CN115120628A - 旋覆花和芝麻的组合物及其用于修复紫外损伤的用途 - Google Patents
旋覆花和芝麻的组合物及其用于修复紫外损伤的用途 Download PDFInfo
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Abstract
本发明提供了一种旋覆花、芝麻组合物及其用于修复紫外损伤的用途。具体地，提供了一种活性成分的组合，所述组合包括：旋覆花提取物；和芝麻提取物。所述组合在预防和/或修复紫外损伤、抗炎等方面具有协同效果。
Description
技术领域
本发明涉及医药领域，具体涉及一种旋覆花和芝麻的组合物及其用于修复紫外损伤用途。
背景技术
紫外线诱导皮肤问题，是皮肤外源性损伤、皮肤老化的主要原因。紫外线对皮肤的损伤包括晒黑、晒伤、导致胶原和弹性蛋白分解变性、加重炎症、诱导自由基形成、抑制皮肤免疫、诱导光化性皮肤病、加重色斑，以及诱导皮肤癌等。
紫外损伤可分为急性损伤和慢性损伤，其中，急性损伤包括晒伤等，而皮肤长期受日光中的紫外线照射后，由于累积性损伤而导致皮肤衰老或加速衰老(即光老化)。
因此，预防和修复紫外诱导的皮肤损伤是保护皮肤、延缓衰老的有效手段。本领域需要提供更多的具有预防和/或修复紫外诱导的皮肤损伤效果的产品。
发明内容
本发明的目的是提供一种能够预防和/或修复紫外损伤的组合物。
本发明第一方面，提供了一种活性成分的组合，所述组合包括：
(a)第一活性成分：旋覆花提取物；和
(b)第二活性成分：芝麻提取物；
其中，以干重计，所述第一活性成分与第二活性成分的重量比为1-10:1-20。
所述第一活性成分与第二活性成分的重量比为1-5:1-5，更佳地1-2：1-2，例如，1:5、2:5、3:5、4:5、5:5、5:1、5:2、5:3、5:4或5:5。
在另一优选例中，所述的旋覆花提取物为花提取物。
在另一优选例中，所述的芝麻提取物为籽提取物，较佳地，从芝麻籽榨取油份之后的残渣提取得到。
在另一优选例中，所述的提取物包括水提取物、醇水溶剂提取物、醇提取物、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)、醇水溶剂、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，所述旋覆花提取物是由以下方法制备得到的，所述方法包括步骤：
(I)将干旋覆花用80-100wt％醇水溶剂提取1-5次，合并提取液，去除溶剂得粗提物；和
(II)将所述粗提物用水重悬，用有机溶剂萃取，收集水相，去除溶剂得所述旋覆花提取物。
在另一优选例中，步骤(I)具有选自以下的一个或多个特征：
(1)所述醇水溶剂中，醇为C1-C6烷基醇，较佳地甲醇、乙醇、异丙醇，或其组合，更佳地90-95％乙醇-水；
(2)所述干旋覆花与醇的用量比为1g：2-10mL，较佳地1g：4-8mL；
(3)所述提取的方法选自下组：浸泡、超声、震荡，或其组合；
(4)所述提取的温度为4-40℃，较佳地20±10℃；
(5)所述提取的时间为1-48h/次，较佳地12-36h/次；
(6)所述提取的次数为1-5次，较佳地2-4次；和/或
(7)所述固液分离选自下组：过滤、离心，或其组合；
在另一优选例中，去除溶剂的方法选自下组：减压蒸发、冻干，或其组合。
在另一优选例中，所述去除溶剂为部分去除(如浓缩)或全部去除。
在另一优选例中，步骤(II)中，所述有机溶剂萃取包括步骤：
根据极性从低至高，使用1-5种(如2-4种)不同极性的有机溶剂(可与水分层的溶剂，如石油醚、乙酸乙酯、正己烷、正丁醇)依次萃取，例如使用石油醚、乙酸乙酯和正丁醇依次萃取。
