三羟甲基丁烷,季戊四醇和二季戊四醇,低级烷基葡糖苷,特别是在烷基残基中具有1-4碳原子的低级烷基葡糖苷,例如甲基和丁基葡糖苷,具有5-12个碳原子的糖醇,例如山梨醇或甘露醇,具有5-12个碳原子的糖,例如葡萄糖或蔗糖,氨基糖,例如葡糖胺。
可以使用的固体例如氧化铁色素、二氧化钛或氧化锌颗粒以及在“UV防晒剂”下额外提到的那些物质。此外,还可以使用产生特殊感官效果的颗粒,例如尼龙-12、一氮化硼、聚合物颗粒如聚丙烯酸酯或聚丙烯酸甲酯颗粒或硅氧烷弹性体。可以使用的填充剂包括淀粉和淀粉衍生物,例如木薯淀粉、磷酸二淀粉、淀粉铝或淀粉钠、辛烯基琥珀酸以及没有主要UV过滤或着色作用的色素,例如Aerosile(CAS No.7631-86-9)。
例如为了改善水抗性,作为成膜剂可以例如使用:聚氨基甲酸酯、二甲基硅油、共聚醇、聚丙烯酸酯或PVP/VA共聚物(PVP=聚乙烯吡咯烷酮,VA=醋酸乙烯酯)。例如以下物质可以用作脂溶性成膜剂:基于聚乙烯吡咯烷酮(PVP)的聚合物、聚乙烯吡咯烷酮共聚物、PVP/十六碳烯共聚物或PVP/二十碳烯共聚物。
例如乙二醇二硬脂酸酯或PEG-3二硬脂酸酯可以用作珠光添加剂。
合适的除臭活性物质例如气味掩蔽剂如通常的香料组分,吸味剂,例如专利Offenlegungsschrift[未经审查的申请的公开]DE 40 09 347所述的层状硅酸盐,其中特别是蒙脱石、高岭石、ilite、贝得石、囊脱石、滑石粉、ilectorite、膨润土、绿土,此外例如蓖麻油酸的锌盐。还适合掺入萌发抑制剂。萌发抑制物质例如2,4,4′-三氯-2′-羟基二苯基醚(Irgasan)、1,6-二-(4-氯苯基双胍基)-己烷(洗必泰)、3,4,4′-trichlorocarbonilide、季铵化合物、丁子香油、薄荷油、百里香油、柠檬酸三乙酯、法呢醇(3,7,11-三甲基-2,6,10-十二碳三烯-1-醇)、乙基己基甘油醚、聚甘油-3辛酸酯(TEGO Cosmo P813,Evonik Goldschmidt GmbH)以及未经审查的专利申请DE 198 55 934、DE 3740 186、DE 39 38 140、DE 42 04 321、DE 42 29 707、DE 42 29 737、DE 4238 081、DE 43 09 372、DE 43 24 219和EP 666 732所述的活性剂。
收敛剂,例如碱性氯化铝如水合氯化铝(“ACH”)和锆-铝-甘氨酸盐(“ZAG”)可以用作止汗活性物质。
例如N,N-二乙基-间甲苯甲酰胺、1,2-戊二醇或驱虫剂3535可以用作驱虫剂。
例如二羟基丙酮和赤藓酮糖可以用作自晒黑剂。
例如单独或几种对羟基苯甲酸烷基酯与苯氧基乙醇的混合物可以用作防腐剂。对羟基苯甲酸烷基酯可以是对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、对羟基苯甲酸丙酯和/或对羟基苯甲酸丁酯。其他醇如苄基醇或乙醇也可以用于代替苯氧基乙醇。此外,还可以使用其他常见的防腐剂,例如山梨酸或苯甲酸、水杨酸、2-溴-2-硝基丙烷-1,3-二醇、氯乙酰胺、二偶氮利定脲、DMDM乙内酰脲、碘丙炔基丁基氨基甲酸酯、羟甲基甘氨酸钠、甲基异噻唑啉、氯甲基异噻唑啉、乙基己基甘油或辛二醇。
例如有机季铵化合物可以用作调节剂,如西曲氯铵、二鲸蜡基二甲基氯化铵、山萮基三甲基氯化铵、二硬脂基二甲基氯化铵、山嵛基三甲基铵甲基硫酸盐、二硬脂酰乙基二甲基氯化铵、棕榈酰胺丙基三甲基氯化铵、瓜尔羟丙基三甲基氯化铵、羟基丙基瓜尔羟丙基三甲基氯化铵或季铵盐-80,或者还有胺衍生物如氨基丙基二甲基硅油或硬脂酰胺丙基二甲基胺。
