CN117982358A - 去甲氧基荚果蕨素促进弹性蛋白生成的应用 - Google Patents
去甲氧基荚果蕨素促进弹性蛋白生成的应用 Download PDFInfo
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Abstract
本发明提供了去甲氧基荚果蕨素促进弹性蛋白生成的应用。本发明还涉及去甲氧基荚果蕨素在制备具有促进弹性蛋白生成作用的皮肤外用剂、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,具体涉及去甲氧基荚果蕨素促进弹性蛋白生成的应用,以及去甲氧基荚果蕨素在制备具有促进弹性蛋白生成作用的皮肤外用剂、药品及保健食品中的应用。
背景技术
成纤维细胞(Fb)是构成皮肤真皮层的主要细胞,是真皮层产生胶原及其他细胞间质的主要细胞,其正常的分化、增殖维系着皮肤正常的结构和生理功能。Fb具有很强的蛋白质合成能力,可以合成并分泌大量弹性蛋白、胶原蛋白、糖胺多糖和糖蛋白等基质成分,进而生成弹性纤维、胶原纤维和网状纤维,并分泌多种细胞修复因子,使得皮肤具有强大的更新与自我修复能力,从而使皮肤真皮层的厚度和密度增加,具有潜在的舒展皱纹、恢复皮肤弹性和光泽的作用。
弹性蛋白(Elastin)是弹性纤维的主要成分。在皮肤结缔组织中特别丰富,与胶原纤维同时存在,赋予组织弹性和抗张力。能让肌肤有伸展和褶合的能力,负责维持与支撑肌肤的弹性。随着年龄增长,肌肤中的弹性蛋白含量降低,从而降低对维持紧致肌肤所需要的网络支持,皮肤开始松弛,并出现细纹等。
去甲氧基荚果蕨素(5,7-二羟基-6,8-二甲氧基二氢黄酮),CAS号:56297-79-1,英文名称:demethoxymatteucinol,分子式:C17H16O4,分子量:284.31,结构式如下:
去甲氧基荚果蕨素是一种二氢黄酮类成分,广泛存在于荚果蕨贯众、中华荚果蕨、浅叶鳞毛蕨、番石榴等植物中。在美容相关功效报道中,仅有少量去甲氧基荚果蕨素抗氧化方面的研究,并未发现关于去甲氧基荚果蕨素促进弹性蛋白生成方面的美容报道。
本发明意外地发现,去甲氧基荚果蕨素能够有效促进弹性蛋白的生成,可以作为功效添加剂,应用于皮肤外用剂、药品及保健食品中。
发明内容
一方面,本发明涉及去甲氧基荚果蕨素促进弹性蛋白生成的应用。
在优选的实施方式中,去甲氧基荚果蕨素的使用浓度至少为0.01μg/mL,优选地至少为0.178μg/mL。
在优选的实施方式中,去甲氧基荚果蕨素的使用浓度为0.089-2.84μg/mL,更优选为0.089 -1.42μg/mL。
另一方面,本发明涉及去甲氧基荚果蕨素在制备具有促进弹性蛋白生成作用的皮肤外用剂、药品及保健食品中的应用。
在优选的实施方式中,去甲氧基荚果蕨素在皮肤外用剂、药品及保健食品中的含量为0.000001-10重量%,优选地为0.00001-5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显示了样品3(0.71μg/mL去甲氧基荚果蕨素)的成纤维细胞形态图。
图2显示了样品4(0.355μg/mL去甲氧基荚果蕨素)的成纤维细胞形态图。
图3显示了样品5(0.178μg/mL去甲氧基荚果蕨素)的成纤维细胞形态图。
图4显示了样品6(0.089μg/mL去甲氧基荚果蕨素)的成纤维细胞形态图。
图5显示了样品8(0.022μg/mL去甲氧基荚果蕨素)的成纤维细胞形态图。
图6显示了溶剂对照组的成纤维细胞形态图。
具体实施方式
本发明涉及去甲氧基荚果蕨素促进弹性蛋白生成的作用,首次发现去甲氧基荚果蕨素能够有效促进弹性蛋白的生成,可作为功效添加剂,应用于皮肤外用剂、药品及保健食品中,以帮助实现维持肌肤紧致、改善皮肤松弛和/或皱纹、抵抗皮肤老化等功效。
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。