在另一优选例中，所述有机溶剂与水不互溶或在水中溶解度≤10wt％。
在另一优选例中，所述芝麻选自下组：黑芝麻、白芝麻，或其组合。
在另一优选例中，所述芝麻提取物是由以下方法制备得到的，所述方法包括步骤：
(i)将榨油后的芝麻残渣用80-100wt％醇水溶剂提取1-5次，合并提取液，去除溶剂得粗提物；和
(ii)将所述粗提物用水重悬，用有机溶剂萃取，收集水相；
(iii)用正丁醇萃取所述水相，收集有机相，去除溶剂得所述芝麻提取物。
在另一优选例中，步骤(i)具有选自以下的一个或多个特征：
(1)所述醇水溶剂中，醇为C1-C6烷基醇，较佳地，乙醇、异丙醇，或其组合，更佳地80-100％乙醇；
(2)所述芝麻残渣与醇水溶剂的用量比为1g：2-10mL，较佳地1g：4-8mL；
(3)所述提取的方法选自下组：浸泡、超声、震荡，或其组合，较佳地超声提取；
(4)所述提取的温度为4-60℃，较佳地20±10℃；
(5)所述提取的时间为0.5-48h/次，较佳地1-6h/次，如2h；和/或
(6)所述提取的次数为1-5次，较佳地2-4次。
在另一优选例中，步骤(ii)中，所述有机溶剂萃取包括步骤：
根据极性从低至高，使用1-5种(如2-4种)不同极性的有机溶剂依次萃取，例如使用正己烷、乙酸乙酯依次萃取。
本发明第二方面，提供了一种组合物，所述组合物包括：如本发明第一方面所述的活性成分的组合，以及化妆品学上或药学上可接受的载体或赋形剂。
在另一优选例中，所述组合物为化妆品组合物，其中，以干重计，所述活性成分的组合的质量百分比为0.0001-15wt％，较佳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，所述组合物为药物组合物，其中，以干重计，所述活性成分的组合的质量百分比为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。
本发明第三方面，提供了如本发明第一方面所述的活性成分的组合或如本发明第二方面所述的组合物的用途，用于制备一化妆品或药物，所述化妆品或药物用于一个或多个选自下组的用途：(a)预防和/或修复紫外损伤；和/或(b)抗炎。
在另一优选例中，所述紫外损伤为紫外诱导的皮肤损伤，较佳地，所述紫外诱导的皮肤损伤选自下组：晒伤(如紫外诱导的红斑)、晒黑(如色素沉着)、光老化、光毒性反应和光敏反应、皮肤肿瘤(如皮肤鳞状上皮癌、基底细胞癌)、或其组合。
在另一优选例中，所述紫外线选自下组：UVA(320-400nm)、UVB(290-320nm)，或其组合。
在另一优选例中，所述晒伤包括：红斑、DNA损伤、紫外诱导的表皮细胞死亡、皮肤表面粗糙、角质增加、肌肤屏障受损，或其组合。
在另一优选例中，所述光老化包括：皱纹、皮肤松弛、毛细血管扩张、皮肤色素不均匀、胶原蛋白和/或弹力纤维的破化。
在另一优选例中，所述紫外诱导的皮肤损伤包括：表皮层损伤(如角质层)和真皮层损伤。
在另一优选例中，所述预防紫外损伤为增强表皮细胞对紫外光的耐受能力。(与对照组(不使用本发明的活性成分的组合的组,如溶媒对照组、单独使用旋覆花提取物的组、或单独使用芝麻提取物的组)相比，经本发明的活性成分的组合处理后的组，经过相同强度紫外光照射后，细胞活性高出10％(即1.1倍以上)、20％、30％、40％、50％、60％、80％或100％以上。
在另一优选例中，所述表皮细胞选自下组：角质形成细胞、树枝状细胞，或其组合。
在另一优选例中，所述药物或化妆品组合物用于防晒和/或抗光老化。
在另一优选例中，所述化妆品或药物用于一个或多个选自下组的用途：
(1)预防紫外诱导的细胞活性降低或提高细胞活性；
(2)抑制紫外诱导的细胞内氧化应激水平升高；
(3)抑制由紫外诱导的细胞内活性氮水平的升高；