天然或合成的香料或其混合物可以用作芳香剂。天然香料为花提取物(百合、薰衣草、玫瑰、茉莉、橙花、依兰)、茎和叶提取物(天竺葵、广藿香、苦橙叶)、果实提取物(茴香、芫荽、葛缕子、杜松)、果皮提取物(佛手柑、柠檬、橙)、根提取物(肉豆蔻、当归、芹菜、小豆蔻、木香、鸢尾、百里香)、针叶和树枝提取物(云杉、冷杉、松树、山松)、树脂和香脂(白松香、榄香、安息香、没药、乳香、opoponax)。此外,可以考虑动物性原料,例如灵猫香和海狸香。典型的香料化合物是酯、醚、醛、酮、醇和烃类型的产物。酯类型的香料化合物例如乙酸苄酯、异丁酸苯氧基乙酯、乙酸对叔丁基环己酯、乙酸里哪醇酯、乙酸二甲基苄基原酯、乙酸苯乙酯、苯甲酸芳樟酯、甲酸苄酯、甘氨酸乙基甲基苯基酯、环己基丙酸烯丙酯、丙酸苏合香酯和水杨酸苄酯。醚包括例如苄基乙基醚,醛包括例如具有8-18个碳原子的线性链烷醛、柠檬醛、香茅醛、香茅基含氧乙醛、仙客来醛、羟基香茅醛、铃兰醛和bourgeonal,酮包括例如紫罗酮、α-异甲基紫罗酮和甲基-柏木酮,醇包括茴香脑、香茅醇、丁子香酚、异丁子香酚、香叶醇、里哪醇、苯基乙基醇和松油醇,烃主要包括萜和香脂。可以使用各种香料的混合物,其一起产生吸引人的香味。一般用作香味组分的低挥发性精油也适合作为香料,例如鼠尾草油、春黄菊油、丁子香油、蜂花油、薄荷油、肉桂叶油、莱姆花油、刺柏油、岩兰油、乳香油、格蓬油、labolanum油和杂薰衣草油。可以单独或以混合物使用香柠檬油、二氢月桂烯醇、铃兰醛、新铃兰醛、香茅醇、苯基乙基醇、α-己基新肉桂醛、香叶醇、苄基丙酮、仙客来醛、里哪醇、Boisambrene Forte、Ambroxan、吲哚、二氢茉莉酮酸甲酯、sandelice、柠檬油、橘子油、橙油、戊基甘醇酸烯丙酯、cyclovertal、杂薰衣草油、香鼠尾草油、β-二氢大马酮、波旁香叶油、水杨酸环己酯、甲基柏木酮、iso-E-super、Fixolide NP、evernyl、iraldein γ、苯基乙酸、牻牛儿醇乙酸酯、乙酸苄酯、玫瑰氧化物、romillate、irotyl以及floramat。
可以使用的染料是适合并允许用于美容目的的物质,例如出版物“Kosmetische ”[Cosmetic colourants]of the Dye Commission ofthe Deutsche Forschungsgemeinschaft[German Research Association],VerlagChemie,Weinheim,1984,p.81 to 106中所列。这些染料通常以相对于总混合物0.001-0.1wt%的浓度使用。
生物源活性物质例如生育酚、生育酚乙酸酯、生育酚棕榈酸酯、抗坏血酸、多酚、脱氧核糖核酸、辅酶Q10、视黄醇、AHA酸、氨基酸、透明质酸、α-羟酸、异黄酮、聚谷氨酸、肌酸(和肌酸衍生物)、胍(和胍衍生物)、假神经酰胺(pseudoceramide)、精油、肽、蛋白水解产物、植物提取物、红没药醇、尿囊素、泛醇、植烷三醇、艾地苯醌、甘草提取物、glycyrrhicidine和艾地苯醌、硬葡聚糖、β-葡聚糖、santalbin acid和维生素复合物。植物提取物的实例为栗提取物、甘菊提取物、迷迭香提取物、黑醋栗和红醋栗提取物、白桦提取物、玫瑰果提取物、海藻提取物、绿茶提取物、芦荟提取物、人参提取物、银杏提取物、葡萄柚提取物、金盏花提取物、樟脑、百里香提取物、山竹提取物、cystus提取物、Terminalia arjuna提取物、燕麦提取物、牛至提取物、树莓提取物、草莓提取物等。
生物源活性物质还可以包括所谓的屏障脂质,例如神经酰胺、植物鞘氨醇和衍生物、鞘氨醇和衍生物、二氢鞘氨醇和衍生物、假神经酰胺、磷脂、溶血磷脂、胆固醇和衍生物、胆固醇酯、游离脂肪酸、羊毛脂和衍生物、角鲨烷、角鲨烯以及相关物质。
在本发明的意义中,生物源活性物质还包括抗痤疮剂,例如苄基过氧化物、植物鞘氨醇和衍生物、烟酰胺羟基苯甲酸酯、烟碱醛、视黄酸和衍生物、水杨酸和衍生物、香茅酸等,以及抗脂肪团剂,例如黄嘌呤化合物如咖啡因、茶碱、可可碱和氨茶碱、肉碱、肌肽、水扬酰植物鞘氨醇、植物鞘氨醇、檀香萜酸(santalbin acid)等,以及去头屑剂,例如水杨酸和衍生物、吡硫锌、硫化硒、硫磺、环吡酮胺、联苯苄唑、甘宝素、羟甲辛吡酮和actirox等,以及收敛剂,例如醇、铝衍生物、鞣酸、水杨酸吡哆素、锌盐如硫酸锌、乙酸锌、氯化锌、乳酸锌、锆盐酸盐等。生物源活性物质还可以包括漂白剂如曲酸、熊果苷、维生素C和衍生物、氢醌、郁金油、肌酸酐、鞘脂、烟酰胺等。
