CN113509478B - 育发生发促进剂 - Google Patents
育发生发促进剂 Download PDFInfo
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- CN113509478B CN113509478B CN202110856186.0A CN202110856186A CN113509478B CN 113509478 B CN113509478 B CN 113509478B CN 202110856186 A CN202110856186 A CN 202110856186A CN 113509478 B CN113509478 B CN 113509478B
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- hair
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- hair papilla
- acid
- papilla cells
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Abstract
本公开提供一种含有式(I)所表示的一种或多种类黄酮类作为有效成分的育发生发促进剂。此外,本公开提供一种含有式(II)所表示的一种或多种苯基丙烷类作为有效成分的育发生发促进剂。
Description
本申请为2016年12月02日提交的申请号为201680034927.4且发明名称为“育发生发促进剂”的专利申请的分案申请。
相关申请的交叉引用
本国际申请要求2015年12月2日在日本专利局提交的日本发明专利申请第2015-235965号的优先权,所述日本发明专利申请的全部内容通过引用而并入本文。
技术领域
本公开涉及育发生发促进剂。
背景技术
毛发的生长按照由生长期、退化期及休止期形成的周期性毛发周期(毛周期)而反复生长及脱落。在毛发周期中,各种细胞发挥作用,而在控制该毛发周期的方面上发挥最重要作用的是毛***细胞。毛***细胞是位于毛根的根干部分的细胞。毛***细胞对存在于毛***细胞周围的毛母细胞产生作用,从而促进毛母细胞的增殖等,在毛发分化中发挥着重要的作用。
此外,会因年龄增长、主要由生活环境所引起的应激、激素平衡的破坏、药物的副作用等产生脱发。若因如上所述的各种原因而使毛***细胞的代谢下降,则会抑制毛母细胞的活化及分化。并且,还会因毛发周期的时间缩短而产生脱发。
近年来,由于应激的增加或饮食生活的变化等各种主要因素,导致因毛发稀疏或脱发而烦恼的男女有增加的倾向,从而提升了对育发生发促进剂的期待。应上述社会需求而开发了各种育发生发促进剂。作为育发生发促进剂的有效成分,例如已知有米诺地尔(化学名:6-(1-哌啶基)-2,4-嘧啶二胺-3-氧化物)或腺苷相关化合物等。
已知米诺地尔通过扩张末梢血管而促进血液循环,从而通过刺激毛***细胞而促进育发。此外,公开有腺苷及其衍生物对毛***细胞的腺苷受体产生作用,促进成纤维细胞生长因子的产生,从而具有使毛母细胞活化的效果(参照专利文献1)。
现有技术文献
专利文献
专利文献1:日本特开第2000-297015号公报
发明内容
发明要解决的问题
然而,关于如上所述的公知的育发生发促进剂,仍未充分地满足需求者的要求。基于上述情况,市场上需要育发生发促进效果更高的育发生发促进剂。
解决问题的技术方案
为了解决如上所述的问题,发明人进行了努力研究,结果发现,以往与育发作用的关系完全未知的类黄酮类及苯基丙烷类具有育发作用。
即,本公开的一个方面提供一种育发生发促进剂,其含有下述式(Ⅰ)所表示的一种或多种类黄酮类作为有效成分。
式(Ⅰ)
其中,上述式(Ⅰ)中,R1为糖基、糖基-糖基-O-芥子酰基或H,R2为O-芥子酰基或OH,R3为H或OH,R4为O-糖基、OH或O-糖基-芥子酰基。
此外,本公开的一个方面提供一种育发生发促进剂,其含有下述式(II)所表示的一种或多种苯基丙烷类作为有效成分。
式(II)
其中,上述式(II)中,R5为H或OH,R6为OH或OCH3。
根据发明者人的研究结果,上述式(Ⅰ)所表示的一种或多种类黄酮类以及上述式(II)所表示的一种或多种苯基丙烷类具有活化毛***细胞的作用。将由下述实施形态阐明具有上述作用。因此,若使用含有上述类黄酮类及上述苯基丙烷类作为有效成分的育发生发促进剂,则可期待极其优异的育发生发效果。
附图说明
图1是表示使用人头发毛***细胞进行异肥皂草苷的细胞毒性试验所得的结果的图。
图2是表示对使用人头发毛***细胞进行异肥皂草苷处理1天后的细胞活化作用进行研究所得的结果的图。
图3是表示对使用人头发毛***细胞进行异肥皂草苷处理3天后的细胞活化作用进行研究所得的结果的图。
图4是表示对使用人头发毛***细胞进行异肥皂草苷处理3天后的VEGF产生促进作用进行研究所得的结果的图。
图5是表示使用人头发毛***细胞进行芹菜素的细胞毒性试验所得的结果的图。
图6是表示使用人头发毛***细胞进行香豆酸的细胞毒性试验所得的结果的图。
图7是表示使用人头发毛***细胞进行咖啡酸甲酯的细胞毒性试验所得的结果的图。
图8是表示对由使用人头发毛***细胞的除异肥皂草苷以外的受验物质所产生的细胞活化作用进行研究所得的结果的图。
图9是表示由使用人头发毛***细胞的除异肥皂草苷以外的受验物质所产生的VEGF产生量的图。
图10是表示由使用人头发毛***细胞的除异肥皂草苷以外的受验物质所产生的FGF-7基因表达量的图。
具体实施方式
本公开的育发生发促进剂含有上述式(Ⅰ)所表示的一种或多种类黄酮类作为有效成分。此外,本公开的育发生发促进剂含有上述式(II)所表示的一种或多种苯基丙烷类作为有效成分。在此,“含有”是指本公开的育发生发促进剂既可以由一种或多种类黄酮类构成,也可以含有除一种或多种类黄酮类以外的其他成分。此外,本公开的“育发生发促进剂”是通过下述机制而发挥育发效果、生发效果、育发效果兼生发效果的物质。育发效果是指使毛发发育,并预防脱发以及减少脱发的效果。生发效果是指使毛发产生的效果。因此,本公开的育发生发促进剂可用作育发剂、生发剂、以及育发剂兼生发剂的任一者。