(4)修复由紫外诱导的细胞内DNA损伤；
(5)抑制由紫外诱导的细胞内炎症因子IL-6水平的升高；和/或
(6)抑制由紫外诱导的细胞内炎症因子TNF-α水平的升高。
在另一优选例中，所述细胞为表皮细胞。
在另一优选例中，所述表皮细胞包括：角质层形成细胞、树枝状细胞，或其组合。
在另一优选例中，所述药物或化妆品组合物用于晒前预防和/或晒后修复。
本发明第四方面，提供了一种护肤的方法，包括步骤：给予有需要的对象本发明第一方面所述的活性成分的组合或包含其的组合物，从而保护皮肤，且所述护肤指(a)预防和/或修复紫外损伤；和/或(b)抗炎。
在另一优选例中，所述对象为哺乳动物，如人、大鼠或小鼠。
在另一优选例中，所述对象为皮肤敏感人群，如老人、儿童或敏感肌人群。
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示出了不同组对细胞的紫外保护作用。
图2示出了不同组对细胞氧化应激水平的作用。
图3示出了不同组对细胞内活性氮水平的作用。
图4示出了不同组对细胞炎症因子IL-6水平的作用。
图5示出了不同组对细胞炎症因子TNF-α水平的作用。
图6示出了不同组对细胞DNA损伤修复的作用。
图1-6中，每个数值来自三次独立实验的平均值±标准误差，****p<0.0001，***p<0.001，**p<0.01。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选和测试，提供了一种旋覆花提取物和芝麻提取物的组合物。令人惊讶地，本发明的组合物在提高细胞活性降低，降低紫外辐射导致的氧化应激、活性氮、细胞炎症因子，细胞内CPDs水平等方面具有显著的协同作用，非常适合用于制备用于防晒、晒后修复、抗炎的化妆品或药物。在此基础上完成了本发明。
术语
除非另有定义，否则本文中所用的全部技术术语和科学术语均具有如本发明所属领域普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文所用，术语“室温”是指温度为4-40℃，较佳地，25±5℃。
活性成分、组合
本发明的第一活性成分为旋覆花(Inula japonica)提取物。长期以来，中医一直使用旋复花的花朵来治疗消化系统疾病，支气管炎和炎症。
本发明的第二活性成分为芝麻(Sesamum indicum)提取物。特别地，本发明的提取物可从芝麻榨油加工后剩下的残渣中提取。芝麻素和芝麻林素是芝麻中特有的木酚素类物质，制取芝麻油后产生的芝麻饼粕中仍然含有相当量的木酚素类化合物。芝麻具有很强的抗氧化活性,也是由于其含木酚素类(lignans,也称木质素、木聚糖)和生育酚类(tocopherols)两大类抗氧化物质。
本发明首次发现了旋覆花提取物和芝麻提取物在(a)预防和/或修复紫外损伤；和/或(b)抗炎等多个方面的协同效果。因而提供了包含旋覆花提取物和芝麻提取物的活性成分的组合物。
提取物、制备方法
本发明中，所述的旋覆花提取物为花提取物。
本发明中，所述的芝麻提取物为籽提取物，较佳地，从芝麻籽榨取油份之后的残渣提取得到。
在本发明的一个优选例中，所述“提取物”或“活性成分”的制备可独立地或共同地通过溶剂提取法、萃取法、超临界萃取法和/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、醇水(混合)溶剂。
在本发明中，所述醇水溶剂指水与其他溶剂如醇(尤其是C1-C4醇，如甲醇、乙醇、异丙醇)所形成的混合溶剂。通常，醇的含量为40-100wt％，较佳地60-95wt％，如80wt％、90wt％、98wt％等，按醇水溶剂的总重量计。
