CN116459172A - 蔓荆子黄素的抗糖化应用 - Google Patents
蔓荆子黄素的抗糖化应用 Download PDFInfo
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- CN116459172A CN116459172A CN202310406775.8A CN202310406775A CN116459172A CN 116459172 A CN116459172 A CN 116459172A CN 202310406775 A CN202310406775 A CN 202310406775A CN 116459172 A CN116459172 A CN 116459172A
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- vitexin
- glycation
- skin
- preparation
- skin external
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Abstract
本发明提供了蔓荆子黄素的抗糖化应用。本发明还涉及蔓荆子黄素在制备具有抗糖化作用的皮肤外用剂、药品及保健食品中的应用。
Description
技术领域
本发明涉及天然药物化学领域,具体涉及蔓荆子黄素的抗糖化应用,及蔓荆子黄素在制备具有抗糖化作用的皮肤外用剂、药品及保健食品中的应用。
背景技术
1912年法国人Louis Camille Maillard在描述甘氨酸与葡萄糖混合加热时形成褐色类黑精的反应时首次提出糖化反应这一概念,因此糖化反应又称美拉德(Maillard)反应。1984年Brownlee首先报道了体内生物大分子存在晚期糖基化终末产物(AdvancedGlycation End-products,AGEs)的现象。AGEs指的是核酸,蛋白质,脂质等生物大分子物质中游离的氨基与还原糖的羰基,在非酶促条件下,经过缩合、重排、裂解、氧化修饰等一系列反应生成的。AGEs呈棕褐色、可发生交联反应且反应不可逆、不怕酶破坏、不易降解、有特别的吸收光谱及荧光特性、能够在体内逐渐累积,可与许多细胞膜特异性受体结合发挥生物学效应。
AGEs是人体内不受控制的缩合反应的有害的产物,对人体健康尤其是皮肤健康具有非常不利的影响,主要有以下几个方面:首先,糖分子会和蛋白质氨基发生反应从而形成糖化蛋白。胶原蛋白和弹性蛋白是皮肤的支架,一旦与糖分子结合,其性质和结构会发生不可逆的改变甚至破坏,导致皮肤失去弹性,发生结构塌陷,最终形成皱纹。糖化的蛋白还会在体内累积,这种棕褐色的物质会影响皮肤的透光性,使皮肤看上去暗黄无光。其次,累积的AGEs不仅可以直接沉积在皮肤导致色素沉着,同时能够通过作用于晚期糖基化终产物受体RAGE(receptor for advanced glycation end products),激活黑素相关的信号通路,上调相关基因的表达和酪氨酸酶的活性,促进黑色素的产生,这进一步的加重了皮肤的色素沉着。最后,AGEs与自由基会相互促进,使体内氧化压力升高,造成肌肤的氧化损伤甚至加剧体内炎性,皮肤问题以至于多种健康问题随之产生。
越来越多的研究希望通过抑制体内的生物大分子的糖化反应,达到改善疾病,延缓衰老,维持肌肤年轻态的目的。目前,主流的抗糖化成分有肌肽,硫辛酸,黄酮类,多酚类,革糖素,葡萄籽提取物等等,他们大致可以被分为人工合成与天然提取两类。天然提取方面尤其以中草药提取物表现最为优异。中草药提取物本身具有天然属性,具有安全性高,毒副作用小,易于利用吸收等优异的性质,具有非常高的研究价值和广阔的应用前景。蔓荆子黄素,可以从青蒿、蔓荆子等植物中分离提纯得到。蔓荆子黄素又称为5-羟基-2-(3-羟基-4-甲氧基苯基)-3,6,7-三甲氧基苯并吡喃-4-酮,英文名是Casticin,CAS号:479-91-4,分子式:C19H18O8,分子量:374.34。具体的蔓荆子黄素的结构式如下:
