CN109152727B - 可可豆的肽和糖水解产物、含有其的化妆品组合物以及该化妆品组合物的化妆品用途 - Google Patents
可可豆的肽和糖水解产物、含有其的化妆品组合物以及该化妆品组合物的化妆品用途 Download PDFInfo
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- CN109152727B CN109152727B CN201780028630.1A CN201780028630A CN109152727B CN 109152727 B CN109152727 B CN 109152727B CN 201780028630 A CN201780028630 A CN 201780028630A CN 109152727 B CN109152727 B CN 109152727B
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- PICXIOQBANWBIZ-UHFFFAOYSA-N zinc;1-oxidopyridine-2-thione Chemical compound [Zn+2].[O-]N1C=CC=CC1=S.[O-]N1C=CC=CC1=S PICXIOQBANWBIZ-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明涉及可可(Theobroma cacao L.)(可可(cocoa))豆的肽和糖水解产物、其制备方法、含有其的化妆品组合物以及该化妆品组合物特别是用于保护皮肤免受蓝光影响和/或对抗皮肤的老化和光老化的迹象出现的化妆品用途。
Description
本发明涉及可可(Theobroma cacao L.)(可可(cocoa))豆的肽和糖水解产物、其制备方法、含有其的化妆品组合物以及该化妆品组合物特别是用于保护皮肤免受蓝光影响的化妆品用途。
蓝光代表可见光中的在400与495nm之间的部分。它是可见光谱的主要高能量部分。接受的大部分蓝光源自太阳。在我们的室内环境中,我们越来越多地暴露于来自在照明设备中以及在智能手机、平板电脑、计算机和电视屏幕中存在的LED(发光二极管)的人造蓝光。
可见光的光子被视网膜细胞上的光感受器捕获,启动光转导机制。这些光感受器(被称为视蛋白)与G蛋白偶联并且含有发色团11-顺式视黄醛,其经历光致异构反应变成全反式视黄醛,从而产生信号转导级联反应。
光的感知通过两种途径作用于生物体:包括视觉效应的初级光学途径;和视网膜下丘脑途径,该途径之后是生物效应,诸如褪黑素的分泌、体温的调控、昼夜节律基因(circadian gene)的表达。
已经对蓝光在视网膜细胞上产生的损伤进行了研究。暴露于可见光会在视网膜的上皮细胞中产生活性氧化物质,诱导线粒体DNA损伤并可能导致这些细胞的凋亡2,3。已表明这些改变与年龄相关性黄斑变性的发展相关联,在此期间在视网膜细胞中发生脂褐素的积聚4。
此外,最近已证明蓝光诱导鼠视网膜细胞中视蛋白的积聚,导致细胞损伤5。
已经报道了因使用诸如触摸板的装置产生的蓝光的另一种影响。在入睡前使用这种类型的装置会改变褪黑素水平并导致昼夜节律的转变6。为了对抗蓝光的有害影响,生产含有LED的装置的几个光学品牌已开发出阻挡对应于蓝色的波长的眼镜或屏幕。
蓝光还对皮肤有影响。已经在人皮肤的角质形成细胞和内皮细胞上证明蓝光发挥抗增殖作用并促进角质形成细胞的分化7。此外,它抑制树突细胞的成熟8。这些特性导致将蓝光用于治疗过度增殖性和/或炎性皮肤病,诸如银屑病、异位性皮炎或湿疹。
此外,具有先前改变的屏障功能的皮肤片段暴露于蓝光显示出皮肤屏障恢复的延迟,该皮肤屏障特别是涉及在角质层与颗粒层之间的界面处分泌的脂质。在可见光谱的其他波长下未观察到这种结果,这表明蓝光的特定活性与皮肤屏障的稳态有关1。
关于痤疮的治疗,暴露于蓝光会产生对痤疮丙酸杆菌(Propionibacterium acne)的细菌卟啉的光激发,导致细菌消除9。
最近,在角质形成细胞和黑素细胞中已经鉴定出视蛋白受体OPN1-SW、OPN2、OPN3和OPN5的表达,这表明它们在皮肤细胞中的光转导中发挥作用10。通过激活皮肤细胞中的视蛋白所产生的信号产生非视觉效应,包括昼夜节律基因的调控11。
考虑到蓝光可能对皮肤产生的有害影响,能够开发可用于保护人体皮肤免受蓝光影响的化妆品组合物似乎是重要的。
出人意料的是,申请人已经开发出从可可豆进行提取的新颖方法,从而能够开发富含肽和糖的提取物,该提取物已被证明展现出通过改善皮肤的屏障功能而具有的关于蓝光的保护活性以及预防皮肤出现老化的活性。
可可树(也称为“可可”)根据经典分类法是梧桐科或根据系统发育学分类是锦葵科的可可属的小型常绿树。可可树是源自墨西哥的热带物种,其在大约3000年前被驯化。它是一种茎生花常绿树,高10至15米,一般修剪至6米或8米。它在第3年开始开花并全年产生花、果实和叶。每年,该树可以产生多达100,000朵白色或略带粉红色的花。花全年出现在树木上的隆起上,该隆起称为花垫。因此,花和果实同时存在于树上。树的果实,称为“豆荚”,是大的细长浆果。每个豆荚的重量可高达400g,长度为15至20cm。它们具有既在主枝上生长,但也直接在树干上生长的特殊特征。它们的特征在不同群体之间,但也在同一群体内是高度可变的。果实的成熟需要5至7个月,这取决于基因型。平均而言,一棵树每年产生大约150个豆荚。豆荚包含许多种子(25个与75个之间),这些种子聚集在穗状花序中并称为可可豆,并且富含淀粉、脂肪和生物碱。
可可豆聚集在豆荚的中心,形成包括五行的团块,该五行对应于胚珠的五个子房室。它们具有可变的卵形扁平形状和约25mm的长度、约15mm的宽度和约8mm的厚度。新鲜时,它们是粘性的,因为它们被称为粘质物的白浆包围。可可豆的内核由大胚形成,该大胚包括两个折叠在其自身上的子叶。它具有范围为从白色到深紫色的可变颜色,这取决于品种。它由粉红色或浅红色的种皮包裹。已知可可豆含有大约50%的脂肪(被称为可可脂)、5%的水、7%的淀粉、4%的纤维素、2%的可可碱、20%的蛋白质和6%的矿物质。
众所周知,可可具有刺激、滋补、营养和抗应激作用,并且由于此类特性,已经从可可中提取了各种物质。特别是在化妆品中,出于对皮肤的滋养作用使用了来自可可树(Theobroma cacao)的可可脂。该脂含有53%的脂肪(主要包含油酸、硬脂酸和棕榈酸的甘油三酯),具有超营养的品质,其特定特征是富含由植物甾醇和角鲨烯组成的不皂化物。首先具有抗氧化、结瘢和舒缓作用(对抗烧伤和皲裂)。其次该脂天然存在于皮肤和皮脂中,有助于角质层的脂质胶结物的再生。这就是为什么可可脂可作为皮肤按摩油以及保湿膏、舒缓膏和保护膏中的成分。它还在护理非常干燥和易乱的毛发以及护理发梢分叉方面具有价值。
此外,从专利文献FR 2810242中已知一种基于可可蛋白质提取物的化妆品和/或皮肤病学组合物,该可可蛋白质提取物至少包含多酚、氨基酸和不皂化物级分。所述组合物可用于治疗和预防皮肤老化迹象。在这个文献中,使用整个可可豆,特别是可可脂,以便在蛋白质的总酶促水解后获得包含0.5%-3%或0.5%-5%不皂化物、氨基酸和多酚的混合物。
此外,从专利文献WO 20080153341中,已知化妆品组合物,其包含用于促进体毛和/或颅毛发的色素沉着的可可豆提取物。制备豆提取物的方法包括消除豆的脂质、核酸和糖。获得豆提取物的方法集中于蛋白质的酶促水解,以便产生多肽、肽和游离氨基酸。
此外,从专利文献US 2013/0035291中,已知生物活性产物,其包含源自可可水解产物的具有5至20个氨基酸的肽,所述产物旨在用于消费和治疗神经变性疾病。该文献中使用的方法使得可以获得特定的纯化的无糖肽级分。
由申请人使用可可豆开发的根据本发明的提取方法与以上引用的文献相比是新颖的。该方法被优化用于在从可可豆获得的水解产物中选择和保证高含量的感兴趣靶分子,该靶分子包括特定肽和糖。根据本发明的肽和糖水解产物不包含不皂化分子或游离氨基酸,并且水解产物中存在的痕量多酚确认了与专利WO 200195872的谱不同的谱。
因此,本发明的第一主题是可可豆的肽和糖水解产物,其主要包含肽和糖。
