CN110882189A - 小花风车子提取物在化妆品中的用途 - Google Patents
小花风车子提取物在化妆品中的用途 Download PDFInfo
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- CN110882189A CN110882189A CN201811052460.3A CN201811052460A CN110882189A CN 110882189 A CN110882189 A CN 110882189A CN 201811052460 A CN201811052460 A CN 201811052460A CN 110882189 A CN110882189 A CN 110882189A
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- Prior art keywords
- extract
- graptopetalum
- cosmetic composition
- pharmaceutical
- repair
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Abstract
本发明提供了小花风车子提取物在化妆品中的用途。具体地，本发明采用溶剂提取法、溶剂萃取法、色谱法等工艺，发现小花风车子植物或其提取物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用。本发明制成的小花风车子提取物可作为化妆品的活性成分添加入化妆品中。所述含有小花风车子提取物的化妆品或药物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等功效，从而达到护肤美容的效果。
Description
技术领域
本发明涉及化妆品和医疗领域，具体涉及小花风车子提取物在化妆品中的用途。
背景技术
化妆品是现代人们生活的巨大需求。化妆品是指以涂敷、揉擦、喷洒等不同方式，涂加在人体皮肤、毛发、口唇等处，起清洁、保护、美化、促进身心愉快等作用的日用化学工业产品。随着社会的进步，人们对于化学合成类化妆品的副作用有了更深的认识，于是人们更加倾向于选择安全、天然的动植物源的天然化妆品。
天然化妆品指的是用天然植物萃取物提炼的成分研制而成的化妆品。但目前市场上的含天然植物萃取物的化妆品效果并不明显。
因此，本领域亟需开发新的安全、能有效护理皮肤的天然化妆品。
发明内容
本发明的目的是提供一种新的安全、能有效护理皮肤的天然化妆品。
本发明的又一目的是提供一种小花风车子提取物在药物或化妆品中的新用途。
本发明第一方面，提供了一种小花风车子植物、或其中药药材、或小花风车子提取物的用途，其特征在于，用于制备药物或化妆品组合物，并且所述药物或化妆品组合物用于：保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化、或其组合。
在另一优选例中，所述药物或化妆品组合物用于抗衰老和/或抗氧化。
在另一优选例中，所述的小花风车子提取物包括小花风车子植物或其同属植物的枝、叶、根、花、果和/或茎的水溶性的和/或脂溶性的提取物。
在另一优选例中，所述的小花风车子提取物包括水提取物、水性溶剂提取物、醇提取物、或其组合。
在另一优选例中，所述的小花风车子提取物为叶的提取物。
在另一优选例中，所述的小花风车子提取物为小花风车子植物或其同属植物的叶的水溶性提取物。
在另一优选例中，所述的提取物含有一种或多种选自下组的组分：多糖(如水溶性多糖)、植物多酚、多酚糖苷类、或其组合。
在另一优选例中，所述的提取物是通过溶剂提取法、萃取法、和/或色谱法而获得的。
在另一优选例中，所述提取物的提取剂选自下组：水、醇类(优选C1-C4醇，如甲醇、乙醇、丙醇)、水性溶剂、或其混合物。
在另一优选例中，所述提取物是通过本发明提取方法提取得到的。