蔓荆子黄素属于多甲氧基黄酮类化合物,具有抗肿瘤、抗炎、舒张血管、抑菌等作用。现有技术对蔓荆子黄素的研究主要基于其药理作用,包括用于风热感冒、齿龈肿痛等。
例如,清华大学在中国专利CN 102908341 A中公开了蔓菁子黄素在抗新血管生成方面的作用;珀莱雅公司在专利CN 108653028 B中研究了蔓菁子黄素作为化妆品防晒成分的应用;中科院武汉病毒研究所在专利CN 109864988 B中公开了蔓菁子黄素在治疗病毒感染产品中的应用。另有一些专利和文献公开了含有蔓菁子黄素的植物组合物对于紧致毛孔的作用。然而,迄今为止,尚未发现蔓荆子黄素具有抗糖化作用的报道。
本发明意外地发现,蔓荆子黄素能够有效降低非酶糖基化反应的终末产物AGEs含量,具有优异的抗糖化作用。
发明内容
本发明提供了蔓荆子黄素的抗糖化应用。
在优选的实施方式中,蔓荆子黄素的使用浓度至少为0.1mg/L,更优选至少1mg/L。
在优选的实施方式中,抗糖化为抑制非酶糖基化反应的终末产物。在优选的实施方式中,抗糖化为抑制糖基化终产物。
本发明还涉及蔓荆子黄素在制备具有抗糖化作用的药品、保健食品、口服剂或皮肤外用剂中的应用。
在优选的实施方式中,蔓荆子黄素在药品、保健食品、口服剂或皮肤外用剂中的含量0.00001-20重量%,更优选0.0001-5重量%。
在优选的实施方式中,所述皮肤外用剂选自:面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾清洁泡、喷雾、沐浴露、或洗面奶。
附图简要说明
图1显示了空白组对照组抗糖化免疫荧光染色(该免疫荧光图片在Leica倒置荧光显微镜20倍物镜视野下拍摄得到,蓝色荧光代表细胞核信号,红色荧光代表CML信号,红色越强,代表糖化程度越明显)。
图2显示了糖化诱导组抗糖化免疫荧光染色(该免疫荧光图片在Leica倒置荧光显微镜20倍物镜视野下拍摄得到,蓝色荧光代表细胞核信号,红色荧光代表CML信号,红色越强,代表糖化程度越明显。与空白对照组相比,糖化诱导组红色明显变强,代表糖化诱导成功)。
图3显示了蔓荆子黄素实施例8抗糖化免疫荧光染色(该免疫荧光图片在Leica倒置荧光显微镜20倍物镜视野下拍摄得到,蓝色荧光代表细胞核信号,红色荧光代表CML信号,红色越强,代表糖化程度越明显。与糖化诱导组相比,红色明显减弱,代表蔓荆子黄素实施例8具有抗糖化作用)。
图4显示了蔓荆子黄素实施例9抗糖化免疫荧光染色(该免疫荧光图片在Leica倒置荧光显微镜20倍物镜视野下拍摄得到,蓝色荧光代表细胞核信号,红色荧光代表CML信号,红色越强,代表糖化程度越明显。与糖化诱导组相比,红色明显减弱,代表蔓荆子黄素实施例9具有抗糖化作用)。
具体实施方式
本发明涉及蔓荆子黄素的抗糖化作用,首次发现蔓荆子黄素可以作为功效添加剂用来制备皮肤外用剂,以达到美白提亮、祛黄、抗衰等护肤效果。在一些实施方式中,蔓荆子黄素可以作为功效添加剂用来制备药品或保健食品。或者,蔓荆子黄素可以作为功效添加剂用来制备口服剂。
为了提供更简明的描述,本文给出的一些数量表述没有用术语“约”修饰。应当理解,无论是否明确地使用了术语“约”,本文所给出的每个量都意在指代实际的给定值,并且还意在指代由本领域的普通技术人员可合理推测出的这些给定值的近似值,包括这些给定值的由实验和/或测量条件所引起的近似值。
为了提供更简洁的描述,本文中一些数量表述被叙述为约X量至约Y量的范围。应当理解,当叙述范围时,该范围并不限制于所叙述的上下界限,而应包括约X量至约Y量的整个范围或它们之间的任何量。
本文所描述的蔓荆子黄素可任选为成品包装形式。在一个实施例中,所述包装为装有蔓荆子黄素的诸如塑料、金属或玻璃管或广口瓶之类的容器。该产品可另外具有用于储存该容器的诸如塑料或纸板盒之类的包装。在一个实施例中,该产品包含蔓荆子黄素并且具有引导使用者将蔓荆子黄素施用至皮肤以处理皮肤的说明书。这种说明书可印刷在容器上、标签插页或任何其它包装上。
