CN103415299B - 用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物 - Google Patents
用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物 Download PDFInfo
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Abstract
本发明涉及用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物，该组合物作为新型皮肤刺激缓解剂包含苦楝皮提取物和莲叶提取物。更具体而言，本发明包含苦楝皮提取物和莲叶提取物，并通过增加是使毒素去毒的酶的NQO1和是排除毒素的酶的GSTT1的表达实现毒素的去毒和排除，通过增加是天然免疫因子的皮肤抗微生物肽hBD3的表达实现对皮肤的保护，以及通过增加抗氧化蛋白GPx1、过氧化氢酶(catalase)、Trx1和Prdx1的表达实现抑制活性氧的产生。因此，本发明具有皮肤保护和老化抑制的优点。
Description
技术领域
本发明涉及用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物，更具体而言，涉及用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物，该组合物包含作为活性成分的苦楝皮和莲叶。
背景技术
皮肤是身体直接暴露于外部环境的部分。其作为屏障保护身体的重要器官，防止过度的失水并保护身体免受感染。虽然皮肤甚至提供强保护防止外界病毒侵袭，但过度暴露于紫外线、污染物等可能造成诸如红斑、水肿、瘙痒等皮肤刺激和炎性反应。由这些环境因素引起的皮肤问题使人没有魅力，并且活性氧增加至细胞的抗氧化保护系统不能处理的水平，对DNA、蛋白质、脂肪和糖是有害的，对细胞的存活、生长和分化产生负面影响，并可能导致细胞死亡。延长的氧化应激导致皮肤老化、癌症和炎症(Ismail等人，Angiopoietin-1reduces H2O2-induced increases in reactive oxygen species and oxidativedamage to skin cells.J Invest Dermatol.130(5)：1307-17，2010)。
亚洲沙尘是起源于中国、蒙古等国沙漠的一种现象，在这些地方，沙子或黄色沙尘随风刮向东并落到地上。每个春天韩国都会受到亚洲沙尘的影响。亚洲沙尘包含有机和无机物质，并且其物理性质和组成是不同的。其甚至包含可能具有生物效应的金属。亚洲沙尘中包含的大颗粒保留在原地或其周围，而直径为10μm或更小的微粒(颗粒物10；PM10)(particulate matter10；PM10)通常到达韩国。据报道，微粒在被吸入时可以深入到达亚段支气管和肺的气体交换区域并可能损伤呼吸系统(Yanagisawa等人，Gene expressionanalysis of murine lungs following pulmonary exposure to Asian sand dustparticles.Exp Biol Med232：1109-1118，2007，Kim等人，Cytotoxicity of yellow sandin lung epithelial cells.J Biosci28(1)：77-81，2003)。已就亚洲沙尘对人呼吸疾病方面的健康影响作了大量的研究。在使用小鼠的实验性研究中，在被灌注亚洲沙尘的小鼠肺中发现氧化应激相关指数增大(Naota 等人，Pathological Study of Acute PulmonaryToxicity Induced by Intratracheally Instilled Asian Sand Dust.ToxicolPathol.2010Sep30.[印刷之前的电子出版物])。