本文所描述的去甲氧基荚果蕨素可任选为成品包装形式。在一个实施例中,所述包装为装有去甲氧基荚果蕨素的诸如塑料、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,该产品包含去甲氧基荚果蕨素并且具有引导使用者将去甲氧基荚果蕨素施用至皮肤以治疗如下文论述的皮肤老化迹象的说明书。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。
本发明发现,去甲氧基荚果蕨素适用于处理皮肤老化迹象。本文所用,“皮肤老化迹象”包括存在细纹和皱纹、损失弹性、肤色不均、斑疤。在一个特别优选的实施例中,老化迹象是存在细纹和皱纹和/或损失弹性。
如本文所用,“处理皮肤老化迹象”指减轻、减少、预防、改善或消除上述皮肤老化的存在或迹象。
本文所用,“皱纹”包括微细纹、微皱纹或粗皱纹。皱纹的例子包括但不限于眼睛周围的微细纹(例如“鱼尾纹”)、前额和颊皱纹、眉心纹和嘴巴周围的笑纹。
如本文所用,“损失弹性”包括皮肤或组织的弹性或结构完整性的损失,包括但不限于松垂、松弛和松散的组织。弹性或组织结构完整性的损失可由多种因素所致,包括但不限于疾病、衰老、激素变化、机械创伤、环境损害,或者是向组织施用诸如美容品或药物之类的产品的结果。
如本文所用,“肤色不均”是指与弥漫状或斑驳状色素沉着有关的皮肤状况,所述色素沉着可分类为色素沉着过度,例如发炎后色素沉着过度。
如本文所用,“斑疤”意指与发红或红斑相关的皮肤状况。
本文所用,“美容品”指能保持、恢复、赋予、刺激或增强体美外观或看起来能增进美貌或年轻的美容物质或制品,尤其是涉及组织或皮肤的外观时。
如本文所用,“美容有效量”意指足以处理一种或多种皮肤老化迹象,但低至足以避免严重副作用的生理活性化合物或组合物的量。化合物或组合物的美容有效量会随以下因素而变:所处理的具体状况、最终使用者的年龄和身体状况、所处理/预防的状况的严重程度、处理的持续时间、其他处理的性质、所采用的具体化合物或产品/组合物、所利用的美容上可接受的具体载体以及类似因素。
去甲氧基荚果蕨素
本发明正是基于以下意外发现:去甲氧基荚果蕨素具有促进成纤维细胞生成弹性蛋白的作用。因此,去甲氧基荚果蕨素可作为功效成分加入到护肤品中使用,以帮助改善皮肤松弛、皱纹问题等。本发明目的在于提供去甲氧基荚果蕨素的促进弹性蛋白生成功效及其在化妆品中应用。
在促进成纤维细胞生成弹性蛋白的应用中,去甲氧基荚果蕨素的使用浓度至少为0.01μg/mL,优选至少为0.08μg/mL。
在一些实施方式中,促进成纤维细胞生成弹性蛋白的应用中,去甲氧基荚果蕨素的使用浓度优选为0.178μg/mL以下。
在一些实施方式中,去甲氧基荚果蕨素的使用浓度为0.01-5μg/mL,优选0.1-1μg/mL,更优选0.01-0.178μg/mL。
在一些实施方式中,去甲氧基荚果蕨素的使用浓度为0.022-2.84μg/mL,优选0.022-1.42μg/mL,更优选0.089-0.178μg/mL。
皮肤外用剂
在一些实施方式中,去甲氧基荚果蕨素可用于皮肤外用剂的制备。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。
去甲氧基荚果蕨素在皮肤外用剂中的含量可以为0.0001-20重量%,优选0.001-10重量%,更优选0.001-5重量%,更优选0.01-5重量%。
所述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。
包含去甲氧基荚果蕨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。
包含去甲氧基荚果蕨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。
尽管优选的是包含去甲氧基荚果蕨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。
包含去甲氧基荚果蕨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。
