CN107427434A - 用于诱导促进细胞复壮的包含染料木黄酮或表没食子儿茶素没食子酸酯的组合物 - Google Patents
用于诱导促进细胞复壮的包含染料木黄酮或表没食子儿茶素没食子酸酯的组合物 Download PDFInfo
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Abstract
本申请公开了染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物或溶剂合物的新用途。含有染料木黄酮、表没食子儿茶素没食子酸酯、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物或其溶剂合物的组合物降低与黑色素生成能力提高有关的因子的表达,降低衰老标记蛋白的表达并调节参与黑色素生成的酶的表达,因此可具有使包括黑色素细胞的细胞复壮的作用。
Description
技术领域
本申请公开了染料木黄酮和表没食子儿茶素没食子酸酯(EGCG)的新用途。
背景技术
通过延缓衰老来预防退行性疾病,并长期健康地生活,是人人都想要的。从过去到现在都在努力寻找延迟衰老的物质。加速衰老的原因包括活性氧如超氧化物,过氧化氢,羟自由基和单线态氧的氧化。
由活性氧代谢产生的自由基通过作用于蛋白质、生物膜、DNA等而在体内产生氧化损伤。为了防止这种现象,在韩国国内外,对现有的或新的抗氧化剂积极地进行了很多的研究。
然而,由于衰老不仅受氧化影响,而且还受到包括端粒、细胞复制活性、遗传损伤、恢复能力等各种因素的影响,所以使用细胞衰老的方法需要评估天然产物和药物预防细胞衰老的能力,而不仅仅是评估其抗氧化作用。
细胞衰老是指在一定数量的分裂后正常体细胞停止分裂的现象。其有助于个体和组织的老化,抑制细胞异常增殖和致癌作用的重要机制。细胞衰老由位于染色体末端的端粒在体细胞重复分裂后缩短、癌基因或致癌基因活性增加、过度氧化应激、紫外线或放射性辐射、细胞毒性物质如抗癌药物、炎症反应等导致。
细胞衰老不仅有助于个体和组织的老化,而且在各种疾病的发病中起重要作用。在类风湿关节炎、骨关节炎、肝炎、慢性皮肤损伤、动脉硬化等炎症组织中经常观察到老化细胞。前列腺增生、肝炎、肝癌等也观察到细胞衰老。
当老化细胞积聚时,由于老化细胞不能很好地分解,损坏的组织不能正确恢复。此外,老化细胞通过分泌降解附近组织的酶,炎性细胞因子等而加速组织损伤,从而促使老化相关疾病的发病。
因为细胞衰老被认为是老化的重要原因,所以已经努力开发延迟细胞衰老的方法。例如,已经努力探索可以调节细胞衰老的物质并利用该物质来预防和治疗疾病。
已经发现调节细胞衰老的代表性物质包括红葡萄酒中富含的且已知增加SIRT1的活性的白藜芦醇、延迟角质形成细胞的细胞衰老的维甲酸等。此外,已经努力鉴定并从植物提取物分离出能够调节细胞衰老的物质以将其用于化妆品、保健、食品等。
然而,以前的研究仅聚焦预防或延缓衰老的方法,从未报道过使老化细胞积极复壮为年轻和健康细胞的方法。
(非专利文献1)Harman,D.,Free radical theory of aging:Role of freeradicals in the origination and evolution of life,aging and diseaseprocesses,3-49.Alan R Liss,New York(1986)。
发明内容
技术问题
一方面,本申请涉及提供染料木黄酮,表没食子儿茶素没食子酸酯(EGCG),其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物及其溶剂合物用于诱导细胞复壮的用途。
另一方面,本申请涉及降低与黑色素生成能力的提高有关的因子的表达。
另一方面,本申请涉及通过调节参与黑色素生成的酶的表达来诱导细胞复壮。
技术方案
一方面,本申请提供了包含选自染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物和其溶剂合物中的一种或多种的组合物。
有益效果
一方面,包含选自染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物及其溶剂合物中的一种或多种的本申请的组合物降低与黑色素生成能力提高相关的包括酪氨酸酶的因子的表达、降低包括p16和p21的衰老标记物的表达,并且调节参与黑色素生成的包括组蛋白脱乙酰酶5和EZH1(zeste同系物1的增强子)的酶的表达,因而可具有使细胞(包括黑色素细胞)复壮的作用。
附图说明
图1A-1D显示在含有染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的培养基中传代培养黑色素细胞后,与黑色素生成能力的提高相关的因子的表达变化的测量结果。