作为上述式(Ⅰ)所表示的类黄酮类,可列举例如:上述式(Ⅰ)中,R1为糖基,R2为OH,R3为H,以及R4为O-糖基所表示的异肥皂草苷[别名:异牡荆黄素4'-O-β-D-吡喃葡萄糖苷(isovitexin4'-O-β-D-glucopyranoside)];上述式(Ⅰ)中,R1为糖基,R2为O-芥子酰基,R3为H,以及R4为O-糖基所表示的7-O-反式-芥子酰基异牡荆黄素4'-O-β-D-吡喃葡萄糖苷(7-O-trans-sinapoylisovitexin4'-O-β-D-glucopyranoside);上述式(Ⅰ)中,R1为糖基,R2为O-芥子酰基,R3为H,以及R4为OH所表示的7-O-反式-芥子酰基异牡荆黄素(7-O-trans-sinapoylisovitexin);上述式(Ⅰ)中,R1为糖基,R2为O-芥子酰基,R3为H,以及R4为O-糖基-芥子酰基所表示的7-O-反式-芥子酰基异牡荆黄素4'-O-(6-O-反式-芥子酰基-β-D-吡喃葡萄糖苷)(7-O-trans-sinapoylisovitexin4'-O-(6-O-trans-sinapoyl-β-D-glucopyranoside));上述式(Ⅰ)中,R1为糖基-糖基-O-芥子酰基,R2为O-芥子酰基,R3为H,以及R4为OH所表示的6’’-O-(2-O-反式-芥子酰基-β-D-吡喃葡萄糖基)-7-O-反式-芥子酰基异牡荆黄素(6’’-O-(2-O-trans-sinapoyl-β-D-glucopyranosyl)-7-O-trans-sinapoylisovitexin);上述式(Ⅰ)中,R1为糖基-糖基-O-芥子酰基,R2为O-芥子酰基,R3为H,以及R4为O-糖基所表示的6’’-O-(2-O-反式-芥子酰基-β-D-吡喃葡萄糖基)-7-O-反式-芥子酰基异牡荆黄素4'-O-β-D-吡喃葡萄糖苷(6’’-O-(2-O-trans-sinapoyl-β-D-glucopyranosyl)-7-O-trans-sinapoylisovitexin4'-O-β-D-glucopyranoside);上述式(Ⅰ)中,R1为糖基,R2为OH,R3为H,以及R4为OH所表示的异牡荆黄素(isovitexin);上述式(Ⅰ)中,R1为糖基,R2为OH,R3为OH,以及R4为OH所表示的异荭草素(isoorientin);上述式(Ⅰ)中,R1为糖基,R2为OH,R3为OH,以及R4为O-糖基所表示的异荭草素4'-O-β-D-吡喃葡萄糖苷(isoorientin4'-O-β-D-glucopyranoside);上述式(Ⅰ)中,R1为H,R2为OH,R3为H,以及R4为OH所表示的芹菜素[别名:2-(4-羟苯基)-5-羟基-7-羟基-4H-1-苯并吡喃-4-酮(2-(4-hydroxyphenyl)-5-hydroxy-7-hydroxy-4H-1-benzopyran-4-one)];上述式(Ⅰ)中,R1为H,R2为OH,R3为OH,以及R4为OH所表示的木犀草素[别名:2-(3-羟基-4-羟苯基)-5-羟基-7-羟基-4H-1-苯并吡喃-4-酮(2-(3-hydroxy-4-hydroxyphenyl)-5-hydroxy-7-hydroxy-4H-1-benzopyran-4-one)]等。进而,上述式(Ⅰ)所表示的类黄酮类优选为异肥皂草苷。此外,异肥皂草苷的基于IUPAC的名称为4'-(β-D-吡喃葡萄糖氧基)-5,7-二羟基-6-β-D-吡喃葡萄糖黄酮。这些类黄酮类可单独使用,亦可多种混合使用。
作为上述式(II)所表示的苯基丙烷类,可列举例如:上述式(II)中,R5为H,R6为OH所表示的香豆酸;R5为OH,R6为OCH3所表示的咖啡酸甲酯等。这些苯基丙烷类可单独使用,亦可多种混合使用。此外,也可以混合使用类黄酮类以及苯基丙烷类。
上述式(Ⅰ)所表示的类黄酮类及上述式(II)所表示的苯基丙烷类可通过化学合成法获得,也可以是从植物中提取精炼而成的。作为所述植物,可列举例如:十字花科植物、石竹科植物(王不留行,学名:Vaccaria segetalis)、伞形科植物、禾本科植物、白花菜科植物、番木瓜科植物、木犀草科植物、旱金莲科植物等。由此,上述式(Ⅰ)所表示的类黄酮类及上述式(II)所表示的苯基丙烷类可以是从上述这些植物中的至少1种提取的成分。例如,上述式(Ⅰ)所表示的类黄酮类及上述式(II)所表示的苯基丙烷类可以是从作为十字花科植物的山萮菜(Wasabia japonica)[别名:山葵]和西洋山萮菜(Armoracia rusticana)[别名:西洋山葵]中的至少一者提取的成分。
作为从所述植物提取精炼的方法,可列举例如:从山萮菜或西洋山萮菜提取上述式(Ⅰ)所表示的类黄酮类和上述式(II)所表示的苯基丙烷类的方法等。尤其是,国际公开第2011/058661号公开了从山萮菜提取异肥皂草苷的方法。与除异肥皂草苷以外的上述式(Ⅰ)所表示的类黄酮类相比,从山萮菜提取的提取物中含有更多的异肥皂草苷。因此,异肥皂草苷可以是从山萮菜提取的成分。
如下述实施例1所示,确认到上述式(Ⅰ)所表示的类黄酮类会活化毛***细胞。
此外,如下述实施例2所示,确认到上述式(Ⅰ)所表示的类黄酮类会增加毛***细胞中的血管内皮细胞生长因子(VEGF:vascular endothelial growth factor)的量。
如下述实施例7所示,确认到上述式(II)所表示的苯基丙烷类会活化毛***细胞,增加VEGF产生量,以及增加FGF-7基因表达量。
在此,已知VEGF对血管内皮细胞起作用,从而促进细胞的增殖、迁移,或者促进血管生成,又或者增加血管通透性。VEGF产生于所有细胞中,亦自毛***细胞产生。自毛***细胞产生的VEGF被认为会促进血管生成,并由此使存在于毛***细胞周围的毛母细胞活化,从而促进生发。因此可以认为,若具有VEGF产生促进作用,则会有助于毛母细胞活化作用、促进生发作用等。
FGF-7是自毛***细胞分泌的成纤维细胞生长因子。FGF-7被认为对毛母细胞起作用而促进其增殖,并由此促进毛发的生长,于毛发周期中的生长期有效。
如上所述,可认为上述式(Ⅰ)所表示的类黄酮类对毛***细胞具有细胞活化作用及VEGF产生促进作用,故而有非常好的育发生发作用。因此,本公开的育发生发促进剂因含有上述式(Ⅰ)所表示的类黄酮类,所以会发挥优异的育发效果、生发效果、以及育发效果兼生发效果。此外,如上所述,可认为上述式(II)所表示的苯基丙烷类具有毛***细胞活化作用、VEGF产生促进作用、以及FGF-7基因促进作用,故而有非常好的育发生发作用。因此,本公开的育发生发促进剂因含有上述式(II)所表示的苯基丙烷类,所以会发挥优异的育发效果、生发效果、以及育发效果兼生发效果。此外,本公开的育发生发促进剂还可用作毛***细胞活化促进剂、以及毛***细胞的VEGF产生促进剂。
上述式(Ⅰ)所表示的类黄酮类可调配于化妆品及药品中。此外,上述式(II)所表示的苯基丙烷类可调配于化妆品及药品中。因此,本公开的育发生发促进剂可用作化妆品及药品。
当以化妆品、药品的形式实施本公开的育发生发促进剂时,也可以用作皮肤外用剂。不过,育发生发促进剂亦可口服摄取,故而并不限定于皮肤外用剂。