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，对于超临界萃取，其所用溶剂没有特别限制，代表性的例子包括(但并不限于)：二氧化碳或丙烷。
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，旋覆花提取物的制备方法为：以旋覆花(干燥的花)为原料，以足量的醇(如1-20倍重量的水)或醇-水混合物溶剂(50-99％醇，优选80-95％乙醇)提取1-5次，从而获得的提取物。此外，本发明中，还可进一步地从上述醇提取物中分离(或去除)脂溶性成分，从而获得水溶性成分含量(或相对含量)更高的旋覆花提取物，或主要含水溶性成分的旋覆花提取物。
在本发明的一个优选例中，芝麻提取物的制备方法为：以芝麻(如榨油后的芝麻残渣)为原料，以足量的醇(如1-20倍重量的水)或醇-水混合物溶剂(50-99％醇，优选80-95％乙醇)提取1-5次，从而获得醇提取物。此外，本发明中，还可进一步地从上述醇提取物中分离(或去除)脂溶性成分(如油脂)，从而获得活性成分含量(或相对含量)更高的芝麻提取物。
组合物及用途
如本文所用，术语“药物或化妆品组合物”包括(a)本发明所述的活性成分的组合；和(b)药学上或化妆品学上可接受的载体。
此外，所述化妆品组合物包括护肤品。
有可能将本发明的活性成分的组合制备成药物组合物，如膏剂、霜剂、凝胶剂、糊剂、贴剂等。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。
有可能将本发明的活性成分的组合制备成化妆品组合物，固体剂型、半固体剂型、或液体剂型，如溶液、凝胶、膏霜、乳液、喷雾、膏剂、霜剂、糊剂、饼、粉剂、贴剂等。
本发明的第一活性成分和第二活性成分可以与载体分别制成单独的剂型，或混合在一起制成单一剂型提供。
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活性成分的组合或本发明所述的组合物。通常，所述方法为用于预防和/或修复紫外诱导的皮肤损伤的方法。
在另一优选例中，所述活性成分的组合的有效浓度范围为1μg/ml–500mg/ml，如5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml或200μg/ml。
如本文所用，术语“有效剂量”是指任何如下所述的量，当单独使用或与另一种治疗剂或化妆品组合使用该量的本发明的活性成分的组合或组合物时，可促进疾病消退，疾病消退表现为疾病症状的严重度降低、无疾病症状期的频率和持续时间增加、或者防止由患病导致的障碍或失能。本发明药物的“治疗有效剂量”也包括“预防有效剂量”，“预防有效剂量”是药物的任何如下所述的量，当将该量的组合物单独施用或者与另一种治疗剂或化妆品组合施用于具有发生疾病或症状的风险或者遭受疾病或症状复发的受试者时，可抑制疾病或症状的发生或复发。
在本发明中，对所述晒伤、晒伤(如紫外诱导的红斑)、晒黑(如色素沉着)、光老化、光毒性反应和光敏反应、皮肤肿瘤等的应用，既包括预防性的应用，也包括事后改善性的应用。例如，对于晒伤，包括在晒之前、之中、和/或之后施用本发明的活性成分的组合或包含其的组合物，以预防和/或修复晒伤。
本发明的主要优点包括：
1.本发明提供了一种具有预防和/或修复紫外损伤、抗炎的活性成分的组合，其中包括旋覆花提取物与芝麻提取物，令人意外地，本发明的旋覆花提取物与芝麻提取物在预防和/或修复紫外损伤、抗炎等方面具有显著的协同作用。
2.本发明的组合物中，所述芝麻提取物可以由榨油后的芝麻残渣提取得到，可对芝麻残渣回收再利用，制备成本低，资源节约。