图2显示在含有染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的培养基中传代培养黑色素细胞后,衰老标记物(p16和p21)变化的结果。
图3显示在含有染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的培养基中传代培养黑色素细胞后,参与黑色素生成的酶的表达变化的观察结果。
图4A-4B显示在含有染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的培养基中传代培养黑色素细胞后,黑色素细胞表型变化的观察结果。图4A显示显微镜观察结果,图4B显示老化细胞与总黑色素细胞的比例。
在每个图中,PA表示每次传代,PA11E是指由第7代在含有EGCG的培养基中培养出的第11代的黑色素细胞,PA11G是指由第7代在含有染料木黄酮的培养基中培养出的第11代的黑色素细胞,PA12E是指由第7代在含有EGCG的培养基中培养出的第12代的黑色素细胞,PA12G是指由第7代在含有染料木黄酮的培养基中培养出的第12代的黑色素细胞。
具体实施方式
以下,对本申请进行详细说明。
一方面,本申请提供用于诱导细胞复壮的组合物,所述组合物包含选自染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物及其溶剂合物中的一种或多种。
在本申请中,“衍生物”可以是通过用另一个原子或原子组取代一部分特定化合物的一部分而获得的化合物。在本申请中,“异构体”特别地不仅包括光学异构体(例如,基本上纯的对映异构体,基本上纯的非对映异构体或其混合物),而且包括构象异构体(即,仅在一个或多个化学键的角度上不同的异构体),位置异构体(特别是互变异构体)或几何异构体(例如顺式-反式异构体)。
在本申请中,“基本上纯的”是指,例如,当与对映异构体或非对映异构体结合使用时,作为对映异构体或非对映异构体的实施例的特定化合物的存在量为约90%(w/w)或以上,特别是约95%或以上,更具体地约97%或以上或约98%或以上,进一步更具体地约99%或以上,甚至更具体地约99.5%或以上。
在本申请中,“药学上可接受的”是指由于当以普通药用剂量使用时可以避免显著的毒性作用,因而其可以被政府或国际组织的管理机构批准或获得核准,或者列在“药典”中,或被认可是用于动物,更具体地用于人的其它一般备用药品。
在本申请中,“药学上可接受的盐”是指本发明的一方面的盐,该盐是药学上可接受的并且表现出其母体化合物的期望的药学活性。该盐可以包括(1)由无机酸或有机酸形成的酸加成盐,该无机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸,有机酸如乙酸、丙酸、己酸、环戊烷丙酸、乙醇酸、丙酮酸、乳酸、丙二酸、琥珀酸、苹果酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、3-(4-羟基苯甲酰基)苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、1,2-乙烷二磺酸、2-羟基乙磺酸、苯磺酸、4-氯苯磺酸、2-萘磺酸、4-甲苯磺酸、樟脑磺酸、4-甲基双环[2,2,2]辛-2-烯-1-羧酸,葡庚糖酸,3-苯基丙酸,三甲基乙酸,叔丁基乙酸,月桂基硫酸,葡萄糖酸,谷氨酸,羟基萘甲酸,水杨酸,硬脂酸和粘康酸;或(2)当母体化合物中的酸性质子被取代时形成的盐。
在本申请中,“前药”是指已经经过化学改变以调节其物理和化学性质的药物,其不表现出生理活性,但通过体内化学或酶促作用转化成原始药物后发挥药用作用。
在本申请中,“水合物”是指与水结合的化合物。该术语以广义使用,包括缺少与水的化学键合的包合物。
在本申请中,“溶剂合物”是指在溶质的分子或离子与溶剂的分子或离子之间形成的高级化合物。
在本申请中,染料木黄酮可以是指作为植物中包含的植物化学化合物的异黄酮之一。它可能存在于大豆、国槐、三叶草或其它植物中。染料木黄酮可以是指具有以下化学式1的结构的物质。
[化学式1]
在本申请中,染料木黄酮衍生物可以为选自,但不限于,下式的化合物:R1至R4中各自独立地选自氢、羟基、烷基或取代的烷基、苯基或取代的苯基、二苯基乙基或取代的二苯基乙基、苯硫基或取代的苯硫基、烷氧基、环烷基、环烷氧基、杂环烷基、芳氧基、卤代烷氧基、芳基、卤代烷基、甲氧基、苄基或取代的苄基、苯甲酰基、氧代、烯炔酸烷基(alkyidenyl)、羰基、酰基、烯基、烷基氨基和二烷基氨基。例如,本申请的染料木黄酮衍生物可以是但不限于6“-O-乙酰基染料木黄酮,6”-O-丙二酰基染料木黄酮,6-羧甲基染料木黄酮或槐苷。