临床上，如果皮肤直接暴露于亚洲沙尘，它可能通过过敏反应导致接触性皮炎。此外，已知干燥和强烈的亚洲沙尘暴通过使皮肤丧失水分导致皮肤干燥、角质化、瘙痒、痤疮、特应性等。近来，关于亚洲沙尘对皮肤的有害作用，首次报道了亚洲沙尘导致角质形成细胞中包括细胞因子白介素6和白介素8的促炎性因子的增加(Choi等人,Asian dust stormparticles induce a broad toxicological transcriptional program in humanepidermal keratinocytes.Toxicol Lett.15；200(1-2)：92-9.2010)。
皮肤最外层中存在的角质形成细胞(角质形成细胞，keratinocyte)通过与成纤维细胞、上皮细胞和皮肤免疫细胞相互作用在皮肤刺激反应的引发、调节和控制方面起重要的作用。皮肤刺激是最常见的与皮下炎性反应有关的有害作用，并且在临床上，70%的接触性皮炎可以用皮肤刺激来解释。红斑、水肿、龟裂、失水、剥脱、瘙痒和疼痛是刺激性或过敏性接触性皮炎的典型症状。它们是细胞内稳态异常和炎性反应的结果。已有少数关于与亚洲沙尘相关的皮肤刺激的研究。然而，随着亚洲沙尘的频率的增加，开发外用于皮肤以预防或治疗由亚洲沙尘引起的皮肤刺激的自然制剂是必要的。
发明内容
技术问题
本发明涉及提供用于抗氧化和减轻由亚洲沙尘对皮肤造成的有害作用的组合物，该组合物作为新型皮肤刺激缓解剂而包含苦楝皮和莲叶。
技术方案
在一个总体方面中，提供了用于抗氧化的组合物，该组合物包含作为活性成分的苦楝皮提取物。
在本发明的一个示例性实施方案中，组合物可以进一步包含莲叶提取物。
在本发明的一个示例性实施方案中，组合物可以抑制活性氧的产生或增加抗氧化蛋白基因的表达。
在另一个总体方面中，提供了用于减轻由亚洲沙尘对皮肤造成的有害作用的组合物，该组合物包含作为活性成分的苦楝皮提取物和莲叶提取物。
在本发明的一个示例性实施方案中，组合物可以增加GST、NQO1或hBD3的表达。
有益效果
根据本发明的组合物实现了下述效果：通过增加使毒素去毒的酶NQO1和排除毒素的酶GSTT1的表达实现毒素的去毒和排除；通过增加天然免疫因子的皮肤抗微生物肽hBD3的表达实现对皮肤的保护；以及通过增加抗氧化蛋白GPx1、过氧化氢酶(catalase)、Trx1和Prdx1的表达来实现抑制活性氧的产生。因此，组合物可以用作预防由亚洲沙尘引起的皮肤问题、保护皮肤以及预防和治疗皮肤老化的药物组合物或美容组合物。
附图说明
图1显示了用根据本发明的示例性实施方案的组合物处理正常人角质形成细胞时活性氧种类的产生。
图2至图4显示了用根据本发明的示例性实施方案的组合物处理正常人角质形成细胞时，通过聚合酶链反应(RT-PCR)分析NQO1(图2)、hBD3(图3)和GSTT1(图4)的表达的结果。
图5显示了用根据本发明的示例性实施方案的组合物处理正常人角质形成细胞时，通过聚合酶链反应(RT-PCR)分析GPx1、过氧化氢酶（catalase）、Trx1和Prdx1的表达的结果。
具体实施方式
现将参照附图在下文中更全面地描述示例性实施方案，从而使本领域普通技术人员可以容易地实施本发明。
本发明提供了用于抗氧化的组合物，该组合物包含作为活性成分的苦楝皮提取物。
苦楝皮是指楝科苦楝(也叫做日本产苦楝(Melia azedarach L.var.japonicaMakino))的树皮或根皮。其用于杀死蛔虫和线虫并用于治疗风疹（風疹）和疥疮（疥癬）。其还用于治疗湿疹并具有利尿和解热作用。虽然报道了对诸如绦虫、蛔虫等肠寄生虫和滴虫阴道炎的药理学作用，但从未报道过抗氧化作用。苦楝皮是弯曲的，具有管形或半管形形状。外表面是淡灰棕色，具有纵向裂缝和横向皮孔。剥皮后的木栓层为红褐色。中断的表面为淡黄白色、具有纤维并且是坚硬的，但易于折断。具有许多皮孔的嫩皮对用作药物是良好的。其也可以叫做苦楝根皮楝皮楝根茎皮金铃子等。
本发明的组合物除了包含苦楝皮提取物之外可以进一步包含莲叶提取物。