下面结合具体实施，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，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor LaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
植物提取物的制备
1.1芝麻提取物的制备将干燥的生黑芝麻(SESAMUM INDICUM)通过压榨去除大部分油脂。取榨油后的芝麻残渣(100g)用95％乙醇(600mL)浸泡，超声1h，反复提取三次。将三次乙醇提取液混合，减压蒸发得到芝麻乙醇粗提物。用水将该芝麻乙醇粗提物重悬，依次用正己烷(保留水相)，乙酸乙酯(保留水相)和正丁醇萃取。将正丁醇萃取所得组分减压蒸发所得即为实验所用的芝麻提取物。
1.2旋覆花提取物的制备在室温下，将风干的旋覆花(INULA JAPONICA)(100g)用95％乙醇(600mL)浸泡24h，随后过滤，滤渣再浸泡，重复三次。将三次滤液混合并减压蒸发得到旋覆花乙醇粗提物。用水将该旋覆花乙醇粗提物重悬，依次用石油醚，乙酸乙酯和正丁醇萃取。将萃取后剩余的水溶液减压蒸发所得即为实验所用的旋覆花提取物。
实施例2
细胞培养，提取物预处理及UVA辐射
人原代角质形成细胞(购自ATCG公司(Rockville,MD,USA))。实验只选择第1-5代的角质形成细胞，用真皮细胞培养液添加角质形成细胞生长试剂盒培养于含5％二氧化碳的37℃恒温培养箱中。在紫外照射前24h，将实验组的细胞培养液换成提前配置好的含不同浓度植物提取物(混合提取物为将芝麻提取物：旋覆花提取物以5：2的比例混合)的培养液，紫外对照组和空白对照组(避光)换为等量的不含任何植物提取物成分的新鲜培养液。
紫外辐射采用UVA荧光灯(Vilber Lourmat,France)，实验中采用的总UVA剂量为4.5J/cm2。进行紫外照射前，用PBS溶液洗涤细胞，并保留微量PBS溶液使得细胞能在实验过程中保持湿润。在整个实验过程中，所有细胞均得到相同处理。
实施例3
细胞活性检测(CCK-8)方法
UVA辐射完毕后，去除剩余的PBS溶液，加入添加了10％CCK-8溶液的培养液，将细胞放回恒温箱继续培养，孵育2h后，用酶标仪检测，在450nm测定吸光度。
实验结果如图1所示，单独使用旋覆花提取物预处理并不能提高细胞活性，而芝麻提取物和本发明的混合提取物对人原代角质形成细胞进行预处理后再进行紫外辐射，可有效提高细胞活性，而与单用芝麻提取物相比，旋覆花提取物与芝麻提取物的混合物在提高紫外诱导的细胞活性方面具有明显的协同作用。
实施例4
细胞氧化应激(ROS)测量
该方法通过DCFA测定细胞内ROS的水平并通过细胞活性校正，以准确评估紫外(UVA)辐射对细胞造成的损伤。将人原代角质形成细胞以1.5×104个/孔的密度接种到96孔板中。接种24h后，将实验组的细胞培养液换成提前配置好的含不同浓度混合提取物的培养液，紫外对照组和空白对照组换为等量的不含任何植物提取物的新鲜培养液。预处理24h后，用PBS溶液将细胞洗涤三次并保留微量PBS溶液使得细胞能在实验过程中保持湿润。用4.5J/cm2UVA照射细胞后，加入含25uM DCFA的PBS溶液，并将细胞放回恒温箱内孵育30min后进行荧光测定，所使用的激发波长为485nm，发射波长为535nm。
实验结果如图2所示，本发明的混合提取物对人原代角质形成细胞进行预处理后再进行紫外辐射，可协同地有效抑制紫外辐射导致的细胞内氧化应激水平升高。
实施例5
细胞活性氮(RNS)测量