包含去甲氧基荚果蕨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、且不影响本发明的去甲氧基荚果蕨素的效果即可。
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如上所述的一种或多种皮肤老化迹象的哺乳动物皮肤。在一个实施例中,可将皮肤外用剂施用至需要处理细纹和皱纹和/或弹性损失的皮肤。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。
本发明实施例中使用的去甲氧基荚果蕨素(纯度≥95%)由中国中医科学院中药研究所提供。
实施例1:去甲氧基荚果蕨素制备
称取28.4mg去甲氧基荚果蕨素,用二甲亚砜(DMSO,Sigma)定容至100mL容量瓶,得到0.284mg/mL去甲氧基荚果蕨素溶液,然后按8个浓度梯度(V/V)用培养基(DMEM,广东博溪生物科技有限公司)来配制实验样品,分别得到待测样品(表1)。
表1:实施例1待测样品信息
测试例1:成纤维细胞细胞毒性测试
细胞接种:皮肤成纤维细胞(Fb20081902,广东博溪生物科技有限公司)按8×103个/孔的接种密度接种细胞至96孔板,培养箱(Thermo,150I)(37℃、5%CO2)中孵育过夜。
试验分组:试验设置调零组、溶剂对照组、阳性对照组(PC)与样品组(实施例1中样品1-8)。每个样品设置3个重复孔。
给药:待96孔板中细胞铺板率达到40%~60%时进行给药。溶剂对照组每孔加入200μL DMEM培养液(广东博溪生物科技有限公司);PC组每孔加入200μL含10% DMSO培养液;样品组每孔加入200μL含有相应浓度样品的培养液;调零组无细胞接种,仅加入200μL细胞培养液。给药完后将96孔板放置在培养箱中培养。
检测:细胞孵育24小时后,倒置显微镜(CKX41,Olympus)10倍物镜下观察不同浓度受试物作用后的细胞状态,倒置显微镜20倍物镜下拍照(图1-6)。拍照结束后弃掉上清,加入0.5mg/mL MTT(Sigma),37℃避光孵育。4小时后弃掉上清,每孔加150mL DMSO,在490nm处读取OD值并计算细胞存活率。
细胞存活率%=(实施例孔OD-调零孔OD)/(溶剂对照孔OD-调零孔OD)*100%。
毒性判断方法:通过MTT检测后样品组的OD值与对照组的OD值进行对比来判断,当筛选出转折点后,在转折点上下各选2个浓度进行形态学拍照,来验证MTT的结果。
实验结果:
表2显示了去甲氧基荚果蕨素成纤维细胞毒性检测结果。结果表示为Mean±SD。
表2
细胞存活率 | |
样品8 | 103.80%±4.58% |
样品7 | 104.82%±5.32% |
样品6 | 103.89%±6.52% |
样品5 | 100.28%±2.51% |
样品4 | 85.45%±3.48% |
样品3 | 81.65%±1.68% |
样品2 | 80.72%±4.28% |
样品1 | 71.08%±4.10% |
PC | 3.52%±0.42% |
根据图1-6成纤维细胞形态图可知,图3(样品5)、图4(样品6)、图5(样品8)中成纤维细胞边缘清楚,形态舒展,细胞密度较大,细胞均质而透明,与图6(溶剂对照组)无明显差异。图2(样品4)成纤维细胞边缘清楚,形态尚可,细胞密度略有降低;图1(样品3)成纤维细胞形态轻微收缩,细胞密度下降,细胞质透明度降低。图1-2与图6差异明显。结合表2毒性检测结果可知,去甲氧基荚果蕨素基于成纤维细胞,在样品5浓度(0.178μg/mL)以下未表现出明显的细胞毒性。
测试例2:基于成纤维细胞的Elastin含量测试
细胞接种:按4×104个/孔的接种密度接种成纤维细胞(Fb20081902,广东博溪生物科技有限公司)至24孔板,培养箱(37℃、5%CO2)中孵育过夜。
配液:分为空白对照组(BC)、阴性对照组(NC)、阳性对照组(PC,0.1μg/mL TGF-β1)和样品组(实施例1中样品5-6)。
给药:待24孔板中细胞铺板率达到40%~60%时进行分组给药。每孔加样2mL,每组设3个复孔。给药完后将24孔板放置在培养箱中培养24小时。