在另一优选例中，所述药物或化妆品组合物还包括选自下组的额外组分：美白或祛斑成分、抗炎成分、抗氧化成分、抗紫外线成分、或其组合。
在另一优选例中，所述药物组合物包括：粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中，所述化妆品组合物的剂型为固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
本发明第二方面，提供了一种可用于制备药物或化妆品组合物的有效部位，其特征在于，所述的有效部位具有以下特征：
(a)该有效部位是提取自小花风车子的枝、叶、根、花、果和/或茎的水溶性、脂溶性、和/或醇提取物；
(b)该有效部位具有选自下组的功能：保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化、或其组合。
在另一优选例中，所述有效部位用于抗衰老和/或抗氧化。
在另一优选例中，所述有效部位含有一种或多种选自下组的组分：多糖(如水溶性多糖)、植物多酚、多酚糖苷类、或其组合。
在另一优选例中，所述有效部位是通过溶剂提取法、萃取法、和/或色谱法而获得的。
在另一优选例中，所述有效部位的提取剂选自下组：水、醇类(优选C1-C4醇，如甲醇、乙醇、丙醇)、水性溶剂、或其混合物。
在另一优选例中，所述有效部位是通过本发明提取方法提取得到的。
本发明第三方面，提供了一种药物或化妆品组合物，其特征在于，含有(a)本发明第二方面所述的有效部位；和(b)药学上或化妆品学上可接受的载体或赋形剂。
在另一优选例中，所述药物或化妆品组合物用于：保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化、或其组合。
在另一优选例中，所述药物或化妆品组合物用于抗衰老和/或抗氧化。
在另一优选例中，在所述化妆品组合物中，(干燥)有效部位的质量百分比为0.0001-10wt％，较佳地0.001-5wt％，以化妆品组合物的总重量计。
在另一优选例中，在所述药物组合物中，(干燥)有效部位的质量百分比为0.001-50wt％，较佳地0.01-20wt％，以药物组合物的总重量计。
在另一优选例中，所述药物或化妆品组合物还包括选自下组的额外组分：美白或祛斑成分、抗炎成分、抗氧化成分、抗紫外线成分、或其组合。
在另一优选例中，所述化妆品学上可接受的载体或赋形剂选自下组：保湿剂、抗氧化剂、抗紫外线剂、防腐剂、成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、抗菌剂、香精、盐类、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分、维生素、氨基酸、核酸、激素、包合化合物或其组合。
在另一优选例中，所述药物组合物包括：粉剂、颗粒剂、胶囊剂、注射剂、酊剂、口服液、片剂或含片。
在另一优选例中，所述化妆品组合物的剂型为固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
在另一优选例中，所述化妆品组合物用于护肤美容。
在另一优选例中，所述化妆品组合物含有本发明第二方面所述的有效部位(小花风车子提取物)作为有效成分(或活性成分)。
本发明第四方面，提供了一种制备药物或化妆品组合物的方法，其特征在于，包括步骤：将本发明第二方面所述的有效部位与药学上或化妆品学上可接受的载体混合，从而形成药物或化妆品组合物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了实施例1中1.7方法制备的提取物的HPLC图谱。
图2显示了小花风车子水提取物减少了UVA诱导的活性氧(ROS)的生成。
图3显示了小花风车子水提取物可以抵抗UVA诱导的硝基酪氨酸的增加。
具体实施方式