作为护理添加剂,可以包含例如乙氧基化甘油脂肪酸酯如PEG-7甘油椰油酸酯,或者阳离子聚合物,例如聚季铵盐-7或聚甘油酯。
作为加脂剂,可以使用以下物质,例如羊毛脂和卵磷脂以及聚乙氧基化或酰化羊毛脂和卵磷脂衍生物、多元醇脂肪酸酯、单甘油酯和脂肪酸链烷醇酰胺,其中后者通常充当泡沫稳定剂。
例如脂肪醇如乙醇、丙醇或1,3-丙二醇,环状碳酸酯如碳酸乙烯酯、碳酸丙烯酯、碳酸甘油酯,单羧酸酯或多羧酸酯如乙酸乙酯、乳酸乙酯、二甲基己二酸和二乙基己二酸、丙二醇、二丙二醇、甘油、碳酸甘油酯或水可以用作溶剂。
作为额外的组分,本发明的护理制剂优选具有选自神经酰胺、胆固醇、脂肪酸的屏障脂质。
此外,作为额外的组分,本发明的护理制剂优选包含脂质调节剂,例如亚麻酸、共轭亚油酸、γ亚麻酸、植物鞘氨醇、水扬酰植物鞘氨醇、短链和中链神经酰胺(C1-C10)、糖醛酸、硫酸胆固醇、植物甾醇、维生素D、白三烯、法呢醇、15-脱氧前列腺素J2、9-羟基十八碳二烯酸(9-HODE),优选选自肌酸、烟酰胺、视黄醇、阿江榄仁酸的脂质调节剂。
本发明的护理制剂可以用作皮肤护理、面部护理、头部护理、身体护理、私密护理、足部护理、毛发护理、指甲护理、口腔卫生、唇部护理或口服卫生产品。毛发护理产品的实例有毛发洗涤剂、护发用品、染发液、发液、发胶、发油、发蜡、发漆、喷发剂、发膏、摩丝、毛膏、去屑洗发水。身体护理产品的实例有沐浴露、沐浴霜、霜胶(cream gel)、沐浴油、沐浴胶、洗涤胶、去角质洗涤液(wash-peeling)、卸妆液、面膜、润肤露、去角质面乳(facial peeling)、眼霜、晚霜、清洁面膜、lotion pad、清理湿巾(cleansing wipe)、卸妆液、卸妆乳、须后胶(aftershave gel)、须后油(aftershavebalm)、晒乳液、晒后产品、自晒黑剂、足部洗液、足部喷雾、身体洗液、身体凝胶、身体喷雾、身体乳、身体去角质液(body peeling)、身体油、身体膏(body butter)。唇部护理产品的实例有唇膏、唇霜、护唇棒。
本发明的护理制剂可以乳液的形式使用,例如水包油(O/W)、油包水(W/O)或硅酮包水(W/S)乳液,多乳液,如W/O/W和O/W/O(也称为水分散体或脂分散体),悬浮液,溶液,霜,软膏,糊剂,凝胶,气雾剂,喷雾剂,清洁产品,化妆或防晒制品或脸部洗液或棒,如脂肪棒或含水棒。
根据本发明制备的酵母提取物还可以用作动物和/或人的食品补充剂。所述提取物可以和食物一起给予。
皮肤病学活性剂或食品补充剂在酵母提取物中必须包含指定最小量的蛋白质。在一优选的实施方案中,酵母提取物包含,以优选的顺序,至少0.5、1、2.5、5、10或25mg蛋白质/mL,或者在0.05-100、0.1-80、0.5-50或1-25mg蛋白质/mL的范围内。
根据本发明制备的酵母提取物可以用于毛发和/或皮肤的美容处理。在一优选实施方案中,本文所用的术语“毛发和/或皮肤”表示人或动物身体的对于皮肤病学物质的外部使用可接近的任何部分。在一优选的实施方案中,本文所用的术语“美容处理”表示非病理学表现的改善,其特别地仅改善美学印象。通过这种类型的处理,可以例如实现皮肤致密、皮肤湿度的改善、均匀肤色或出汗的改善。
本发明人令人惊讶地发现,用根据本发明制备的酵母提取物处理皮肤细胞适合于治疗疾病。
通过以下附图和非限制性实施例进一步示例本发明,其中可见本发明进一步的特征、实施方案、方面和优势。
附图说明
图1示出耶氏酵母属、毕赤酵母属和酵母属在pH2下随时间的生长。