[化学式2]
在本申请中,表没食子儿茶素没食子酸酯可指具有化学式3的结构的物质。
[化学式3]
此外,本申请的表没食子儿茶素没食子酸酯的衍生物可以但不限于选自(-)-表儿茶素没食子酸酯(ECG),没食子酸(GA),(-)-表没食子儿茶素(EGC),(-)-表儿茶素(EC)和没食子儿茶素(GC)。
在本申请的一方面,细胞系可以是黑色素细胞。
在本申请中,“细胞的复壮”可指将老化细胞恢复为年轻和健康的细胞。
在本申请中,“黑色素细胞细胞系”可指可以通过培养、对细胞系进行连续分裂或增殖的一系列克隆的黑色素细胞。
在本申请中,“传代培养体系”可指包括黑色素细胞母细胞系和通过将特定细胞系传代培养1至数代而获得的细胞系中的一种或多种的体系。
在本申请中,“黑色素细胞母细胞系”是指在传代培养之前的黑色素细胞细胞系。
在本申请中,“早期”和“晚期”基于细胞系的传代培养的次数进行分类。例如,“早期细胞系”可指作为黑色素细胞母细胞系的PA1细胞系和分别通过传代培养黑色素细胞母细胞系1、2、3、4和5次获得的PA2、PA3、PA4、PA5和PA6细胞系中的至少一种,且“晚期细胞系”可指分别通过传代培养黑色素细胞母细胞系6、7、8、9、10和11次的PA7、PA8、PA9、PA10、PA11和PA12细胞系中的至少一种。早期细胞系可以包括第一早期黑色素细胞细胞系和第二早期黑色素细胞细胞系,并且晚期黑色素细胞细胞系可以包括第一晚期黑色素细胞细胞系和第二晚期黑色素细胞细胞系。第一早期黑色素细胞细胞系可以包括PA1、PA2和PA3细胞系中的至少一种,第二种早期黑色素细胞细胞系可以包括PA4、PA5和PA6黑色素细胞细胞系中的至少一种。此外,第一晚期黑色素细胞细胞系可以包括PA7、PA8和PA9细胞系中的至少一种,第二晚期黑色素细胞细胞系可以包括PA10、PA11和PA12黑色素细胞细胞系中的至少一种。
在本申请中,第n代或PAn是指通过将母细胞系继代培养n-1次获得的细胞系。例如,第3代或PA3是指通过传代培养母细胞系2次获得的细胞系。如本文所用,n为整数。
在这方面,细胞的复壮可指使晚期细胞系的细胞恢复成与早期细胞系的细胞相同或相似的细胞。例如,这可指意味着使PA12细胞系恢复成PA1细胞系,PA12细胞系恢复成PA2细胞系、PA12细胞系恢复成PA3细胞系、PA12细胞系恢复成PA4细胞系、PA12细胞系恢复成PA5细胞系、PA12细胞系恢复成PA6细胞系、PA11细胞系恢复成PA1细胞系、PA11细胞系恢复成PA2细胞系、PA11细胞系恢复成PA3细胞系、PA11细胞系恢复成PA4细胞系、PA11细胞系恢复成PA5细胞系、PA11细胞系恢复成PA6细胞系、PA10细胞系恢复成PA1细胞系、PA10细胞系恢复成PA2细胞系、PA10细胞系恢复成PA3细胞系、PA10细胞系恢复成PA4细胞系、PA10细胞系恢复成PA5细胞系、PA10细胞系恢复成PA6细胞系、PA9细胞系恢复成PA1细胞系、PA9细胞系恢复成PA2细胞系、PA9细胞系恢复成PA3细胞系、PA9细胞系恢复成PA4细胞系、PA9细胞系恢复成PA5细胞系或PA9细胞系恢复成PA6细胞系。
在根据本申请的一个方面的组合物中,所述组合物可以将对应于已经传代培养6次或更多次的细胞系的细胞恢复成对应已经传代培养5次或更少次的包括在传代培养体系中的细胞系的细胞系的细胞。传代培养体系可以包括但不限于包含选自PA1、PA2、PA3、PA4、PA5和PA6细胞系中的一种或多种细胞系的早期黑色素细胞细胞系,其中PA1为黑色素细胞母细胞系,且通过分别传代培养母细胞系1、2、3、4和5次获得PA2、PA3、PA4、PA5和PA6;而晚期黑色素细胞细胞系包含选自分别通过传代培养黑色素细胞母细胞系6、7、8、9、10和11次而获得的PA7、PA8、PA9、PA10、PA11和PA12细胞系中的至少一种细胞系。
在根据本申请的一个方面的组合物中,传代培养体系可以包括但不限于PA1、PA2、PA3、PA4、PA5、PA6、PA7、PA8、PA9、PA10、PA11和PA12细胞系。
在这方面,传代培养的细胞系可以是登录号为KCTC 12906BP或KCTC 12907BP的细胞系。例如,早期黑色素细胞细胞系可以是登录号为KCTC 12906BP的细胞系,而晚期黑色素细胞细胞系可以是登录号为KCTC 12907BP的细胞系。具体地,PA3细胞系可以是登录号为KCTC 12906BP的细胞系,而PA11细胞系可以是登录号为KCTC 12907BP的细胞系。