莲（Nelumbo nucifera）是具有黄色花朵的多年生水生植物。其在东亚、尤其是在中国广泛用作食物，并且也用作各种药物。具体地说，莲的种子用于治疗组织炎症、癌症和麻风病；用于解毒等。而且，据报道莲的胚芽在体内（in vivo）缓解急性炎症。而且，莲叶在中国传统医学中用于止血并已知缓解实验动物的高脂血症。
本发明所用的苦楝皮或莲叶提取物的提取方法不受具体限制。可以使用水或有机溶剂获得提取物。本发明中所用的有机溶剂可以是选自乙醇、甲醇、丁醇、乙醚、乙酸乙酯和氯仿中的一种或多种。而且，可以使用这些有机溶剂的混合物。在下面所述的实施例中，提取物获自韩国植物提取物库。
具体地说，可以以基于组合物总重量的1-30wt%的量包括苦楝皮提取物或莲叶提取物。如果提取物的总量小于1wt%，可能达不到期望的效果。并且，如果量超过30wt%，可能产生稳定性问题。
包含苦楝皮提取物作为活性成分的组合物和包含苦楝皮提取物和莲叶提取物的组合物具有抗氧化作用。
如将在下文描述的测试实施例1和图1中所见，可以通过抑制活性氧的产生来实现抗氧化作用。活性氧(Reactive oxygen species,ROS)是具有自由电子的氧化合物。为了使它们的不稳定状态稳定，它们攻击细胞膜的脂蛋白或者细胞内DNA，从而导致细胞损伤、细胞凋亡等并引起各种疾病和老化。因此，通过减少活性氧可以预防皮肤老化。
如将在下文描述的测试实施例3和图5中所见，可以通过增加抗氧化蛋白基因的表达来实现抗氧化作用。抗氧化蛋白基因包括GPx1、过氧化氢酶（catalase）、Trx1和Prdx1，但其不限于此。
谷胱甘肽过氧化物酶1(Glutathione peroxidase1,GPx1)是许多谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)中的一种，在人体中由GPx1基因编码。谷胱甘肽过氧化物酶参与过氧化氢的去毒作用(detoxification)并且在人体中是最重要的抗氧化酶。举例来说，该蛋白可以具有序列Hs00829989_Gh。
过氧化氢酶（catalase）是在几乎所有生命有机体中发现的常见酶。其催化过氧化氢分解成水和氧气。举例来说，该蛋白可以具有序列Hs00156308_m1。
硫氧还蛋白1(thioredoxin，Trx1)是保护细胞抵抗氧化和还原应激的硫氧化蛋白系统中的一种细胞质硫氧还蛋白同工酶。举例来说，该蛋白可以具有序列Hs01555212_g1。
过氧化物还原酶1(peroxiredoxin1，Prdx1)在人体中是由PRDX1基因编码的蛋白。举例来说，该蛋白可以具有序列Hs00602020_Mh。
本发明还提供了用于减轻由亚洲沙尘对皮肤造成的有害作用的组合物，该组合物包含作为活性成分的苦楝皮提取物和莲叶提取物。更具体地说，本发明的组合物可以减轻对皮肤中的正常角质形成细胞（keratinocyte）的有害作用。
如将在下文描述的测试实施例2和图2至图4中所见，与单独包括莲叶提取物或苦楝皮提取物时相比，在包括苦楝皮提取物和莲叶提取物时GST、NQO1和hBD3基因的表达显著增加。GSTT1是排除毒素的酶，NQO1是使毒素去毒的酶，hBD3是皮肤抗微生物肽和天然免疫因子。如果包括GST、NQO1或hBD3的与去毒相关的酶的表达增加，可以通过缓解皮肤刺激来实现皮肤保护。
谷胱甘肽S-转移酶(Glutathione S-transferase，GST)被分成5类。其中的一类是谷胱甘肽S-转移酶θ-1(Glutathione S-transferase theta-1，GSTT1)，其是由GSTT1基因编码的酶，并催化还原型谷胱甘肽结合多种亲电子化合物和疏水化合物。举例来说，该蛋白可以具有序列Hs00184475_m1。
醌氧化还原酶1(quinine oxidoreductase1，NQ01)催化醌的2电子还原反应。该酶提供保护防止醌介导的致癌作用并激活生物还原型烷基化剂。举例来说，该蛋白可以具有序列Hs01045994_m1。