图2示出利用所述方案获得的具有芯片数据的两个代表性组。数据通过分析用耶氏酵母属细胞的提取物处理的人成纤维细胞的标记获得,所述耶氏酵母属细胞正常地在pH5.4下培养(图2a)或者暴露于pH2的应激(图2b)。
实施例
实施例1:各种酵母菌株在暴露于低pH培养基的应激下的生长
为了研究是否可能通过暴露于低pH培养基来应激酵母菌株,并且研究哪种酵母菌株可能在低pH下表现出合适或有利的生长性质,在pH=2下研究三个属(即酵母属、毕赤酵母属和耶氏酵母属)的每一个的一个代表的生长。
将要研究的各种酵母菌株的预培养物在标准培养基(每升:33g葡萄糖一水合物、0.88g硫酸镁x7H2O、2g氯化钙x2H2O、4.83g氯化铵、0.06g氯化钠、1g磷酸二氢钾、0.059g肌醇、20g MOPS、0.3mL微量元素溶液(每kg:100g 8M H2SO4、50g柠檬酸一水合物、48g FeSO4x7H2O、16.7gZnSO4x7H2O、2.5g CuSO4x5H2O、1.88g MnSO4xH2O、2g H3BO3、2gNaMoO4x2H2O、0.5g KI)和0.3mL维生素溶液(每升:5g烟酸、5g D-泛酸钙、5g硫胺、3.33g对氨基苯甲酸酯、0.5g吡哆醇、0.0167生物素)中过夜培养,其pH用硫酸调节为2。通过分光光度法连续监控光密度。
测试的结果示于图1中。可见,所有使用的菌株在这些条件下表现出可检测的生长,但是耶氏酵母属的代表于pH=2下在相同条件下表现出比比较的属更快的生长,并且达到更高的光密度。
实施例2:利用芯片阵列,通过升高的温度应激、用过氧化氢处理或暴露于低pH培养基的酵母细胞对人成纤维细胞的作用的比较表征
研究暴露于低pH培养基的应激是否对酵母细胞施加与通过升高的温度或用过氧化氢处理处理应激相同的效果,以及不同地应激的酵母细胞或它们的提取物是否有不同。酿酒酵母(Saccharomyces cerevisiae)选作模式生物。
制备酵母提取物
如上文所述获得预培养物并调节至5.4的pH。将培养基接种在无挡板的摇瓶中,并且添加消泡剂。温度为30℃,并且速度为180rpm(幅度:2.5cm)。预先测定光密度,其进行连续测量,并且在OD>35时,产生各自的应激状况,即通过升高温度至37℃,通过添加硫酸至期望的pH或者通过添加过氧化氢。
然后,通过离心收获细胞并冷冻,并且通过水性裂解裂解细胞。为此,将提取物悬浮并通过高压均质器处理。将悬浮液过滤或离心,并且水溶液为终产物。
芯片阵列实验的测试原则和实行
在这些测试中,研究酵母提取物对人真皮成纤维细胞行为的效果,所述成纤维细胞是公认的用于人皮肤的模型。为此目的,将提取物处理后的细胞裂解,分离它们的RNA,并通过用于皮肤病学相关标记表达的调节的芯片来研究通过逆转录获得的DNA。专门地使用上调或下调指示对皮肤的形态和生理的积极效果的标记,特别是Wnt-途径和ECM的标记。Wnt基因编码分泌蛋白的大家族。目前为止,在人中已经鉴定了19种Wnt蛋白。个体物种中Wnt的各种亚组以及不同物种之间Wnt的比较表现出非常高的同源性。例如,在人中,个体Wnt表现出27-83%的一致性(Miller,2001)。典型的Wnt信号级联的中心分子是β-联蛋白(βCat),其稳定性受Wnt蛋白调节。胚胎和出生后皮肤的研究证实,Wnt在毛囊发育中起到重要作用。特别地,蛋白Wnt10a、Wnt10b和Wnt5a在毛囊发育的早期节段特异性地表达(Reddy et al.,2001)。
在皮肤中发现了大部分已知的联系机制。通过Wnt途径,基细胞优选地保持干细胞特性。随着细胞老化,细胞和分子损伤积累,导致组织和器官再生能力的降低。这种降低的再生能力部分地受脂肪组织的组织特异性成体干细胞的自我更新能力和分化潜力的影响。已知WNT信号途径,这是一种已知的信号途径,调节许多干细胞自我更新潜能和分化潜能,因此对干细胞的生长具有效果。此外,通过Wnt和β-联蛋白表达,表皮增殖、分化和迁移增加,从而加速伤口愈合。
