CN117412760A - 永生花固体残余物的油性提取物作为亮肤剂的美容用途 - Google Patents
永生花固体残余物的油性提取物作为亮肤剂的美容用途 Download PDFInfo
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- CN117412760A CN117412760A CN202280036709.XA CN202280036709A CN117412760A CN 117412760 A CN117412760 A CN 117412760A CN 202280036709 A CN202280036709 A CN 202280036709A CN 117412760 A CN117412760 A CN 117412760A
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Abstract
本发明涉及永生花固体残余物的油性提取物作为亮肤剂的美容用途。本发明还涉及一种美容组合物，所述美容组合物在生理上可接受的介质中包含永生花残余物的油性提取物和己基间苯二酚；以及该组合物的用途。
Description
技术领域
本发明涉及永生花蒸馏残余物的油性提取物作为亮肤剂的美容用途。本发明还涉及美容组合物，所述美容组合物在生理上可接受的介质中包含永生花蒸馏残余物的油性提取物和己基间苯二酚，并且还涉及该组合物的用途。
背景技术
皮肤色素沉着的机制是涉及由黑素细胞产生黑色素的复杂过程，黑素细胞是位于表皮的基底层中的特化细胞。示意性地，该机制涉及以下主要步骤：
酪氨酸->多巴->多巴醌->多巴色素->黑色素，
由该反应序列中涉及的关键酶酪氨酸酶催化。
皮肤颜色主要由黑素细胞产生的黑色素的类型和浓度决定。黑素细胞可以通过内部或外部刺激来活化，以在称为黑素体的特化溶酶体样细胞器中产生黑色素。成熟后，沿着微管和肌动蛋白细胞骨架将核周黑素体转运至黑素细胞树突的外周。然后将含有黑色素的黑素体转移至相邻角质形成细胞。
在该复杂过程中的任何步骤的损害可导致皮肤色素沉着障碍。特别地，活性黑素细胞数量的增加和/或黑色素产生的增加和/或黑素体从黑素细胞到角质形成细胞的转移的增加可能导致皮肤色素沉着过度。
因此，环境、遗传和/或激素因素可调节皮肤和/或毛发中黑色素的量、类型和分布，具有显著的美容和心理后果。因此，在太阳下，更暗和/或更多的色斑可能出现在皮肤上，更具体地出现在某些人的手上。其它类型的由于黑素细胞过度活性引起的区域性色素沉着过度是在妊娠(“妊娠斑”或黄褐斑)或雌激素孕激素避孕期间发生的特发性黑斑病。相反地，皮肤色素沉着过低可以在疤痕暴露于太阳的情况下观察到，或者在某些白斑例如白癜风中观察到。在这些情况下，如果不可能重新使受损的皮肤进行色素沉着，则将正常皮肤的剩余区域进行脱色素以给予皮肤作为整体的均匀白色色调。
特别寻求使用具有良好功效的无害局部脱色素物质以治疗所有这些色素沉着过度。如果物质直接作用于发生黑色素生成的表皮黑素细胞的活力，和/或如果它干扰黑色素生物合成的步骤之一，通过抑制黑色素生成中涉及的酶之一，特别是酪氨酸酶，或通过嵌入黑色素合成链中的一种化学化合物的结构类似物然后可以阻断该链，从而确保脱色素并因此确保皮肤提亮，则该物质被认为是漂白的或脱色素的。
各种美容活性剂，例如维生素C及其衍生物、熊果苷、壬二酸、乙醇酸、正丁基间苯二酚(或芸香酚)、己基间苯二酚以及谷胱甘肽及其衍生物，目前用于治疗这些色素沉着过度问题。这些化合物具有不同的作用模式，包括抑制酪氨酸酶活性，减少形成的黑色素的量，甚至刺激角质形成细胞中的黑色素消除。然而，这些化合物中的一些可能导致副作用，例如烧伤、红斑和皮肤干燥。
因此，仍然需要一种新型人皮肤漂白剂，其与已知的那些皮肤漂白剂同样有效，但没有其缺点，即对皮肤无刺激、无毒和/或无过敏性。