本发明人经过广泛而深入的研究，首次意外发现小花风车子植物、或其中药材、或小花风车子提取物在药物或化妆品(如护肤品)中的用途。具体地，本发明采用溶剂提取法、溶剂萃取法、色谱法等工艺，发现小花风车子植物或其提取物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用。本发明制成的小花风车子提取物可作为化妆品的活性成分添加入化妆品中。所述含有小花风车子提取物的化妆品或药物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等功效，从而达到护肤美容的效果。在此基础上完成了本发明。
小花风车子
小花风车子，Combretum micranthum(又称kinkeliba)，是一种在西非虎丛林地区发现的无驯化的灌木物种。它是一种浓密的灌木或藤蔓，常见于耕地和休耕地，主要位于撒哈拉以南非洲，塞内加尔，马里和布基纳法索的产量较高。在几个热带西非大草原国家中，人们会从野生种群的小花风车子获取它的叶子用作流行的传统草本茶。小花风车子作为灌木茶，具有令人愉快的味道，茶水浅至深绿褐色。该茶也被当地用作一种传统的万能药具有利尿和帮助消化问题的功能，如肠胃问题，绞痛和呕吐。
以前的植物化学研究中发现小花风车子茶叶的提取物中富含黄酮类化合物包括牡荆素，异牡荆素，荭草素(orientin)，异荭草素(homoorientin)，杨梅素-3-O-葡萄糖苷和杨梅素-3-O-芸香糖苷；生物碱包括水苏碱，羟基水苏碱和胆碱；和糖醇，包括间肌醇和山梨糖醇；和黄烷类生物碱，包括kinkeloids A，B，C和D。有报道显示小花风车子茶叶的乙酸乙酯提取物中所富含的黄酮类化合物具有降血糖活性。
本发明首次发现小花风车子植物或其提取物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用。本发明制成的小花风车子提取物可作为化妆品的有效成分添加入化妆品中。
有效部位
如本文所用，术语“本发明的提取物”和“本发明的有效部位”可互换使用，均指提取自小花风车子植物的，具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用的提取物。
在本发明中，本发明有效部位可用小花风车子植物为原料进行提取。
此外，虽然本发明有效部位可来自于小花风车子植物的全株，优选从小花风车子植物的地上部分如叶等部位提取。
如本文所用，术语“提取物”或“有效部位”包括水溶性的和/或脂溶性的提取物。该术语还包括醇提取物、或水提取物、或水性溶剂提取物。此外，还包括有效部位群，即含有脂溶性有效部位和水溶性有效部位的提取物或其混合物。
如本文所用，“水溶性提取物”指的是可溶于极性溶剂(优选地，水)的提取物。
在另一优选例中，本发明提取物是通过本发明提取方法提取得到的。
经分析，本发明有效部位所含的化学成分至少包括一种或多种选自下组的物质：多糖(如水溶性多糖)、植物多酚、多酚糖苷类、或其组合。本发明有效部位具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用。
在本发明的一个实施方式中，所述的提取物具有图1所示的HPLC图谱。
可用于制备本发明的小花风车子提取物的方法没有特别限制。可以用常规方法，以小花风车子植物为原料，获得水溶性的和/或脂溶性的提取物。
在本发明的一个优选例中，有效部位的制备通过溶剂提取法、萃取法、和/或色谱法进行。
在本发明中，提取次数可以是一次或多次(如2次、3次、4次、5次)。当多次提取时，可将多次的提取物合并后进行后续处理。
在本发明中，对于溶剂提取法，其所用的溶剂没有特别限制，代表性的例子包括(但并不限于)：水、乙醇、甲醇、水性溶剂中的一种或几种溶剂的混合溶剂。
在本发明中，水性溶剂指水与其他溶剂如醇(尤其是C1-C4醇，如乙醇、丙醇)所形成的混合溶剂。通常，在水性溶剂中，醇溶剂的含量为0.01-80wt％，较佳地5-60wt％，更佳地为10-30wt％，按水性溶剂的总重量计。
在本发明中，对于溶剂萃取法，其所用的溶剂没有特别限制，代表性的例子包括(但并不限于)：正丁醇、二氯甲烷、氯仿、C5-C7烷烃、环己烷、石油醚中的一种或几种溶剂的混合溶剂。萃取次数可以是一次或多次。