在根据本申请的一个方面的组合物中,包含选自染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物及其溶剂合物中的一种或多种的所述组合物可以降低(但不限于)选自酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1(TRP-1)、酪氨酸酶相关蛋白-2(TRP-2)、EZH1(zeste同系物1的增强子)、p16和p21中的一种或多种的表达。在这方面,尽管包含酪氨酸酶的上述物质的表达水平可能随传代重复而增加,但是在含有根据本申请的一个方面的组合物的培养基中传代培养的细胞系(例如,第7代的细胞系)可表现出选自酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1、酪氨酸酶相关蛋白-2、EZH1、p16和p21中的一种或多种的表达水平相当于或低于早期细胞系(例如,第2代的细胞系)的表达水平。
在本申请的一方面,所述组合物可以降低选自p14、p19、p27、细胞周期蛋白D、细胞周期蛋白E、细胞周期蛋白依赖性激酶4(CDK4)、与卡介素相关的抗微生物肽(CRAMP)、微管解聚蛋白(stathmin)、EF-1a和几丁质酶3样蛋白3中的一种或多种的表达。在这方面,尽管包含p14的上述物质的表达水平可随着传代培养重复而增加,但已在含有根据本申请的一个方面的组合物的培养基中传代培养的细胞系(例如,第7代的细胞系)可表现出与早期细胞系的表达水平相当或更低的p14等的表达水平。
在根据本申请的一个方面的组合物中,组合物可以增加组蛋白脱乙酰酶5的表达。
表达变化的检测可以在组织、细胞(例如黑色素细胞)或体液(例如血液或组织液)中进行,但不限于此。
在根据本申请的一个方面的组合物中,所述组合物可以是药物、保健食品或化妆品组合物。
具体地,可以根据药物或化妆品领域中常用的方法以适当的单位剂型向患者施用所述组合物。适于此目的的剂型包括注射剂、用于局部应用的制剂等作为胃肠外制剂。具体地说,用于注射的制剂可以为等渗水溶液或悬浮液。然而,也可以包括常用的稀释剂或赋形剂,例如填料、增量剂、粘合剂、润湿剂、崩解剂、表面活性剂等。根据常用方法,本申请的组合物可以肠胃外施用,例如直接进入特定部位。可以使用注射器进行组合物的注射。应当理解,考虑各种相关因素,包括施用途径、体重、患者年龄和性别等确定活性成分的实际施用剂量。包含在单一单位剂量中的组合物的浓度可以是例如1μM。然而,本申请的范围不受通过任何方式施用的剂量的限制。
在根据本申请的一个方面的组合物中,组合物可以是用于局部应用的组合物。
在根据本申请的一个方面的组合物中,组合物可以是化妆品组合物。具体地,化妆品组合物可以是底妆化妆品、彩妆化妆品、发用化妆品、身体美容等形式,其制剂没有特别限制,并且可以根据目的适当选择。
例如,所述化妆品组合物可以配制成但不限于溶液、悬浮液、乳液、糊剂、凝胶、霜剂、洗剂、粉末、肥皂、含表面活性剂的清洁剂、油、粉末、乳液粉底、蜡底、喷雾剂等。更具体地,所述化妆品组合物可以配制成基础化妆品如软化洗剂、滋养洗剂、洗剂、身体洗剂、滋养霜、按摩霜、保湿霜、护手霜、精华液、眼霜、清洁霜、清洁泡沫、洁肤水、面膜、凝胶、贴剂、水包油(O/W)或油包水(W/O)乳液等,着色化妆品如口红、底妆、粉底等,清洁剂如洗发剂、漂洗剂、洁肤露、牙膏、漱口水等,或发用化妆品,例如护发素、发胶、发用摩丝、生发剂、染发剂等。
化妆品组合物含有美容上可接受的介质或碱。其可以以适于局部施用的任何制剂的形式提供,例如溶液、凝胶、固体、无水糊剂、水包油乳液、悬浮液、微乳液、微胶囊、微粒、或离子(脂质体)和/或非离子型囊泡分散体,或以霜、皮肤洗剂、洗剂、粉末、软膏、喷雾剂或隐形棒的形式提供。这些制剂可以通过本领域常用的方法制备。
当本申请的制剂为溶液或乳液时,使用溶剂、增溶剂或乳化剂作为载体成分。例如,使用水、乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇油、甘油脂肪酸酯、聚乙二醇或脱水山梨醇的脂肪酸酯。
当本申请的制剂为悬浮液时,液体稀释剂如水、乙醇或丙二醇,悬浮剂如乙氧基化异硬脂醇、聚氧乙烯山梨糖醇酯和聚氧乙烯脱水山梨醇酯、微晶纤维素、偏氢氧化铝(aluminum metahydroxide)、膨润土、琼脂、或黄蓍胶等可用作载体成分。
当本申请的制剂为糊剂、霜或凝胶、动物油、植物油、蜡、石蜡、淀粉、黄蓍胶、纤维素衍生物、聚乙二醇、硅、膨润土、二氧化硅、滑石、锌氧化物等可以用作载体成分。
当本发明的制剂为粉末或喷雾剂时,可以使用乳糖、滑石、二氧化硅、氢氧化铝、硅酸钙,聚酰胺粉末等作为载体成分。特别地,当制剂是喷雾剂时,其还可以含有推进剂如氯氟烃、丙烷/丁烷或二甲醚。