如本文所用,“局部施用”意指例如利用手或诸如擦拭物、辊或喷雾器之类的施用装置,直接涂抹或散布在外部皮肤、头皮或毛发上。
如本文所用,“美容上可接受的”意指该术语所描述的成分适用于与组织(例如皮肤或毛发)接触而无不当的毒性、不相容性、不稳定性、刺激性、变应性反应或类似症状。
蔓荆子黄素
本发明涉及化合物蔓荆子黄素的抗糖化作用,在研究中发现了蔓荆子黄素能够有效降低非酶糖基化反应的终末产物AGEs生成,具有优越的抗糖化作用。因此,蔓荆子黄素可以作为功效添加剂,用于制备药品、保健食品、口服剂或皮肤外用剂,以达到美白提亮、祛黄、抗衰等护肤效果。
在抗糖化的应用中,蔓荆子黄素的使用浓度至少为0.1mg/L,更优选至少为1mg/L。
在一些实施方式中,蔓荆子黄素的使用浓度为0.1-100mg/L。在一些实施方式中,蔓荆子黄素的使用浓度为0.1-50mg/L,优选1-50mg/L,优选1-20mg/L,更优选1-10mg/L。
含蔓荆子黄素的药品、保健食品、口服剂或皮肤外用剂
本发明的蔓荆子黄素可以作为功效添加剂应用于药品、保健食品、口服剂或皮肤外用剂中。
在一些实施方式中,蔓荆子黄素在药品、保健食品、口服剂或皮肤外用剂中的含量为0.00001-20重量%,更优选0.0001-5重量%。
在一些实施方式中,所述药品选自:片剂、胶囊、乳剂、混悬剂、粉末剂、颗粒剂、溶液剂、以及本领域已知的各种药品剂型。根据剂型的不同类型添加不同的用量。
保健食品亦称功能性食品。它具有调节人体功能的作用,但不以治疗疾病为目的,适于特定人群食用。在一些实施方式中,所述保健食品可以是粉末剂型。
在一些实施方式中,皮肤外用剂选自:面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾(清洁泡)、喷雾、沐浴露和洗面奶。根据制剂的不同类型添加不同的用量。
皮肤外用剂是通常用于皮肤外部的所有成分的统称概念,例如可以是化妆品组合物。所述化妆品组合物中可以是基础化妆品、面部妆容化妆品、身体用化妆品、头发护理用化妆品等,对其剂型无特殊限制,根据不同目的可合理选择。所述化妆品组合物中根据剂型和目的的不同还含有不同的化妆品学层面允许的介质或基质赋形剂。
包含蔓荆子黄素的皮肤外用剂可局部施用至人皮肤和/或毛发。该皮肤外用剂还可包含美容上可接受的局部用载体,该局部用载体可为皮肤外用剂的约50重量%至约99.99重量%(如皮肤外用剂的约80重量%至约99重量%)。在本发明的一个优选的实施例中,美容上可接受的局部用载体包括水。美容上可接受的局部用载体可包括一种或多种选自润湿剂、润肤剂、油脂、湿润剂以及相似物质。在一个实施例中,美容上可接受的局部用载体包括诸如无纺布或膜材料之类的基材。
可将皮肤外用剂制备成多种产品类型,包括但不限于洗剂、霜剂、凝胶剂、棒剂、喷雾剂、油膏剂、清洁液体洗剂和固体皂剂、洗发剂和毛发调理剂、毛发固定剂、糊剂、泡沫剂、粉末剂、摩丝、剃须膏、擦拭物、贴剂、水凝胶、成膜产品、面膜和皮肤膜剂、膜剂和化妆品如粉底以及睫毛膏。这些产品类型可含有几种类型的美容上可接受的局部用载体,包括但不限于溶液、悬浮液、乳液(例如微乳液和纳米乳液)、凝胶、固体和脂质体。
包含蔓荆子黄素的皮肤外用剂可配制成溶液剂。溶液剂通常包含水性溶剂或有机溶剂(例如,约50%至约99.99%或约90%至约99%的美容上可接受的水性溶剂或有机溶剂)。合适的有机溶剂的例子包括丙二醇、聚乙二醇、聚丙二醇、甘油、1,2,4-丁三醇、山梨醇酯、1,2,6-己三醇、乙醇以及它们的混合物。
皮肤外用剂可配制成包含润肤剂的溶液。此类皮肤外用剂优选包含约2%至约50%的一种或多种润肤剂。如本发明所用,“润肤剂”指用于预防或减轻干燥,例如通过防止皮肤水分经皮肤损失的物质。润肤剂的例子包括但不限于植物油、矿物油、脂族酯等。
洗剂可由这种溶液制备。洗剂通常含有约1%至约20%(例如,约5%至约10%)的一种或多种润肤剂和约50%至约90%(例如,约60%至约80%)的水分。
可以由溶液配制的另一类型的产品是霜剂。霜剂通常含有约5%至约50%(例如,约10%至约20%)的一种或多种润肤剂和约45%至约85%(例如,约50%至约75%)的水分。