在本发明中，对于柱色谱法，其柱色谱没有特别限制，代表性的例子包括(但并不限于)：活性碳、硅胶、反相硅胶、大孔树脂、葡聚糖凝胶中的一种或几种的组合。
在本发明的一个优选例中，以小花风车子植物叶(干燥或新鲜的叶)为原料，以足量的水(如5-200倍重量的水)或水性溶剂提取1-5次，从而获得水性提取物。此外，本发明中，还可进一步地从水性提取物中分离(或去除)脂溶性成分，从而获得水溶性成分含量(或相对含量)更高的小花风车子提取物，或主要含水溶性成分的小花风车子提取物。
在本发明中，一种去除脂溶性成分的优选方法包括：用脂溶性溶剂(如乙酸乙酯、己烷等)对提取物进行萃取处理，经脂溶性溶剂萃取后剩余的水溶液为优选的有效部位。经测试，该有效部位具有显著更低或没有细胞毒性。
在本发明的一个实施方式中，所述提取方法包括以下步骤：
a.提供小花风车子，优选地小花风车子叶；
b.用提取剂提取所述小花风车子；
c.任选至少部分地除去提取剂以获取浓提取物；
d.任选去除提取物中脂溶性成分；
e.任选至少部分地除去提取剂以获取浓提取物；
f.任选干燥以获得提取物。
在另一优选例中，所述提取剂为水、乙醇、甲醇、丙醇、水性溶剂、或其混合物。
在另一优选例中，步骤b中所述提取是1次或多次提取。
在另一优选例中，所述提取在5-100℃(优选20-95℃)下进行。
在另一优选例中，所述干燥为喷雾干燥或真空干燥(如真空冷冻干燥)。
应用
如本文所用，术语“药物或化妆品组合物”包括(a)本发明所述的有效部位；和(b)药学上或化妆品学上可接受的载体或赋形剂。此外，所述药物组合物还包括保健品组合物，所述化妆品组合物包括护肤品。
有可能将本发明的小花风车子提取物制备成药物组合物，诸如片剂、胶囊、粉剂、微粒剂、溶液剂、锭剂、胶冻、乳膏制剂、醑剂、悬液、酊、泥敷剂、搽剂、洗剂、和气雾剂之类的剂型。药物能够由通常已知的制备技术来制备，并且合适的药物添加剂能够被添加到该药物中。
药物添加剂的例子包括赋形剂、粘结剂、分解剂、润滑剂、流动助剂、悬浮剂、乳化剂、稳定剂、保温(润湿)剂、防腐剂、溶剂、增溶剂、防腐剂、调味剂、增甜剂、染料、香料、推进剂等，这些药物添加剂可以进行选择并以在不影响本发明的效果的范围内的合适量添加。
有可能将本发明的小花风车子提取物制备成化妆品组合物，如乳剂、液体、膏剂、霜剂、糊剂、饼、粉剂之类的剂型。
在不防碍本发明的效果的范围内，本发明的化妆品中可以添加通常的化妆品中使用的其它成分，例如成膜剂、油溶性凝胶化剂、有机改性粘土矿物、树脂、保湿剂、防腐剂、抗菌剂、香精、盐类、抗氧化剂、pH调节剂、螯合剂、清凉剂、抗炎剂、皮肤美化用成分(美白剂、细胞活性剂、皮肤粗糙改善剂、血液循环促进剂、皮肤收敛剂、抗脂漏剂等)、维生素类、氨基酸类、核酸、激素、包合化合物等。
油溶性凝胶化剂是选自硬脂酸铝、硬脂酸镁、肉豆蔻酸锌等金属皂；N-月桂酰基-L-谷氨酸、α，γ-二-正丁基胺等氨基酸衍生物；环糊精棕榈酸酯、环糊精硬脂酸酯、环糊精2-乙基己酸棕榈酸酯等环糊精脂肪酸酯；蔗糖棕榈酸酯、蔗糖硬脂酸酯等蔗糖脂肪酸酯；一亚苄基山梨醇、二亚苄基山梨醇等山梨醇的亚苄基衍生物；二甲基苄基十二烷基铵蒙脱石粘土、二甲基二十八烷基铵蒙脱石粘土等有机改性粘土矿物等的凝胶化剂，可以根据需要使用一种，或者使用二种或以上。
保湿剂有：甘油、山梨醇、丙二醇、双丙甘醇、1，3-丁二醇、葡萄糖、木糖醇、麦芽糖醇、聚乙二醇、透明质酸、硫酸软骨素、吡咯烷酮羧酸盐、聚氧乙烯甲基葡糖苷、聚氧丙烯甲基葡糖苷等。
抗菌防腐剂有：对羟基苯甲酸烷基酯、苯甲酸、苯甲酸钠、山梨酸、山梨酸钾、苯氧基乙醇等，抗菌剂有：苯甲酸、水杨酸、石炭酸、山梨酸、对羟基苯甲酸烷基酯、对氯间甲酚、六氯酚、苯扎氯铵、氯化洗必泰、三氯-N-碳酰苯胺、三氯生、感光素、苯氧基乙醇等。