该方法通过硝基酪氨酸ELISA试剂盒测定细胞内活性氮水平。将人原代角质形成细胞以1.5×105个/孔的密度接种到24孔板中。接种24h后，用PBS溶液洗涤细胞，并保留微量PBS溶液使得细胞能在实验过程中保持湿润。用4.5J/cm2 UVA照射细胞后，去除剩余的PBS溶液，向实验组细胞加入提前配置好的含不同浓度混合提取物的培养液，紫外对照组和空白对照组加入等量的不含任何植物提取物的新鲜培养液。将细胞放回恒温箱培养16h后收集细胞。将细胞沉淀物置于提取缓冲液中，并在冰上温育20min。离心后，将上清液转移至干净的试管中，并通过BCA蛋白测定法测量样品的蛋白浓度。将3NT BSA用作标准阳性对照以验证测定。在上样之前，将样品的蛋白质浓度调整到大约相同的水平。将五十微升的每种稀释的标准品或样品与2X HRP检测器抗体一起添加到预涂板的每个孔中，并在室温下孵育2h。洗涤四次并完全除去液体后，将100微升HRP显影溶液添加到每个孔中。测量OD值并根据标准曲线计算硝基酪氨酸浓度。
实验结果如图3所示，在紫外辐射后用混合提取物对人原代角质形成细胞进行修复处理，可协同地有效抑制由紫外辐射导致的细胞内活性氮水平的升高。
实施例6
细胞炎症因子IL-6和TNF-α的测量
细胞炎症因子的测量均通过ELISA试剂盒完成。将人原代角质形成细胞以1.5×105个/孔的密度接种到24孔板中。接种24h后，用PBS溶液洗涤细胞，并保留微量PBS溶液使得细胞能在实验过程中保持湿润。用4.5J/cm2 UVA照射细胞后，去除剩余的PBS溶液，向实验组细胞加入提前配置好的含不同浓度混合提取物的培养液，紫外对照组和空白对照组加入等量的不含任何植物提取物的新鲜培养液。将细胞放回恒温箱培养24h后收集细胞培养液。离心后取培养基上清液作为样品，将50微升样品或标准品添加至ELISA的96孔板中，同时在每个孔内加入等量的抗体混合物。在室温下孵育1h后，洗涤四次，向每个孔中添加100微升TMB底物并在黑暗中反应显色10min，然后添加终止溶液。测量OD值并根据标准曲线计算细胞炎症因子浓度。
IL-6水平测量结果如图4所示，在紫外辐射后用混合提取物对人原代角质形成细胞进行修复处理，可有效抑制由紫外辐射导致的细胞炎症因子IL-6水平的升高。
TNF-α水平测量结果如图5所示，在紫外辐射后用混合提取物对人原代角质形成细胞进行修复处理，可协同地有效抑制由紫外辐射导致的细胞炎症因子TNF-α水平的升高。
实施例7
DNA损伤CPDs测量
DNA损伤水平使用ELISA试剂盒测定CPDs。将人原代角质形成细胞以3×105个/孔的密度接种到12孔板中。接种24h后，用PBS溶液洗涤细胞，并保留微量PBS溶液使得细胞能在实验过程中保持湿润。用4.5J/cm2 UVA照射细胞后，去除剩余的PBS溶液，向实验组细胞加入提前配置好的含不同浓度混合提取物的培养液，紫外对照组和空白对照组加入等量的不含任何植物提取物的新鲜培养液。将细胞放回恒温箱培养1h后用0.05％胰蛋白酶处理并收集细胞。使用DNeasy血液和组织试剂盒提取DNA样品。在冷AE缓冲液中将每个DNA样品稀释至4μg/mL。通过将DNA在95℃下孵育10min并随后在冰上快速冷却10min，将样品和标准品转换为单链DNA。将50微升样品或标准液添加到DNA高结合板的孔中。将50微升的DNA结合溶液加入到每个孔中，并在室温下孵育过夜。除去DNA溶液并用PBS洗涤两次后，将200微升Assay Diluent加入每个孔中，并在室温下孵育1h。之后，将100微升稀释的抗CPD抗体添加到所有孔中，并在室温下孵育1h。随后使用洗涤缓冲液洗涤五次，并将100微升稀释的第二抗体-HRP偶联物添加到所有孔中，并在室温下孵育1h。再洗涤五次后，将100微升TMB底物添加到每个孔中，并在室温下孵育15min，然后添加终止溶液。读取450nm处的吸光度并根据标准曲线计算细胞中CPDs的浓度。