在本申请的示例性实施例中,化妆品组合物还可以含有增稠剂。包括在本申请的化妆品组合物中的增稠剂可以为甲基纤维素、羧甲基纤维素、羧甲基羟基鸟嘌呤、羟甲基纤维素、羟乙基纤维素、羧乙烯基聚合物、聚季铵盐、鲸蜡硬脂醇、硬脂酸、角叉菜胶等。优选地,可以使用羧甲基纤维素、羧基乙烯基聚合物和聚季铵盐中的一种或多种。最优选地,可以使用羧乙烯基聚合物。
在本申请的示例性实施方案中,如果需要,所述化妆品组合物可以含有各种基质和添加剂。普通技术人员可轻易地选择这些成分的种类和量。如果需要,所述化妆品组合物还可以包括本领域常用的可接受的佐剂,例如,防腐剂、颜料、添加剂等。
具体地,防腐剂可以是苯氧基乙醇、1,2-己二醇等,且香料可以是合成香料。
并且,在本申请的一个示例性实施例中,所述化妆品组合物可以包含选自水溶性维生素、油溶性维生素、多肽、多糖、鞘脂和海藻提取物中的一种或多种。另外,还可以加入、润肤剂、表面活性剂、有机或无机颜料、有机粉末、紫外线吸收剂、防腐剂、杀菌剂、抗氧化剂、植物提取物、pH控制剂、醇、颜料、香料、血液循环促进剂、冷却剂、止汗剂、纯净水等作为化合物
所述保健食品可指但不限于通过使用日常饮食可能不足的营养物或对人体有用的成分(功能成分)而制备,并且通过维持人体的正常功能或刺激生理功能来维持和改善健康的食品。所述保健食品可以但不限于以如下形式制备和加工:片剂、胶囊、粉末、颗粒、液体、丸剂等。所述保健食品可以根据法定形式制备和加工:
在本申请的一方面,所述保健饮料组合物除了含有必需的活性成分之外还可以含有其它成分。除了以有针对性的比例含有作为必要成分的上述化合物之外,所述保健饮料组合物对含有其它成分没有特别限制。如在普通饮料中,所述保健饮料组合物可以含有各种口味,天然碳水化合物等。天然碳水化合物可以为单糖、多糖、糖如环糊精等,或糖醇如木糖醇、山梨糖醇、赤藓糖醇等。可以使用除了上述调味料以外的调味料,天然调味料(索马甜(thaumatin)、甜叶菊提取物(例如,莱鲍迪苷A、甘草甜素等))和合成香料(例如,糖精、阿斯巴甜(aspartame)等)。
通常,通过保健食品组合物施用的活性成分的量可以为0.0001mg/kg/天至约1000mg/kg/天,更具体地为0.02mg/kg//天至100mg/kg/天。一天可以施用一次或若干次。
另外含有的成分不限于上述成分,且也可以在不对本申请的目的和效果产生负面影响的范围内添加任何成分。
在根据本申请的一个方面的组合物中,所述组合物可以包括但不限于0.001-2.0μM或0.005-1.5μM的染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物或其溶剂合物。具体而言,在本申请的一方面,组合物中包含的染料木黄酮、表没食子儿茶素没食子酸酯、其衍生物、其异构体、其药学上可接受的盐、前体药物、其水合物或其溶剂合物的浓度可以是但不限于至少0.0001μM、至少0.001μM、至少0.002μM、至少0.003μM、至少0.004μM、至少0.005μM、至少0.006μM、至少0.007μM、至少0.008μM、至少0.009μM、至少0.01μM、至少0.15μM、至少0.15μM、至少0.2μM、至少0.25μM、至少0.3μM、至少0.35μM、至少0.4μM、至少0.45μM、至少0.5μM、至少0.6μM、至少0.7μM、至少0.8μM、至少0.9μM、至少1.0μM、至少1.1μM、至少1.2μM、至少1.3μM、至少1.4μM、至少1.5μM、至少1.6μM、至少1.7μM、至少1.8μM、至少1.9μM,或至少2.0μM,并且可以是但不限于至多2.1μM、至多2.0μM、至多1.9μM、至多1.8μM、至多1.7μM、至多1.6μM、至多1.5μM、至多1.4μM、至多1.3μM、至多1.2μM、至多1.1μM、至多1.0μM、至多0.9μM、至多0.85μM、至多0.80μM、至多0.75μM、至多0.70μM、至多0.65μM、至多0.60μM、至多0.55μM、至多0.50μM、至多0.45μM、至多0.40μM、至多0.35μM、至多0.30μM、至多0.25μM、至多0.20μM、至多0.15μM、至多0.10μM、至多0.05μM、至多0.04μM、至多0.03μM、至多0.02μM、至多0.01μM、至多0.005μM、至多0.004μM、至多0.003μM、至多0.002μM、至多0.001μM、至多0.0005μM。
以下,通过实施例对本申请进行详细说明。然而,以下实施例仅用于说明目的,本申请的范围不受实施例的限制。
[实施例1]黑色素细胞的传代培养
将人原代黑色素细胞(Life Technologies,CA,USA)在补充有人黑色素细胞生长添加物(HMGS;Gibco BRL,NY,USA)的M-254培养基(Gibco BRL,NY,USA)中,置于5%CO2培养箱,室温下在100mm培养板上培养,每隔一天更换培养基直到细胞占板面积的80%,然后以1:4的比例在另一个100-mm培养板上传代培养。