抗氧化剂有：生育酚、丁基羟基茴香醚、二丁基羟基甲苯、植酸等，pH调节剂有：乳酸、柠檬酸、乙醇酸、琥珀酸、酒石酸、dl-苹果酸、碳酸钾、碳酸氢钠、碳酸氢铵等，螯合剂有丙氨酸、乙二胺四乙酸钠盐、多磷酸钠、偏磷酸钠、磷酸等，清凉剂有：L-薄荷醇、樟脑等，抗炎剂有：尿囊素、甘草亭酸、甘草酸、凝血酸、甘葡环烃(Azulene)等。
皮肤美化用成分有：胎盘提取液、熊果苷、谷胱甘肽、虎耳草提取物等美白剂；蜂王浆、感光素、胆甾醇衍生物、小牛血液提取液等细胞活性剂；皮肤粗糙改善剂；壬酸缬草酰胺、烟酸苄酯、烟酸β-丁氧基乙酯、辣椒素、姜油酮、斑蝥酊、鱼石脂、咖啡因、鞣酸、α-冰片、烟酸生育酚、六烟酸肌醇脂、环扁桃酯、桂利嗪、妥拉唑啉、乙酰胆碱、维拉帕米、千金藤素、γ-谷维醇等血液循环促进剂；氧化锌、鞣酸等皮肤收敛剂；硫、等抗脂漏剂等，维生素类有：维生素A油、松香油、乙酸松香油、棕榈酸松香油等维生素A类；核黄素、丁酸核黄素、黄素腺嘌呤核苷酸等维生素B2类；吡多辛盐酸盐、吡多辛二辛酸酯、吡多辛三棕榈酸酯等维生素B6类，维生素B12及其衍生物，维生素B15及其衍生物等维生素B类；L-抗坏血酸、L-抗坏血酸二棕榈酸酯、L-抗坏血酸-2-硫酸钠、L-抗坏血酸磷酸二酯二钾等维生素C类；麦角钙化醇、胆钙化醇等维生素D类；α-生育酚、β-生育酚、γ-生育酚、乙酸dl-α-生育酚、烟酸dl-α-生育酚、琥珀酸dl-α-生育酚等维生素E类；维生素H；维生素P；烟酸、烟酸苄酯、烟酰胺等烟酸类；泛酸钙、D-泛醇、泛酰基乙基醚、乙酰基泛酰基乙基醚等泛酸类；生物素等。
氨基酸类有：甘氨酸、缬氨酸、亮氨酸、异亮氨酸、丝氨酸、苏氨酸、苯丙氨酸、精氨酸、赖氨酸、天冬氨酸、谷氨酸、胱氨酸、半胱氨酸、甲硫氨酸、色氨酸等，核酸有脱氧核糖核酸等，激素有雌二醇、乙烯基雌二醇等。
本发明的化妆品的优选例子包括：皮肤护理化妆品、彩妆化妆品、防紫外线化妆品。例如有乳液、霜、露、防晒剂、面膜材料、洗面奶、精华液等基础化妆品；粉底、白粉、腮红等彩妆化妆品等。
对于产品的形态没有特别限定，可以是液状、乳液状、霜状、固体状、糊状、凝胶状、粉末状、多层状、摩丝状(mousse)、喷雾状等。
本发明还提供了一种皮肤护理方法，所述方法包括步骤：对需要的个体施用本发明的所述的小花风车子提取物或本发明所述的有效部位。
在另一优选例中，所述小花风车子提取物的有效浓度范围为100μg/ml–500mg/ml。
在另一优选例中，所述方法为保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等方法。
本发明的主要优点包括：
(a)首次发现了小花风车子对人体(如表皮)能明显保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化，该发现对于新化妆品或药物的开发具有重要意义。
(b)小花风车子植物早已长期广泛使用，为药食两用植物，是一种安全的低毒副作用的常用中药材。
(c)制备有效部位的工艺简单实用，获得有效部位在低浓度时即能明显保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化。
(d)本发明所述的药物或化妆品组合物的活性成分为小花风车子提取物，更天然安全、低刺激，并且具有多种功效，能够更有效地护理皮肤。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1小花风车子提取物的制备方法
1.1取500g(干重)的小花风车子叶，加入足量水后保持85-100℃浸泡30min以上。若需要，可重复加水浸泡1-4次并合并提取液。将提取液浓缩至原体积的1/3-1/4。加入乙酸乙酯，萃取后去除乙酸乙酯相，收集水相。减压浓缩后冻干成粉，待用。
1.2 95％乙醇(1:10)回流提取三次，1h/次。合并三次提取液，40℃以下减压浓缩至无乙醇，得小花风车子粗提物浸膏。小花风车子粗提物浸膏中加入纯化水(1：1)，超声混悬，转移至合适的分液漏斗中，然后依次用石油醚，乙酸乙酯，正丁醇分别萃取3次，分别合并石油醚层溶液，乙酸乙酯层溶液，正丁醇层溶液以及水溶液，40-60℃以下减压浓缩，得四组粗提物浸膏。