UVA辐照:对NC组、PC组和样品组进行30J/cm2的UVA辐照,置于培养箱中继续培养24小时。
收样:24小时后,收集细胞培养上清液于EP管中,置于-80℃冰箱冷冻保存。
ELISA检测:根据Elastin ELISA试剂盒(Human Elastin ELISA Kit,Abcam公司)的操作说明书进行检测。Elastin含量结果表示为Mean±SD。
弹性蛋白(Elastin)实验结果:
表3
实验分组 | Elastin含量(ng/mL) |
BC | 522.08±22.82 |
NC | 265.78±9.99 |
PC | 890.37±51.30 |
样品5 | 278.11±22.43 |
样品6 | 292.56±3.53 |
与BC组相比,NC组的Elastin含量由522.08ng/mL降低至265.78ng/mL,说明本次测试刺激条件有效。
与NC组相比,阳性对照组的Elastin含量由265.78ng/mL提高至890.37ng/mL,说明本次测试阳性对照有效。
与NC组相比,样品5(0.178μg/mL去甲氧基荚果蕨素溶液)作用后成纤维细胞Elastin含量由265.78ng/mL提高至278.11ng/mL;样品6(0.089μg/mL去甲氧基荚果蕨素溶液)作用后成纤维细胞Elastin含量由265.78ng/mL提高至292.56ng/mL。说明去甲氧基荚果蕨素能够很好的促进成纤维细胞生成Elastin,达到紧致功效。
去甲氧基荚果蕨素可用于药品、保健食品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。实施例1中样品1-8去甲氧基荚果蕨素,在皮肤外用剂中的重量百分比为0.000001%-10%(w/w)。优选的重量百分比为0.00001%-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)。
以下是去甲氧基荚果蕨素在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。具体应用例如下:
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
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
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应用例11:洗面奶的制备
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Claims (9)
1.去甲氧基荚果蕨素促进弹性蛋白生成的应用。
2.如权利要求1所述的应用,其特征在于,所述去甲氧基荚果蕨素的使用浓度至少为0.01μg/mL。
3.如权利要求2所述的应用,其特征在于,所述去甲氧基荚果蕨素的使用浓度至少为0.178μg/mL。
4.如权利要求2所述的应用,其特征在于,所述去甲氧基荚果蕨素的使用浓度为0.089-2.84μg/mL。
5.如权利要求2所述的应用,其特征在于,所述去甲氧基荚果蕨素的使用浓度为0.089-1.42μg/mL。
6.去甲氧基荚果蕨素在制备具有促进弹性蛋白生成作用的皮肤外用剂、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,所述去甲氧基荚果蕨素在皮肤外用剂、药品及保健食品中的含量为0.000001-10重量%。
8.如权利要求6所述的应用,其特征在于,所述去甲氧基荚果蕨素在皮肤外用剂、药品及保健食品中的含量为0.00001-5重量%。
9.如权利要求6-8中任一项所述的应用,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。
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