在该上下文中，已经提出使用各种永生花提取物作为漂白剂，特别是通过使用诸如乙醇的溶剂提取露头蜡菊(Helichrysum gymnocephalum(DC)Humbert)的地上部分获得的提取物。该提取物的特征在于存在查耳酮或二氢查尔酮的单萜衍生物，特别是gymnochalcone(FR2970416)。也可以提及WO 2007/015232，其建议在消除疤痕和皮肤色素沉着的过程中使用沙生蜡菊(Helichrysum arenarium)的(未定义的)提取物，以及CN111973544，其公开了蜡菊(Helichrysum bracteatum)的水-二醇提取物的脱色素效果。漂白性质也被发现于白叶蜡菊(Helichrysum angustifolium)精油(CN108670926)。此外，若干不同的永生花提取物的组合已被建议用于其抗老化性质，并且还能够减少色素斑(FR3003167)。最后，专利申请WO 2019/086602表明，包含咖啡酰奎尼酸的意大利蜡菊(Helichrysum italicum)的开花顶部的亚临界水提取物可能具有脱色素性质并且用作抗瑕疵美容活性剂。
申请人现已证明，相当令人惊讶地，对于其刺激角质形成细胞分化的性质及其恢复皮肤屏障的能力(WO 2019/180368)已知的另一种永生花提取物即使在低浓度下也显示出良好的脱色素活性，并且同时是良好耐受的。
发明内容
准确地说，本发明的主题是局部施用于皮肤的意大利蜡菊物种的永生花蒸馏残余物的油性提取物作为用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的试剂的美容用途。
本发明的另一个主题是一种用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的试剂，所述试剂由意大利蜡菊物种的永生花蒸馏残余物的油性提取物组成。
本发明的另一个主题涉及一种美容组合物，其相对于所述组合物的总重量，在生理上可接受的介质中包含0.01重量％至1重量％的如上定义的永生花蒸馏残余物的油性提取物，和0.001重量％至5重量％的己基间苯二酚。
本发明的主题也是上述美容组合物的美容用途，用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现，包括将该组合物局部施用于皮肤。
本发明的主题也是一种用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的美容方法，包括将上述组合物局部施用于皮肤。
具体实施方式
本发明使用意大利蜡菊物种优选是科西嘉岛(Corsican)来源的意大利蜡菊物种的永生花蒸馏残余物的油性提取物。
术语“蒸馏残余物”指的是在水蒸馏或蒸汽蒸馏永生花植物中的任何部分并分离产生的精油和水溶胶之后剩余的固体残余物。因此，根据本发明使用的永生花蒸馏残余物根据包括以下步骤的方法获得：对所有或部分植物、优选植物的地上部分、特别是其花或开花顶部进行水蒸馏或蒸汽蒸馏的步骤，随后回收蒸馏或蒸汽蒸馏残余物的步骤。
然后可以经由用于从蒸馏残余物中提取油的任何方法，并且更具体地通过使用超临界流体(例如二氧化碳、一氧化氮、二甲醚、丙烷、乙烯或甲醇，该列表是非限制性的)提取蒸馏残余物而获得来自蒸馏残余物的油性提取物。因此可以使用超临界状态的二氧化碳。本领域技术人员知道如何根据期望的提取速度和产量来调节提取参数，特别是温度、压力和流体流速。例如，使用超临界CO2的提取可以在250至300巴的压力和40至80℃，优选50至70℃的温度下进行。在提取之后，超临界流体通过减压返回到气态并且通常再循环。由此获得的植物提取物有利地经受分子蒸馏步骤，例如在180-250℃，优选200-220℃的温度下，以便浓缩提取物中存在的不皂化物质。在该分子蒸馏步骤之前，植物提取物可以任选地在油性溶剂中稀释，特别是基于植物油如葵花油的油性溶剂。在分子蒸馏步骤期间除去该油性溶剂。
根据优选的实施方案，根据本发明使用的永生花蒸馏残余物的油性提取物是根据包括以下步骤的方法获得的：用超临界流体、优选在超临界状态下的二氧化碳提取蒸馏残余物的步骤，任选的随后的分子蒸馏步骤。
因此获得的馏出物特别包含30重量％至50重量％，更特别地35重量％至45重量％的不皂化物质和1重量％至10重量％，更特别地3重量％至7重量％的甾醇，基本上β-谷甾醇。