结缔组织的物理性质和相关的皮肤致密性由细胞外基质决定,从而细胞在它们的分化、迁移和增殖上受到影响。基质由位于其中的细胞形成(成纤维细胞、成肌纤维细胞、成脂细胞、成骨细胞和成软骨细胞)。细胞外基质由大分子构成,其由糖胺聚糖和纤维蛋白组成。细胞外基质的淋巴系统与过量脂肪积累的相互作用涉及皮肤致密性。总身体脂肪由脂肪细胞的从头分化、生长和凋亡(自溶)之间的平衡决定。脂肪细胞分化是复杂的过程,其出生时开始并且持续整个生命。
所用的标记的完整列表示于表1。
表1:芯片实验所考虑的标记的列表
人原代细胞
使用体外细胞培养系统形式的人真皮成纤维细胞或人皮下前脂肪细胞作为生物学测试系统。两种细胞类型都是可商购的(Lifeline公司或CellApplications公司)。
人真皮成纤维细胞一般而言获得自新生儿供体(包茎(phimoses))或者获得自成体供体的皮肤。它们通常是非感染性供体材料,其通过各自的供应商从体内组织敷料分离并且在相应的细胞培养基中单独地扩增。在接种在合适的细胞培养基中后,冷冻保存的人真皮成纤维细胞可以生长至16个倍增循环而不破坏其典型的生理和形态。人真皮成纤维细胞存在于所有结缔组织结构中并且在体外测试系统中良好表征。它们体外释放细胞外基质蛋白并且适合于体外研究,特别是用于研究成纤维细胞生长、研究成纤维细胞的分化以及研究伤口愈合情况中的胶原蛋白代谢。已经通过各种方式证实,人真皮成纤维细胞适合于体外重建的群体、三维皮肤等同物以及体外重建的人皮肤模型。
人前脂肪细胞(HPAd)通常获得自未感染供体的人脂肪组织。虽然人脂肪细胞不适合于低温保存从而不适合于在从供体组织提取细胞后进行可能的再接种,但是在相应的提取后人前脂肪细胞可以很好地低温保存并且可以进行体外培养。然而,它们无法传代超过两次。在那之后,它们分化为人脂肪细胞。人前脂肪细胞的特征在于非常类似于人真皮成纤维细胞的形态的形态。从人前脂肪细胞的体外分化获得的人脂肪细胞可以用作体外测试系统,其用于研究胰岛素刺激的葡萄糖转运、表征激素控制脂质裂解以及研究脂肪组织中的基因表达。HPAd/Had是非常好表征的用于研究肥胖和II型糖尿病体外测试系统。
使人细胞与酵母提取物接触
人原代成纤维细胞在培养基199中扩增(Souto LR,Rehder J,Vassallo J,Cintra ML,Kraemer MH,Puzzi MB(2006)Sao Paulo Med J.124(2),71-6)。人皮肤的体外重建模型包括相关的真皮和表皮;E.Pinney,K.Liu,B.Sheemanand J.Mansbridge(2001):用10% FCS(胎牛血清)和2%青霉素(Pen)/链霉素(Strep)至75%亚汇合的人三维成纤维细胞培养物表达血管形成活性(J CellPhysiol 183,74–82)。细胞收获后,将细胞整合入细胞外胶原蛋白基质(ECM)中,并且在包含5%FCS和2% Pen/Strep的M199培养基的存在下培养。将细胞在ECM中接种24-48h的时间段后,用活性物质(酵母提取物)暴露4-5天。在培养期间利用标准MTT测试监控细胞成活力(Mosmann,Tim ″Rapidcolorimetric assay for cellular growth and survival:application to proliferationand cytotoxicity assays″.Journal of Immunological Methods 65(1–2):55–63)。选择正常细胞培养条件(5%CO2;37℃,最大湿度)。
在正常细胞培养条件下扩增人前脂肪细胞并且利用特定生长培养基(供应商:Cell Applications)进行至60-75%亚汇合。抑制细胞分化为脂肪细胞。收获细胞并再接种在6孔板(>10000细胞/cm2)后,将细胞在培养基的存在下培养24-48h,然后利用特定分化培养基(供应商:Cell Applications)进行分化。分化在酵母提取物的存在下进行10-14的时间段(每5-7天连续的“重复剂量”)。