1.3 95％乙醇(1:10)室温浸泡三次，24h/次。合并三次提取液，40℃以下减压浓缩至无乙醇，得小花风车子粗提物浸膏。小花风车子粗提物浸膏中加入纯化水(1：1)，超声混悬，转移至合适的分液漏斗中，然后依次用石油醚，乙酸乙酯，正丁醇分别萃取3次，分别合并石油醚层溶液，乙酸乙酯层溶液，正丁醇层溶液以及水溶液，40-60℃以下减压浓缩，得四组粗提物浸膏。
1.4水回流提取(1:10)三次，1h/次。合并三次提取液，60℃以下减压浓缩至原体积的1/4，得小花风车子粗提浓缩液，转移至合适的分液漏斗中，然后依次用石油醚，乙酸乙酯，正丁醇分别萃取3次，分别合并石油醚层溶液，乙酸乙酯层溶液，正丁醇层溶液以及水溶液，40-60℃以下减压浓缩，得四组粗提物浸膏。
1.5水室温浸泡(1:10)三次，24h/次。合并三次提取液，60℃以下减压浓缩至原体积的1/4，得小花风车子粗提浓缩液，转移至合适的分液漏斗中，然后依次用石油醚，乙酸乙酯，正丁醇分别萃取3次，分别合并石油醚层溶液，乙酸乙酯层溶液，正丁醇层溶液以及水溶液，40-60℃以下减压浓缩，得四组粗提物浸膏。
1.6沸水浸三次(6.5:350)，16min/次。合并三次提取液，60℃以下减压浓缩至原体积的1/4，得小花风车子粗提浓缩液，转移至合适的分液漏斗中，然后依次用石油醚，乙酸乙酯，正丁醇分别萃取3次，分别合并石油醚层溶液，乙酸乙酯层溶液，正丁醇层溶液以及水溶液，40-60℃以下减压浓缩，得四组粗提物浸膏。
1.7将干燥的小花风车子叶(20g)用1.5L沸水浸泡16分钟，此过程重复3次，合并水溶液冷却至室温。通过旋转蒸发(60℃)浓缩水溶液至1.5L。然后依次用乙酸乙酯和正己烷(各1.5L，重复三次)萃取，收集水层并真空蒸发至所需量，冷冻干燥，并在无水的条件下保持在室温下待定分析。
在具有DAD检测器的Waters 2695 HPLC系统上进行HPLC分析。用C18柱色谱分离待分析样品，流动相由水(A)和乙腈(B)组成。梯度程序如下：0-10分钟，含25-46％乙腈(B)；11-20分钟，含46-10％乙腈(B)；21-35分钟，用10％乙腈(B)冲洗，流动相的流速保持在1mL/min。样品溶液的注射量为10μL。上述1.7方法制备的提取物具有图1所示的HPLC图谱。
实施例2小花风车子水提取物的效果
1.细胞培养，小花风车子水提取物处理和UVA照射
人原代表皮角质形成细胞购自美国模式培养物保藏所(ATCC)(Rockville，MD，USA)。第1-5代的角质形成细胞在真皮细胞基础培养基中于37℃，5％CO 2中培养。培养基补充角质形成细胞生长试剂盒，其中含有0.4％牛垂体提取物(BPE)，0.5ng/mL rh TGF-α，6mML-谷氨酰胺，100ng/mL氢化可的松，5μg/mL胰岛素，1.0μM肾上腺素和5μg/mL载脂蛋白-转铁蛋白。将小花风车子水提取物稀释至不同浓度，并根据不同的实验装置在UV照射之前或之后加入细胞培养基中。使用UVP CL-1000L荧光灯(Fisher Scientific，USA)照射细胞，总共4.5J/cm2UVA。在整个过程中对所有细胞进行相同的处理。在照射前，用PBS洗涤细胞，用薄的PBS层覆盖，并在没有塑料盖的情况下用UVA照射。使用UVA-365辐射计(Lutron Co，Taiwan)测量UVA照射的强度。
2.细胞存活率测量
根据制造商的说明，通过CCK-8测定法测量细胞存活率。在通过UVA照射或小花风车子水提取物的处理后，用PBS洗涤细胞三次。随后，向每个孔中加入含有10％的CCK-8溶液的培养基。温育2小时后，使用Victor X4Multilabel Plate Reader(PerkinElmer，MA，USA)在450nm处测量吸光度。
3.细胞氧化应激(ROS)测量