当以化妆品的形式实施本公开的育发生发促进剂时,化妆品中的类黄酮类的含量优选为0.001μmol/L~5000μmol/L,更优选为0.1μmol/L~1000 μmol/L。当以药品的形式实施本公开的育发生发促进剂时,药品中的类黄酮类的含量优选为0.1mg~5.0g,更优选为0.2mg~1.0g。此外,该药品的成人每人的日剂量优选为1mg~10g,更优选为20mg~1.0g。
本公开的育发生发促进剂中,除了作为有效成分的上述式(Ⅰ)所表示的类黄酮类,亦可在无损本公开的效果的范围内,视需要适当调配通常用于化妆品及药品中的各种任意成分。此外,本公开的育发生发促进剂中,除了作为有效成分的上述式(II)所表示的苯基丙烷类,亦可在无损本公开的效果的范围内,视需要适当调配通常用于化妆品及药品中的各种任意成分。例如,可任意地选择并用下列例示的成分或添加剂。并且,基于增强育发生发效果的目的而视需要调配抗氧化剂等任意的药效成分、生理活性物质、及一般已知对育发生发效果有效的成分等,由此与本公开的构成成分发挥协同效应,从而带来超出通常预期的优异的使用效果。
例如,作为油脂类,可列举:大豆油、月见草油、米胚芽油、米糠油、小麦胚芽油、鳄梨油、葡萄籽油、山茶油、荷荷芭油、苏子油、橄榄油、芝麻油、可可油、春黄菊油、胡萝卜油、黄瓜籽油、牛油脂肪酸、椰果油、红花油、玉米油、菜籽油、蓖麻油、棉籽油、花生油、貂油、蛋黄油、棕榈油、棕榈仁油、椰子油、牛油、猪油、乳木果油、角鲨烯、角鲨烷、姥鲛烷或这些油脂类的氢化物(硬化油等)等。
例如,作为蜡类,可列举:蜂蜡、白蜂蜡、巴西棕榈蜡、鲸蜡、羊毛脂类、小烛树蜡、褐煤蜡、紫胶蜡、米糠蜡等。
例如,作为矿物油,可列举:液状石蜡、凡士林、石蜡、地蜡、纯地蜡、微晶蜡等。
例如,作为脂肪酸,可列举:月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、山萮酸、油酸、亚油酸、亚麻酸、二十二碳六烯酸、二十碳五烯酸、羊毛脂脂肪酸等天然脂肪酸、异戊酸等脂肪酸等。
例如,作为醇类,可列举:乙醇、异丙醇、乙二醇、月桂醇、十六烷醇、硬脂醇、油醇、羊毛脂醇、胆固醇、苯氧乙醇、2-己基癸醇、异硬脂醇、2-辛基十二烷醇等,作为多元醇类,可列举:乙二醇、二甘醇、三甘醇、乙二醇单***、乙二醇单丁醚、聚乙二醇、环氧丙烷、丙二醇、聚丙二醇、1,3-丁二醇、戊二醇、丙三醇、季戊四醇、苏糖醇、阿糖醇、木糖醇、半乳糖醇、山梨糖醇、甘露醇、乳糖醇、麦芽糖醇等。
例如,作为酯类,可列举:肉豆蔻酸异丙酯、棕榈酸异丙酯、硬脂酸丁酯、月桂酸己酯、肉豆蔻酸肉豆蔻酯、油酸油醇酯、油酸癸酯、肉豆蔻酸辛基十二烷基酯、二甲基辛酸己基癸酯、乳酸鲸蜡酯、乳酸肉豆蔻酯、邻苯二甲酸二乙酯、邻苯二甲酸二丁酯、乙酰化羊毛脂、乙二醇单硬脂酸酯、丙二醇单硬脂酸酯、丙三醇单硬脂酸酯、丙二醇二油酸酯等。
例如,作为金属皂类,可列举:硬脂酸铝、硬脂酸镁、硬脂酸锌、硬脂酸钙、棕榈酸锌、肉豆蔻酸镁、月桂酸锌等。
例如,作为胶质、糖类或水溶性高分子化合物,可列举:***胶、黄原胶、瓜尔胶、刺梧桐树胶、琼脂、酪蛋白、乳糖、果糖、蔗糖或其酯、海藻糖或其衍生物、糊精、明胶、果胶、淀粉、鹿角菜胶、甲壳素或壳聚糖类、海藻酸或其盐、透明质酸或其盐、硫酸软骨素或其盐、肝素、乙基纤维素、甲基纤维素、羧甲基纤维素、羧乙基纤维素、结晶纤维素、β-葡聚糖、聚乙烯醇、聚乙烯甲醚、聚丙烯酸盐、聚环氧烷或其交联聚合物、羧乙烯聚合物等。
例如,作为表面活性剂,可列举:烷基羧酸盐、烷基磺酸盐、烷基硫酸酯盐、烷基磷酸酯盐等阴离子表面活性剂、烷基胺盐、烷基四级铵盐等阳离子表面活性剂、羧酸型两性表面活性剂、硫酸酯型两性表面活性剂、磺酸型两性表面活性剂、磷酸酯型两性表面活性剂、非离子表面活性剂等两性表面活性剂、天然表面活性剂、蛋白质水解物的衍生物、高分子表面活性剂、含钛、硅的表面活性剂、氟化碳系表面活性剂等其他表面活性剂等。
例如,作为维生素类,可列举:视黄醇、视黄醛、脱氢视黄醛、胡萝卜素、番茄红素等维生素A群、盐酸硫胺素、硫酸硫胺素、核黄素、吡哆醇、氰钴氨素、叶酸类等维生素B群、维生素C、生物素、泛酸、磷酸抗坏血酸镁盐、磷酸抗坏血酸钠盐、抗坏血酸四己基癸酸酯等维生素C衍生物、维生素D群、维生素E群及其衍生物、维生素K群,此外可列举:必需脂肪酸、肉碱、阿魏酸、γ-谷维素、维生素P类、维生素U类等。
例如,作为氨基酸,可列举:缬氨酸、白氨酸、异白氨酸、苏氨酸、甲硫胺酸、苯丙氨酸、色氨酸、赖氨酸、甘氨酸、丙氨酸、天冬酰氨、谷氨酰氨、丝氨酸、半胱氨酸、胱氨酸、酪氨酸、脯氨酸、羟基脯氨酸、天冬氨酸、谷氨酸、精氨酸、鸟氨酸、组氨酸等、或它们的硫酸盐、磷酸盐、硝酸盐、柠檬酸、或者吡咯烷酮羧酸等氨基酸衍生物等。
例如,作为紫外线吸收/阻断剂,可列举:β-异丙基呋喃酮衍生物、尿刊酸、尿刊酸乙酯、对二甲基苯甲酸辛酯、2-羟基-4-甲氧基二苯甲酮、2-羟基-4-甲氧基二苯甲酮-5-磺酸等二苯甲酮衍生物、对氨基苯甲酸、对氨基苯甲酸乙酯等对氨基苯甲酸衍生物、对甲氧基肉桂酸乙酯、对甲氧基肉桂酸异丙酯等甲氧基肉桂酸衍生物、水杨酸衍生物、邻氨苯甲酸衍生物、尿刊酸衍生物、香豆素衍生物、氨基酸系化合物、苯并***衍生物、四唑衍生物、咪唑啉衍生物、嘧啶衍生物、二噁烷衍生物、樟脑衍生物、呋喃衍生物、吡喃酮衍生物、核酸衍生物、尿囊素衍生物、烟酸衍生物、维生素B6衍生物、伞形酮、马栗树皮苷、肉桂酸苄酯、西诺沙酯、氧苯酮、二羟苯酮、辛苯酮、磺异苯酮、苯酰间苯二酚、熊果苷、愈创奥、紫草宁、黄芩苷、黄芩素、小檗碱、Neo Heliopan、Escalol系列紫外线吸收剂、氧化锌、氧化钛、滑石、高岭土等。
例如,作为代谢促进作用/细胞活化物质,可列举:对苯二酚、乳酸菌提取物、胎盘提取物、灵芝提取物、维生素A、维生素E、尿囊素、脾脏提取物、胸腺提取物、酵母提取物、发酵乳提取物、植物提取物(芦荟、黄芩、问荆、龙胆、牛蒡、紫草、胡萝卜、金缕梅属、啤酒花、薏苡、短柄野芝麻、当药、当归、金盏花、土常山、小连翘、黄瓜、百里香、迷迭香、欧芹)等。
例如,作为活性氧清除剂,也可以使用琥珀酸、丹宁酸、尿囊素、氯化锌、硫酸锌等收敛剂,可列举:超氧化歧化酶(SOD)、过氧化氢酶、谷胱甘肽过氧化物酶等。
例如,作为抗氧化剂,可列举:维生素C或其盐、硬脂酸酯、维生素E或其衍生物、去甲二氢愈创木酸、二丁基羟基甲苯(BHT)、丁基羟基茴香醚(BHA)、羟基酪醇、对羟基苯甲醚、没食子酸丙酯、芝麻酚、芝麻素、棉子酚、蜂胶等。
例如,作为过氧化脂质生成抑制剂,可列举:β-胡萝卜素、植物提取物(芝麻、土常山、小连翘、金缕梅属、丁香、蜜蜂花、日本香茶菜、白桦、鼠尾草、迷迭香、南天竹果、玫瑰果、银杏、绿茶)等。
例如,作为抗炎症剂,可列举:鱼石脂、吲哚美辛、高岭土、水杨酸、水杨酸钠、水杨酸甲酯、乙酰水杨酸、盐酸苯海拉明、d-樟脑、dl-樟脑、氢化可的松、愈创奥、母菊奥、马来酸氯苯那敏、甘草酸或其盐、甘草次酸或其盐、甘草提取物、紫草提取物、玫瑰果提取物、虎耳草提取物、紫苏提取物、薏苡提取物、蜂胶等。