在每种情况下,将共培养中的细胞如上文所述扩增。成纤维细胞整合入ECM中(如上文所述)后48小时,将人前脂肪细胞施用于胶原蛋白基质上。在正常细胞培养条件下利用(前)脂肪细胞再培养整个构建物48h。然后,前脂肪细胞的分化在活性物质的存在下利用分化培养基进行。再次利用标准MTT测试监控两种细胞类型的细胞成活力。定量确定细胞内乳酸脱氢酶(LDH)以评价细胞膜的完整性。
人表皮角质形成细胞在Cascade Biologics(Invitrogen Company)的Medium 154中扩增。达到70-80%亚汇合后,收获细胞并在上文所述的培养基的存在下于聚碳酸酯膜(<0.4μM孔大小)上浸没培养>48h的时间段。培养基额外补充有FCS和BSA(牛血清白蛋白):脂肪酸复合物以及维生素E、L-丝氨酸和L-肉碱。然后将细胞转换至空气/培养基边界层,并且转移至无血清分化培养基,其具有增加的Ca2+离子浓度并且添加了维生素C。从这个气升(airlift)培养的第7天至第14天在活性物质的存在下(每天连续的“重复剂量”)培养细胞。在这种情况下也选择正常细胞培养条件。
从成纤维细胞分离RNA
利用RNeasy Mini Kit从成纤维细胞分离总RNA。对于分析,将冷冻的成纤维细胞用5mm钢球、300μL RLT缓冲液并且用10μl/mL的Tissuelyser中的β巯基乙醇以16000Hz均质化3min。将样品简单离心以使得Eppendorf管中的泡沫沉降。加入590μL无RNase水和10μL蛋白酶K后,将溶液在恒温摇床中于55℃下消化10min。将约900μL的上清转入新的Eppendorf管中,并加入450μL的96%乙醇。取出700μL,转移至RNeasyMini Spin Column并在室温下以10000rev/min离心15s,可以弃去液体。用剩余的液体重复这一步骤。然后在RNeasy Mini Spin Column中加入700μL缓冲液RW1,并在室温下以10000rev/min离心15s,可以弃去液体。将RNeasy Mini Spin Column放置在新管(2.2mL)上,加入500μL缓冲液RPE并以10000rev/min再离心15s,弃去液体。现在加入500μL缓冲液RPE,将其在室温下以10000rev/min离心2min,弃去液体。将RNeasy MiniSpin Column放置在新管(2.2mL)上,并在室温下以13000rev/min离心1min直至干燥。将RNeasy Mini Spin Column放在Eppendorf管(1.5mL)上,并将30μL的无RNase水直接放置在膜上。然后将其以10000rev/min离心1min。对于测量,在生物分析仪上研究3μL的RNA溶液。分装剩余的溶液(5μL份),并保存在-80℃冰箱中。
从脂肪细胞分离RNA
利用RNeasy Mini Kit从脂肪细胞分离总RNA。为此,通过滗析从脂肪细胞定量除去分化/暴露培养基。对于细胞裂解和释放胞浆RNA,在RT下,在包含盐酸胍的缓冲液(V=600μL;Qiagen buffer RLT)的存在下,将脂肪细胞在6孔细胞培养板中培养2min的时间段。对于细胞的完全破坏,通过1mL一次性注射器和一次性注射套管(0.60x30mm;23Gx1 1/4)剪切细胞悬浮液。将无细胞提取物定量转移至2-mL反应管。通过加入1体积的乙醇(70%)来沉淀总RNA。将700μL沉淀的裂解物转移至RNeasy Mini Column并以≥8000xg离心15s。总RNA结合至柱材料。弃去流过物质(flow-through)。用剩余体积的裂解物进行相同的程序。加入700μL洗涤缓冲液(Qiagen缓冲液RW1),并将其以≥8000x g离心15s。再次弃去流过物质。然后将柱用500μL含乙醇的缓冲液(Qiagen缓冲液RPE)洗涤两次。