首次置于100-mm培养板上的黑色素细胞被认为是第1代(PA1),传代培养进行11次(PA12)。
[实施例2]观察与黑色素细胞的黑色素生成能力提高相关的因子的表达的变化
基于已知与黑色素生成相关的因子(酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2))的表达水平的变化,调查了染料木黄酮和表没食子儿茶素没食子酸酯(EGCG)对晚期黑色素细胞细胞系的影响。
作为对照组,将黑色素细胞传代培养至第11代和第12代而无需任何处理,作为试验组,将细胞在与对照组相同的环境下在含有1μM染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的培养基中传代培养至第6代,然后从第7代传代培养至第11代和第12代。
具体地,为了研究已知与黑色素生成有关的因子(酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2))的表达水平的变化的关系进行Q-PCR。从分离自人类黑色素细胞的RNA合成cDNA,并使用各自的引物通过Q-PCR(Applied Biosystems,7500Fast)测量mRNA的量的变化。以95℃进行15秒且60℃下进行60秒,重复40个循环进行Q-PCR实验。测量相对于对照组增加的基因表达。使用AppliedBiosystems制造的以下TaqMan引物:酪氨酸酶(TYR,商品编号:Hs01099965_m1),MITF(商品编号:Hs01117294_m1),TRP1(商品编号:Hs00167051_m1),TRP2(商品编号:Hs01095856_m1)。
结果(图1A-1D),查明试验组的早期细胞系酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1(TRP-1)和酪氨酸酶相关蛋白-2(TRP-2)的mRNA表达水平显著下降,而对照组增加(图中纵坐标为作为相应的基因的相对表达水平的每个基因的基因表达水平/GAPDH mRNA表达水平)。
[实施例3]黑色素细胞衰老标记物的观察
研究老化细胞中高水平存在的p16和p21的表达水平是否受到染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的影响。
对照组和试验组与实施例2相同。
具体地,由不同传代的黑色素细胞的RNA合成cDNA,并使用p16和p21的特异引物通过Q-PCR(Applied Biosystems,7500Fast)测量mRNA的量的变化。使用Applied Biosystems制造的以下TaqMan引物:p16(商品编号:Hs00923894_m1),p21(商品编号:Hs00355782_m1)。
结果(图2),在晚期细胞系中,p16和p21的表达在对照组中大大增加,但在试验组中显著降低(图中纵坐标为作为相应基因的相对表达水平的每个基因的基因表达水平/GAPDH mRNA表达水平)。
[实施例4]参与黑色素生成的酶的表达变化的观察
为了研究EZH1(zeste同系物1的增强子)对黑色素生成的影响,使用siRNA抑制EZH1蛋白的表达。结果,黑色素的生成大大减少,这表明EZH1抑制黑色素生成(图3,底部)。
还研究了EZH1的表达水平是否受染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)的影响。
对照组和试验组与实施例2相同。
具体地,从对照组和试验组的黑色素细胞的RNA合成cDNA,使用EZH1特异引物,通过Q-PCR(Applied Biosystems,7500Fast)测定mRNA的量的变化。使用Applied Biosystems制造的以下TaqMan EZH1引物:EZH1(商品编号:Hs00940463_m1)。
结果(图3),晚期细胞系中EZH1的表达在对照组中显著增加,但在试验组中显著降低(图中纵坐标为作为相应基因的相对表达水平的每个基因的基因表达水平/GAPDH mRNA表达水平)。
[实施例5]黑色素细胞的表型的观察
为了研究染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)是否影响黑色素细胞的表型,用光学显微镜(放大规格:400X)观察对照组和试验组的黑色素细胞的表型。用光学显微镜以40倍放大倍数观察培养基中包含的细胞的表型。