尽管优选的是包含蔓荆子黄素的皮肤外用剂包含水,但作为另一种选择该皮肤外用剂可以是无水的或为不包含水而是有机和/或有机硅溶剂、油脂、类脂和蜡的油膏剂。油膏剂可含有动物或植物油或半固体烃的简单基料。油膏剂可含有约2%至约10%的一种或多种润肤剂和约0.1%至约2%的一种或多种增稠剂。
可将该皮肤外用剂配制为乳剂。如果局部用载体是乳液,则约1%至约10%(如约2%至约5%)的该局部用载体含有一种或多种乳化剂。乳化剂可以为非离子型、阴离子型或阳离子型。合适的乳化剂的例子包括个人护理和化妆品配方领域中通常鉴定为合适乳化剂的那些。
洗剂和霜剂可配制成乳剂。通常这类洗剂含有0.5%至约5%的一种或多种乳化剂。这种霜剂通常含有约1%至约20%(如约5%至约10%)的一种或多种润肤剂;约20%至约80%(如30%至约70%)的水;以及约1%至约10%(如约2%至约5%)的一种或多种乳化剂。
水包油和油包水型单乳液护肤制剂,例如洗剂和霜剂是化妆品领域所熟知的,并且可用于本发明。多相乳状液皮肤外用剂(例如水包油包水型和油包水包油型)也可用于本发明。通常,此类单相乳状液或多相乳状液含有水分、润肤剂和乳化剂作为其基本成分。
包含蔓荆子黄素的皮肤外用剂还可配制成凝胶剂(例如,使用合适的胶凝剂的水性凝胶、醇类凝胶、醇/水型凝胶或油性凝胶)。用于水性凝胶和/或醇类凝胶的适当胶凝剂包括但不限于天然树胶、丙烯酸和丙烯酸酯聚合物和共聚物以及纤维素衍生物(例如羟甲基纤维素和羟丙基纤维素)。用于油(例如矿物油)的适当胶凝剂包括但不限于氢化丁烯/乙烯/苯乙烯共聚物和氢化乙烯/丙烯/苯乙烯共聚物。这种凝胶剂通常含有约0.1重量%和5重量%之间的这类胶凝剂。
包含蔓荆子黄素的皮肤外用剂也可配制成固体制剂(例如蜡基棒剂、条皂、粉剂或含有粉剂的擦拭物)。
除了上述组分外,可用于本发明的皮肤外用剂还可含有多种在常规上以其技术领域确定的水平用于在皮肤和毛发上使用的皮肤外用剂中的其他油溶性物质和/或水溶性物质。
本发明的皮肤外用剂可以包含在皮肤护理组合物中通常能找到的附加组分,例如润肤剂、皮肤调节剂、乳化剂、防腐剂、抗氧化剂、香料、螯合剂等,只要它们与皮肤外用剂中其他的组分在物理上和化学上相容、且不影响本发明的蔓荆子黄素的效果即可。
在本发明的皮肤外用剂的一些实施方式中,可使用一种或多种防腐剂。适宜的防腐剂包括对羟基苯乙酮、C1-C4对羟基苯甲酸烷基酯和苯氧基乙醇。基于组合物总重量,防腐剂的用量为大约0.5至大约2重量%,优选大约0.5至1重量%。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种抗氧化剂。适宜的抗氧化剂包括丁化羟基甲苯(BHT)、抗坏血酸棕榈酸酯(BHA)、丁羟茴醚、苯基-α-萘基胺、氢醌、没食子丙酯、去甲二氢愈创木酸、维生素E或维生素E的衍生物、维生素C及其衍生物、泛酸钙、绿茶提取物和混合的多酚,以及上面所述的物质的混合物。所使用的抗氧化剂是组合物总重量的大约0.02至0.5重量%,更最优选的是从大约0.002至0.1重量%的用量范围。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种润肤剂,通过其保持在皮肤表面上或在角质层中的能力,起到润滑剂的作用,以减少剥落,改善皮肤的外观。典型的润肤剂包括脂肪酯、脂肪醇、矿物油、聚醚硅氧烷共聚物等等。适宜的润肤剂的例子非限定地包括,聚丙二醇(“PPG”)-15十八烷基醚,PPG-10十六烷基醚,Steareth-10,Oleth-8,PPG-4十二烷基醚,维生素E醋酸酯,羊毛脂,鲸蜡醇,鲸蜡硬脂醇乙基己酸酯,十六十八醇,甘油硬脂酸酯,羟基硬脂酸辛脂,二甲基聚硅氧烷,以及它们的组合。鲸蜡醇,鲸蜡硬脂醇乙基己酸酯,十六十八醇,甘油硬脂酸酯以及它们的组合是优选的。使用时,润肤剂是基于组合物总重量的大约0.1至大约30重量%,优选大约1至大约30重量%的用量范围。