实验结果如图6所示，在紫外辐射后用混合提取物对人原代角质形成细胞进行修复处理，可协同地有效修复由紫外辐射导致的细胞DNA损伤。
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)
1.一种活性成分的组合，其特征在于，所述组合包括：
(a)第一活性成分：旋覆花提取物；和
(b)第二活性成分：芝麻提取物；
其中，以干重计，所述第一活性成分与第二活性成分的重量比为1-10:1-20。
2.如权利要求1所述的组合，其特征在于，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。
3.如权利要求1所述的组合，其特征在于，所述旋覆花提取物是由以下方法制备得到的，所述方法包括步骤：
(I)将干旋覆花用80-100wt％醇水溶剂提取1-5次，合并提取液，去除溶剂得粗提物；和
(II)将所述粗提物用水重悬，用有机溶剂萃取，收集水相，去除溶剂得所述旋覆花提取物。
4.如权利要求1所述的组合，其特征在于，步骤(I)具有选自以下的一个或多个特征：
(1)所述醇水溶剂中，醇为C1-C6烷基醇，较佳地甲醇、乙醇、异丙醇，或其组合，更佳地90-95％乙醇-水；
(2)所述干旋覆花与醇的用量比为1g：2-10mL，较佳地1g：4-8mL；
(3)所述提取的方法选自下组：浸泡、超声、震荡，或其组合；
(4)所述提取的温度为4-40℃，较佳地20±10℃；
(5)所述提取的时间为1-48h/次，较佳地12-36h/次；
(6)所述提取的次数为1-5次，较佳地2-4次；和/或
(7)所述固液分离选自下组：过滤、离心，或其组合。
5.如权利要求1所述的组合，其特征在于，所述芝麻提取物是由以下方法制备得到的，所述方法包括步骤：
(i)将榨油后的芝麻残渣用80-100wt％醇水溶剂提取1-5次，合并提取液，去除溶剂得粗提物；和
(ii)将所述粗提物用水重悬，用有机溶剂萃取，收集水相；
(iii)用正丁醇萃取所述水相，收集有机相，去除溶剂得所述芝麻提取物。
6.如权利要求1所述的组合，其特征在于，步骤(i)具有选自以下的一个或多个特征：
(1)所述醇水溶剂中，醇为C1-C6烷基醇，较佳地，乙醇、异丙醇，或其组合，更佳地80-100％乙醇；
(2)所述芝麻残渣与醇水溶剂的用量比为1g：2-10mL，较佳地1g：4-8mL；
(3)所述提取的方法选自下组：浸泡、超声、震荡，或其组合，较佳地超声提取；
(4)所述提取的温度为4-60℃，较佳地20±10℃；
(5)所述提取的时间为0.5-48h/次，较佳地1-6h/次，如2h；和/或
(6)所述提取的次数为1-5次，较佳地2-4次。
7.一种组合物，其特征在于，所述组合物包括：如权利要求1所述的活性成分的组合，以及化妆品学上或药学上可接受的载体或赋形剂。
8.如权利要求7所述的组合物，其特征在于，所述组合物为化妆品组合物，其中，以干重计，所述活性成分的组合的质量百分比为0.0001-15wt％，较佳0.0001-10wt％，较佳地0.001-5wt％，以化妆品组合物的总重量计。
9.如权利要求1所述的活性成分的组合或如权利要求2所述的组合物的用途，其特征在于，用于制备一化妆品或药物，所述化妆品或药物用于一个或多个选自下组的用途：(a)预防和/或修复紫外损伤；和/或(b)抗炎。
10.如权利要求9所述的用途，其特征在于，所述紫外损伤为紫外诱导的皮肤损伤，较佳地，所述紫外诱导的皮肤损伤选自下组：晒伤(如紫外诱导的红斑)、晒黑(如色素沉着)、光老化、光毒性反应和光敏反应、皮肤肿瘤(如皮肤鳞状上皮癌、基底细胞癌)、或其组合。
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