通过用CCK-8测定法校正的DCFA评估细胞中ROS的形成，以解释UVA辐射后的细胞损失，测试前24小时，以每孔15,000个角质形成细胞的密度接种96孔板。用不同浓度的小花风车子水提取物预处理HEK细胞24小时，然后在UVA照射之前用PBS洗涤三次。在用4.5J/cm2UVA照射后，100μL含有DCFA(25μM)的PBS加入细胞在37℃下培育30分钟后进行细胞内ROS的荧光测定。将平板置于Victor X4 Multilabel Plate Reader(PerkinElmer，MA，USA)中。使用485nm的激发波长和535nm的发射波长监测荧光。在汇集之前用CCK-8测定法所测定的细胞存活率校正个体吸光度值。
紫外线辐射是对皮肤的主要环境威胁。它通过产生活性氧导致光氧化损伤的增加。结果如图2所示，与对照组相比，UVA组显着增加细胞活性氧的产生。同时，用小花风车子水提物预处理减少了UVA诱导所增加的活性氧。结果表明，小花风车子具有修复紫外所引起的氧化损伤作用。
4.硝基酪氨酸的测量
使用竞争性ELISA试剂盒ab113848(Abcam，Cambridge，MA，USA)中提供的试剂进行硝基酪氨酸的检测。3NT BSA用作标准阳性对照以验证测定。简言之，在测试前24小时将2×105/孔角质形成细胞接种在24孔板中。在UVA照射后，随后用小花风车子水提取物处理16小时后，通过刮擦收集贴壁细胞.在冰上，将细胞沉淀溶解于提取缓冲液中20分钟。在4℃下离心20分钟后，收集上清液。将每个样品稀释并通过蛋白质浓度测定将其调节至大致相同的蛋白质浓度(Bio-Rad，Hercules，CA)。将50微升每种稀释的标准品或样品与50μL的2×HRP检测抗体一起加入到经硝酸酪氨酸包被的96孔微孔板的每个孔中，并在室温下温育2小时。洗涤四次后，向每个孔中加入100μLHRP开发溶液。根据制造商的说明测量OD值。硝基酪氨酸的浓度通过3NT BSA产生的标准曲线计算。
结果如图3所示，在UVA诱导过后，与对照细胞相比，UV组的硝基酪氨酸含量显着增加。与UV组相比，用小花风车子水提物处理的角质形成细胞中的硝基酪氨酸水平显着降低。
5.8-OHdG的测量
作为氧化性DNA损害副产品之一，8-OHdG是一种普遍存在的氧化应激因子。使用OxiSelect Oxidative DNA Damage ELISA Kit(8-OhdG Quantitation)(Cell Biolabs,San Diego,USA)，实现了DNA样品中8-OHdG的测量。接种角质形成细胞在6孔板中，密度为8×105/孔。24小时孵育后，将细胞暴露于4.5J/cm2UVA。照射后，细胞在有或没有用小花风车子水提取物处理的情况下在37℃下孵育60分钟。随后，使用0.05％胰蛋白酶-EDTA溶液收获细胞。使用DNeasy血液和组织试剂盒(Qiagen，CA，USA)提取DNA样品。将每种DNA样品在冷PBS中稀释至40μg/mL。然后通过将DNA样品在95℃下孵育10分钟，在冰上快速冷却10分钟，把DNA样品转化为单链DNA。与10单位核酸酶P1在37℃，20mM乙酸钠(pH 5.2)中孵育2小时后，变性的DNA样品将被消化成核苷，然后用5单位碱性磷酸酶在37℃，100mM的Tris(pH7.5)下处理1小时。然后将反应混合物在6000g下离心5分钟，并将上清液用于8-OHdG ELISA测定。每个样品中8-OHdG的量将通过将其吸光度与已知标准曲线的吸光度进行比较来确定。
6.CPD的测量
使用OxiSelectTMOxidative UV-induced DNA Damage ELISA试剂盒(CellBiolabs，San Diego，USA)实现DNA样品中CPD的检测和定量。将角质形成细胞以4×105/孔的密度接种在12孔板中。在用或不用小花风车子水提取物处理孵育24小时后，将细胞暴露于4.5J/cm 2UVA。照射后，在37℃下温育，细胞进行或不进行处理60分钟，90分钟和120分钟。随后，使用0.05％胰蛋白酶-EDTA溶液收获细胞。DNA样本使用DNeasy血液和组织试剂盒(Qiagen，CA，USA)提取。将每种DNA样品在冷PBS中稀释至2μg/mL。然后通过将DNA样品在95℃温育10分钟并在冰上快速冷却10分钟，将DNA样品和CPD-DNA标准品转化为单链DNA。将100μL稀释在冷PBS中的变性DNA样品或CPD-DNA标准品添加到DNA高结合板的孔中用于进一步的ELISA测定。每个样品中的CPD数量将通过比较其吸光度与已知CPD-DNA标准曲线的吸光度来确定。