使用超临界流体或由分子蒸馏步骤产生的馏出物获得的提取物可以任选地在油性溶剂中稀释到1重量％至10重量％的浓度，例如，特别是3重量％至5重量％，以便于处理。适用于美容用途的任何油性溶剂可用于该目的，特别是脂肪酸甘油三酯，例如辛酸/癸酸甘油三酯。
申请人通过耦合到质谱法的气相色谱法表征永生花蒸馏残余物的油性提取物。因此证明，如通过气相色谱法测定的，永生花蒸馏残余物的油性提取物含有小于10％的挥发性馏分。
还显示，根据本发明使用的永生花蒸馏残余物的油性提取物在其组成方面与通过超临界CO2获得的永生花开花顶部的提取物的区别在于其基本上不含有(即小于10重量％，或甚至小于5重量％，或甚至小于2重量％)(如果有的话)棕榈油酸、芳樟醇、橙花醇、2-癸烯醛、2-十三烷酮、十七烷、8-十七烯、异丁酸、异戊酸、十六碳烯醇、月桂酸、十五烷酸、癸酸和异硬脂酸。更一般地，该提取物不包含含有超过10个碳原子的饱和脂肪酸。相反，根据本发明使用的永生花蒸馏残余物的油性提取物含有尚未在通过超临界CO2获得的永生花开花顶部的提取物中识别的成分，即2(Z)-癸烯酸和4(Z)-癸烯酸。
此外，与永生花精油相比，根据本发明使用的永生花蒸馏残余物的油性提取物含有甾醇、基本上β-谷甾醇、直链羧酸、C7-C10直链饱和或不饱和羧酸(特别是庚酸、辛烯酸和癸烯酸)、苯甲酸和C8-C18直链饱和或不饱和脂肪酸的乙酯(特别是辛酸酯、癸酸酯和亚油酸酯)。
它还不同于通过使用葵花油进行提取而获得的永生花油性提取物，不同之处在于它不含有任何柠檬烯并含有不存在于上述油性提取物中的脂肪酸和/或其乙酯(庚酸、2-辛烯酸、4-辛烯酸、壬酸、2-癸烯酸、4-癸烯酸、亚油酸)和苯甲酸。
最后，根据本发明使用的永生花蒸馏残余物的油性提取物多酚少，并且特别是不含二咖啡酰奎尼酸和查耳酮。
相对于根据本发明的组合物的总重量，永生花蒸馏残余物的油性提取物有利地占0.01％至1％。
根据本发明的永生花蒸馏残余物的油性提取物包含在美容组合物中，所述美容组合物含有生理上可接受的介质，特别是美容上可接受的介质，即不引起与美容用途不相容的任何刺痛或发红的介质。该介质优选含有水相和/或脂肪相。优选地，所述组合物为乳液形式，特别是水包油乳液或油包水乳液，或无水油性组合物。术语“无水组合物”是指包含小于5重量％的水、优选小于2重量％的水或甚至根本不包含水的组合物。
当存在时，水相包含水和任选的选自多元醇和水性胶凝剂的至少一种组分。有利地，水占组合物总重量的40％至80％，例如50％至70％。多元醇可以特别地选自甘油、丙二醇、丁二醇、戊二醇及其混合物，并且可以占组合物总重量的5％至30％，优选15％至25％。
术语“水性胶凝剂”表示能够通过水合来固定水分子并因此增加水相粘度的聚合物化合物。此类胶凝剂可选自：多糖，例如：纤维素及其衍生物、改性淀粉、角叉菜胶、琼脂、黄原胶和植物胶，例如瓜尔胶或刺槐豆胶；合成聚合物，特别是任选交联的丙烯酸钠均聚物，以及丙烯酸类共聚物，特别是丙烯酸钠和/或(甲基)丙烯酸烷基酯和/或(甲基)丙烯酸羟烷基酯和/或(甲基)丙烯酸(聚乙氧基)烷基酯共聚物，任选地具有至少一种其它单体，有利地为2-丙烯酰胺基-2-甲基丙磺酸(AMPS)，这些共聚物任选地交联；及其混合物。
对于其部分，当存在时，脂肪相可以包含一种或多种挥发性和/或非挥发性油。挥发性油的实例是支链烷烃，例如异十二烷，和直链C10-C13烷烃。可特别提及的非挥发性油包括：
-酸和一元醇酯，选自：饱和线性C2-C10(优选C6-C10)酸和饱和线性C10-C18(优选C10-C14)一元醇的单酯和聚酯，饱和线性C10-C20酸和支链或不饱和C3-C20(优选C3-C10)一元醇的单酯和聚酯；支链或不饱和C5-C20酸和支链或不饱和C5-C20一元醇的单酯和聚酯；支链或不饱和C5-C20酸和直链C2-C4一元醇的单酯和聚酯；
-C6-C12脂肪酸的甘油三酯，例如辛酸和癸酸甘油三酯和三庚酸甘油酯；
-支链和/或不饱和C10-C20脂肪酸(如油酸和亚油酸)；
-支链和/或不饱和C10-C20脂肪醇(例如辛基十二烷醇和油醇)；
-烃，例如角鲨烷(C30)，特别是从橄榄油中提取的植物角鲨烷，和半角鲨烷(C15)；