此外,本发明的第二主题是用于制备根据本发明的可可豆的肽和糖水解产物的方法,该方法包括根据以下项的下列步骤:
a)将粉碎的可可豆分散在水相中;
b)对步骤a)中获得的水性分散体进行酶促处理;
c)通过固/液分离进行酶促水解产物的回收;
d)通过超滤和纳米过滤纯化水解产物;然后任选地
e)对步骤d)中获得的水解产物进行冻干。
本发明的第三主题是化妆品组合物,其特征在于该化妆品组合物包含作为活性保护剂的有效量的根据本发明的可可豆水解产物以及生理学上可接受的介质。
最后,本发明的第四主题是根据本发明的组合物用于保护皮肤免受蓝光的有害影响、特别是免受因蓝光产生的氧化应激以便改善皮肤的屏障功能和对抗皮肤老化和皮肤光老化的迹象出现的化妆品用途。
在阅读下面的根据附图撰写的描述和非限制性实施方案后将更好地理解本发明和由其产生的优点,在附图中:
-图1表示:
-根据本发明的水解产物和根据FR 2810242的富含多酚的水-醇提取物的多酚谱的比较以及
-根据本发明的水解产物的氨基酸谱与文献FR 2810242中给出的氨基酸谱的比较,本发明的水解是通过用强酸诸如盐酸(6N)对肽进行总水解24至48小时来进行。
-图2表示在暴露于蓝光(425nm和470nm)之后视蛋白-1、视蛋白-2和视蛋白-3的水平
-图3A表示通过根据本发明的可可水解产物进行的对胶原蛋白1的表达的定量
-图3B表示通过根据本发明的可可水解产物进行的对弹性蛋白的表达的定量
-图3C表示通过根据本发明的可可水解产物进行的对原纤蛋白-1的表达的定量
-图3D表示根据本发明的可可水解产物对DNA甲基化的效果。
在本描述中,除非在正文中另外说明,否则应理解:
-当给出区间时,其包括所述区间的上限和下限,
-除非另外指明,否则正文中的%表示为重量/重量。
此外,在本说明书中,表述“可可豆的肽和糖水解产物”或“肽和糖水解产物”或“根据本发明的可可水解产物”或“水解产物”可互换使用。
在本发明中,
√“有效量”应理解为意指获得所需结果,即特别是使得可能实现保护皮肤免受蓝光影响所需的活性分子的量,而所述量是无毒性的。
√“主要为肽和糖”应理解为意指肽和糖的量为干物质重量的大于50%、优选大于60%、甚至更优选大于70%且可能达到约90%(重量/重量),优选90%。
√“肽和糖水解产物”应理解为意指主要或基本上包含肽和糖(单糖或寡糖)的水解产物。天然存在于豆中的蛋白质和多糖已水解为肽、寡糖和单糖,该水解有利地是酶促水解。
√“局部施用”表示将根据本发明的活性物质或含有它的组合物施用或涂抹在皮肤、粘膜或角质化皮肤附属物的表面上的事实。
√“化妆品上可接受的”应理解为意指根据本发明的活性物质或含有它的组合物适于与皮肤或粘膜接触而不引起毒性或不耐受反应。
√“生理学上可接受的”应理解为意指适于与人体皮肤接触的局部使用或适于通过其他给予途径(例如口服途径或注射到皮肤中)使用,而没有毒性、不相容性、不稳定性、过敏性反应的任何风险。
因此,本发明的第一主题是可可豆的肽和糖水解产物,其主要包含肽和糖。
根据本发明的水解产物是由可可豆作为原料获得的,该可可豆可包括单独的豆或豆及其壳;优选使用包括豆及其壳的豆。
预先将使用的豆进行干燥,并且该豆可以是经发酵的或未经发酵的。在根据本发明的优选实施方案中,使用的豆是未经发酵的干燥的可可豆。
收获的经洗涤且经干燥的可可豆优选来自秘鲁。它们可以源自各种品种的混合物,或来自Criollo、Forastero和Trinitario三个品种中的至少一种。
在根据本发明的优选实施方案中,豆源自Criollo品种且更具体地源自Porcelana亚品种。此外,可可豆不经历巧克力生产过程的基本步骤,即发酵和焙烧,因为这些步骤对用于该应用的蛋白质和糖具有有害影响。
在根据本发明的优选实施方案中,根据本发明的肽和糖水解产物是通过用至少一种碳水化合物酶和至少一种蛋白酶进行的酶促处理获得的。酶促处理分两步进行,第一次处理使用碳水化合物酶并主要产生二糖和单糖,然后第二次处理使用蛋白酶以获得肽。
在本发明甚至更优选的实施方案中,碳水化合物酶选自果胶酶、纤维素酶、阿拉伯聚糖酶、半纤维素酶、木聚糖酶和β-葡聚糖酶,并且蛋白酶是碱性、中性或酸性类型的蛋白酶,优选是具有内肽酶活性的碱性类型的蛋白酶。
有利地,碳水化合物酶是碳水化合物酶的混合物,其至少包括聚半乳糖醛酸酶、果胶甲基酯酶、β葡聚糖酶、β甘露聚糖酶、β葡糖苷酶、纤维素酶、半纤维素酶、阿拉伯聚糖酶和木聚糖酶。
根据本发明的未冻干可可水解产物包含20%至70%的肽和5%至40%的糖。根据本发明的冻干的不含干燥支撑物的可可水解产物包含超过90%的干物质,该干物质包含20%至70%的肽和5%至40%的糖。
根据本发明的另一个非常优选的实施方案,存在于根据本发明的水解产物中的肽和糖具有200Da至10kDa的分子量。
有利地,根据本发明的水解产物不含不皂化物。
还有利地,根据本发明的水解产物不包含游离氨基酸。
最后,根据本发明的水解产物包含最多0.05%的多酚,其主要是甲基黄嘌呤类型的分子,如图1中所指出的。
本发明的第二主题是用于制备根据本发明的可可豆的肽和糖水解产物的方法,该方法包括根据以下项的下列步骤:
a)将粉碎的可可豆分散在水相中;
b)对步骤a)中获得的水性分散体进行酶促处理;
c)将该酶通过热处理变性
d)通过固/液分离进行酶促水解产物的回收,
e)通过超滤和纳米过滤纯化该水解产物;然后任选地
f)对步骤e)中获得的水解产物进行冻干。
首先对根据本发明的方法的步骤a)中使用的粉碎的可可豆进行收获、洗涤、干燥,然后通过压榨进行脱脂以产生可可豆饼。将该饼用于进行根据本发明的方法的步骤a)。
干燥应理解为意指收获后的阶段,在该阶段中豆被脱水以便充分降低豆的含水量以保证有利于豆的储存或随后转化的条件。
例如,可以将豆通过暴露于开放空气(在阳光下或在阴凉处)自然干燥持续至多10至20天的时间,或者有利地,可以将它们在具有吹出的热空气的、伴有相对湿度调控的干燥器中在40℃与60℃之间的温度下温和地人工干燥。
优选地,在有利地洗涤之后,将豆干燥,然后粉碎或冷冻粉碎并脱脂。
脱脂豆是指用于消除可可豆的全部或部分脂质的所有方法,诸如用挥发性溶剂进行固/液提取、用超临界流体提取、或压榨。
在根据本发明的方法的步骤a)中,豆并且特别是压榨饼优选以10%干物质溶解于水中。
根据本发明方法的步骤b)的酶促处理有利地分两步进行,第一次处理使用碳水化合物酶以水解植物细胞的壁,然后第二次处理使用蛋白酶以水解蛋白质。
在根据本发明的方法的优选实施方案中,碳水化合物酶选自果胶酶、纤维素酶、阿拉伯聚糖酶、半纤维素酶、木聚糖酶和β-葡聚糖酶,并且碱性、中性或酸性类型的蛋白酶优选是具有内肽酶活性的碱性类型的蛋白酶。
有利地,碳水化合物酶是碳水化合物酶的混合物,其至少包括聚半乳糖醛酸酶、果胶甲基酯酶、β葡聚糖酶、β甘露聚糖酶、β葡糖苷酶、纤维素酶、半纤维素酶、阿拉伯聚糖酶和木聚糖酶。
相对于待处理的豆的重量,根据本发明的方法的步骤b)中使用的酶按重量计有利地占0.1%至3.0%。
在根据本发明的优选实施方案中,将使用比纤维素酶更多的果胶酶。因此,优选使用的果胶酶相对于纤维素酶的比率为2:1。
酶的变性步骤c)是在80℃与90℃之间的温度下进行15分钟至2小时的持续时间,热处理条件优选为85℃持续45分钟。
优选地,在根据本发明的方法的不同步骤a)至d)期间,温度是在20℃与90℃之间并且pH是在3.0与11.0之间。
根据本发明方法的步骤c)进行的酶促水解产物的回收是通过以下方式进行的:通过本领域技术人员已知的不同方法诸如离心、旋转、过滤,以获得不含固体颗粒的提取物的这样的方式进行固/液分离。有利地,通过离心回收液相。
该固液分离步骤之后是使用截止阈值在10与15kDa之间、优选10kDa的膜进行超滤纯化的步骤d)以便清除残留的蛋白质和变性的酶,以及渗透物的回收。有利地通过稀释渗余物来进行渗滤,以便改善分离性能并因此回收最大量的感兴趣的化合物。
“渗滤”应理解为意指用体积等于渗余物的体积的水冲洗该渗余物,以便促进通过并因此回收分子量低于过滤阈值的最多分子。
该步骤d)还可包括纳米过滤,以便任选地清除游离氨基酸和矿物盐。例如,截止阈值将在100Da(道尔顿)与300Da之间,有利地在130Da与300Da之间,并且通常为200Da。