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种保湿剂。保湿剂也称为湿润剂,其有助于提高润肤剂的效果,减少剥落,刺激组成的鳞皮的去除和提高皮肤触感。可使用多元醇作为保湿剂,包括但并不限于,甘油,聚亚烷基二醇,亚烷基多元醇及其衍生物,包含丁二醇、丙二醇、双丙二醇,聚丙三醇,聚乙二醇及其衍生物,山梨醇,羟丙基山梨醇,己二醇,1,3-二丁二醇,1,2,6-己三醇,乙氧基化甘油,丙氧基化甘油,以及它们的组合。使用时,基于组合物的总重量,保湿剂的用量为大约0.1至大约20重量%,优选是大约1至大约15重量%。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种乳化剂。乳化剂可在有效稳定量的范围内使用。优选地,在组合物总重量的基础上,以大约1.0至大约10.0重量%,更优选的是大约3.0至大约6.0重量%的用量来使用乳化剂。可以使用任何与组合物中的组分相容的乳化剂。适宜的乳化剂包括硬脂酸,鲸蜡醇,甘油硬脂酸酯,卵磷脂,十八烷醇,Steareth-2,Steareth-20,丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物,以及它们的组合。
在本发明的皮肤外用剂的一个实例中,可使用一种或多种pH调节剂。本发明的皮肤外用剂中有益的pH调节剂包括氨丁三醇。使用时,基于组合物的总重量pH调节剂的用量为大约0.1至大约2重量%,优选是大约0.1至大约1重量%。
在本发明的一个具体实施方式中,皮肤外用剂包含丙烯酸(酯)类/C10-30烷醇丙烯酸酯交联聚合物、甘油、对羟基苯乙酮、甘油硬脂酸酯和卵磷脂、十六/十八醇、鲸蜡硬脂醇乙基己酸酯、氨丁三醇或它们的组合。
另外的美容活性剂
在一些实施方式中,皮肤外用剂还可包含另外的美容活性剂。如本文所用,“美容活性剂”是对皮肤或毛发具有美容或治疗效果的化合物(如合成化合物或从天然来源或天然提取物分离的化合物),包括但不限于抗痤疮剂、控油剂、抗微生物剂、抗炎剂、抗真菌剂、抗寄生虫剂、外用止痛剂、防晒剂、光防护剂、抗氧化剂、角质层分离剂、表面活性剂、保湿剂、营养剂、维生素、能量增强剂、防汗剂、收敛剂、除臭剂、固化剂、抗硬角质剂和用于毛发和/或皮肤调理的试剂。
在一个实施例中,这些美容活性剂选自(但不限于):羟基酸、过氧化苯甲酰、D-泛醇、甲氧基肉桂酸辛酯、二氧化钛、水杨酸辛酯、胡莫柳酯、阿伏苯宗、类胡萝卜素、自由基清除剂、自旋捕获剂、胺、诸如视黄醇和视黄基棕榈酸酯之类的类视色素、神经酰胺、多不饱和脂肪酸、必需脂肪酸、酶、酶抑制剂、矿物质、诸如雌激素之类的激素、诸如氢化可的松之类的类固醇、2-二甲基氨乙醇、诸如氯化铜之类的铜盐、诸如Cu:Gly-His-Lys之类的含铜肽、辅酶Q10、肽、诸如脯氨酸之类的氨基酸、维生素、乳糖酸、乙酰基-辅酶A、烟酸、核黄素、硫胺素、核糖、诸如NADH和FADH2之类的电子传递物质和诸如芦荟、小白菊、麦片之类的其他植物提取物以及它们的衍生物和混合物。以本发明皮肤外用剂的重量计,美容活性剂通常以约0.001%至约20%,例如约0.005%至约10%如约0.01%至约5%的量存在。
维生素的例子包括但不限于维生素A、维生素B(例如维生素B3、维生素B5和维生素B12)、维生素C、维生素K和维生素E的不同形式(如α、β、γ或Δ生育酚)或它们的混合物以及它们的衍生物。
羟基酸的例子包括(但不限于)乙醇酸、乳酸、苹果酸、水杨酸、柠檬酸和酒石酸。
抗氧化剂的例子包括但不限于:水溶性抗氧化剂例如巯基化合物和它们的衍生物(如,焦亚硫酸钠和N-乙酰基-半胱氨酸)、硫辛酸和二氢硫辛酸、白藜芦醇、乳铁蛋白和抗坏血酸以及抗坏血酸衍生物(如抗坏血酸棕榈酸酯和抗坏血酸多肽)。适用于本发明的皮肤外用剂中的油溶性抗氧化剂包括(但不限于)丁基化羟基甲苯、类视色素(如,视黄醇和视黄基棕榈酸酯)、生育酚类(如,生育酚乙酯)、生育三烯酚类和泛醌。含有适用于本发明皮肤外用剂的抗氧化剂的天然提取物包括但不限于:含有黄酮类和异黄酮类以及它们的衍生物(如,染料木素和二联玉米蛋白)的提取物、含有白藜芦醇的提取物等。这种天然提取物的例子包括葡萄籽、绿茶、松树皮和蜂胶。