例如,作为抗菌、杀菌、消毒药,可列举:桧木醇、利凡诺、硫磺、葡萄糖酸钙、葡萄糖酸氯己定、氨磺酰、汞溴红、乳铁蛋白或水解物、氯化烷基二胺基乙基甘氨酸液、三氯生、次氯酸钠、氯胺T、漂白粉、碘化合物、碘仿、己二烯酸或盐、丙酸或盐、水杨酸、脱氢乙酸、苯甲酸、苯甲酸钠、对羟基苯甲酸酯类、十一碳烯酸、硫胺素十二烷基硫酸盐、硫胺素十二烷基硝酸盐、苯酚、甲酚、对氯苯酚、对氯间二甲苯酚、对氯间甲酚、百里香酚、苯乙醇、O-苯基苯酚、玉洁新CH3565、卤卡班、六氯芬、氯己定、乙醇、甲醇、异丙醇、苄醇、乙二醇、丙二醇、苯氧乙醇、1,2-戊二醇、吡啶硫酮锌、氯丁醇、异丙基甲基苯酚、聚氧乙烯十二烷基醚等非离子表面活性剂、两性表面活性剂、十二烷基硫酸钠等阴离子表面活性剂、苯扎氯铵等阳离子表面活性剂、甲醛、乌洛托品、感光素101号、感光素201号、感光素401号、N-长链酰基碱性氨基酸衍生物及其酸加成盐、氧化锌、桧木醇、苦参根、蜂胶、油溶性甘草提取物等。
例如,作为保湿剂,可列举:丙三醇、丙二醇、1,3-丁二醇、聚乙二醇、辛酸/癸酸甘油三酯、乙醇酸(-羟酸)、透明质酸或其盐、硫酸软骨素或其盐、水溶性甲壳素或其衍生物或壳聚糖衍生物、吡咯啶酮羧酸或其盐、乳酸钠、尿素、山梨糖醇、氨基酸或其衍生物、橄榄油、春黄菊油、蓖麻油等油脂类(包含硬化油)、蜂蜡、羊毛脂、紫胶蜡等蜡类、液状石蜡、凡士林、石蜡等矿物油、纳豆菌代谢物、纳豆提取物、丝纤维提取物、春黄菊、芦荟等植物提取物等。
例如,作为角质溶解剂,可列举:二异丙基氟磷酸、植物提取物(黄芩、小连翘、苦参、桑叶、桂皮、老鹳草、聚合草、鼠尾草、西洋接骨木、菩提树、牡丹皮)、海藻提取物等弹性蛋白酶活性抑制剂、维生素E或其衍生物、当药提取物、大蒜提取物、胡萝卜提取物、芦荟提取物、龙胆提取物、当归提取物、千金藤素、卡普氯铵、米诺地尔等末梢血管血流促进剂,还可列举:辣椒酊、壬酸香草酰胺、斑蝥酊、姜酊、薄荷油、1-薄荷醇、樟脑、烟酸苄酯等刺激剂、吡哆醇或其衍生物、硫磺、维生素B6等抗脂溢剂、间苯二酚、水杨酸、乳酸、尿素等。
例如,作为香料,可列举:麝香等天然动物性香料、薄荷醇、绿薄荷、胡椒薄荷、大茴香精油、柑橘精油、小豆蔻精油、愈创木精油、小茴香精油、桂皮精油、肉桂精油、天竺葵精油、芫荽精油、紫苏精油、雪松精油、香茅精油、茉莉精油、姜草精油、杉精油、绿薄荷精油、欧洲薄荷精油、橙花精油、冬青精油、玫瑰精油、扁柏精油、扁柏叶精油、檀香精油、月桂精油、佛手柑精油、桉树精油、莱姆精油、熏衣草精油、柠檬精油、迷迭香精油、日本薄荷精油等植物性香料、其他合成香料等。
例如,作为激素类,可列举:***及其酯、雌酮、炔雌醇、可的松及其酯、氢化可的松及其酯、***、***龙等。
例如,作为一般已知对育发生发效果有效的成分,可列举:十五烷酸甘油酯、锦紫苏提取物、龙胆提取物、松球提取物、蜂王浆提取物、山白竹提取物、t-黄烷酮、6-苄基氨基嘌呤、当药提取物、卡普氯铵、米诺地尔、非那雄胺、腺苷、烟酰胺、桑根提取物、地黄提取物、5-氨基乙酰丙酸等。
作为其他成分,可列举:金属离子螯合剂、pH调整剂、螯合剂、防腐防霉剂、清凉剂、稳定剂、乳化剂、动植物蛋白质及其分解物、动植物性多糖类及其分解物、动植物性糖蛋白及其分解物、血流促进剂、抗炎症剂、消炎剂、防腐剂、抗过敏剂、创伤治疗剂、增泡剂、增稠剂、酶、色素、纯化水、口腔用剂、消炎除臭剂、苦味剂等。
此外,本公开的育发生发促进剂的使用形式随意,并无限定。具体而言,作为化妆品、药品,可列举例如:内用外用剂、化妆水、乳液、乳霜、软膏、洗液、油、发膜、皮肤清洁剂、洗发精、润发精、护发素、发乳、润发油、喷雾式整发剂、整发剂、烫发剂、生发油、染发剂等。
当本公开的育发生发促进剂以化妆品、药品的形式加以实施时,剂型并无限定,可制成安瓿剂、胶囊剂、粉末剂、颗粒剂、丸剂、片剂、固体剂、液体剂、凝胶剂、气泡剂、乳液剂、乳霜剂、软膏剂、膜剂、摩丝等多种剂型。
以下为了更详细地对本公开进行说明而列举实施例,但本公开并不限于此。
[实施例1:毛***细胞活化作用的评价]
(1-1)人头发毛***细胞的培养方法
试验对象细胞使用作为广泛用于育发剂药效评价的细胞的人头发毛***细胞(目录编号:CA60205a,批号:2548,白色人种,来自47岁女性,销售商:东洋纺株式会社)。使用毛***细胞增殖培养基(目录编号:TMTPGM-250,销售商:东洋纺株式会社),在二氧化碳培养箱(5%CO2,37℃)内对人头发毛***细胞进行培养。利用I型胶原蛋白对用于培养的烧瓶进行涂覆后加以使用。细胞的继代使用毛***细胞专用继代培养套件(目录编号:CA090K,销售商:东洋纺株式会社),将细胞***瓶剥离而使用。
(1-2)人毛***细胞毒性试验
在本实施例中,将异肥皂草苷作为受验物质,评价其对人头发毛***细胞有无毒性。
将人头发毛***细胞以5×103个/孔(本试验中为0.1mL/孔)的方式接种到涂覆I型胶原蛋白的96孔板中。在二氧化碳培养箱(5%CO2,37℃)内培养1天后(50%汇合度),置换为含异肥皂草苷的试验培养基,然后,培养3天。利用活细胞数量测量试剂SF(目录编号:07553,销售商:Nacalai Tesque株式会社)对细胞的增殖性进行比较研究。考虑到在测定活细胞数量时于测量吸光度时附着于细胞的析出物可能会对吸光度造成影响,在3天的培养结束后,将培养液中的上清液去除,并利用磷酸盐缓冲生理盐水(略称:PBS)对细胞进行洗涤。利用PBS洗涤后,对每孔添加100μL含10%的活细胞测量试剂SF的培养基。添加后,于30分钟后及90分钟后,对培养上清液的吸光度(测量波长450nm,参照波长595nm)进行测量。由90分钟的值以及30分钟的值算出每1小时的吸光度ABS的变化量(单位:ABS/hr)。实施3次试验。
如图1所示,若异肥皂草苷为1000μmol/L以下,则并未对人头发毛***细胞表现出毒性。因此,在后述的异肥皂草苷的评价实验中,使用0.1μmol/L、10μmol/L、1000μmol/L的异肥皂草苷试验浓度。该试验浓度由使异肥皂草苷(分子量594.52)以100mmol/L而溶解于毛***细胞增殖培养基而获得的储备溶液配制而成。
(1-3)人头发毛***细胞活化作用的评价