在另一个实施方案中,在10kDa超滤的第一步骤后,进行通过5kDa膜超滤的第二步骤。该步骤使得可以回收含有其分子量在5kDa与10kDa之间的肽和糖的渗余物级分。
最后,又通过纳米过滤纯化该5kDa超滤的渗透物,以清除游离氨基酸和矿物质并获得包含200Da与5kDa之间的肽和糖的水解产物。
此外,然后可以将由此获得的水解产物在水或任何含水的溶剂混合物中稀释。因此,根据本发明的可可水解产物可以有利地稀释于一种或多种生理学上可接受的溶剂中,该溶剂诸如水、甘油、乙醇、丙二醇、丁二醇、二丙二醇、乙氧基化或丙氧基化二甘醇、环状多元醇或这些溶剂的任何混合物。
根据本发明的第三主题涉及化妆品组合物,其特征在于该化妆品组合物包含作为活性保护剂的有效量的根据本发明的可可豆水解产物以及生理学上可接受的介质。
在根据本发明的优选实施方案中,相对于组合物的总重量,可可水解产物以0.001%至20%,优选0.1%至10%、更优选0.2%至5%、甚至更优选0.5%至1.5%的浓度存在于组合物中。为了制备组合物,根据本发明的可可水解产物可以是液体或冻干形式。
根据本发明的组合物更特别地旨在用于通过局部途径给予。因此,这些组合物必须含有化妆品上可接受的介质,即与皮肤和角质化皮肤附属物相容的介质,并且它们覆盖所有化妆品形式。这些组合物可以特别是呈霜、水包油乳液或油包水或多重乳液、溶液、悬浮液、凝胶、乳、洗剂、棒或还有粉末的形式,并且适于应用于皮肤、嘴唇和/或角质化的皮肤附属物。这些组合物包含其配制所必需的赋形剂,诸如溶剂、增稠剂、稀释剂、表面活性剂、抗氧化剂、染料、防腐剂、香料。它们可用作护肤品或皮肤化妆品。
此外,根据本发明的组合物包含所考虑的应用领域中常用的任何添加剂以及其配制所需的佐剂,诸如溶剂、增稠剂、稀释剂、抗氧化剂、染料、防晒剂、仿晒黑剂(self-tanning agent)、颜料、填料、防腐剂、香料、气味吸收剂、化妆品或药物活性成分、精油、维生素、必需脂肪酸、表面活性剂、成膜聚合物等。
由“个人护理产品理事会公司(the Personal Care Products Council,Inc.)”(华盛顿哥伦比亚特区)出版的INCI字典和手册(“国际化妆品成分命名法(InternationalNomenclature of Cosmetic Ingredients),第13版,2010”)描述了各种各样但非限制性的在皮肤护理行业中常规使用的化妆品和药物成分,其适合用作根据本发明的组合物中的附加成分。
这些类别的附加成分的非限制性例子包括:结瘢剂、抗老化剂、抗皱剂、抗萎缩剂、保湿剂、软化剂、抗细菌剂、抗寄生虫剂、抗真菌剂、杀真菌剂、抑真菌剂、杀细菌剂、抑细菌剂、抗微生物剂、抗炎剂、止痒剂(antiprurigenous agent)、麻醉剂、抗病毒剂、角质层分离剂、抗自由基剂、抗皮脂溢剂、去头皮屑剂、调节皮肤分化、增殖或色素沉着的剂、渗透促进剂、脱屑剂、刺激或抑制黑色素合成的剂、漂白剂、除色素沉着剂或亮白剂、促色素沉着剂、仿晒剂、NO合成酶抑制剂、抗氧化剂、自由基捕获剂和/或抗大气污染剂、抗糖化剂、紧致剂、刺激皮肤或表皮大分子合成的药剂和/或能够预防或抑制该大分子降解的剂、胶原蛋白合成刺激剂、弹性蛋白合成刺激剂、核心蛋白聚糖合成刺激剂、层粘连蛋白合成刺激剂、防御素合成刺激剂、分子伴侣合成刺激剂、水通道蛋白合成刺激剂、透明质酸合成刺激剂、刺激脂质和角质层的组分(神经酰胺、脂肪酸…)合成的剂、胶原降解抑制剂、弹性蛋白降解抑制剂、成纤维细胞增殖刺激剂、角质形成细胞增殖刺激剂、脂肪细胞增殖刺激剂、黑素细胞增殖刺激剂、角质形成细胞分化刺激剂、脂肪细胞分化刺激剂、乙酰胆碱酯酶抑制剂、糖胺聚糖合成刺激剂、DNA修复剂、DNA保护剂、抗瘙痒剂、用于治疗和/或护理敏感皮肤的剂、紧致剂、抗妊娠纹剂、收敛剂、皮脂产生调控剂、皮肤松弛剂、愈合佐剂、再上皮化刺激剂、再上皮化佐剂、细胞因子生长因子、舒缓剂、抗炎剂、作用于毛细血管循环和/或微循环的剂、血管生成刺激剂、血管通透性抑制剂、作用于细胞代谢的剂、旨在用于改善真皮-表皮交界处的剂、诱导颅毛发和/或体毛生长的剂、抑制或减缓颅毛发和/或体毛生长的剂、肌肉松弛剂、抗污染和/或抗自由基剂、脂解刺激剂、减肥剂、消脂肪团剂、作用于微循环的剂、作用于细胞代谢的剂、清洁剂、毛发定型剂、毛发生长刺激剂、防晒油、防晒霜、化妆剂、洗涤剂、药物产品、乳化剂、润肤剂、有机溶剂、防霉剂、除臭活性剂、生理学上可接受的介质、表面活性剂、研磨剂、吸收剂、美学组分(诸如香料、颜料、着色剂、染料和天然染料)、精油、纹理剂、化妆品收敛剂、抗痤疮剂、抗凝剂、防沫剂、抗氧化剂、配体、生物添加剂、酶、酶抑制剂、酶诱导剂、辅酶、螯合剂、植物提取物和植物衍生物、精油、海洋提取物、源自生物发酵和/或生物技术过程的剂、矿物盐、细胞提取物、防晒油(对紫外线A和/或B辐射具有活性的有机或矿物光保护剂)、神经酰胺、肽、缓冲剂、容量剂、螯合剂(chelating agent)、化学添加剂、染料、化妆品杀生物剂、变性剂、药用收敛剂、外用镇痛剂、膜产生剂诸如用于增强组合物的膜产生特性和亲和性的聚合物、季化衍生物、亲和性增加剂、遮光剂、pH调节剂和调控剂(例如三乙醇胺)、推进剂、还原剂、螯合剂(sequestering agent)、皮肤漂白剂和/或亮白剂、皮肤调理剂(即保湿剂,包括混杂的和包藏的)、保留水分的物质、α羟基酸、β羟基酸、保湿剂、表皮水解剂、舒缓剂和/或成瘢剂、皮肤治疗剂、抗皱剂、能够减少或治疗眼袋的剂、去角质剂、增稠剂、软化剂、胶凝聚合物、维生素及其衍生物、润湿剂、剥离剂、舒缓剂、皮肤固化剂、木脂素、防腐剂(即苯氧乙醇和对羟基苯甲酸酯)、抗紫外线剂、细胞毒剂、抗肿瘤剂、粘度调节剂、非挥发性溶剂、粒化剂、防汗剂、脱毛剂、疫苗、香水、皮肤重构剂、赋形剂、填充剂、矿物质、抗分枝杆菌剂、抗过敏剂、H1或H2抗组胺剂、抗刺激剂、免疫系统刺激剂、免疫系统抑制剂、驱虫剂、润滑剂、色素或染料、色素减退剂、光稳定剂及其混合物,前提条件是它们与组合物的其他成分、尤其是与本发明的活性物质在物理和化学上相容。
此外,这些附加成分的性质决不能不可接受地改变本发明活性物质的益处。这些附加成分可以是合成的或天然的,例如像植物提取物,或者它们可以源自生物发酵过程。另外的例子可见于INCI字典和手册。
此类附加成分可选自包括以下项的组:氨基糖、葡糖胺、D-葡糖胺、N-乙酰基葡糖胺、N-乙酰基-D-葡糖胺、甘露糖胺、N-乙酰基甘露糖胺、半乳糖胺、N-乙酰基半乳糖胺、维生素B3及其衍生物、烟酰胺、脱氢乙酸钠、脱氢乙酸及其盐、植物甾醇、水杨酸化合物、己脒定、二烷酰基化合物的二羟基脯氨酸、大豆提取物和衍生物、雌马酚(equol)、异黄酮、黄酮类、植烷三醇、法呢醇、香叶醇、红没药醇、肽及其衍生物、二-、三-、四-、五-和六肽及其衍生物、lys-thr-thr-lys-ser、棕榈酰-lys-thr-thr-lys-ser、肌肽、N-酰基氨基酸化合物、类视黄醇、丙酸视黄酯、视黄醇、棕榈酸视黄酯、乙酸视黄酯、视黄醛、视黄酸、水溶性维生素、抗坏血酸盐、维生素C、抗坏血酸葡萄糖苷、抗坏血酸棕榈酸酯、抗坏血酸磷酸镁、抗坏血酸磷酸钠、维生素及其盐和衍生物、维生素原及其盐和衍生物、乙基泛醇、维生素B及其衍生物、维生素B1、维生素B2、维生素B6、维生素B12、维生素K及其衍生物、泛酸及其衍生物、泛醇基乙基醚、泛醇及其衍生物、乙基泛醇、右泛醇、生物素、氨基酸及其盐和衍生物、水溶性氨基酸、天冬酰胺、丙氨酸、吲哚、谷氨酸、水不溶性维生素、维生素A、维生素E、维生素F、维生素D及其化合物、单-、二-和三萜类化合物、β-紫罗兰醇、雪松醇及其衍生物、水不溶性氨基酸、酪氨酸、色胺、微粒物质、丁羟甲苯、丁羟茴醚、尿囊素、生育酚烟酸酯、生育酚、生育酚酯、棕榈酰-gly-his-lys、植物甾醇、羟基酸、乙醇酸、乳酸、乳糖酸、酮酸、丙酮酸、植酸、溶血磷脂酸、芪、肉桂酸、白藜芦醇、激动素、玉米素、二甲氨基乙醇、天然肽、大豆肽、酸性糖盐、葡萄糖酸锰、葡萄糖酸锌、吡罗克酮乙醇胺盐、3,4,4'-三氯碳酰苯胺、三氯卡班、吡啶硫酮锌、氢醌、曲酸、抗坏血酸、抗坏血酸磷酸镁、抗坏血酸葡糖苷、吡哆醇、芦荟、萜烯醇、尿囊素、红没药醇、甘草酸二钾、甘油、山梨糖醇、季戊四醇、吡咯烷酮的酸及其盐、二羟基丙酮、赤藓酮糖、甘油醛、tartaraldehyde、丁香精、薄荷脑、樟脑、桉树精、丁子香酚、乳酸薄荷酯、金缕梅馏分、二十碳烯和乙烯基吡咯烷酮共聚物、碘丙基丁基氨基甲酸酯、多糖、必需脂肪酸、水杨酸酯、甘草次酸、类胡萝卜素、神经酰胺和类神经酰胺、复合脂质、通常天然来源的油(诸如乳木果油、杏油、野生驴油(onager oil)、李油、棕榈油、大溪地萃取油(monoi油)、卡海油(kahai oil))、氢醌、HEPES、丙半胱氨酸、O-辛酰基-6-D-麦芽糖、甲基甘氨酸二乙酸二钠盐、类固醇诸如薯芋皂素和DHEA衍生物、DHEA脱氢表雄酮和/或化学或生物前体或衍生物、N-乙基羰基-4-对氨基苯酚、蓝莓提取物、植物激素、酿酒酵母(Saccharomycescerevisiae)酵母提取物、藻类、大豆、羽扇豆、玉米和/或豌豆的提取物、阿尔维林及其盐(特别是柠檬酸阿尔维林)、假叶树和七叶树提取物及其组合、金属蛋白酶抑制剂、秘鲁乳香树(Schinus molle)提取物。