从图4A-4B中可以看出,还查明晚期细胞系中的黑色素细胞的表型变得平坦,细胞体尺寸增加,树突数量和异质性增加(由于老化增加而形状变化的细胞数量增加;细胞体直径至少为3μm的细胞数量增加),然而长时间暴露于染料木黄酮或表没食子儿茶素没食子酸酯(EGCG)后,细胞类似地恢复为传代培养早期的细胞。
在下文中,将通过制剂实施例详细描述本申请。然而,以下制剂实施例仅用于说明目的,本申请的范围不受该制剂实施例的限制。
[制剂实施例1]软膏
【表1】
成分 | 含量(wt%) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 0.1 |
甘油 | 8.0 |
丁二醇 | 4.0 |
液态石蜡 | 15.0 |
β-葡聚糖 | 7.0 |
卡波姆 | 0.1 |
辛酸/癸酸甘油三酯 | 3.0 |
角鲨烷 | 1.0 |
鲸蜡硬脂基葡糖苷 | 1.5 |
脱水山梨醇硬脂酸酯 | 0.4 |
鲸蜡硬脂醇 | 1.0 |
蜂蜡 | 4.0 |
防腐剂、颜料和香料 | 适量 |
纯净水 | 余量 |
[制剂实施例2]按摩霜
【表2】
成分 | 含量(wt%) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 1.5 |
甘油 | 8.0 |
丁二醇 | 4.0 |
液态石蜡 | 45.0 |
β-葡聚糖 | 7.0 |
卡波姆 | 0.1 |
辛酸/癸酸甘油三酯 | 3.0 |
蜂蜡 | 4.0 |
鲸蜡硬脂基葡糖苷 | 1.5 |
失水山梨醇倍半油酸酯 | 0.9 |
凡士林 | 3.0 |
石蜡 | 1.5 |
防腐剂、颜料和香料 | 适量 |
纯净水 | 余量 |
[制剂实施例3]软化洗剂(皮肤洗剂)
【表3】
成分 | 含量(wt%) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 0.1 |
甘油 | 3.0 |
丁二醇 | 2. |
丙二醇 | 2.0 |
聚羧乙烯 | 0.1 |
PEG-12壬基苯基醚 | 0.2 |
聚山梨酯80 | 0.4 |
乙醇 | 10.0 |
三乙醇胺 | 0.1 |
防腐剂、颜料和香料 | 适量 |
纯净水 | 余量 |
[制剂实施例4]滋润洗剂(牛奶洗剂)
【表4】
成分 | 含量(wt%) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 0.5 |
甘油 | 3.0 |
丁二醇 | 3.0 |
丙二醇 | 3.0 |
聚羧乙烯 | 0.1 |
蜂蜡 | 4.0 |
聚山梨酯60 | 1.5 |
辛酸/癸酸甘油三酯 | 5.0 |
角鲨烷 | 5.0 |
失水山梨醇倍半油酸酯 | 1.5 |
液态石蜡 | 0.5 |
鲸蜡硬脂醇 | 1.0 |
三乙醇胺 | 0.2 |
防腐剂、颜料和香料 | 适量 |
纯净水 | 余量 |
[制剂实施例5]护发膜
【表5】
成分 | 含量(wt%) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 0.1 |
甘油 | 4.0 |
聚乙烯醇 | 15.0 |
透明质酸提取物 | 5.0 |
β-葡聚糖 | 7.0 |
尿囊素 | 0.1 |
壬基苯基醚 | 0.4 |
聚山梨酯60 | 1.2 |
乙醇 | 6.0 |
防腐剂、颜料和香料 | 适量 |
纯净水 | 余量 |
[制剂实施例6]注射剂
【表6】
成分 | 含量(ppm) |
染料木黄酮或表没食子儿茶素没食子酸酯 | 10 |
用于注射剂的无菌蒸馏水 | 适量 |
pH控制剂 | 适量 |
虽然已经展示并阐述了示例性实施例,但是本领域技术人员会理解,在不脱离由所附权利要求限定的本申请的精神和范围的情况下,可以对形式和细节进行各种改变。
【保藏号】
保藏单位:韩国生物科学和生物技术研究所
登录号:KCTC 12906BP
登录日期:2015年9月17日
保藏单位:韩国生物科学和生物技术研究所
登录号:KCTC 12907BP
登录日期:2015年9月17日
中文译文
国际承认的用于专利程序的微生物保藏的布达佩斯条约
国际版
由本页下端的保藏机构根据第7条第1款规定所发出的
原始保藏证明
至:李恩憬
株式会社爱茉莉太平洋技术研究院
韩国京畿道龙仁市器兴区龙句大路1920
BP/4表(KCTC版17)
单页
中文译文
国际承认的用于专利程序的微生物保藏的布达佩斯条约
国际版
由本页下端的保藏机构根据第7条第1款规定所发出的
原始保藏证明
至:李恩憬
株式会社爱茉莉太平洋技术研究院
韩国京畿道龙仁市器兴区龙句大路1920
BP/4表(KCTC版17)
单页
Claims (11)