-碳酸二烷基酯，例如碳酸二辛酯和碳酸二乙基己酯；
-二烷基醚，例如二辛基醚；和
-其混合物。
还可提及含有一种或多种上述成分的植物油。
作为酸和一元醇的酯，特别可以提及单酯，例如椰油酰癸酸酯/辛酸酯混合物、马来酸乙酯、乳木果油乙酯、异硬脂酸异硬脂酯、异壬酸异壬酯、异壬酸乙基己酯、新戊酸己酯、新戊酸乙基己酯、新戊酸异硬脂酯、新戊酸异癸酯、肉豆蔻酸异丙酯、肉豆蔻酸辛基十二烷酯、棕榈酸异丙酯、棕榈酸乙基己酯、月桂酸己酯、月桂酸异戊酯、壬酸鲸蜡硬脂酯、辛酸丙基庚酯及其混合物。可使用的其它酯包括酸和一元醇的二酯，例如己二酸二异丙酯、己二酸二乙基己酯、癸二酸二异丙酯和癸二酸二异戊酯。
植物油的实例特别是小麦胚芽油、葵花油、阿甘油、木槿油、芫荽油、葡萄籽油、芝麻油、玉米油、杏仁油、蓖麻油、乳木果油、鳄梨油、橄榄油、大豆油、甜杏仁油、棕榈油、菜籽油、棉籽油、榛子油、澳洲坚果油、霍霍巴油、苜蓿油、御米油、南瓜油、乳南瓜油、黑加仑油、月见草油、薰衣草油、琉璃苣油、小米油、大麦油、藜麦油、黑麦油、红花油、烛果油、西番莲油、玫瑰果油、蓝蓟油、亚麻籽油和山茶油。
脂肪相还可以包含至少一种脂肪相结构化剂。术语“脂肪相结构化剂”是指能够增稠组合物中包含的油的化合物，特别是选自蜡、脂肪相胶凝剂和糊状脂肪物质，及其混合物。
根据一个优选的实施方式，该组合物还含有除了永生花蒸馏残余物的油性提取物之外的至少一种脱色素活性剂，其可以特别选自：植物提取物，特别是甘草提取物；维生素C及其衍生物，特别是抗坏血酸、抗坏血酸葡糖苷、抗坏血酸乙基醚、抗坏血酸四异棕榈酸酯和抗坏血酸磷酸酯镁；熊果苷；阿魏酸；曲酸；间苯二酚及其衍生物，例如己基间苯二酚和4-(1-苯乙基)-1,3-苯二酚，特别是由Symrise公司以商品名Symwhite 销售的；水杨酸、其盐和其酯；绣线菊和鸢尾草花(Iris florentina)提取物；甘草酸二钾；氨甲环酸；鞣花酸；烟酸衍生物，例如烟酰胺；由Gattefossé销售的猕猴桃(Actinidia chinensis)提取物；由Ichimaru Pharcos以商品名Liquid销售的牡丹根(Paeonia suffruticosa)提取物；由SEPPIC以商品名销售的脂氨基酸(用十一碳烯酸改性的苯丙氨酸)；意大利蜡菊精油；及其混合物。
在本发明的优选实施方案中，相对于组合物的总重量，美容组合物在生理上可接受的介质中包含0.01重量％至1重量％，优选0.05重量％至1重量％，更优选0.05重量％至0.3重量％的如前述定义的永生花蒸馏残余物的油性提取物和0.001重量％至5重量％的己基间苯二酚。
己基间苯二酚或4-己基-1,3-苯二酚指以下化学结构的化合物：
已知己基间苯二酚的脱色素和护肤性质。优选地，引入本发明美容组合物中的己基间苯二酚是纯化的并几乎不含间苯二酚(后一种化合物不稳定并且对皮肤具有刺激性作用)。为此目的，可以使用经由在Sythéon专利EP 2152 685中描述的方法获得的产品，或者直接使用已知包含超过99.5％己基间苯二酚的商业产品HR，以抑制体外黑色素产生(Funasaka A.H.等，J.Lipid Res.1999)和在人志愿者皮肤上具有漂白活性(EP 2152685)。
如下文实施例所示，根据本发明的永生花蒸馏残余物的油性提取物已显示为增强己基间苯二酚的脱色素效果。
作为变体或另外，该美容组合物可包含选自以下的至少一种活性剂：自由基清除剂、保湿剂、用于刺激角质形成细胞和/或成纤维细胞的分化和/或增殖的试剂；用于刺激糖胺聚糖和/或胶原蛋白和/或真皮表皮锚定原纤和/或弹性纤维的合成的试剂；用于防止胶原蛋白和/或糖胺聚糖和/或真皮表皮锚定原纤和/或弹性纤维降解的试剂；抗糖化剂；及其混合物。