将人头发毛***细胞以1.2×104个/孔(本试验中为0.3mL/孔)的方式接种到48孔板中。在二氧化碳培养箱内培养1天后,置换为以最终浓度0.1μmol/L、10μmol/L、1000μmol/L而溶解有异肥皂草苷的毛***细胞增殖培养基。其后,培养1天以及3天,利用活细胞数量测量试剂SF对各个细胞的增殖性进行比较研究。1天及3天的培养分别结束后,将培养液中的上清液去除,并利用PBS对细胞进行洗涤。利用PBS洗涤后,对每孔添加300μL含10%的活细胞测量试剂SF的培养基。添加后,于30分钟后及90分钟后,将每孔的培养上清液100μL移至另一96孔板,并通过酶标仪对吸光度(测量波长450nm,参照波长595nm)进行测量。由90分钟的值及30分钟的值算出每1小时的吸光度ABS的变化量(单位:ABS/hr),以对细胞活化作用进行评价。实施5次试验。其中,使用无添加对照区作为异肥皂草苷的比较对照区。
作为阳性对照,对添加最终浓度100μmol/L的腺苷(目录编号:016-10493,销售商:和光纯药工业株式会社)及最终浓度30μmol/L的米诺地尔(目录编号:M4145-25MG,销售商:Sigma-Aldrich Japan株式会社)的情形,也进行相同的试验。其中,腺苷是在溶解于二甲基亚砜(略称:DMSO)后,以使得DMSO的最终浓度为0.1%的方式而添加至试验培养基中的。作为溶剂对照,设置0.1%DMSO试验区。米诺地尔是在溶解于乙醇后,以使得乙醇的最终浓度为0.1%的方式添加至试验培养基中的。一般而言,在毛***细胞中,0.1%以下的乙醇浓度不会对细胞功能造成影响,故而使用无添加对照区作为米诺地尔的比较对照区。
将添加作为受验物质的异肥皂草苷后并培养1天的测定结果示于图2中,将添加异肥皂草苷后并培养3天的测定结果示于图3中。于比较试验区间进行差异显著性检验。检验以Student T-检验的形式进行,将P<0.05(零假设小于5%)判断为具有显著性,并记载于图中。
由图2可明确,在进行了处理时间为1天的异肥皂草苷处理的组中,在0.1μmol/L~1000μmol/L的范围内虽然确认到较无添加对照区仅为稍许差异但却对人头发毛***细胞具有显著的细胞活化作用。此外,由图3可明确,在进行了处理时间为3天的异肥皂草苷处理的组中,在0.1μmol/L~1000μmol/L的范围内确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。并确认到,该细胞活化作用是实施米诺地尔处理时的约3倍的作用。
另一方面,作为阳性对照物的腺苷,经3天的处理时间确认到较0.1%DMSO试验区对人头发毛***细胞具有显著的细胞活化作用。此外,作为阳性对照物的米诺地尔,经1天的处理时间虽然确认到较无添加对照区仅为稍许差异但却对人头发毛***细胞具有显著的细胞活化作用。而米诺地尔经3天的处理时间确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。
由该结果表明,通过将异肥皂草苷应用于头皮等,可发挥极其优异的毛***细胞活化作用,从而可期待育发效果及生发效果。
[实施例2:异肥皂草苷的VEGF产生促进作用的评价]
通过对实施了异肥皂草苷处理的人头发毛***细胞所产生的VEGF量进行测定,来评价异肥皂草苷对人头发毛***细胞的VEGF产生促进作用。
通过ELISA套件(制品名:Human VEGF Quantikine ELISA,目录编号:DVE00,销售商:R&D system公司)测定对人头发毛***细胞进行了3天异肥皂草苷处理所得的培养上清液中的VEGF量。进行了3天异肥皂草苷处理所得的培养上清液使用了上述(1-3)项中培养了3天时的上清液。具体而言,在上述(1-3)项中,为了对吸光度进行测量,在添加异肥皂草苷3天后,会将培养液中的上清液去除,但在VEGF产生促进作用的评价试验中对添加异肥皂草苷3天后的培养上清液进行回收,并将该上清液作为被检体而使用。实施5次试验。
以下详细叙述VEGF的测定方法。其中,在VEGF的测定中,不稀释上清液,而使用原液。
步骤1-1.以VEGF标准原液(2000pg/mL)为基础,使用反应缓冲液制备1000、500、250、125、62.5、31.2、15.6pg/mL的VEGF标准溶液。
步骤1-2.将VEGF标准溶液及被检体(培养上清液)200μL添加到已向各孔中添加了50μL的稀释用液的VEGF固相化微孔板中,于室温下反应2小时(一次反应)。
步骤1-3.将孔内的溶液去除,并利用洗涤液进行3次洗涤。
步骤1-4.向各孔均添加200μL西洋山萮菜过氧化物酶结合VEGF抗体溶液,并在室温下使其反应2小时(二次反应)。
步骤1-5.将孔内的溶液去除,并利用洗涤液洗涤3次。
步骤1-6.向各孔中添加200μL基质溶液,于室温下使其反应20分钟。
步骤1-7.向各孔中均添加50μL反应终止溶液,利用板混合器混合1分钟后,利用酶标仪对各孔的吸光度进行测量(测量波长450nm)。
步骤1-8.由标准曲线算出被检体中的VEGF浓度。
此外,使用无添加对照区作为异肥皂草苷的比较对照区。
作为阳性对照,对添加最终浓度30μmol/L的米诺地尔的情形也进行相同的试验。使用无添加对照区作为米诺地尔的比较对照区。
将对受验物质进行了3天处理所得的培养上清液中的VEGF量的测定结果示于图4中。与上述(1-3)项相同,进行差异显著性检验,并记载于图中。
由图4可明确,于异肥皂草苷1000μmol/L浓度区中,与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。另一方面,作为阳性对照物的米诺地尔与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。
由该结果表明,通过将异肥皂草苷应用于头皮等,可提高VEGF产生量,从而可期待优异的育发效果及生发效果。
[实施例3:化妆品的制造]
将100kg山萮菜用水清洗后,切成1cm宽,并通过切割搅拌器将其粉碎。向经粉碎的山萮菜中添加50%丁二醇200kg,并搅拌1小时。然后,进行压榨而获得180kg的压榨液。进而,于90℃下对该压榨液进行30分钟的加热杀菌,从而获得提取物。使用以此方式获得的山萮菜的提取物,制造具有下列成分的头发用化妆品。下述成分表示为将总量设为100g。在该头发用化妆品100g中含有异肥皂草苷1.5mg。
水 86.48g
丁二醇 5.0g
丙三醇 2.0g
山萮菜的提取物 5.0g
海藻糖 1.0g
苯氧乙醇 0.5g
柠檬酸钠 0.01g
柠檬酸 0.01g
[实施例3的变形例]
于上述实施例3中,作为化妆品,列举了于头发用化妆品100g中含有异肥皂草苷1.5mg的示例,但并不限于此。例如,亦可于100mL化妆品中含有异肥皂草苷5μg(0.1μmol/L)~59.0mg(1000μmol/L)。
此外,在上述实施例3中,列举了于头发用化妆品中含有异肥皂草苷的示例,不过也可以含有苯基丙烷类。具体而言,可于100mL头发用化妆品中含苯基丙烷类1.0mg。此外,苯基丙烷类的含量并不限于此。例如,也可以于100mL化妆品中含有苯基丙烷类0.001~100mg。
[实施例4:胶囊的制造(之一)]