人β防御素3(human beta defensin3，hBD3)诱导抗原递呈细胞（antigenpresenting cells）的迁移和活化并充当免疫增强剂（immune enhancer）。举例来说，该蛋白可以具有序列hs00218678_m1。
根据本发明的组合物可以是美容组合物、药物组合物或保健食品组合物。
美容组合物的剂型不受具体限制。例如，可以将其配制成软化洗剂、收敛洗剂、滋养洗剂、滋养霜、按摩霜、精华素、眼霜、眼部精华素、清洁霜、清洁洗剂、清洁泡沫、清洁水、面膜、美容精华素、粉、身体洗剂、身体乳霜、身体护理油、身体精华素、身体清洁剂、染发剂、生发剂、头发滋养洗剂、头发精华素、头发修复液、头皮护理、头发护理、头发调理剂、洗发香波、头发洗剂、牙膏、漱口剂、头发定型剂、生发剂、洗剂、软膏剂、凝胶剂、乳膏剂、贴剂、喷雾剂等。
药物组合物可以进一步包含诸如防腐剂、稳定剂、润湿剂、乳化促进剂的药物佐剂和/或用于控制渗透压的盐和/或缓冲液等，和其他治疗用物质，并且可以根据常用方法将该药物组合物制备成用于口服或肠胃外给药的各种剂型。
用于口服给药的剂型可以包括例如片剂、丸剂、硬胶囊和软胶囊、液体剂、悬浮剂、乳化剂、糖浆剂、颗粒剂等，并且这些剂型除了包含有效成分之外还可以进一步包含稀释剂(例如乳糖、右旋糖、蔗糖、甘露醇、山梨醇、纤维素或甘氨酸)和润滑剂(例如二氧化硅、滑石粉、硬脂酸及其镁盐或钙盐或聚乙二醇)。片剂可以包含粘合剂，诸如硅酸镁铝、淀粉糊、明胶、黄蓍胶、甲基纤维素、羧甲基纤维素钠或聚乙烯吡咯烷酮，并且必要时，其可以包含药物添加剂，诸如崩解剂(例如淀粉、琼脂、海藻酸或其钠盐)、吸收剂、着色剂、调味剂、甜味剂等。片剂可以通过普通的混合、制粒或包衣方法制备。
而且，用于肠胃外给药的剂型可以是外用于皮肤的制剂的形式。例如，其可以是洗剂、软膏剂、凝胶剂、乳膏剂、贴剂或喷雾剂的形式，但并不限于此。
保健食品组合物的剂型不受具体限制。举例来说，可以将其配制成片剂、颗粒剂、饮料、糖果、食物棒等。各种剂型的保健食品组合物除了包含活性成分外，还可以包含相关领域中常用的其他成分，本领域技术人员可以毫不费力地对这些其他成分进行选择。额外添加的成分可以提供协同效应。
活性成分给药剂量的确定处于本领域技术人员的水平范围内。根据包括受试者的年龄和身体状况、是否存在并发症、待治疗的病症的阶段和严重程度等各种因素，每日给药剂量将有所不同。对成年人来说，组合物的一般没日给药剂量可以为1-500mg/kg，具体为30-200mg/kg。可以一天一次或者一天二次进行给药。然而，上述给药剂量并不以任何方式限制本发明的范围。
实施例
下文将通过实施例详细地描述本发明。然而，下列实施例仅用于举例说明的目的，并且对于本领域技术人员而言，本发明的范围不受实施例的限制是显然的。
测试实施例1由亚洲沙尘引起的人角质形成细胞中的活性氧产生的变化
如推荐的那样，在37℃和5%CO2的条件下在CO2培养箱(CO2incubator)中对购自龙沙公司（Lonza,Inc.，美国马里兰州沃克斯维尔（Walkersville）)的正常人角质形成细胞(人表皮新生角质形成细胞，human epidermal neonatal keratinocyte cells)进行继代培养。根据龙沙公司的说明书对细胞培养物进行孵育。具体地说，将细胞培养在500mL的补充有KGM-2（Bullet试剂盒：2mL BPE(牛垂体提取物，bovine pituitary extract)，0.5mLhEGF(人表皮生长因子，human epidermal growth factor)，0.5mL胰岛素（insulin），0.5mL氢化可的松（hydrocoitisone），0.5mL转铁蛋白（transferrin），0.5mL肾上腺素（epinephrine），0.5mL硫酸庆大霉素硫酸盐+两性霉素B(Gentamycin slufate+amphotericin B:GA-1000)的KBM-2培养基(Clonetics CC-3103)中。