这样的抗衰老活性剂的实例特别是：抗坏血酸，及其盐、醚和酯，特别是抗坏血酸葡糖苷；腺苷；核糖；蜂蜜提取物；蛋白质和糖蛋白，特别是从甜杏仁中提取的；水解的植物蛋白质，特别是衍生自大米、木槿种子或羽扇豆；多肽和伪二肽，例如脱羧肌肽盐酸盐、分别以商品名3000和Synthe'6销售的棕榈酰五肽-4(Pal-Lys-Thr-Thr-Lys-Ser)和棕榈酰三肽-38，以及由Lucas Meyer公司以商品名销售的棕榈酰三肽-8，由Lipotec公司以商品名Solution销售的五肽-18，由Sandream公司以商品名和由Infinitec公司以商品名X50 Powder销售的棕榈酰六肽-52；硅烷，例如甲基硅烷醇甘露糖醛酸；特别是从黑麦面粉中提取的阿拉伯木聚糖和特别是来自落叶松的阿拉伯半乳聚糖；透明质酸及其盐；多酚，特别是从含羞草中提取的；α-羟基酸，包括从柠檬提取的那些和由Naturex以商品名Hibiscus 销售的那些；植物的提取物(通常是水性的)，所述植物例如水苜蓿、野生三色堇、马尾草、马齿苋(Acmellaoleracea)、大翅蓟(Onopordum acanthium)、蓍(Achillea millefolium，特别包含在来自BASF公司的产品中)、酸藤子(Embelia concinna，由SEPPIC公司销售)、刺梨(Opuntia ficus indica，特别由Mibelle AG Biochemistry以商品名销售)、鼠尾草(Salvia officinalis，特别由Provital Group销售)、Vitex negundo(由Laboratoires Expanscience以商品名销售)、栗子、番木瓜、阿甘、燕麦、葵花、雏菊、牡丹或莳萝；水性藻类提取物，特别是珊瑚、细珠珊瑚草(slender-beaded coralweed)、裙带菜(Ungaria pinnatifada)、翅藻(Alaria esculenta)或微绿球藻(Nannochlorosis oculata)；精油，特别是桃金娘精油；锌和/或铜葡糖酸盐；及其混合物。
根据本发明的组合物还可含有可分散在脂肪相中和/或在水相中的各种成分，只要这些成分与局部施用于皮肤相容即可。
因此，它可以含有至少一种通常非离子的水包油或油包水乳化剂，例如聚氧乙烯酯、任选的聚乙氧基化脱水山梨糖醇酯、任选的甘油的聚乙氧基化脂肪酸酯、糖的脂肪醇醚，例如烷基葡糖苷，及其混合物。乳化剂可以占组合物总重量的2％至10％，优选4％至6％。
根据本发明的组合物还可包含一种或多种粉状填料，其有利地为多孔或中空的，优选多孔的微粒形式。这些微粒原则上是基本上球形的。这些填料可以特别选自：
-有机填料，例如：多糖特别是天然淀粉、改性淀粉或纤维素的粉末；丙烯酸类聚合物例如聚(甲基丙烯酸甲酯)、聚酰胺或聚烯烃的粉末；干燥的藻类例如珊瑚藻(Corallinaofficinalis)的粉末；
-矿物填料如二氧化硅、粘土、珍珠岩和滑石；
-及其混合物。
二氧化硅优选用作矿物填料。
这些填料可以占组合物总重量的1重量％至5重量％。
根据本发明的组合物还可以包含特别选自有机和/或无机光保护剂的添加剂，其在蓝光和/或UVA和/或UVB中是活性的；基于多糖的成膜聚合物，其能够形成防污保护膜，例如由Solabia以商品名和出售的产品；香料；抗氧化剂；多价螯合剂；pH调节剂；防腐剂；色素；着色剂；及其混合物。
该组合物可以是适于局部施用于皮肤的任何形式，特别是乳、乳膏、流体、洗剂、凝胶、糊剂、油或薄膜的形式。它通常是免洗型组合物，特别是护肤组合物。
作为变体，根据本发明的组合物可以是用于护肤的洗去组合物，特别是用于面部和可能的身体。在这种情况下，其可以用作例如面膜或去角质糊剂。
根据本发明的组合物可以施用于具有由环境因素介导的皮肤颜色不均匀性、特别是色素斑、黄褐斑和/或由于疤痕或痤疮痕迹引起的色素沉着缺陷的人的皮肤。其通常被施用于相关的身体的至少一个区域，并且更具体地，被施用于面部、领口、颈部、胳膊、手和/或腿的皮肤，期望提亮这些区域和/或统一皮肤颜色。该组合物可以一天一次或多次施用，例如早晨和/或晚上。
在任何情况下，以足以抑制黑色素生成的量向皮肤施用永生花蒸馏残余物的油性提取物。例如，可将0.5至5mg/cm2的量的美容组合物每日施用于相关的皮肤区域，所述美容组合物含有0.01重量％至1重量％的根据本发明的永生花蒸馏残余物的油性提取物。
附图说明
图1说明了与两种阳性对照相比，根据本发明的提取物对小鼠B16黑素细胞的黑色素产生的抑制效果。
