CN101277713A - 用于促进ⅰ型胶原蛋白产生的组合物 - Google Patents
用于促进ⅰ型胶原蛋白产生的组合物 Download PDFInfo
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- CN101277713A CN101277713A CNA2006800361751A CN200680036175A CN101277713A CN 101277713 A CN101277713 A CN 101277713A CN A2006800361751 A CNA2006800361751 A CN A2006800361751A CN 200680036175 A CN200680036175 A CN 200680036175A CN 101277713 A CN101277713 A CN 101277713A
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Abstract
本发明以提供促进人体皮肤纤维芽细胞的I型胶原蛋白产生的组合物为目的，涉及含有水飞蓟宾及促进胶原蛋白产生的肽(Gly－Pro－Hyp、Gly－His－Lys、Lys－Thr－Thr－Lys－Ser或Gly－Glu－Pro－Arg的氨基酸序列所组成的肽)的用于促进I型胶原蛋白产生的组合物。
Description
技术领域
本发明涉及具有促进人体皮肤纤维芽细胞产生的I型胶原蛋白产生的效果的组合物。
背景技术
胶原蛋白约占生物体内蛋白质的1/3，大量存在于血管、皮肤、骨骼中，作为细胞的基础对这些组织的形成、构建起重要作用。胶原蛋白因基本不被消化酶分解，所以曾被认为是营养价值低的蛋白质，但有如下报道指出，通过摄取胶原蛋白可促进新陈代谢(专利文献1)、促进头发的生发(非专利文献1)、作为关节病治疗用药剂利用(专利文献2)等，因此其有效性被重新认识。且因胶原蛋白随年龄增加而减少，所以血管的脆化、皮肤弹性、柔软性的减少是引起皱纹、松弛产生的原因之一。
利用胶原蛋白的热变性体明胶的高度的粘性、凝固性及保水性，将其作为食品的口感改良剂和化妆品(专利文献3)利用。但是，未经处理的胶原蛋白及明胶因粘性高、易凝固，所以多使用经部分水解处理后的物质。且因其是蛋白质，所以具有抗原性，在过敏体质的人的摄取上还存在问题。为此，有如下报道，将胶原蛋白通过胶原酶低分子化，来用于失去抗原性的面向过敏患者的蛋白资源或输液制剂成分(专利文献4)。对于通过胶原酶分解的胶原蛋白分解物的生理活性，已知有血纤维蛋白凝集抑制活性(专利文献5)、麻醉作用(非专利文献2)。且有如下报道，对于通过来自溶组织梭菌(Clostridium Histolyticum)的胶原酶分解的胶原蛋白的分解产物，其生成的三肽的36％是Gly-Pro-Hyp，为最多(非专利文献3)。
以抗皱等抗皮肤老化为目的，尝试使用以包括Gly-Pro-Hyp在内的通过胶原酶分解的胶原蛋白分解物为有效成分的皮肤外用剂(专利文献6)。
且开发出含有包括Gly-Pro-Hyp在内的通过胶原酶分解的胶原蛋白分解物、特别是分子量为400以下的物质的生物体胶原蛋白产生促进剂(专利文献7)。
通过胶原酶分解的胶原蛋白分解物，成为生物体胶原蛋白的原料，促进胶原蛋白合成。其他，作为促进胶原蛋白产生的肽，开发出了来自α1胶原蛋白分解物的五肽Lys-Thr-Thr-Lys-Ser(非专利文献4)、来自α2胶原蛋白的分解物的三肽Gly-His-Lys(非专利文献5)、来自免疫球蛋白G(immunoglobulin G)的分解物的四肽Gly-Glu-Pro-Arg(非专利文献6)等。
另外，还发现了不作为生物体胶原蛋白的原料起作用，而是促进胶原蛋白产生的成分。例如，开发出维甲酸、其衍生物视黄醇(非专利文献7)及来自水飞蓟提取物的成分水飞蓟素(专利文献8)等。且已证明了维甲酸、其衍生物视黄醇(非专利文献8)及来自水飞蓟提取物的成分水飞蓟素(专利文献9)抑制作为胶原蛋白分解酶的胶原酶。
因维甲酸、其衍生物视黄醇在以皮肤刺激性为首的安全性方面存在问题(非专利文献9)，且稳定性差，所以一般不实用。
虽开发出为使生物体胶原蛋白增加的各种成分及组合物，但均未达到兼具安全性且十分有效的作用。
本发明发现与各自单独使用相比，并用水飞蓟宾及各种肽具有协同促进I型胶原蛋白产生的作用，从而完成了本发明。
专利文献1：日本特开平7-278012
专利文献2：日本特开昭63-39821
专利文献3：日本特开昭52-111600
专利文献4：日本特开平7-82299
专利文献5：日本特开平6-46875
专利文献6：日本专利第3504205号
专利文献7：日本特开2001-1310844
专利文献8：WO 2004/085429
专利文献9：日本特开2002-173424
专利文献10：日本特开平5-286864号公报
专利文献11：日本专利第2948818号
专利文献12：日本特开2000-169328号公报
专利文献13：日本特开2000-169332号公报
专利文献14：日本特愿2002-255448号
专利文献15：日本特公平5-9406号公报
专利文献16：日本特公昭63-41396号公报
专利文献17：日本特开2002-255847号公报
非专利文献1：Nutrition Reports International，13，579，1976
非专利文献2：Br.J.Pharmacol.，69，551，1980
非专利文献3：J.Biochem.，50，486，1961
非专利文献4：J.Biol.Chem.，268(14)，9941-9944，1993
非专利文献5：Ann.NY Aced.Sci.，580，p573
非专利文献6：Int J Pept Protein Res.，17(4)，p430-435.，1981
非专利文献7：J.Invest.Dermatol.，Vol.94，No.5，p717-723，1990
非专利文献8：Varani J.，et al.，J Invest.Dermatol.，116，480-486，2000
非专利文献9：Zouboulis，C.C.，Retinoids：Is there a New Approach？，IFSCC Magazine，Vol.3，No.3，2000
非专利文献10：Wagner，H.，etal.，Arznein.Forsch，24，466，1974.