在所有情况下,本领域技术人员必须确保选择这些佐剂及其比例,以免损害根据本发明的组合物的所需有利特性。
根据本发明的第四主题涉及根据本发明的组合物用于保护皮肤免受蓝光的有害影响和改善皮肤的屏障功能、以及对抗皮肤的老化和光老化的迹象出现的化妆品用途。
“皮肤老化的迹象”应理解为意指由于实足年龄而对皮肤外观的任何改变,诸如皱纹和小皱纹、龟裂、眼袋、眼周环、干皱、皮肤的弹性和/或色调丧失、暗淡或缺乏光泽、色素缺陷以及皮肤的未系统性地表现在改变外观中的任何内部改变(例如像真皮变薄和密度损失、角质层增厚)。
“皮肤光老化的迹象”应理解为意指暴露于UV辐射后皮肤的任何劣化,诸如眼睛和口腔周围细皱纹以及前额上的表情皱纹的过早出现;鼻子、脸颊和颈部的毛细血管扩张(小的扩张血管);各种色素斑点,诸如红斑点和日光性着色班、肤色不规则性、皮肤色调的普遍丧失、紧绷感增加、唇部失色或唇部皮肤变薄,皮肤也可能变厚且变得粗糙。还包括皮肤的未系统性地表现在改变外观中的任何内部改变,例如像血管壁的增厚、成纤维细胞形状的改变、胶原蛋白合成的减缓和胶原原纤维的组织的破坏、含有弹性蛋白的异常和无定形物质的积聚(日光性弹力组织变性)。为了说明,下面描述本发明的实施方案。
实施例1:从可可豆制备可可水解产物
使用的豆包括其壳,并且是源自秘鲁的Criollo品种并且更特别地是Porcelana亚品种。
该程序如下:
a)将通过压榨脱脂的可可豆饼以10%干物质(DM)溶解在水中;
b)通过纤维素酶(0.5%/DM)和果胶酶(1.0%/DM)的混合物的作用在pH 4.5和55℃温度下将碳水化合物酶促水解2小时,该混合物包括聚半乳糖醛酸酶、果胶甲基酯酶、β葡聚糖酶、β甘露聚糖酶、β葡糖苷酶、纤维素酶、半纤维素酶、阿拉伯聚糖酶和木聚糖酶;
c)然后通过内肽酶类型的碱性蛋白酶(0.5%DM)在pH 9.0和55℃温度下进行酶促水解4小时;
d)在85℃下热处理45分钟以使酶变性;
e)离心并回收液相;
f)通过10kDa膜对液相进行超滤和渗滤并回收渗透物;
g)通过200Da膜对来自超滤的渗透物进行纳米过滤并回收渗余物。
在步骤g)中的渗余物中获得的可可水解产物含有3.0%与4.0%之间的干物质。
此干物质具体地包含按重量计20%至70%的肽和5%至40%的糖,该肽和该糖具有在200Da与10kDa之间的分子量。
然后可以将水解产物稀释于水中或任何含水的溶剂混合物中。因此,根据本发明的可可水解产物可以有利地稀释于一种或多种生理学上可接受的溶剂中,该溶剂诸如水、甘油、乙醇、丙二醇、丁二醇、二丙二醇、乙氧基化或丙氧基化二甘醇、环状多元醇或这些溶剂的任何混合物。
实施例2:从可可豆制备可可水解产物
步骤a)至g)与实施例1中进行的那些相同。然后,进行以下程序:
h)将纳米过滤渗余物冻干,该纳米过滤渗余物相对于此渗余物的干提取物具有按重量计50%的麦芽糖糊精(干燥支撑物)。
在步骤h)的渗余物中获得的可可肽水解产物含有94.0%至98.0%的干物质。
此干物质具体地包含按重量计50%的麦芽糖糊精、10%至35%的肽和2%至20%的糖,该肽和该糖具有在200Da与10kDa之间的分子量。
实施例3:从可可豆制备可可水解产物
步骤a)至g)与实施例2中进行的那些相同。然后,进行以下程序:
h)在不含干燥支撑物的情况下将纳米过滤的渗余物冻干。
在步骤h)中获得的可可肽水解产物含有94.0%与98.0%之间的干物质。
此干物质具体地包含按重量计20%至70%的肽和5%至40%的糖,该肽和该糖具有在200Da与10kDa之间的分子量。
实施例4:从可可豆制备可可水解产物
步骤a)至f)与实施例1中进行的那些相同以回收超滤液1。然后,进行以下程序:
g)通过5kDa膜对超滤的渗透物1进行超滤并回收渗余物。
h)在不含干燥支撑物的情况下将超滤的渗余物2冻干。
步骤h)中获得的可可的肽水解产物含有94.0%至98.0%的干物质,其具有在5kDa与10kDa之间的分子量。
该干物质具体地包含按重量计20%至70%的肽和5%至40%的糖。
实施例5:从可可豆制备可可水解产物
步骤a)至f)与实施例1中进行的那些相同以回收10kDa超滤的渗透物(超滤液1)。然后进行以下程序:
g)通过5kDa膜对超滤液1进行超滤2并回收渗透物(超滤液2);
h)通过200Da膜对超滤液2进行纳米过滤并回收渗余物。
步骤h)中获得的可可的肽和糖水解产物含有3.0%至4.0%的干物质,其具有在200Da与5kDa之间的分子量。
该干物质具体地包含按重量计20%至70%的肽和5%至40%的糖。
实施例6:根据本发明的水解产物与根据FR 2810242获得的提取物的比较
作为现有技术引用的专利FR 2810242提出了化妆品组合物,其含有按重量计0.05%至5%的从可可中提取的氨基酸、按重量计0.05%至5%的从可可中提取的多酚和按重量计0.05%至3%的不皂化物浓缩物的组合。
作为对比提取物,如下制备根据文献FR 2810242的富含多酚的水-醇提取物(在以下实施例中称为富含多酚的提取物):
通过在50℃下将具有10%干提取物的脱脂饼在70%乙醇中浸渍1小时提取多酚,然后在真空下蒸发乙醇。
然后通过福林酚(Folin Ciocalteu)方法测定提取物,该方法使得可以测定所有多酚。该方法使用没食子酸作为标准物,并且结果以没食子酸当量的重量%表示。
两种提取物的定量比较如下。
根据本发明的肽和糖水解产物的特征在于:
-0.5%没食子酸当量的多酚含量。含有10%的根据本发明的水解产物的组合物包含最多0.05%的多酚。这对应于FR 2810242的在0.5%与5%之间的最小含量以至最接近的当量(没食子酸的分子量与水解产物中存在的多酚的分子量相比)。
-它含有很少或不含游离氨基酸。它们处于肽的形式(蛋白质的部分水解)。
-它不含不皂化的化合物。
两种提取物的定性比较
重要的是要注意,根据本发明的肽和糖水解产物含有很少或不含游离氨基酸。它们处于肽的形式(蛋白质的部分水解)。因此,在比较方面,比较氨基酸含量和肽含量是不合理的。然而,比较相对的氨基酸谱(两种提取物中呈现的氨基酸的相对图谱)是有趣的。
根据本发明的水解产物的多酚谱和氨基酸谱在图1中给出。该图包括:
-根据本发明的水解产物和根据FR 2810242的富含多酚的水-醇提取物的多酚谱以及
-根据本发明的完全水解的水解产物的氨基酸谱和FR 2810242中给出的氨基酸谱结果。
根据FR 2810242的富含多酚的提取物具有复杂的多酚谱,其中可能存在三个分子家族:类黄酮(例如,儿茶素和衍生物)、类苯基丙烷(例如阿魏酸和衍生物)和甲基黄嘌呤(例如,咖啡因和可可碱)。相比之下,根据本发明的水解产物的多酚谱不再包含类黄酮,但仍包含潜在的甲基黄嘌呤和类苯基丙烷。
结论:
根据本发明的水解产物与根据FR 2810242的富含多酚的水-醇提取物的定性比较显示出在多酚和氨基酸(游离的或不是游离的)组成方面的显著差异。
实施例7:评价根据实施例1的可可的肽和糖水解产物对因蓝光而在培养物中的角
质形成细胞上产生的细胞活性氧化物质水平的影响:
该研究的目的是评价根据实施例1的可可水解产物的处理对因暴露于蓝光而在培养物中的正常人角质形成细胞中产生的氧化应激水平的影响。同时,使用根据实施例6的富含多酚的可可水解产物进行处理。
将由LED组成的蓝光源用于在415nm和470nm处产生应激。通过荧光探针在细胞水平上测定由这种暴露产生的活性氧化物质(ROS)。
方案:
将培养物中的正常人角质形成细胞通过在特定培养基中稀释至0.1%体积/体积(或稀释至1/1000)的根据实施例1的水解产物进行预处理,每天两次,而将对照培养物维持在未处理条件下。24小时后,将对照培养物和经处理的培养物的一部分暴露于蓝光(415和470nm,在3mW/cm2下持续18分钟),而将其他部分维持为避光保护。在该暴露之后,再次将水解产物每天应用两次,持续24小时。然后,再次重复对蓝光的暴露,接着进行ROS检测的测试。
出于此目的,将细胞在荧光探针(绿色试剂,Lifetechnologies)的存在下于37℃放置30分钟。在固定和冲洗后,在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下观察细胞。基于所获得的照片,借助于图像分析软件(PerkinElmer,Inc.)进行荧光强度的定量,该荧光强度与存在的ROS的量成比例。
还进行了在根据实施例4和5获得的可可水解产物的存在下的处理。出于此目的,预先稀释水解产物以便获得与实施例1的水解产物可比的浓度。
结果:
在角质形成细胞上用0.1%的根据实施例1的水解产物处理使得可以显著降低因暴露于蓝光而产生的ROS的水平(与未经处理的对照相比,暴露于蓝光的细胞中的+90%强度相对于经处理和暴露的细胞中的+34%)。此外,观察到的效应大于根据实施例6的富含多酚的提取物对这些相同细胞所获得的效应。
通过根据实施例4和5获得的水解产物处理也使得可以观察到因暴露于蓝光而产生的ROS的水平降低。
结论:
根据实施例1、4或5之一获得的可可水解产物的应用使得可以限制因暴露于蓝光而产生的氧化应激,如通过用水解产物处理的角质形成细胞中细胞内ROS的水平降低所表明的。
实施例8:评价根据实施例1的可可水解产物对因蓝光而在培养物中的角质形成细 胞上产生的线粒体活性氧化物质的水平的影响:
该研究的目的是评价根据实施例1的可可水解产物的处理对因暴露于蓝光而在培养物中的正常人角质形成细胞中产生的在线粒体水平上的氧化应激水平的影响。实际上,线粒体是ROS的优先靶标,并且在视网膜的上皮细胞暴露于可见光之后已经观察到线粒体DNA的损伤13。同时,对这些相同细胞用根据实施例6的富含多酚的可可水解产物进行处理。
方案:
处理细胞和暴露于蓝光的方案与实施例7中描述的方案相同。在这些步骤之后,通过荧光探针(MitoSOXTM红色线粒体超氧化物指示剂,Invitrogen)在37℃下与细胞接触15分钟来揭示线粒体ROS。在冲洗和固定后,在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下观察细胞。基于所获得的照片,借助于图像分析软件(PerkinElmer,Inc.)进行荧光强度的定量,该荧光强度与存在的ROS的量成比例。
还进行了在根据实施例4和5获得的可可水解产物的存在下的处理。出于此目的,预先稀释水解产物以获得与实施例1的水解产物可比的浓度。
结果:
暴露于蓝光导致线粒体ROS增加(与未经处理的对照相比为+156%)。在根据实施例1的可可水解产物以0.1%存在的情况下,观察到线粒体ROS水平的强烈降低(与未经处理的对照相比为+8%)。观察到的效应大于根据实施例6的富含多酚的可可水解产物对这些相同细胞所获得的效应。
用根据实施例4和5获得的水解产物处理也使得可以观察到因暴露于蓝光而产生的ROS的水平降低。
结论:
除了实施例7中报道的结果之外,该测试还可以得出结论:根据实施例1、4或5的可可水解产物的应用使得可以限制因暴露于蓝光而产生的氧化应激,如通过线粒体ROS水平降低所表明的。
实施例9:根据实施例1的可可水解产物对暴露于蓝光后视蛋白-1、视蛋白-2和视 蛋白-3的水平的维持的影响。
本研究的目的是观察如实施例7和8中所定义的蓝光暴露对通过根据实施例1的可可水解产物处理的正常人角质形成细胞中光感受器视蛋白-1、视蛋白-2和视蛋白-3的水平的影响。这些光感受器是由角质形成细胞表达OPN1-SW、OPN2和OPN3基因产生的,并且能够在每种视蛋白的特定蓝光波长存在下诱导信号传导通路12。
方案:
通过稀释至0.1%体积/体积(或稀释至1/1000)的根据实施例1的水解产物处理培养物中的正常人角质形成细胞,然后根据实施例1中描述的方案将其暴露于蓝光。第二次暴露后3小时,使用间接免疫荧光技术检测视蛋白-1、视蛋白-2和视蛋白-3。
出于此目的,将细胞冲洗、用3.7%的多聚甲醛固定,并用0.1%的Triton透化10分钟。将非特异性位点用1%的牛血清白蛋白饱和10分钟后,将细胞与针对视蛋白-1(OPSNShort)、视蛋白-2(视紫红质)或视蛋白-3(全整蛋白)的抗体溶液一起孵育一小时,然后与偶联至荧光染料(Alexa488,Invitrogen)的抗兔二抗溶液一起孵育。然后在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下检查细胞。基于所获得的照片,借助于图像分析软件(PerkinElmer,Inc.)进行荧光的定量。
还进行了在根据实施例4和5获得的可可水解产物的存在下的处理。出于此目的,预先稀释水解产物以获得与实施例1的水解产物可比的浓度。
结果:
在蓝光的存在下在确定用于模拟培养物中角质形成细胞过度暴露的条件下,视蛋白-1的标记强度在与该视蛋白敏感的波长相对应的415nm处暴露时显著降低(与对照相比-33%)。在用0.1%的实施例1的可可水解产物处理细胞的条件下,维持视蛋白-1的标记(与对照相比为-10%)。在相同条件下,不能观察到根据实施例6的富含多酚的可可水解产物对视蛋白-1水平的保护作用(与未经处理的对照相比为-33%)。
在相同的处理条件下,当角质形成细胞暴露于蓝光(470nm)时,视蛋白-2和视蛋白-3的标记强度降低(与未经处理的对照相比分别为-43%和-27%)。在根据实施例1的可可水解产物以0.1%存在的情况下,视蛋白的标记表现为得以维持(与未经处理的对照相比分别为-28%和-10%)。在相同条件下,不能观察到根据实施例6的富含多酚的可可水解产物对视蛋白-2和视蛋白-3水平的保护作用(与未经处理的对照相比分别为-42%和-30%)。
结果呈现于图2中。
用根据实施例4和5获得的水解产物处理也使得可以观察到在暴露于蓝光后维持 视蛋白-1、视蛋白-2和视蛋白-3的水平。
结论:
根据实施例1、4或5之一获得的可可水解产物使得可以在模拟过度暴露于蓝光的应激存在下维持视蛋白-1、视蛋白-2和视蛋白-3的水平。这些结果证明,针对蓝光,根据本发明的水解产物具有保护作用。
实施例10:根据实施例1的可可水解产物对暴露于蓝光的角质形成细胞上的昼夜 节律蛋白CRY-1和PER-1的表达水平的影响:
本研究包括使用经历暴露于蓝光的角质形成细胞,在先前定义的用于对这些细胞产生应激的条件下,评价根据实施例1的可可水解产物的处理对昼夜节律蛋白CRY-1和PER-1的水平的维持的影响。
昼夜节律基因的调节是在视网膜细胞水平上激活光感受器的后果之一。视网膜细胞中视蛋白的表达本身依赖于时钟基因的表达,该表达受环境因素(诸如暴露于光)的调控13。
方案:
用稀释至0.1%体积/体积(或稀释至1/1000)的根据实施例1的可可水解产物处理培养物中的正常人角质形成细胞,然后根据实施例7中描述的方案将其暴露于蓝光。将在相同条件下处理的培养物维持在黑暗中。第二次暴露后6小时,根据如实施例9中所述的标准方案,通过免疫细胞化学技术揭示昼夜节律蛋白CRY-1和PER-1的表达水平。
在冲洗、固定和将非特异性位点饱和后,将细胞与针对蛋白质CRY-1和PER-1的一抗一起孵育,然后与偶联至荧光染料(Alexa488,Invitrogen)的二抗一起孵育。然后在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下检查细胞。基于所获得的照片,借助于图像分析软件(PerkinElmer,Inc.)进行荧光的定量。