将山萮菜100kg用水清洗后,切成1cm宽,并通过切割搅拌器将其粉碎。向经粉碎的山萮菜中添加50%乙醇200kg,并搅拌1小时。然后,进行压榨而获得180kg的压榨液。进而,将该液体浓缩而去除醇成分后,于90℃下进行30分钟的加热处理,由此进行杀菌。添加10kg纤维素并进行混合搅拌后,进行干燥、粉碎,从而获得山萮菜的提取物粉末。将以此方式而获得的提取物粉末200mg填充于硬胶囊(3号)中,从而制造出胶囊(例如营养补充剂等)。该胶囊以1mg/粒而含有异肥皂草苷。
[实施例5:片剂的制造(之二)]
使用实施例4中所制作的提取物粉末150mg,以如下所示的调配比制备原料组合物,并进行压片,从而制造出片剂(例如营养补充剂等)。该片剂中的异肥皂草苷的含量为0.75mg/粒。
片剂 1.5g/粒(5粒/天)
砂糖 1.20g
浓缩果汁 0.045g
柠檬酸 0.075g
香料 0.03g
山萮菜的提取物粉末 0.15g
[实施例6:片剂的制造(之三)]
将山萮菜100kg洗涤并切碎,添加50%乙醇200kg,从而获得180kg的提取液。将所获得的提取液浓缩而去除醇成分,添加30kg纤维素并进行干燥,并将其粉末化。对所获得的粉末进行压片,从而制成1片200mg的片剂。于1片中含1.0mg的苯基丙烷类。
[实施例4、5、6的变形例]
在上述实施例5中,作为片剂,列举了于1.5g/粒的片剂中以0.75mg/粒而含有异肥皂草苷的示例,但并不限于此。例如,也可以是于250mg/粒的片剂中以0.5mg~100mg/粒而含有异肥皂草苷。
此外,在上述实施例5中,列举了片剂的制造方法的示例,但并不限于此。例如,也可以混合异肥皂草苷与糊精,并进行压片,从而制造片剂。此外,例如,也可以将实施例4中所制作的提取物粉末与糊精进行混合,并进行压片,从而制造片剂。
此外,在上述实施例6中,列举了于1片中含有1.0mg的苯基丙烷类的示例,但并不限于此。例如,也可以通过改变浓缩率,而使得于1片中含有1.0mg~100mg的苯基丙烷类。
[实施例7:除异肥皂草苷以外的受验物质的评价]
在实施例7中,在分别添加了表1所示的受验物质的培养液中对人头发毛***细胞进行培养后,对活细胞数量的测定,VEGF产生量、以及FGF-7基因表达量进行了验证。
[表1]
受验物质 | 溶剂 | 储备溶液的浓度 |
芹菜素 | DMSO | 200mmol/L |
香豆酸 | 乙醇 | 200mmol/L |
咖啡酸甲酯 | 乙醇 | 200mmol/L |
作为受验物质的芹菜素,使用和光纯药工业株式会社的目录编号012-18913的芹菜素。作为受验物质的香豆酸,使用Sigma-Aldrich公司的目录编号C9008的香豆酸。作为受验物质的咖啡酸甲酯,使用Alfa Aesar公司的目录编号J63876的咖啡酸甲酯。
此外,将为制作各受验物质的储备溶液而使用的溶剂及储备溶液的浓度示于表1中。各储备溶液在过滤灭菌后用于试验。
对于作为阳性对照的腺苷,将DMSO作为溶剂制作100mmol/L的储备溶液,以使得腺苷的最终浓度为100μmol/L的方式将储备溶液添加至试验培养基中(使用0.1%DMSO试验区作为溶剂对照)。对于米诺地尔,使用50%乙醇制作浓度15mmol/L的储备溶液,以使得米诺地尔的最终浓度为30μmol/L的方式将储备溶液添加至试验培养基中。由于此时的溶剂(乙醇)浓度为0.2%,故而将0.2%乙醇试验区作为对照区。
(7-1)人头发毛***细胞的培养方法
试验对象细胞使用人头发毛***细胞(目录编号:CA602t05a,批号:2868,白色人种,来自51岁男性,头顶部,销售商:东洋纺株式会社)。以与实施例1的(1-1)项所记载的方法相同的方式,对人头发毛***细胞进行培养。
(7-2)人毛***细胞毒性试验
以与实施例1的(1-2)项所记载的方法相同的方式,评价受验物质对人头发毛***细胞有无毒性。将其结果示于图5~图7中。
如图5所示,芹菜素为0.08mmol/L以上时,对人头发毛***细胞表现出了毒性。如图6所示,香豆酸为2mmol/L以上时,对人头发毛***细胞表现出了毒性。如图7所示,咖啡酸甲酯为0.016mmol/L以上时,对人头发毛***细胞表现出了毒性。基于细胞毒性试验的结果,本试验中的试验浓度设定为如表2所示。
[表2]
受验物质 | 溶剂 | 试验浓度 | 溶剂浓度 |
芹菜素 | DMSO | 0.001、0.01、0.1、1、10μmol/L | 0.005% |
香豆酸 | 乙醇 | 0.001、0.01、0.1、10、100μmol/L | 0.05% |
咖啡酸甲酯 | 乙醇 | 0.001、0.01、0.1、1、10μmol/L | 0.01% |
由于各受验物质的试验最高浓度下的溶剂浓度为0.1%以下,故而判断本试验中不存在溶剂的影响,在差异显著性比较中将无添加对照区作为比较基准。此外,检验以Student T-检验的形式进行,将P<0.1、P<0.05、P<0.01、以及P<0.001判断为具有显著性,并记载于图中。
(7-3)人头发毛***细胞活化作用及VEGF产生促进作用的评价
将人头发毛***细胞以1.2×104个/孔(于本试验中为0.3mL/孔)的方式接种于48孔板。在二氧化碳培养箱(5%CO2,37℃)内培养1天后,置换为含受验物质的培养基。然后,培养3天,并利用活细胞数量测量试剂SF对各个细胞的增殖性进行比较研究。实施5次试验。3天的培养结束后,回收培养液中的上清液,用ELISA套件以与实施例2相同的方式对该上清液中的VEGF产生量进行测定。
然后,去除该上清液,并在利用PBS对细胞进行洗涤后,添加含10%的活细胞测量试剂SF的培养基(300μL/孔)。添加后,在30分钟后以及90分钟后将每孔的培养上清液100μL移至另一96孔板,并通过酶标仪对吸光度(测量波长450nm,参照波长595nm)进行测量。由90分钟的值以及30分钟的值算出每1小时的吸光度ABS的变化量(单位:ABS/hr),从而对细胞活化作用进行评价。
对于由差异显著性检验判断为具有显著性的受验物质,将活细胞数量的测定结果示于图8中,将VEGF蛋白产生量的结果示于图9中。
如图8所示,在进行了芹菜素处理的组中,于0.001μmol/L~1μmol/L的范围内确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。此外,在进行了芹菜素处理的组中,在10μmol/L并未表现出显著性。因此,对于进行了芹菜素处理的组,可以认为在0.001μmol/L~9μmol/L的范围内确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。