第一洗涤操作通过以≥8000xg离心(弃去流过物质)进行15s,并且第二洗涤操作通过以≥8000xg离心(也弃去流过物质)进行2min。对于柱材料的完全干燥,将提取柱转移至新的收集容器并以8000xg离心1min。然后将提取柱转移至1.5-mL反应容器并使结合至柱的总RNA通过加入50μL水(无RNase)再水合。总RNA的最终洗脱通过另一离心步骤(1min,≥8000xg)。对于测量,向10μL RNA溶液中加入90μL水并测量260nm波长处的吸收。1.00的吸收(A260)对应于50μg/mL双链DNA、33μg/mL短的单链DNA或40μg/mL RNA的浓度。将剩余的40μL溶液分装(10μL份)并保存在-80℃冰箱中。
芯片技术
通过生物芯片研究分离的mRNA。对于这个生物芯片,选择500个生物标记(参见表1)并且研究它们的特定途径。生物芯片由Heidelberg的FebitBiomed GmbH公司开发。对于每个生物芯片,制备5种不同的样品,并且对每个样品有6个技术重复,从而对于每个生物标记有30个重复。Geniom由Geniom One Instrument利用寡核苷酸合成的标准试剂盒合成。光活化的原位寡核苷酸合成通过数字Micromirror单元连接至玻璃-硅-玻璃夹心上的活化三维反应载体上的Geniom One Instrument。通过Agilent 2100生物分析仪利用RNA 6000 Nano Kit研究mRNA的量。
利用所述的方案获得的具有芯片数据的代表性组的实例示于图2a和2b。数据通过分析用耶氏酵母属细胞的提取物处理的人成纤维细胞的标记获得,所述耶氏酵母属细胞正常地在pH5.4下培养(图2a)或者暴露于pH2的应激(图2b)。
测试的结果总结于表2。
表2:用酿酒酵母培养物的提取物处理后的皮肤病学相关成纤维细胞标记的调节,所述酿酒酵母通过升高的温度应激,用过氧化氢处理或者暴露于低pH的培养基
酵母菌株 | 应激条件 | 诱导或抑制标记的总数 | 强诱导或抑制标记(≥2)的数目 |
酿酒酵母 | T | 273 | 1 |
酿酒酵母 | pH | 32 | 14 |
酿酒酵母 | H2O2 | 292 | 3 |
用各种应激的酵母的提取物处理的细胞的不同表达谱证实,酵母提取物具有不同的性质,因此它们是不同且区分的产品。
实施例3:不同酵母属的pH应激的细胞的提取物对人成纤维细胞的作用的比较研究
研究了不同酵母属的细胞适合于产生皮肤病学特别有效的提取物的程度。
测试条件对应于实施例2中所列的用于制备pH应激的细胞的提取物的条件,除了使不仅一种菌株而是来自毕赤酵母CBS1991、解脂耶氏酵母和酿酒酵母的提取物与人成纤维细胞接触并比较它们的效果。
表3:用不同酵母菌株的培养物的提取物处理后的皮肤病学相关成纤维细胞标记的调节,所述酵母菌株通过暴露于低pH培养基进行应激
表3中所总结的结果证实,pH-应激的耶氏酵母属的酵母细胞对皮肤具有特别有利的效果,与人成纤维细胞接触时,它们积极地调节大量的皮肤标记。
实施例4:加工成提取物并施用于人成纤维细胞后,不同应激条件对耶氏酵母属的酵母细胞的皮肤病学效力的效果的比较
鉴定耶氏酵母属的酵母细胞为生物技术和皮肤病学特别有利的皮肤施用的酵母提取物来源之后,比较其各种应激条件和组合,提供以下结果以发现制备这些酵母提取物的特别有利的方法。
在每种情况中,利用基于水和基于醇的溶剂制备提取物,从而能够比较各自的效果。
表4:用耶氏酵母属培养物的提取物处理后,皮肤病学相关成纤维细胞标记的调节,所述耶氏酵母属培养物已经在各种应激调节及其组合下进行应激
表4中所总结的结果证实,首先,利用水溶液裂解细胞优于利用醇溶液,因为在第一种情况中,多得多的皮肤病学相关成纤维细胞标记以皮肤病学有利的方向被诱导或抑制。
此外可见,暴露于低pH培养基然后暴露于温度升高的应激因素的组合带来皮肤病学相关标记的最高积极调节,因此优于其他应激条件或其组合。