图2说明了与两种阳性对照相比，意大利蜡菊的油性提取物对小鼠B16黑素细胞的黑色素产生的抑制效果。
图3说明了与这两种活性剂中的每一种以及两种阳性对照相比，根据本发明的提取物与己基间苯二酚的组合对小鼠B16黑素细胞的黑色素产生的抑制效果。
实施例
在以下实施例的基础上将更清楚地理解本发明，这些实施例仅通过说明的方式给出，并不旨在限制由权利要求书限定的本发明的范围。
实施例1：提取物的制备和表征-与开花顶部的油性提取物的比较
1.1-提取物的制备
由意大利蜡菊的开花顶部的水蒸馏产生了精油和水溶胶，随后在60℃的温度和285巴的压力下使用超临界CO2分离和提取水蒸馏残余物(蒸馏残余物)。将由此获得的不皂化物质在葵花油中稀释，然后在200℃下进行分子蒸馏的步骤，并且通过将所得馏出物在辛酸/癸酸甘油三酯中稀释至3-5重量％而标准化。
将该提取物的多酚组合物与如下获得的意大利蜡菊的油性提取物的多酚组合物进行比较：将9重量％的干燥和磨碎的植物粉末(开花顶部)与91％的超声除臭的葵花油在冷却至10℃的容器中混合。在超声下，以500rpm搅拌进行提取30分钟。在0.4L/min氮气流下微波继续提取。通过倾析分离获得的固体和液体油性相，然后将油性相过滤并储存在惰性气氛下，避光。
1.2-多酚的表征
材料
-超高性能液相色谱仪(Agilent Technologies，USA)
-1290串联自动注射器
-1260串联二极管阵列检测器(DAD)
-质谱仪：配备有电喷电离(ESI)源的Equire 6000(Brüker Daltonics，Bremen)。
样品的制备
通过将2g蒸馏残余物提取物溶解在1mL己烷中制备根据本发明的提取物。将该油性溶液用2mL MeOH/H2O混合物(60/40)萃取三次。然后将MeOH/H2O相合并并在MeOH/H2O混合物(60/40)中稀释1/2。
分析方法
将稀释/萃取后得到的溶液通过PTFE过滤器(0.45μm)过滤，然后注射(1μL)到Agilent C18柱上(2.1mm x 100mm；1.8μm)，温度保持在25℃，流速为0.4mL/min。所用溶剂为：A＝H2O/HCOOH(0.1％)和B＝ACN/HCOOH(0.1％)。多酚洗脱梯度如下：
[表1]
在离开二极管阵列检测器后，将洗脱液注入质谱仪。分析以负或正模式进行。
在100至1400的整个质量范围(m/z)上采集LC-MS谱。然后使用Hystar版本3.0软件收集和处理所有数据。
结果
[表2]
n.d：未检测到；<LQ：检测到但低于定量限制
1.3-查耳酮的表征
上文所述的蒸馏残余物提取物中四种查耳酮(linderatin、linderachalcone、甲基-linderatin、gymnochalcone)的存在通过使用具有1H/13C HSQC光谱的2D NMR法来评估。
材料和方法：将蒸馏残余物提取物的等分试样(15mg)溶解在700μL DMSO-d6中，以便在298Kelvin下通过2D NMR分析。
使用配备有NMR探头的Brüker Avance AVIII-600光谱仪进行2DNMR分析。获得的NMR值与使用ACD Labs软件计算的NMR值进行比较。
结果：样品中未检测到查耳酮。
1.4-其它组分的表征
平行地，在上述两种提取物中评价各种有机化合物的存在，并且还在以与根据本发明的提取物相似的方式获得但是使用对永生花开花顶部而不在它们的水蒸馏残余物上进行的工艺的提取物中评价各种有机化合物的存在。
材料
-微量GC超气体色谱仪(ThermoElectron SAS)
-质谱仪：在E1模式中使用的El LT Large T ISQ质谱仪(ThermoElectron SAS)
样品的制备
通过在6mL异辛烷中稀释0.3g提取物制备样品。加入0.6mL甲醇氢氧化钾，混合物涡旋30秒。
加入1g NaHSO4，混合物涡旋30秒。
然后使混合物通过沉降分离。
取上层清液进行色谱分析。
分析方法
在以下条件下分析样品：
TR WAX 60m x 0.25μm x 0.25mm极性色谱柱
H2流速：1mL/min
烘箱程序：50℃(2分钟)，然后10℃/分钟直至250℃(18分钟)