1.一种用于诱导细胞复壮的组合物,其包含选自染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物和溶剂合物中的一种或多种作为活性成分。
2.根据权利要求1所述的组合物,其特征在于,所述细胞为黑色素细胞。
3.根据权利要求1或2所述的组合物,其特征在于,所述组合物将对应于已经传代培养至少6次的细胞系的细胞恢复为在包含于传代培养体系中的细胞系中对应于已传代培养至多5次的细胞系的细胞,
所述传代培养体系包括:
早期黑色素细胞细胞系,其包含选自PA1、PA2、PA3、PA4、PA5和PA6细胞系中的一种或多种细胞系,其中PA1为黑色素细胞母细胞系,PA2、PA3、PA4、PA5和PA6通过将所述母细胞系分别传代培养1次、2次、3次、4次和5次而获得;和
晚期黑色素细胞细胞系,其包含通过将母细胞系分别传代培养6、7、8、9、10和11次而获得的PA7、PA8、PA9、PA10、PA11和PA12细胞系中的一种或多种细胞系。
4.根据权利要求3所述的组合物,其特征在于,所述传代培养体系由PA1、PA2、PA3、PA4、PA5、PA6、PA7、PA8、PA9、PA10、PA11和PA12细胞系组成。
5.根据权利要求3所述的组合物,其特征在于,通过传代培养获得的细胞系为登录号KCTC12906BP或KCTC 12907BP的细胞系。
6.根据权利要求1或2所述的组合物,其特征在于,所述组合物降低选自酪氨酸酶、小眼畸形相关转录因子(MITF)、酪氨酸酶相关蛋白-1(TRP-1)、酪氨酸酶相关蛋白-2(TRP-2)、EZH1(zeste同系物1的增强子)、p16和p21中的一种或多种的表达。
7.根据权利要求1或2所述的组合物,其特征在于,所述组合物增加组蛋白脱乙酰酶5的表达。
8.根据权利要求1或2所述的组合物,其特征在于,所述组合物为药物、保健食品或化妆品组合物。
9.根据权利要求1或2所述的组合物,其特征在于,所述组合物施用于皮肤或局部注射。
10.根据权利要求1或2所述的组合物,其特征在于,所述组合物含有0.001~2.0μM的染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物或其溶剂合物。
11.根据权利要求1或2所述的组合物,其特征在于,所述组合物含有0.005~1.5μM的染料木黄酮、表没食子儿茶素没食子酸酯(EGCG)、其衍生物、其异构体、其药学上可接受的盐、其前药、其水合物或其溶剂合物。
Applications Claiming Priority (3)
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PCT/KR2015/012881 WO2016085302A1 (ko) | 2014-11-28 | 2015-11-27 | 제니스테인 또는 에피갈로카테친 갈레이트를 포함하는 세포 회춘 촉진 유도용 조성물 |
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EP (1) | EP3225238B1 (zh) |
JP (1) | JP2017537072A (zh) |
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CN109825518A (zh) * | 2019-02-25 | 2019-05-31 | 佐登妮丝(广州)美容化妆品有限公司 | 3′-羟基染料木黄酮的制备方法及其用途 |
CN112912057A (zh) * | 2018-11-06 | 2021-06-04 | 株式会社爱茉莉太平洋 | 含有聚酯型儿茶素a的化妆料组合物 |
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CN115252519A (zh) * | 2022-08-03 | 2022-11-01 | 浙江熙正霖生物科技有限公司 | 一种具有生物活性成分的护发精华液及其制备方法 |
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EP3225238A4 (en) | 2018-07-18 |
JP2017537072A (ja) | 2017-12-14 |
EP3225238B1 (en) | 2021-12-29 |
WO2016085302A1 (ko) | 2016-06-02 |
KR20160065035A (ko) | 2016-06-08 |
US20170304175A1 (en) | 2017-10-26 |
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