还进行了在根据实施例4和5获得的可可水解产物的存在下的处理。出于此目的,预先稀释水解产物以便获得与实施例1的水解产物可比的浓度。
结果:
在将细胞维持在黑暗中的条件下,与未经处理的对照相比,0.1%的根据实施例1的可可水解产物使得可以将蛋白质CRY-1和PER-1的表达水平分别增加+19%和+17%。暴露于蓝光(415nm)导致CRY-1显著降低(与未经暴露的细胞相比为-25%),该降低通过根据实施例1的可可水解产物的处理来抵消(与未经暴露和未经处理的对照相比为+20%)。关于PER-1的表达水平,它在暴露于蓝光(415nm)的细胞中也降低(与未经暴露的细胞相比为-11%),并且在这些相同的条件下,在暴露和用水解产物处理的条件下显现为维持为高于对照(与未经暴露和未经处理的对照相比为+27%)。
用根据实施例4和5获得的水解产物处理也可以观察到相同的结果。
结论:
用根据实施例1、4或5之一获得的可可水解产物处理使得可以维持暴露于蓝光的角质形成细胞中的昼夜节律蛋白CRY-1和PER-1的水平。这些结果证明,针对蓝光,根据本发明的水解产物具有保护作用。
实施例11:根据实施例1的可可水解产物对暴露于UVB应激或蓝光的离体皮肤活检 样品中的原纤蛋白-1网络的维持的影响:
该研究的目的是评价根据实施例1的可可水解产物在因UVB或因暴露于蓝光而产生的应激的存在下对原纤蛋白-1网络的潜在保护作用。
弹性纤维网络显现是光老化的特定靶标,特别是关于富含原纤蛋白-1的微原纤维结构的变化。该蛋白质是存在于真皮浅层中的耐酸水解性纤维的组分。在光老化的情况下,弹性蛋白的微原纤维在真皮-表皮交界处丧失其特有的枝状烛台形状14。
方案:
将维持在培养物中的人皮肤活检样品用在PBS中稀释至1%体积/体积(或稀释至1/100)的根据实施例1的可可水解产物每天处理2次,持续24小时,然后将样品用UVB(100mJ/cm2)照射或暴露于蓝光(470nm,3mW/cm2,持续26min)。同时,将活检样品保持在黑暗中。每天同样应用处理2次,持续24小时,然后使活检样品经历对UVB或蓝光的第二次暴露。在再24小时的处理后,收集活检样品以用于通过免疫组织化学检测原纤维蛋白-1。
使用在抗原纤蛋白-1抗体(鼠单克隆抗体)的存在下孵育的冷冻切片进行该技术。孵育1小时接着冲洗后,将切片在偶联至荧光团(Alexa488,Invitrogen)的抗小鼠二抗的存在下孵育。然后在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下检查切片。然后观察存在于真皮-表皮交界处的弹性蛋白微原纤维网络。
还进行了在根据实施例4和5获得的可可水解产物的存在下的处理。出于此目的,预先稀释水解产物以获得与实施例1的水解产物可比的浓度。
结果:
对于保持在黑暗中的活检样品,与未经处理的活检样品相比,在1%的根据实施例1的可可水解产物的存在下,用原纤蛋白-1标记的微原纤维显现为更长。
在活检样品暴露于UVB的条件下,微原纤维网络看起来明显减小并且不再可能区分纤维的垂直取向。与未经处理、经暴露的活检样品相比,在将活检样品用1%的根据实施例1的可可水解产物处理并暴露于UVB的条件下,观察到垂直于真皮-表皮交界处的纤维更长。在暴露于蓝光的情况下也观察到这些效果。
用根据实施例4和5获得的水解产物处理也可以观察到相同的结果。
结论:
这些结果指示了在UVB应激和暴露于蓝光的情况下根据实施例1、4或5之一获得的可可水解产物对于原纤蛋白-1的保护作用,这表明了在预防光老化方面的优势。
实施例12:根据实施例1的可可水解产物对离体皮肤活检样品中多配体蛋白聚糖- 4的表达水平的影响:
本研究的目的是评价根据实施例1的可可水解产物对人皮肤活检样品中的多配体蛋白聚糖-4的水平的潜在影响。
多配体蛋白聚糖-4是细胞表面存在的具有硫酸乙酰肝素的蛋白聚糖,在细胞表面处起细胞相互作用介导剂的作用,从而调控细胞粘附、迁移、增殖、内吞作用和机械转导的机制15。
方案:
用在PBS中稀释至1%体积/体积(或稀释至1/100)的根据实施例1的可可水解产物处理人皮肤活检样品,每天2次,总共持续72小时。
然后通过免疫组织化学揭示这些活检样品切片上的多配体蛋白聚糖-4。出于此目的,使切片与针对多配体蛋白聚糖-4的一抗(兔多克隆抗体)接触。孵育和冲洗后,将偶联至荧光团(Alexa488,Invitrogen)的抗兔二抗应用于切片。然后在落射荧光显微镜(Zeiss Axiovert 200M显微镜)下检查切片。基于所获得的照片,借助于图像分析软件(PerkinElmer,Inc.)进行荧光的定量。
结果:
与未经处理的对照活检样品相比,根据实施例1的可可水解产物使得可以将多配体蛋白聚糖-4的水平增加+18%。用根据实施例6制备的富含多酚的可可水解产物观察到这种效果的程度较小(+12%)。
结论:
根据实施例1的可可水解产物对皮肤活检样品中的多配体蛋白聚糖-4具有活性,这表明对角质形成细胞与其细胞外环境之间的相互作用的潜在影响。
实施例13:证明根据实施例2的可可水解产物的抗老化和复苏活性的体外测试
真皮除了作为向表皮供给的元件之外,还为表皮提供固体支撑。它主要由成纤维细胞和细胞外基质组成,该细胞外基质主要由胶原蛋白、弹性蛋白和被称为基本物质的物质组成。这些组分由成纤维细胞合成。表皮与真皮之间的内聚力是由真皮-表皮交界确保的。
胶原蛋白是皮肤细胞外基质的主要蛋白质。迄今为止,已鉴定出20种胶原蛋白类型并将其标记为I至XX。整个真皮中主要存在的胶原蛋白是I型和III型胶原蛋白,其形成整个真皮的细胞外基质(这些胶原蛋白占真皮干重的70%-80%)。随着年龄的增长,真皮变得更薄并且皮肤表面上出现皱纹。因此,考虑到胶原蛋白在确保皮肤完整性及其对机械外部攻击的抵抗力方面的重要作用,刺激这些胶原蛋白且特别是I型胶原蛋白的合成显现是缓解皮肤老化迹象的有效手段。(由Tzaphlidou M.,Micron 35(2004)173-177综述的)。
1-年轻细胞相对于老化细胞的研究
以下研究使得可以研究根据实施例2的可可水解产物(在图中称为水解产物)对作为真皮细胞外基质的成分的胶原蛋白I、弹性蛋白和原纤蛋白1的表达的影响,以便评价其抗老化和抗光老化活性及其复苏活性。还通过研究根据实施例2的可可水解产物对DNA甲基化即表观遗传靶标的影响来评价后一种活性。
在老化过程中,胶原纤维和弹性纤维由于合成减少和降解增加而改变,导致皮肤质量降低,伴有色调减少和弹性丧失。
为了允许细胞复苏的声称,我们对在胶原蛋白I和弹性蛋白的情况下内在老化(复制性衰老)的细胞内、或者在原纤蛋白1的情况下通过反复UV照射而外在老化(光老化)的细胞内的涉及这些纤维结构的蛋白质的表达进行定量。
为了理解观察到的蛋白质表达再激活的机制,我们对内在老化细胞进行了DNA甲基化分析。实际上,细胞衰老的特征之一是在二核苷酸CpG中包含的胞嘧啶水平上DNA内甲基化区域的积聚。这导致启动子的失活和某些基因表达的降低。
·细胞毒性的初步评价
在NHDF上评价分子的细胞毒性。
在可溶于0.1%DMSO的水性溶液中的测试的最大浓度下和在1/2log至1/2log的7个稀释度下评价细胞毒性。
该试剂含有紫色指示剂四唑盐。该试剂被代谢活性细胞裂解形成黄色指示剂甲臜。因此,黄色着色的水平与活细胞的数量成比例。吸光度测量是在450nm下进行。
该测试考虑小于对照的90%的值指示产物的潜在细胞毒性(在图形中用绿线标示)。这也可表明细胞的代谢活性降低。小于75%的值指示显著的细胞毒性(在图表中用红线标示)。
此外,在显微镜下观察细胞以观察和比较它们的外貌。
根据实施例2的可可水解产物在1000ppm下展现出高细胞毒性,而在200与20ppm之间展现出较弱但显著的细胞毒性。根据实施例2的可可水解产物在2ppm与20ppm之间没有展现出显著的细胞毒性。
保留用于测试的浓度:
对根据实施例2的可可水解产物在以下浓度20-10-5ppm下进行测试。
-胶原蛋白I靶标
胶原蛋白I是主要的胶原蛋白,其赋予皮肤机械阻力。
这种蛋白质占脊椎动物胶原蛋白的90%。它构成骨骼(与钢筋混凝土的增强材料相当)且更通常地构成常见结缔组织的框架。它存在于骨骼、皮肤、肌腱、角膜和内脏中。