非专利文献11：Tittel，G.，etal.，J.Chromatogr.，135，499，1977.
非专利文献12：Tittel，G.，etal.，J.Chromatogr.，153，227，1978.
非专利文献13：Quercia，V.，etal.，Chromatography in Biochemistry，Medicine and Environmental Research，Frigerio A.(Ed).，ElsevierScientificPublIshing Company，Amsterdam，1983，p1.
非专利文献14：Lowry，O.et.al.，J.Biol.Chem.，193，265，1951
非专利文献15：M.J.Barttek，etal.，J.Invest.Dermatol.，58，114，1972
发明内容
本发明以提供促进人体皮肤纤维芽细胞的I型胶原蛋白产生的组合物为课题。
本发明者进行了为更有效地提高I型胶原蛋白的产生的研究。其结果，发现并用作为胶原蛋白的原料起作用的促进胶原蛋白产生的来自胶原蛋白的肽及水飞蓟宾时，可协同促进I型胶原蛋白的产生，从而完成了本发明。
即本发明涉及1.用于促进I型胶原蛋白产生的组合物，其特征在于，含有水飞蓟宾及促进胶原蛋白产生的肽；2.本发明1中所述的组合物，其中，促进胶原蛋白产生的肽是从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上；3.本发明1～2中任一项所述的组合物，其用于预防及/或改善皱纹、松弛、暗淡、斑点。4.化妆料，其特征在于，含有水飞蓟宾及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser、或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上。5.食品，其特征在于，含有水飞蓟宾及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上。6.本发明1或2中所述的组合物，其用于动物。
本发明可提供通过并用水飞蓟宾和促进胶原蛋白产生的肽，在促进人体皮肤纤维芽细胞的I型胶原蛋白产生上效果优异的组合物。
通过将本发明的组合物用于化妆料或作为食品摄取，可使皮肤的张力、弹性提高，期待能预防、防止、改善皱纹和松弛。
且本发明的组合物对皮肤来说安全性非常高。
附图说明
图1是在水飞蓟宾、水飞蓟宁、水飞蓟亭的三维皮肤模型中通过Western印迹法检测I型胶原蛋白产生的示意图。
图2是将图1的结果通过图像分析处理后数值化的示意图。
图3是在Gly-Pro-Hyp(记为G-P-Hyp)序列的三肽的三维皮肤模型中通过Western印迹法检测I型胶原蛋白产生的示意图。
图4是将图3的结果通过图像分析处理后数值化的示意图。将Gly-Pro-Hyp记为G-P-Hyp。
图5是在Gly-Pro-Hyp(记为G-P-Hyp)序列的三肽及水飞蓟宾的三维皮肤模型中通过Western印迹法检测I型胶原蛋白产生的示意图。
图6是将图5的结果通过图像分析处理后数值化的示意图。将Gly-Pro-Hyp记为G-P-Hyp。
图7是在Lys-Thr-Thr-Lys-Ser(记为K-T-T-K-S)、Gly-His-Lys(记为G-H-K)、Gly-Glu-Pro-Arg(记为G-Q-P-R)序列的肽及水飞蓟宾的三维皮肤模型中通过Western印迹法检测I型胶原蛋白产生的示意图。
图8是将图7的结果通过图像分析处理后数值化的示意图。将Lys-Thr-Thr-Lys-Ser记为K-T-T-K-S、将Gly-His-Lys记为G-H-K、将Gly-Glu-Pro-Arg记为G-Q-P-R。
图9是在并用Lys-Thr-Thr-Lys-Ser(记为K-T-T-K-S)、Gly-His-Lys(记为G-H-K)、Gly-Glu-Pro-Arg(记为G-Q-P-R)序列的肽及水飞蓟宾时的三维皮肤模型中通过Western印迹法检测I型胶原蛋白产生的示意图。