使用未处理的人角质形成细胞作为对照组。向苦楝皮提取物处理的测试组中加入10ppm苦楝皮提取物(获自韩国植物提取物库)。另外，向莲叶提取物处理的测试组中加入10ppm莲叶提取物(获自韩国植物提取物库)。向苦楝皮提取物和莲叶提取物处理的测试组中加入5ppm苦楝皮提取物和5ppm莲叶提取物。预处理30分钟后，加入25μg/mL亚洲沙尘样品，并将每组细胞孵育24小时。亚洲沙尘样品是在亚洲沙尘季节期间在爱茉莉太平洋研发中心(韩国龙仁市)的屋顶上收集的。用25μg/mL亚洲沙尘处理24小时后，将细胞用10mL磷酸缓冲盐(phosphate buffered saline，PBS)洗涤两次，并使用Glomax20/20发光检测仪在加入1mM鲁米诺和20单位/mL辣根过氧化物酶(HRP)1分钟后在2mL PBS中测量化学发光。结果在图1中示出。
如从图1中所见，苦楝皮提取物和莲叶提取物抑制由亚洲沙尘增加的正常人角质形成细胞中活性氧的产生。特别是，当细胞用苦楝皮提取物和莲叶提取物处理时，抑制作用更显著。
测试实施例2在用苦楝皮提取物或莲叶提取物处理后的毒素去毒和排除因子和作为天然免疫因子的皮肤抗微生物肽的表达增加
如推荐的那样，在37℃和5%CO2的条件下在CO2培养箱(CO2incubator)中对购自龙沙公司的正常人角质形成细胞(人表皮新生角质形成细胞，human epidermal neonatalkeratinocyte cells)进行继代培养。根据龙沙公司的说明书对细胞培养物进行孵育。具体地说，将细胞培养在500mL的补充有KGM-2（Bullet试剂盒：2mL BPE(牛垂体提取物，bovinepituitary extract)，0.5mL hEGF(人表皮生长因子，human epidermal growth factor)，0.5mL胰岛素（insulin），0.5mL氢化可的松（hydrocoitisone），0.5mL转铁蛋白（transferrin），0.5mL肾上腺素（epinephrine），0.5mL硫酸庆大霉素硫酸盐+两性霉素B(Gentamycin slufate+amphotericin B:GA-1000)的KBM-2培养基(Clonetics CC-3103)中。
使用未处理的人角质形成细胞作为对照组。向苦楝皮提取物处理的测试组中加入10ppm苦楝皮提取物(获自韩国植物提取物库)。另外，向莲叶提取物处理的测试组中加入10ppm莲叶提取物(获自韩国植物提取物库)。向苦楝皮提取物和莲叶提取物处理的测试组中加入5ppm苦楝皮提取物和5ppm莲叶提取物。预处理30分钟后，加入25μg/mL亚洲沙尘样品，并将每组细胞孵育24小时。用亚洲沙尘处理24小时后，将细胞用10mL磷酸盐缓冲液洗涤两次，并使用TRIzol(Invitrogen)从细胞中分离出总RNA（total RNA）。再次使用RNA试剂盒(Qiagen)对分离出的RNA进行纯化并使用Bioanalyzer2100(Agilent)分析RNA的质量（quality）。使用Superscript逆转录酶(RT)II试剂盒(Superscript reversetranscriptase(RT)II kit)由分离出的RNA合成cDNA，并通过实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction，Q-RT-PCR)对cDNA进行定量分析。使用TaqMan基因表达分析试剂盒(TaqMan gene expression assay kit，Applied Biosystems)对GSTT1(Hs00184475_m1)、NQO1(Hs01045994_m1)和hBD3(hs00218678_m1)的表达模式的变化进行评价。结果在图2至图4中示出。
如从图2至图4所见，苦楝皮提取物和莲叶提取物增加使毒素去毒的NQO1(图2)、是天然免疫因子的皮肤抗微生物肽hBD3(图3)和是排除毒素的酶的GSTT1(图4)在正常人角质形成细胞中的表达。当用苦楝皮提取物和莲叶提取物一起进行处理时，这种效应更显著。