·评价根据实施例2的可可水解产物对胶原蛋白I表达的影响的方法
将年轻NHDF(正常人真皮成纤维细胞)或通过复制性衰老老化的NHDF接种在96孔板中并在37℃、5%CO2下孵育24h。
在测试产品的存在下处理细胞24h。
然后用福尔马林固定细胞,并且通过免疫荧光检测蛋白质的表达。
通过自动显微镜(Arrayscan CellomicsTM)对荧光标记物进行成像和定量。通过生物学应用软件Compartimental Analysis定量荧光。
·评价根据实施例2的可可水解产物对胶原蛋白I表达的影响
结果在图3A中给出。
与复制性老化的细胞相比,根据实施例2的可可水解产物使得可以再激活胶原蛋白I的表达。实际上,在根据实施例2的可可水解产物的三种测试浓度下获得的靶蛋白的蛋白质表达百分比高于用老化细胞模型获得的那些。
结论:
根据实施例2的可可水解产物是作用于皮肤老化靶标胶原蛋白I的成分,该作用是通过与未经处理的老化细胞相比重新启动蛋白质表达以及通过与年轻对照相比部分重建蛋白质表达来进行的。
-弹性蛋白靶标
弹性蛋白是参与ECM组成的结构糖蛋白(如层粘连蛋白和纤连蛋白)。弹性蛋白是结构型纤维蛋白家族的蛋白质。它基本上在生长期由成纤维细胞分泌,具有弹性特性。其合成随着年龄的增长而减少,并且发现弹性蛋白被不可延展的胶原蛋白替代。妊娠纹是该过程的可见例子,其与机械应力相关联。皮肤老化是这个的第二个例子。
在细胞外基质中,弹性蛋白由成纤维细胞在细胞外空间中合成并分泌,首先呈前弹性蛋白的形式,然后呈原弹性蛋白的形式。除原纤蛋白外,弹性蛋白是弹性纤维的主要组分(高达90%)。因此,通过横向共价键与形成弹性纤维的弹性蛋白和原纤蛋白缔合的胶原蛋白是细胞外基质的主要成分。弹性蛋白的全部产生在青春期左右停止。此后,弹性蛋白的可用量将随着时间的推移而减少。
弹性蛋白的降解与弹性蛋白酶的作用有关,弹性蛋白酶是由成纤维细胞分泌的酶。α1-抗胰蛋白酶抑制弹性蛋白酶的酶促作用。抑制降解创建了平衡,从而增加了弹性蛋白的稳定性。
独特特征赋予弹性蛋白特色:弹性蛋白允许细胞连接并使生物组织形成。因此,皮肤、肺、血管、结缔组织以及某些肌腱和软骨的适当功能发挥与弹性蛋白的特征密切相关。顾名思义,弹性蛋白具有弹性。在相同的直径下,它比橡皮筋的弹性强5倍。在断裂之前,它可以拉伸至其静止长度的150%。因此,它允许组织被拉伸并在拉伸后恢复其初始状态,这赋予了组织柔韧性。
弹性蛋白存在于皮肤的充当支撑物的真皮中。在衰老过程中,例如,真皮的弹性和色调丧失不再能够抵抗下方肌肉收缩的作用,从而引起皱纹的出现。此外,暴露于紫外线辐射会增加弹性蛋白的降解。
·评价根据实施例2的可可水解产物对弹性蛋白表达的影响的方法
将年轻NHDF(正常人真皮成纤维细胞)或通过复制性衰老老化的NHDF接种在96孔板中并在37℃、5%CO2下孵育24h。
在测试产品的存在下处理细胞24h。
然后用福尔马林固定细胞,并且通过免疫荧光检测蛋白质的表达。
通过自动显微镜(Arrayscan CellomicsTM)对荧光标记物进行成像和定量。通过生物学应用软件Compartimental Analysis定量荧光。
·评价根据实施例2的可可水解产物对弹性蛋白表达的影响
结果在图3B中给出。
与老化细胞相比,根据实施例2的可可水解产物使得可以略微再激活弹性蛋白的表达。
结论:
根据实施例2的可可水解产物是作用于皮肤老化靶标弹性蛋白的成分,该作用是通过与未经处理的老化细胞相比在三种测试浓度下略微重新启动蛋白质表达并通过与年轻对照相比部分部分重建蛋白质表达进行的。
-原纤蛋白1靶标
原纤蛋白1是构成微原纤维的蛋白质,该微原纤维与弹性纤维缔合并参与其组装。
·评价可可的肽水解产物对原纤蛋白1表达的影响的方法
将年轻NHDF(正常人真皮成纤维细胞)或通过辐射外在老化的NHDF接种在96孔板中并在37℃、5%CO2下孵育24h。
将用于外在老化的细胞用UVA照射3次,每次照射间隔24h,并且用UVB照射2次,每次照射间隔48h。
在测试产品的存在下处理细胞24h。
然后用福尔马林固定细胞,并且通过免疫荧光检测蛋白质的表达。
通过自动显微镜(Arrayscan CellomicsTM)对荧光标记物进行成像和定量。通过生物学应用软件Compartimental Analysis定量荧光。
·评价根据实施例2的可可水解产物对原纤蛋白1表达的影响
结果在图3C中给出。
与老化细胞相比,根据实施例2的可可水解产物使得可以再激活原纤蛋白1的表达。
·结论
根据实施例2的可可水解产物是作用于皮肤老化靶标原纤蛋白1的成分,该作用是通过与未经处理的老化细胞相比在三种测试浓度下重新启动原纤蛋白1的蛋白质表达并通过与年轻对照相比几乎完全重建蛋白质表达进行的。
-DNA甲基化靶标
甲基化是对组蛋白N末端的修饰。它可以在赖氨酸上或在精氨酸上发生,并且可以通过添加一个、两个或三个甲基来实现。根据甲基化残基和添加的基团数,它与转录的激活或抑制相关联。被认为是长期静止的组蛋白甲基化显示是动态过程中涉及的可逆修饰,但它比乙酰化和磷酸化更稳定。已经鉴定出越来越多的组蛋白去甲基化酶。通常,这种类型的修饰对乙酰化具有拮抗作用,并且赖氨酸的脱乙酰化必须先于其甲基化。这种拮抗作用导致异染色质结构域(通常不能表达并在某些关键氨基酸上甲基化)与常染色质结构域(通常可以表达并被乙酰化)之间的某种动态平衡的发展。例如,已知甲基化的组蛋白H3的赖氨酸9与周围染色质的抑制相关联。该甲基化被蛋白质HP1识别,因此HP1与甲基化的H3结合。HP1进而吸引组蛋白甲基转移酶蛋白质Suv39,从而能够甲基化相邻核小体的组蛋白H3的赖氨酸9,依此类推。因此,可以看到,组蛋白H3将如何逐步甲基化以及染色质将如何浓缩。然而,如果遇到的H3的赖氨酸9已经被乙酰化,则将停止这种异染色质入侵。因此,在表达的染色质结构域与抑制的染色质结构域之间形成竞争平衡。组蛋白尾部的修饰起到表观遗传“标记”的作用,导致不同类别的蛋白质的募集,这是因为乙酰化或甲基化的赖氨酸被不同的蛋白质结构域识别。此外,在染色质水平上募集某些因子需要先前存在对组蛋白和已经结合的蛋白质的修饰。因此,组蛋白的编码在与染色质相关联的其他因子的背景下得以解释,并且它是修饰的组蛋白与其他因子之间的相互作用的组合,所述其他因子决定蛋白质是否被募集以用于染色质。生物体的所有组织都受到老化的影响。如许多出版物中所述,该过程与表观遗传修饰,诸如DNA特异性胞嘧啶残基水平上的甲基化变化有关。表观遗传修饰对老化的作用(细胞分裂和恶化大分子的积聚)促成老化的表型。此外,环境和随机事件可以通过表观遗传机制(诸如DNA甲基化和组蛋白甲基化和乙酰化)的中间媒介来改变该表型。表观遗传修饰的潜在可逆性使其成为治疗老化相关病理状况的有吸引力的靶标。
·评价根据实施例2的可可水解产物对DNA甲基化的影响的方法
将年轻NHDF(正常人真皮成纤维细胞)或通过复制性衰老老化的NHDF接种在96孔板中并在37℃、5%CO2下孵育24h。
在测试产品的存在下处理细胞24h。
然后在胰蛋白酶的存在下将细胞解离并裂解。用乙醇沉淀基因组DNA。
使用Enzo试剂盒:5-甲基胞嘧啶DNA ELISA试剂盒通过ELISA测定DNA的甲基化水平。为了更好地使结果可视,这些值从50%开始表示。
·评价根据实施例2的可可水解产物对DNA甲基化的影响
结果在图3D中给出。
根据实施例2的可可水解产物使得可以将复制性老化的NHDF中的甲基化水平降低至10和5ppm,以便接近在年轻细胞中观察到的水平。
·结论
根据实施例2的可可水解产物是对DNA甲基化水平(其是表观遗传老化的靶标)的降低起作用的成分,因此它具有复苏活性潜力。
-关于抗老化活性和复苏活性的一般结论
在老化过程中,胶原纤维和弹性纤维由于合成减少和降解增加而改变。为了证明根据实施例2的可可水解产物对细胞复苏起作用,对在胶原蛋白I和弹性蛋白的情况下内在老化(复制性衰老)的细胞内、或者在原纤蛋白1的情况下通过反复UV照射而外在老化(光老化)的细胞内的涉及这些纤维结构的蛋白质的表达进行定量。
根据实施例2的可可水解产物使得可以重新启动3种标记物的表达。