分离比：100
注射体积：1μL
注射器温度：250℃
质量范围：50-650amu
结果
值得注意的是，根据本发明的永生花蒸馏残余物的提取物与开花顶部的油性提取物的不同之处在于，其不含有柠檬烯，含有显著更多的β-桉叶醇并且含有脂肪酸和/或其乙酯(庚酸、2-辛烯酸、4-辛烯酸、壬酸、2-癸烯酸、4-癸烯酸和亚油酸)和苯甲酸，它们不存在于永生花开花顶部的油性提取物中。
此外，根据本发明的蒸馏残余物提取物明显不同于开花顶部的超临界CO2提取物，因为其不包含含有超过10个碳原子的饱和脂肪酸，并且不同之处在于它含有少得多的(如果有的话)萜类化合物(特别是单萜和倍半萜)，特别是它不含橙花醇和芳樟醇，并且含有少得多的(如果有的话)重烷烃(含有超过16个碳原子)，并且不含棕榈油酸、异丁酸和异戊酸、2-癸烯醛和十六碳烯醇。此外，根据本发明的蒸馏残余物提取物含有不存在于开花顶部的超临界CO2提取物中的特征分子，即(2)-(Z)-癸烯酸和4-(Z)-癸烯酸。
实施例2：根据本发明的永生花蒸馏残余物提取物对黑色素生成的影响
测试实施例1中所述的永生花蒸馏残余物提取物和开花顶部的油性提取物，以评估它们抑制黑色素产生的能力。
2,1-材料和方法
将黑素细胞(小鼠B16细胞系)在含有10％胎牛血清、2mM谷氨酰胺、1mM丙酮酸钠和100μg/ml降粘蛋白的无酚红DMEM培养基中以2000细胞/孔接种于96孔板中。第二天，用0.1μM的NDP-MSH处理细胞以诱导色素沉着。在测试条件下，在NDP-MSH存在下，将3mM的曲酸和0.5％的维生素C(抗坏血酸葡萄糖苷)用作色素沉着抑制的阳性对照，并以几种浓度测试待评估的提取物。所有条件均三份测试。在提取物和阳性对照存在下，将细胞在37℃、5％CO2下孵育72h。然后使用XTT方法评估细胞活力，并且仅分析黑色素量的非细胞毒性条件(其活力大于未处理对照的活力的85％)。
对于黑色素测定，抽吸各孔中的培养基，并通过向细胞群中加入50μl 1M NaOH来裂解细胞。将板在室温下摇动30分钟，然后将裂解液转移到玻璃烧瓶中并在95℃下孵育过夜。孵育后，将裂解物以2000rpm离心以除去细胞碎片。在405nm处读取上清液的吸光度，反映每个孔中的黑色素的量，并且针对每种条件确定三次重复的平均值。未用NDP-MSH处理的细胞的孔用作对照，并且其OD值从其它孔的所有OD值中减去。然后根据下式计算黑色素抑制的百分比：
100-((测试条件中的黑色素量/未处理对照中的黑色素量)x 100)
使用单向Anova检验(对于蒸馏残余物提取物和开花顶部的油性提取物)或Student′s t检验(对于曲酸和维生素C)评估结果的显著性。
2.2-结果
如图1和2所示，通过证明曲酸和维生素C的功效(***p<0.001；****p<0.0001)验证测试。
图1还示出根据本发明的永生花蒸馏残余物提取物在浓度为0.05％或以上(0.1％，****p<0.0001；0.05％，*p<0.05)时以剂量依赖性方式显著抑制黑素细胞色素沉着。在0.1％的浓度下，根据本发明的蒸馏残余物提取物具有比最高浓度的维生素C高得多的功效，维生素C构成化妆品中常规使用的漂白活性剂。其功效接近作为基准脱色素剂的曲酸的功效，但是已知曲酸牵涉严重的过敏和刺激风险。
因此，本实施例证明根据本发明的永生花蒸馏残余物提取物构成了用于皮肤提亮的美容活性剂的选择。
相比之下，图2示出了永生花开花顶部的油性提取物对色素沉着的效果与对照的效果不显著不同。
实施例3：己基间苯二酚和根据本发明的永生花蒸馏残余物提取物的组合对黑色素生成的影响
重复实施例2中描述的测试，将己基间苯二酚添加到根据本发明的永生花蒸馏残余物提取物中。初步的细胞毒性研究定义了可以施用于细胞的最大非细胞毒性剂量，即10μg/mL的己基间苯二酚和1％的根据本发明的永生花蒸馏残余物提取物。将这两种活性剂以0.035％永生花蒸馏残余物提取物和5μg/mL的己基间苯二酚的浓度进行测试。
将结果与单独使用根据本发明的提取物和单独使用的己基间苯二酚获得的效果进行比较，以相同剂量使用。