因此，可见苦楝皮提取物和莲叶提取物的联合处理提供了改进毒素的去毒和排除并增加天然免疫因子的表达的协同作用。
测试实施例3在用苦楝皮提取物和莲叶提取物处理后的抗氧化蛋白基因的表达增加
如推荐的那样，在37℃和5%CO2的条件下在CO2培养箱(CO2incubator)中对购自龙沙公司的正常人角质形成细胞(人表皮新生角质形成细胞，human epidermal neonatalkeratinocyte cells))进行继代培养。根据龙沙公司的说明书对细胞培养物进行孵育。具体地说，将细胞培养在500mL的补充有KGM-2（Bullet试剂盒：2mL BPE(牛垂体提取物，bovine pituitary extract)，0.5mL hEGF(，human epidermal growth factor，人表皮生长因子)，0.5mL胰岛素（insulin），0.5mL氢化可的松（hydrocoitisone），0.5mL转铁蛋白（transferrin），0.5mL肾上腺素（epinephrine），0.5mL硫酸庆大霉素硫酸盐+两性霉素B(Gentamycin slufate+amphotericin B:GA-1000)的KBM-2培养基(Clonetics CC-3103)中。
使用未处理的人角质形成细胞作为对照组。向苦楝皮提取物处理的测试组中加入10ppm苦楝皮提取物(获自韩国植物提取物库)。另外，向莲叶提取物处理的测试组中加入10ppm莲叶提取物(获自韩国植物提取物库)。向苦楝皮提取物和莲叶提取物处理的测试组中加入5ppm苦楝皮提取物和5ppm莲叶提取物。处理24小时后，将细胞用10mL磷酸盐缓冲液洗涤两次，并使用TRIzol(Invitrogen)从细胞中分离出总RNA（total RNA）。再次使用RNA试剂盒(Qiagen)对分离出的RNA进行纯化并使用Bioanalyzer2100(Agilent)分析RNA的质量（quality）。使用Superscript逆转录酶(RT)II试剂盒(Superscript reversetranscriptase(RT)II kit，Invitrogen)由分离出的RNA合成cDNA，并通过实时反转录聚合酶链反应(real-time reverse transcription polymerase chain reaction，Q-RT-PCR)对cDNA进行定量分析。使用TaqMan基因表达分析试剂盒(TaqMan gene expression assaykit，Applied Biosystems)对GPx1(Hs00829989_gH)、过氧化氢酶(Hs00156308_m1)、Trx1(Hs01555212_g1)和PRDX1(Hs00602020_mH)的表达模式的变化进行评价。
当用苦楝皮提取物处理正常人角质形成细胞时，是清除活性氧的抗氧化酶的GPx1、过氧化氢酶、Trx1和Prdx1的相对表达分别约为2.5、2.0、1.9和1.9。当用莲叶提取物处理细胞时，相对表达分别约为1.9、1.7、1.6和1.7。当用苦楝皮提取物和莲叶提取物一起处理细胞时，GPx1、过氧化氢酶、Trx1和Prdx1的表达最高分别约为3.0、2.43、2.48和2.4(图5)。
因此，可见当单独用苦楝皮提取物处理细胞时抗氧化酶的表达增加，并且当用苦楝皮提取物和莲叶提取物一起处理细胞时抗氧化酶的表达进一步增加。
在下文将描述根据本发明的包含苦楝皮提取物和莲叶提取物的组合物的剂型实施例。然而，下列剂型实施例仅用于举例说明的目的，并且对于本领域普通技术人员而言，本发明的范围不受剂型实施例的限制是显然的。
剂型实施例1洗剂
剂型实施例2乳膏剂
剂型实施例3面膜
剂型实施例4美容精华素
Claims (2)
1.苦楝皮提取物和莲叶提取物的组合在制备用于抗皮肤氧化的组合物中的用途。
2.苦楝皮提取物和莲叶提取物的组合在制备用于减轻由亚洲沙尘对皮肤造成的有害作用的组合物中的用途。
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