在进行了香豆酸处理的组中,在0.1μmol/L确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。此外,在进行了香豆酸处理的组中,在0.01μmol/L及10μmol/L并未表现出显著性。由此,对于进行了香豆酸处理的组,可以认为在0.011μmol/L~9μmol/L,优选在0.011μmol/L~5μmol/L,确认到较无添加对照区对人头发毛***细胞具有显著的细胞活化作用。
另一方面,对于作为阳性对照物的腺苷,确认到较0.1%DMSO试验区对人头发毛***细胞具有显著的细胞活化作用。此外,对于作为阳性对照物的米诺地尔,确认到较0.2%乙醇试验区对人头发毛***细胞具有显著的细胞活化作用。
由上述结果表明,通过将芹菜素及香豆酸应用于头皮等,可发挥优异的毛***细胞活化作用,从而可期待育发效果及生发效果。
此外,如图9所示,在进行了芹菜素处理的组中,在0.001μmol/L~0.01μmol/L的范围内,与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。此外,在进行了芹菜素处理的组中,在0.1μmol/L并未表现出显著性。由此,对于进行了芹菜素处理的组,可以认为在0.001μmol/L~0.09μmol/L的范围内,与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。
在进行了咖啡酸甲酯处理的组中,在1μmol/L,与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。此外,在进行了咖啡酸甲酯处理的组中,在0.1μmol/L及10μmol/L并未表现出显著性。由此,对于进行了咖啡酸甲酯处理的组,可以认为在0.11μmol/L~9μmol/L,优选在0.011μmol/L~5μmol/L,与无添加对照区相比,显著地增加了人头发毛***细胞所产生的VEGF量。
另一方面,作为阳性对照物的腺苷,虽较0.1%DMSO试验区仅有稍许差异,但却显著地增加了人头发毛***细胞所产生的VEGF量。此外,作为阳性对照物的米诺地尔,较0.2%乙醇试验区显著地增加了人头发毛***细胞所产生的VEGF量。
(7-4)FGF-7基因表达的评价
将人头发毛***细胞以1×105个/孔(于本试验中为0.3mL/孔)的方式接种到涂覆胶原蛋白的48孔板中。在二氧化碳培养箱内(5%CO2,37℃)培养1天后(确认100%汇合),置换为含受验物质(3个阶段浓度:无毒性浓度区域)的培养基。然后,培养2小时,并回收总RNA,并在反转录为cDNA后,通过实时PCR法测定FGF-7基因表达。使用GAPDH基因作为内部标准,作为与阴性对照组的相对值算出FGF-7基因表达,实施3次试验。各方法的详细内容如下所示。
首先,按照FastLane Cell RT-PCR套件的步骤进行从细胞回收总RNA以及cDNA化。
步骤7-1.向每孔的利用PBS洗涤的细胞中添加500μL的缓冲剂FCW,并立即去除抽吸缓冲剂FCW。
步骤7-2.向每孔添加200μL的缓冲剂FCP,并在室温(25℃)下培养5分钟后,将FastLane溶解产物移至96孔的PCR管中。在进行下列cDNA化操作之前以-80℃保存管。
步骤7-3.于室温下将步骤7-2的溶解产物溶解。向新的PCR管中添加2μL的gDNAWipeout Buffer、1μL的FastLane溶解产物、11μL的无RNase水,并于42℃下反应5分钟。然后,立即移至冰上(实际上,向装有溶解产物的管中适量添加gDNA Wipeout Buffer与无RNase水的预混试剂)。
步骤7-4.向步骤7-3中所获得的反应液14μL中添加6μL反转录反应预混试剂(1μL的Quantiscript Reverse Transcriptase、4μL的Quantiscript RT Buffer、1μL的RTPrimer Mix/管),并在42℃下培养30分钟。
步骤7-5.通过在95℃下对反应液进行3分钟处理而使反转录酶失活。将该反应液作为合成cDNA而用于实时PCR。并在用于解析前以-80℃进行保存。
接下来,如下所示地在实时PCR专用管中调整反应液,并进行PCR。作为PCT反应,进行60个循环的95℃10秒和60℃30秒的反应。
dsH2O 2.4μL
SYBER Premix Ex Taq 4.0μL
正向引物(20μmol/L) 0.3μL
反向引物(20μmol/L) 0.3μL
合成cDNA 1.0μL
总计 8.0μL
用于试验的FGF-7的特异性引物、以及作为内部标准的GADPH的特异性引物如下所示。
FGF-7的引物
正向引物:tctgtcgaacacagtggtacctgag(序列编号1)
反向引物:gccactgtcctgatttccatga(序列编号2)
GADPH的引物
正向引物:catccctgcctctactggcgctgcc(序列编号3)
反向引物:ccaggatgcccttgagggggccctc(序列编号4)
以如下方式算出各基因的相对表达量。由各基因的扩增曲线与阈值线的交点算出Ct值(PCR循环数)。计算自目的基因的Ct值减去内标准GAPDH基因的Ct值,即Ct(目的基因)-Ct(GAPDH2)=ΔCt值。然后,计算自ΔCt值减去空白组的平均ΔCt值,即ΔCt(样本处理区)-ΔCt(空白组区)=ΔΔCt值。将ΔΔCt值代入2的指数项而得的2-ΔΔCt值为相对表达量。
对于由差异显著性检验而判断为具有显著性的受验物质,将FGF-7基因表达量的结果示于图10中。
如图10所示,在进行了香豆酸处理的组中,在0.001μmol/L,与无添加对照区相比,对人头发毛***细胞显著地表现出了FGF-7基因表达的上升。此外,在进行了香豆酸处理的组中,在0.01μmol/L并未表现出显著性。由此,对于进行了香豆酸处理的组,可以认为在0.001μmol/L~0.009μmol/L,优选在0.001μmol/L~0.005μmol/L,与无添加对照区相比,对人头发毛***细胞显著地表现出FGF-7基因表达的上升。
另一方面,作为阳性对照物的腺苷,与0.1%DMSO试验区相比,对人头发毛***细胞显著地表现出了FGF-7基因表达的上升。
以上对育发生发促进剂的实施例进行了说明,不过,可对上述实施例中例示的构成进行各种变更。例如,在上述实施例中,作为上述式(Ⅰ)所表示的类黄酮类的示例,示出了使用异肥皂草苷及芹菜素的示例,不过不限于此。除了异肥皂草苷及芹菜素以外,亦可使用已于本说明书中例示的其他类黄酮类,当使用这些类黄酮类时,亦可期待与使用异肥皂草苷的情形相同的育发生发促进效果。
此外,在上述实施例中,作为化妆品的原料,列举了利用山萮菜的示例,不过不限于此。化妆品原料例如也可以为西洋山萮菜,还可以一并使用山萮菜与西洋山萮菜。