如图3所示，通过证实曲酸和维生素C(****p<0.0001)的功效并证实己基间苯二酚的脱色素功效来验证测试。
此外，所测试的组合比单独使用两种活性剂时更强烈地抑制色素沉着。该功效不仅与未处理的对照物显著不同，而且与以相同的浓度单独使用的每种活性剂显著不同(“单向Anova检验”，$$p<0.01对比蒸馏残余物提取物；Δp<0.05对比己基间苯二酚)。
因此，该测试证明，己基间苯二酚对黑色素生成的效果通过添加根据本发明的提取物而显著增强，这是出乎预料的。
实施例4：美容组合物
通过以所指示的重量比例混合以下成分，以本领域技术人员常规方式制备以下组合物。
护手霜
面油和身体油油
Claims (17)
1.局部施用于皮肤的意大利蜡菊(Helichrysum italicum)物种的永生花蒸馏残余物的油性提取物作为用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的试剂的美容用途。
2.根据权利要求1所述的用途，其特征在于，如通过气相色谱法测定的，所述永生花蒸馏残余物的油性提取物含有小于10％的挥发性馏分。
3.根据权利要求1或2所述的用途，其特征在于，所述永生花蒸馏残余物的油性提取物含有直链饱和或不饱和的C7-C10羧酸，包括2-(Z)-癸烯酸和4-(Z)-癸烯酸；和直链饱和或不饱和C8-C18脂肪酸的乙酯。
4.根据权利要求3所述的用途，其特征在于，所述直链饱和或不饱和的C7-C10羧酸包括庚酸、辛烯酸和癸烯酸；并且所述直链饱和或不饱和C8-C18脂肪酸的乙酯包括辛酸乙酯、癸酸乙酯和亚油酸乙酯。
5.根据权利要求1至4中任一项所述的用途，其特征在于，所述永生花蒸馏残余物的油性提取物含有甾醇。
6.根据权利要求5所述的用途，其特征在于，所述甾醇主要由β-谷甾醇组成。
7.根据权利要求1至6中任一项所述的用途，其特征在于，所述意大利蜡菊物种的永生花为科西嘉岛来源。
8.根据权利要求1至7中任一项所述的用途，其特征在于，根据包括以下步骤的方法获得所述永生花蒸馏残余物的油性提取物：用超临界流体、优选在超临界状态下的二氧化碳提取所述蒸馏残余物的步骤，任选的随后的分子蒸馏步骤。
9.根据权利要求8所述的用途，其特征在于，通过提取和任选的分子蒸馏获得的所述永生花蒸馏残余物的油性提取物在油性溶剂如辛酸/癸酸甘油三酯中稀释。
10.根据权利要求8所述的用途，其特征在于，根据包括以下步骤的方法获得所述永生花蒸馏残余物：对所有或部分植物、优选所述植物的地上部分、特别是其花或开花顶部进行水蒸馏或蒸汽蒸馏的步骤，随后回收蒸馏或蒸汽蒸馏残余物的步骤。
11.根据权利要求1至10中任一项所述的用途，其特征在于，所述永生花蒸馏残余物的油性提取物施用于面部、领口、颈部、胳膊、手和/或腿的皮肤。
12.根据权利要求1至11中任一项所述的用途，其特征在于，所述永生花蒸馏残余物的油性提取物以足以抑制黑色素生成的量施用于皮肤。
13.根据权利要求1至12中任一项所述的用途，其特征在于，所述永生花蒸馏残余物的油性提取物施用于具有由环境因素介导的皮肤颜色不均匀性、特别是色素斑、黄褐斑和/或由于疤痕或痤疮痕迹引起的色素沉着缺陷的人的皮肤。
14.一种美容组合物，其相对于所述组合物的总重量，在生理上可接受的介质中包含0.01重量％至1重量％的如权利要求1至10中任一项所定义的永生花蒸馏残余物的油性提取物，和0.001重量％至5重量％的己基间苯二酚。
15.根据权利要求14所述的组合物用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的美容用途，包括将所述组合物局部施用于皮肤。
16.一种用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的试剂，所述试剂由意大利蜡菊物种的永生花蒸馏残余物的油性提取物组成。
17.一种用于漂白皮肤和/或提亮肤色和/或用于均质化皮肤颜色和/或减少或防止色素斑出现的美容方法，包括将根据权利要求14所述的组合物局部施用于皮肤。
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