使用方法
可将本发明的皮肤外用剂局部施用至需要处理的一种或多种皮肤的哺乳动物的皮肤。在一个实施例中,可将皮肤外用剂施用至需要处理美白提亮、祛黄、抗衰的皮肤。可根据合适的处理方案,如每月、每周、每隔一天、每天、每天两次等,将皮肤外用剂施用至需要这种处理的皮肤。
在某些实施例中,本发明的皮肤外用剂还可用用于处理与皮肤相关的其他需要。例如,本发明的皮肤外用剂可用于处理发炎后色素沉着过度、用于减少毛孔大小、用于减少皮脂产生以及用于缓和疤痕。在某些其他实施例中,可将本发明的皮肤外用剂与机械或物理表皮剥脱处理(例如微晶磨皮处理)同时施用或在该处理的数小时内施用,或与化学剥脱剂或角质层分离剂例如水杨酸同时施用。在某些其他实施例中,可将本发明的皮肤外用剂施用至粘膜或其它组织例如阴道组织、口腔组织或眼组织。在某些其他实施例中,可将本发明的皮肤外用剂施用至轻度伤口或术后部位以促进愈合、施用至昆虫叮咬处、施用至毒藤皮肤病症或相似皮肤状况,或通常用于减轻发痒。
实施例
下面结合具体实施例,以进一步阐述本发明。有必要在此指出的是,实施例只用于对本发明进行进一步的说明,不能理解为对本发明保护范围的限制,该领域的技术熟练人员可以根据上述本发明的内容做出一些非本质的改进和调整。下列实施例中未注明具体条件的试验方法,通常按照常规条件,或按照制造厂商所建议的条件。除非另有说明,所有的百分比和份数按重量计。
本发明使用的蔓荆子黄素,纯度>90%,来源于青蒿,由中国中医科学院中药研究所提供。
实施例1:样品制备
称取37.4mg蔓荆子黄素,用二甲亚砜(Sigma,DMSO)溶解,补加适量DMSO并定容至100mL容量瓶,得到374mg/L蔓荆子黄素溶液,备用。
实施例2:样品制备
用移液枪吸取2mL实施例1蔓荆子黄素溶液,补加适量磷酸缓冲液至10mL容量瓶并定容,得到74.8mg/L蔓荆子黄素溶液,备用。
实施例3:样品制备
用移液枪吸取5mL实施例2蔓荆子黄素溶液,补加适量磷酸缓冲液至10mL容量瓶并定容,得到37.4mg/L蔓荆子黄素溶液,备用。
实施例4:样品制备
用移液枪吸取5mL实施例3蔓荆子黄素溶液,补加适量磷酸缓冲液至10mL容量瓶并定容,得到18.7mg/L蔓荆子黄素溶液,备用。
实施例5:样品制备
用移液枪吸取5mL实施例4蔓荆子黄素溶液,补加适量磷酸缓冲液至10mL容量瓶并定容,得到9.36mg/L蔓荆子黄素溶液,备用。
实施例6:样品制备
用移液枪吸取5mL实施例5蔓荆子黄素溶液,补加适量磷酸缓冲液至10mL容量瓶并定容,得到4.68mg/L蔓荆子黄素溶液,备用。
实施例7:样品制备
称取187.2mg蔓荆子黄素,用二甲亚砜(Sigma,DMSO)溶解,补加适量DMSO并定容至100mL容量瓶,得到1872mg/L蔓荆子黄素溶液,备用。
实施例8:样品制备
在生物安全柜内用移液枪吸取10μL实施例7蔓荆子黄素溶液,补加适量细胞培养液至10mL,得到1.87mg/L蔓荆子黄素细胞培养液,备用。
实施例9:样品制备
在生物安全柜内用移液枪吸取50μL实施例7蔓荆子黄素溶液,补加适量细胞培养液至10mL,得到9.36mg/L蔓荆子黄素细胞培养液,备用。
测试例1:抗糖化作用测试
1.测试原理:
糖化反应是指多余的糖分在血液中游离,附着在蛋白等生物大分子上发生缩合、重排及链延长反应生成高反应性的糖化中间产物;继续作用进而生成不可逆转的糖化终产物(Advanced glycation end-products,AGEs)。在生化实验中将葡萄糖溶液与牛血清白蛋白(BSA)溶液混合,用氨基胍作为阳性对照,磷酸缓冲液作为空白对照。将混合以后的样品,于55℃下避光反应4天,通过荧光酶标仪测定溶液产物在激发波长370nm,发射波长440nm条件下的荧光强度并计算其抑制率。通过抑制率判断样品的抗糖化作用。
2.测试材料:
2.1主要试剂:
牛血清蛋白(Sigma,M26HS122527)、葡萄糖(Sigma,SLBR0902V)、PBS缓冲液(博士德,WH0621P201)、氨基胍盐酸盐(Aladdin,G1909194)、二甲亚砜(DMSO,国药集团化学试剂有限公司)。
2.2主要设备:
酶标仪(BioTek,Epoch)、涡旋振荡仪(上海启前电子科技,MZX-2500)、恒温培养箱(天津市泰斯特仪器有限公司,DH400BII)、电子天平(赛多利斯,BSA124S-CW)、生物安全柜(孚约安防设备(上海)有限公司,BSC-1604IIA2)。
3.测试分组与浓度:
注:由于受到测试体系的影响,配制的测试样品浓度在进行的测试时会被进一步稀释2倍,因此配制浓度为测试终浓度的2倍。在进行测试结果说明时涉及到的浓度也指的是测试终浓度。
4.测试方法:
4.1糖化反应液的配制:
准确称取8g牛血清蛋白和24g的葡萄糖,加入到100mL的磷酸盐缓冲溶液(PBS,0.1mol/L)中,充分搅拌溶解之后,即为糖化反应液,过0.22μm的滤头除菌之后备用。
4.2样品溶液的配制:
准确称取3.74mg的蔓荆子黄素溶解于10mLDMSO中,配制成374mg/L的初始样品溶液。用0.1mol/L的PBS缓冲液将初始样品溶液稀释5倍,浓度为74.8mg/L,之后采用1/2稀释法对样品进行梯度稀释,得到5个测试溶液。
按照下表配制各组反应体系。各组反应体系中牛血清白蛋白终浓度为40mg/mL,葡萄糖终浓度为120mg/mL,样品浓度见3测试分组与浓度。
各组测试样品混合均匀后置于55℃下孵育4天。以PBS/样品溶剂作为阴性对照或溶剂对照、以1%氨基胍盐酸盐作为阳性对照、以PBS代替糖基化反应液作为对照体系。反应结束后,将孵育的溶液冷却至室温测定各管的荧光强度,依次取反应液200μL加入96孔板,每个样品设置3个复孔,使用荧光酶标仪测试样品溶液在激发波长370nm,发射波长440nm条件下的荧光强度,根据下面的计算方法计算AGEs抑制率。
5.AGEs抑制率的计算
式中:
A-样品组的糖化体系荧光强度
B-样品组的PBS溶液荧光强度
C-对照组的糖化体系荧光强度
D-对照组的PBS溶液荧光强度
6.结果判定
样品在某一浓度下与糖化反应液反应后,与对照组相比AGEs抑制率>0%,则样品在该浓度下具有抑制AGEs生成的能力。
7.测试结果
测试结果如表1所示。
表1
由AGEs抑制实验可知,与空白对照组相比,蔓荆子黄素在2.34mg/L,4.68mg/L,9.36mg/L,18.7mg/L以及37.4mg/L浓度下,均能够显著抑制AGEs,且在较低浓度2.34mg/L时抑制率也可以达到84.62%,说明蔓荆子黄素的抗糖化作用突出。
测试例2:抗糖化作用测试
1.测试原理:
糖化是一种非酶化学反应,蛋白质与羰基化合物(葡萄糖果糖和乙二醛等)发生非酶糖基化反应最终可形成一系列不可逆的糖基化终产物(Advanced Glycation Endproducts,AGEs),AGEs可与蛋白质发生交联改变蛋白质的生理活性引起一系列病理生理改变。这些修饰同时影响细胞外和细胞内的蛋白质,导致细胞和组织功能障碍,从而加速皮肤的老化过程。羧甲基赖氨酸(Carboxymetyl Lysin,CML)是晚期糖基化终末产物代表之一,实验中采用乙二醛诱导人皮肤原代成纤维细胞糖基化,采用免疫荧光染色法对CML的表达进行定量分析。
本测试旨在评估蔓荆子黄素在细胞模型上的潜在的抗糖化作用。
2.测试材料:
2.1主要试剂:
人原代真皮成纤维细胞和培养基购自于 Cell Technology公司;青霉素-链霉素-两性霉素B溶液购自于Bioind,DPBS购自于Gibco公司,CCK试剂购自于同仁生物(40203ES80);96孔细胞培养板采购自Thermo公司;CML抗体购自于Abcam公司,Alexa568 Donkey Anti-MouseIgG购自Invitrogen公司,牛血清白蛋白BSA购自于碧云天,乙二醛购自于Adamas公司,氨基胍(Aladdin,A151036)。