Claims (6)
1.异肥皂草苷在制备毛***细胞活化促进剂中的用途,
所述毛***细胞活化促进剂含有异肥皂草苷作为有效成分,
所述毛***细胞活化促进剂用作化妆品或药品,
所述化妆品以及所述药品中的所述异肥皂草苷的含量为:0.1μmol/L~1000μmol/L,
所述药品中含有的所述异肥皂草苷的成人每人的日剂量为1mg~10g。
2.异肥皂草苷在制备毛***细胞活化促进剂中的用途,
所述毛***细胞活化促进剂含有异肥皂草苷作为有效成分,
所述毛***细胞活化促进剂用作化妆品或药品,
所述化妆品以及所述药品中的所述异肥皂草苷的含量为:0.1μmol/L~1000μmol/L。
3.根据权利要求1或2所述的异肥皂草苷在制备毛***细胞活化促进剂中的用途,
在育发生发促进剂中使用所述毛***细胞活化促进剂。
4.异肥皂草苷在制备毛***细胞的血管内皮细胞生长因子产生促进剂中的用途,
所述毛***细胞的血管内皮细胞生长因子产生促进剂含有异肥皂草苷作为有效成分,
所述毛***细胞的血管内皮细胞生长因子产生促进剂用作化妆品或药品,
所述化妆品以及所述药品中的所述异肥皂草苷的含量为:0.1μmol/L~1000μmol/L,
所述药品中含有的所述异肥皂草苷的成人每人的日剂量为1mg~10g。
5.异肥皂草苷在制备毛***细胞的血管内皮细胞生长因子产生促进剂中的用途,
所述毛***细胞的血管内皮细胞生长因子产生促进剂含有异肥皂草苷作为有效成分,
所述毛***细胞的血管内皮细胞生长因子产生促进剂用作化妆品或药品,
所述化妆品以及所述药品中的所述异肥皂草苷的含量为:0.1μmol/L~1000μmol/L。
6.根据权利要求4或5所述的异肥皂草苷在制备毛***细胞的血管内皮细胞生长因子产生促进剂中的用途,
在育发生发促进剂中使用所述毛***细胞的血管内皮细胞生长因子产生促进剂。
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KR102417319B1 (ko) * | 2017-09-28 | 2022-07-06 | (주)아모레퍼시픽 | 용해도가 향상된 아피게닌을 포함하는 탈모 완화 조성물 |
JP6964290B2 (ja) * | 2017-10-17 | 2021-11-10 | 国立大学法人北海道大学 | Atp産生促進用剤 |
JP6965111B2 (ja) * | 2017-11-09 | 2021-11-10 | 三井製糖株式会社 | 髪質改善用組成物 |
JPWO2020013179A1 (ja) * | 2018-07-09 | 2021-08-02 | 国立大学法人九州大学 | ミノキシジルの放出が制御された経皮吸収組成物 |
JP7079920B2 (ja) * | 2019-02-28 | 2022-06-03 | 芳弘 二村 | 血管内皮細胞増殖作用を呈するヒドロキシピラン誘導体及びヒト口腔細胞の培養工程からなるその製造方法 |
JPWO2021132529A1 (zh) * | 2019-12-27 | 2021-07-01 | ||
CN111110618B (zh) * | 2020-02-11 | 2022-09-27 | 张运适 | 一种用于促进毛发生长的组合物 |
JP2022042891A (ja) * | 2020-09-03 | 2022-03-15 | 株式会社サニープレイス | 抗菌水用組成物 |
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US11813351B2 (en) * | 2020-11-23 | 2023-11-14 | Stella Grace Newman | Beauty product composition for eyebrows |
US11744788B2 (en) * | 2021-10-27 | 2023-09-05 | James D. Welch | Method of encouraging growth and regrowth of hair in human males technical area |
JP7442833B2 (ja) * | 2021-11-19 | 2024-03-05 | 株式会社サニープレイス | 抗菌方法 |
CN116251032A (zh) * | 2021-12-09 | 2023-06-13 | 高露洁-棕榄公司 | 个人护理组合物 |
CN114392263B (zh) * | 2021-12-20 | 2023-11-14 | 亿利耐雀生物科技有限公司 | 一种甘草新苷防脱育发组合物及其制备方法 |
CN115120522A (zh) * | 2022-08-03 | 2022-09-30 | 浙江熙正霖生物科技有限公司 | 木犀草素及衍生物作为有效成分促进生发的组合物及应用 |
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JP7054242B2 (ja) | 2022-04-13 |
CN108135879A (zh) | 2018-06-08 |
JPWO2017094905A1 (ja) | 2018-04-05 |
US20200108005A1 (en) | 2020-04-09 |
KR20190133803A (ko) | 2019-12-03 |
HK1255026A1 (zh) | 2019-08-02 |
BR112017027147A2 (pt) | 2018-08-14 |
KR102466224B1 (ko) | 2022-11-10 |
US11033473B2 (en) | 2021-06-15 |
JP2020055848A (ja) | 2020-04-09 |
KR20180006963A (ko) | 2018-01-19 |
WO2017094905A1 (ja) | 2017-06-08 |
US20180250213A1 (en) | 2018-09-06 |
TWI740861B (zh) | 2021-10-01 |
EP3300731A1 (en) | 2018-04-04 |
CN108135879B (zh) | 2022-11-08 |
EP3300731A4 (en) | 2019-06-19 |
CN113509478A (zh) | 2021-10-19 |
TW201825082A (zh) | 2018-07-16 |
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