在DNA甲基化分析的背景下,根据实施例2的可可水解产物在该实验中具有均一的结果,它在测试的3个浓度中的2个浓度下减少了甲基化。这种效果提供了关于该产物使得可以再激活细胞外基质蛋白质的表达从而使其具有复苏作用的作用方式的信息。
因此,根据实施例2的可可水解产物是具有抗老化和复苏生物抗老化活性的活性成分或物质。通过与老化细胞中观察到的蛋白质表达相比重新启动蛋白质表达,它一致地作用于皮肤老化的3种靶标。它还具有表观遗传学的新颖活性,如通过其对成熟细胞内DNA甲基化的作用所证明的。最后,由于其对细胞外基质蛋白质合成的功效,它展示出深度抗老化作用。
实施例14:护理霜
程序
1.将A相倒入主容器中并且开始均化。加热至70℃-75℃。
2.喷淋到UltraThix P-100中并且充分混合大约30min。
3.将D相的成分添加到第二烧杯并且在70℃-75℃下加热。
4.将预混合的C相添加到A相,直到完全均化。
5.在70℃-75℃下,将D相加入到主容器并且充分混合。乳液必须是完全均匀的。
6.开始冷却。
7.当混合物达到约50℃时,添加预混合的E相并且充分混合。
8.在环境温度下,添加F相并且混合直到获得均匀混合物。
9.在25℃停止。
特性:
外观:白色霜
pH:5.2-5.6
粘度(D0)25,000-50,000(Brookfield RVT/转子B/5 RPM/1分钟/25℃)
通经28天的双重功效测试验证了该配方的保存。
然而,防腐剂未优化至其最低有效水平。
实施例15:用于外部活动的保护性流体(体外日晒防护SPF 30)
程序
1.在室温下将水添加到主容器并且添加A相的成分
2.喷淋到A相中并且混合至均匀
3.将C相的成分添加到相邻容器并且通过混合加热至65℃-70℃,然后冷却至室温
4.在室温下,均化C相+D相
5.将CD添加到主容器并且充分混合,直到获得均匀乳液
6.将E相预混合并添加到主容器,同时进行混合
7.将F相预混合至50℃,添加到主容器,同时进行混合
8.将G相的成分逐一添加并且充分混合至均匀
9.在25℃停止。
特性:
外观:黄色流体
pH:5.5-6.0
粘度(D0)Brookfield RVT/转子3/5 RPM/1分钟/25℃:5000-11,000cps
通过经28天的双重功效测试验证了该配方的保存。
然而,防腐剂未优化至其最低有效水平。
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Claims (17)
1.一种可可(Theobroma cacao L.)豆的肽和糖水解产物,其特征在于该肽和糖具有在200Da与10kDa之间的分子量,并且所述水解产物是通过包括以下步骤的方法获得的:
a)将未发酵的、干燥的且粉碎的可可豆分散在水相中;
b)分两步对步骤a)中获得的水性分散体进行酶促处理,在第一次处理中使用至少一种碳水化合物酶,并且然后在第二次处理中使用至少一种蛋白酶,该碳水化合物酶选自果胶酶、纤维素酶或半纤维素酶,并且该蛋白酶是具有内肽酶活性的碱性类型的蛋白酶,其中所述碳水化合物酶包括按重量计占总干物质重量的0.5%的纤维素酶和按重量计占总干物质重量的1%的果胶酶;
c)将该酶通过热处理变性;
d)通过固/液分离进行该酶促水解产物的回收;
e)通过超滤和纳米过滤纯化该水解产物;然后任选地
f)对步骤e)中获得的水解产物进行冻干。
2.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于此外,步骤a)的豆是脱脂的。
3.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于步骤b)的碳水化合物酶是碳水化合物酶的混合物,该混合物包括至少一种聚半乳糖醛酸酶、果胶甲基酯酶、β葡聚糖酶、β甘露聚糖酶、β葡糖苷酶、阿拉伯聚糖酶和木聚糖酶。
4.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于相对于待处理的豆的重量,该方法的步骤b)中使用的总酶浓度按重量计在0.1%与3.0%之间。
5.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于该方法的步骤a)至d)是在20℃与90℃之间的温度和在3.0与11.0之间的pH进行。
6.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于步骤d)的固/液分离是通过选自旋转和过滤的方法进行。
7.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于步骤d)的固/液分离是通过离心的方法进行的。
8.根据权利要求1所述的可可豆的肽和糖水解产物,其特征在于在步骤e)中,该超滤是以5与15kDa之间的截止阈值进行,并且该纳米过滤是以100Da与300Da之间的截止阈值进行。
9.一种化妆品组合物,其特征在于该化妆品组合物包含作为活性剂的有效量的根据权利要求1所述的可可豆的水解产物以及生理学上可接受的介质。
10.根据权利要求9所述的组合物,其特征在于相对于该组合物的总重量,该可可豆的水解产物以0.001%至20%的浓度存在于该组合物中。
11.根据权利要求10所述的组合物,其中相对于该组合物的总重量,该可可豆的水解产物的浓度为0.1%至10%。
12.根据权利要求10所述的组合物,其中相对于该组合物的总重量,该可可豆的水解产物的浓度为0.2%至5%。
13.根据权利要求10所述的组合物,其中相对于该组合物的总重量,该可可豆的水解产物的浓度为0.5%至1.5%。
14.根据权利要求9所述的组合物,其特征在于该组合物被配制用于局部应用于皮肤。
15.根据权利要求9所述的组合物在制备用于保护皮肤免受蓝光的有害影响的化妆品组合物方面的用途。
16.根据权利要求15所述的用途,其为在制备用于保护皮肤免受因暴露于蓝光产生的氧化应激的化妆品组合物的方面的用途。
17.根据权利要求9所述的组合物在制备用于对抗皮肤的老化和光老化的迹象出现的化妆品组合物方面的用途。
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PCT/EP2017/056084 WO2017157998A1 (fr) | 2016-03-16 | 2017-03-15 | Hydrolysat peptidique et osidique des fèves de cacao, compositions cosmetiques le comprenant et leurs utilisations cosmétiques |
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FR3075620A1 (fr) * | 2017-12-22 | 2019-06-28 | L V M H Recherche | Composition cosmetique pour le traitement de la peau grasse |
FR3075621B1 (fr) * | 2017-12-22 | 2020-01-17 | L V M H Recherche | Composition cosmetique comprenant un extrait de caesalpinia spinosa, un extrait de kappaphycus alvarezii, et un hydrolysat de feves de theobroma cacao l |
FR3075647B1 (fr) * | 2017-12-22 | 2020-05-22 | L V M H Recherche | Composition de maquillage comprenant un hydrolysat de feves de theobroma cacao l, et au moins un prebiotique et un probiotique |
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