COX-2，TNF-α，IL-6和IL-8表达的测量将使用PathScan Total Cox2SandwichELISA Kit#7291(Cellsignal),Human TNFαELISA Kit(ab181421),Human IL-6 ELISA Kit(ab178013),Human IL-8ELISA Kit(ab214030)试剂盒进行。在使用前24小时，在有或没有小花风车子水提取物处理的情况下将密度为2×105/孔角质形成细胞接种在24孔板中。随后的测量将根据制造商的说明进行。结果表明，小花风车子具有抗炎作用。
综上所述，小花风车子植物具有保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化等作用。因此可作为一种新的活性成分添加于护肤品以及药品中。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
Claims (10)
1.一种小花风车子植物、或其中药药材、或小花风车子提取物的用途，其特征在于，用于制备药物或化妆品组合物，并且所述药物或化妆品组合物用于：保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化、或其组合。
2.如权利要求1所述的用途，其特征在于，所述的小花风车子提取物包括小花风车子植物或其同属植物的枝、叶、根、花、果和/或茎的水溶性的和/或脂溶性的提取物。
3.如权利要求1所述的用途，其特征在于，所述的小花风车子提取物为叶的提取物。
4.如权利要求1所述的用途，其特征在于，所述的提取物是通过溶剂提取法、萃取法、和/或色谱法而获得的。
5.如权利要求1所述的用途，其特征在于，所述提取物的提取剂选自下组：水、醇、水性溶剂、或其混合物。
6.一种可用于制备药物或化妆品组合物的有效部位，其特征在于，所述的有效部位具有以下特征：
(a)该有效部位是提取自小花风车子的枝、叶、根、花、果和/或茎的水溶性、脂溶性、和/或醇提取物；
(b)该有效部位具有选自下组的功能：保湿、抗炎、晒后修复、修复皮肤屏障、修复紫外诱导DNA损伤、美白、淡斑、抗糖化、或其组合。
7.如权利要求6所述的有效部位，其特征在于，所述有效部位含有一种或多种选自下组的组分：多糖、植物多酚、多酚糖苷类、或其组合。
8.一种药物或化妆品组合物，其特征在于，含有(a)权利要求6所述的有效部位；和(b)药学上或化妆品学上可接受的载体或赋形剂。
9.如权利要求8所述的药物或化妆品组合物，其特征在于，在所述化妆品组合物中，干燥有效部位的质量百分比为0.0001-10wt％，较佳地0.001-5wt％，以化妆品组合物的总重量计。
10.一种制备药物或化妆品组合物的方法，其特征在于，包括步骤：将权利要求6所述的有效部位与药学上或化妆品学上可接受的载体混合，从而形成药物或化妆品组合物。
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| CARA RENAE WELCH: "Chemistry and Pharmacology of kenkeliba(Combretum Mircranthum), A west African Medicinal Plant", A DISSERTATION SUBMITTED TO UNIV. RUTGERS STATE NEW JERSEY * |
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