Claims (20)
1.用于制备皮肤病学活性酵母提取物的方法,其包括以下步骤:
a)提供酵母细胞的预培养物,
c)将所述细胞在1.8-4的pH下培养至少15分钟,
d)收获所述细胞,以及
e)将所述细胞裂解。
2.权利要求1的方法,其还包括:
b)将所述细胞在34-39℃的温度和pH>5下培养至少1小时
3.权利要求1或2之一的方法,其中步骤e)利用基于水的裂解剂进行。
4.权利要求2-3之一的方法,其中首先进行步骤b),然后进行步骤c)。
5.权利要求1-4之一的方法,其中步骤c)在34-39℃的温度下进行。
6.权利要求2-5之一的方法,其中步骤b)进行3-5小时。
7.权利要求1-6之一的方法,其中步骤c)进行45-75分钟。
8.权利要求2-7之一的方法,其中步骤b)和c)在36-38℃的温度下进行。
9.权利要求1-8之一的方法,其中步骤c)在1.9-2.2的pH下进行。
10.权利要求1-9之一的方法,其中其为包含以下的属的组的酵母细胞:耶氏酵母属(Yarrowia)、酵母属(Saccharomyces)和毕赤酵母属(Pichia),优选耶氏酵母属。
11.权利要求2-10之一的方法,其中步骤b)在36-38℃的温度下进行3-5小时,步骤c)在1.9-2.2的pH和36-38℃的温度下进行45-75分钟,并且所用的酵母细胞为耶氏酵母属的酵母细胞。
12.可通过权利要求1-11之一的方法获得的酵母提取物。
13.皮肤病学活性剂,其包含耶氏酵母属的酵母细胞的酵母提取物或者权利要求12的酵母提取物。
14.权利要求13的皮肤病学活性剂,其中所述活性剂具有皮肤和/或组织致密作用。
15.食品补充剂,其包含权利要求12的酵母提取物。
16.权利要求13或14的皮肤病学活性剂或者权利要求15的食品补充剂,其中所述酵母提取物中蛋白质的比例为0.5-50mg/L。
17.美容处理皮肤和/或毛发的方法,其包括局部使用权利要求12的酵母提取物或者权利要求13、14或16之一的皮肤病学活性剂。
18.权利要求12的酵母提取物,其用于制备医药产品。
19.权利要求12的酵母提取物,其用于制备针对肥胖、糖尿病、动脉粥样硬化、炎性疾病或心血管疾病或者用于伤口处理的医药产品。
20.权利要求12的酵母提取物在体外刺激角质形成细胞、成纤维细胞和/或脂肪细胞的增殖中的用途。
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EP11171953A EP2540170A1 (de) | 2011-06-29 | 2011-06-29 | Dermatologisch wirksamer Hefeextrakt |
EP11171953.0 | 2011-06-29 | ||
PCT/EP2012/061263 WO2013000717A2 (de) | 2011-06-29 | 2012-06-14 | Dermatologisch wirksamer hefeextrakt |
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US (1) | US9655934B2 (zh) |
EP (1) | EP2540170A1 (zh) |
JP (1) | JP2014520769A (zh) |
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US9655934B2 (en) | 2017-05-23 |
EP2540170A1 (de) | 2013-01-02 |
BR112013033841A2 (pt) | 2016-08-16 |
US20140127257A1 (en) | 2014-05-08 |
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