2.2主要设备:
细胞培养箱,倒置荧光显微镜(Leica),生物安全柜,酶标仪。
3.测试分组与浓度:
4.测试方法:
4.1细胞接种:
将细胞从瓶中消化后接种到已经加入细胞爬片的24孔板中,每孔1.5*104个细胞。
4.2糖化诱导:
48小时后,采用0.5 mM乙二醛处理细胞诱导糖化反应作为阴性对照组,同时设空白组(NT)和样品组(乙二醛+样品),处理细胞48小时。
4.3固定染色:
将诱导后的细胞用PBS冲洗一次,用多聚甲醛(PFA)固定后,进行CML荧光染色。经过BSA封闭,一抗孵育和二抗孵育之后,最后采用含DAPI水性封片剂封片。荧光显微镜拍摄荧光图像,使用Image J对图像进行量化分析。
3.3统计方法
所有结果以Mean±SD表示。
4.实验结果
用实施例8和实施例9处理细胞并进行荧光染色分析,如图3和4所示。图像分析表明,1.87 mg/L和9.36 mg/L的蔓荆子黄素对糖化抑制率分别为26.35%和23.81%。CML荧光图像定量详细数据见表2。
表2
免疫荧光实验可知,与对照组相比,蔓荆子黄素在1.87mg/L浓度下,能够显著抑制AGEs,抑制率为26.35%,说明蔓荆子黄素的抗糖化作用较为突出。
本发明所述的蔓荆子黄素可以作为中间体原料或功效添加剂用于药品、保健食品、口服剂或皮肤外用剂的制备,所述皮肤外用剂优选为化妆品组合物,包括但不限于面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾(清洁泡)、喷雾、沐浴露和洗面奶等剂型的产品的制备,实施例1-6蔓菁子黄素,在药品、保健食品、口服剂或皮肤外用剂中的重量百分比为0.00001%-20%(w/w)。优选的重量百分比为0.0001%-10%(w/w)。更优选的重量百分比为0.0001%-5%(w/w)。最优选的重量百分比为0.001%-5%(w/w)。
以下是蔓菁子黄素在药品、保健食品、口服剂或皮肤外用剂中的具体应用例,以及这些剂型的配方和制备方法。以下各表中“-”表示无添加。
应用例1:面霜的制备
应用例2:乳液的制备
应用例3:啫喱的制备
应用例4:化妆水的制备
应用例5:精华液的制备
应用例6:面膜的制备
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应用例7:眼霜的制备
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应用例8:气雾(清洁泡)的制备
应用例9:喷雾的制备
应用例10:沐浴露的制备
应用例11:洗面奶的制备
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Claims (11)
1.蔓荆子黄素的抗糖化应用。
2.如权利要求1所述的应用,其特征在于,蔓荆子黄素的使用浓度至少为0.1mg/L。
3.如权利要求2所述的应用,其特征在于,蔓荆子黄素的使用浓度至少为1mg/L。
4.如权利要求1所述的应用,其特征在于,所述抗糖化为抑制非酶糖基化反应的终末产物。
5.如权利要求1所述的应用,其特征在于,所述抗糖化为抑制糖基化终产物。
6.蔓荆子黄素在制备具有抗糖化作用的皮肤外用剂、药品及保健食品中的应用。
7.如权利要求6所述的应用,其特征在于,蔓荆子黄素在皮肤外用剂、药品及保健食品中的含量0.00001-20重量%。
8.如权利要求7所述的应用,其特征在于,蔓荆子黄素在皮肤外用剂、药品及保健食品中的含量为0.0001-5重量%。
9.如权利要求6所述的应用,其特征在于,所述皮肤外用剂选自:面霜、乳液、啫喱、化妆水、精华液、面膜、眼霜、气雾清洁泡、喷雾、沐浴露、或洗面奶。
10.如权利要求6所述的应用,其特征在于,所述抗糖化为抑制非酶糖基化反应的终末产物。
11.如权利要求6所述的应用,其特征在于,所述抗糖化为抑制糖基化终产物。
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