图10是将图9的结果通过图像分析处理后数值化的示意图。将Lys-Thr-Thr-Lys-Ser记为K-T-T-K-S、将Gly-His-Lys记为G-H-K、将Gly-Glu-Pro-Arg记为G-Q-P-R。
具体实施方式
水飞蓟素(Silymarin；CAS No.65666-07-1)是从菊科水飞蓟(学名Silibum marianum Gaertn、别名大蓟、大翅蓟、乳蓟；CAS No.84604-20-6)中提取的黄酮木脂素的总称，是含有用分子式C25H22O10表示的水飞蓟宾(Silybin；CAS No.22888-70-6)、水飞蓟宁(Silydianin；CAS No.29782-68-1)、水飞蓟亭(Silychristin；CAS No.33889-69-9)、异水飞蓟宾(Isosilybin；CASNo.72581-71-6)等的组合物(非专利文献10)。本发明中，与以往技术相同，将含有水飞蓟提取物中所含有的这些黄酮木脂素的组合物称为水飞蓟素。且水飞蓟素如上所述是黄酮木脂素的混合物，水飞蓟素在植物提取物、植物中的含量可通过分光光度计测定的方法(非专利文献11)、通过薄层色谱法的方法(非专利文献9)、通过高效液相色谱法的方法(非专利文献12～14)测定。这些测定法中，通过分光光度计测定的方法之一2，4-二硝基苯肼分析法已被德国药局[关于水飞蓟(Silybum marianum)果实的专题论文]报道，并广泛应用。
本发明中，上述成分的混合组合物的定量使用2，4-二硝基苯肼分析法，用换算成水飞蓟宾的质量％标记。
水飞蓟素在欧洲从古至今都用于预防及治疗肝脏疾病，且作为抗氧化剂广为人知。作为对皮肤有效的组合物，已开发出干癣及特应性皮炎的治疗制剂(专利文献10)，含有以黄酮木脂素和磷脂的复合物为活性成分，对红斑、烫伤、皮肤或粘膜的营养不良状态、皮炎等的治疗、皮肤的老化防止及对放射线、风、太阳等外部环境刺激的保护非常有效的组合物(专利文献11)，表皮渗透屏障强化剂(专利文献12)，皮脂分泌抑制剂(专利文献13)，预防、防止、改善表皮扁平化的用于防止皮肤老化的组合物(专利文献14)，利用抗氧化能力的用于防止皮肤老化的化妆料(专利文献15)，人体皮肤纤维芽细胞及三维皮肤模型中用于促进I型胶原蛋白产生的组合物(专利文献8)。
将水飞蓟素从水飞蓟的果实中高纯度分离的方法，已报道的有以70～80％的纯度分离的方法及以90～96％的纯度分离的方法(专利文献16)。水飞蓟素通常是用乙醇、乙酸乙酯、丙酮等从水飞蓟的种实中提取，再通过喷雾干燥制成干燥粉末后作为提取物原料市售。本发明中使用的水飞蓟宾可直接使用如上述制备、市售的含有水飞蓟宾的水飞蓟素，也可使用从水飞蓟中浓缩水飞蓟宾后的提取物及将水飞蓟宾分离、精制后作为化合物使用。
本发明中含有水飞蓟宾的植物体，可使用叶、茎、芽、花、木质部、木皮部(树皮)等的地上部、根、块茎等的地下部、种子、树脂等的所有部位。
本发明中水飞蓟宾及含有水飞蓟宾的植物体，可使用使其自身干燥后的干燥物及使用各种溶剂将其溶解后的溶解物。例如，可使用以水或乙醇、甲醇等的醇类、丙二醇、1，3-丁二醇等的多元醇、醚、丙酮、乙酸乙酯等的有机溶剂溶解的溶解物。
本发明中含有水飞蓟宾的植物体，可直接使用天然干燥、热风干燥、冷冻干燥或发酵处理后的产物。制备植物提取物时可依照通常方法，使用通过提取、浓缩、粉末化等处理后获得的产物。
本发明的促进胶原蛋白产生的肽，优选Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽。
已知Gly-Pro-Hyp是将胶原蛋白成分或明胶成分通过胶原酶特异性分解后得到的肽组合物，可比以往的胶原蛋白、明胶或其水解物更快更有效地被消化吸收(专利文献17)。如前所述的报道，对于通过来自溶组织梭菌(Clostridium Histolyticum)的胶原酶分解的胶原蛋白的分解产物，其生成的三肽的36％是Gly-Pro-Hyp，为最多(非专利文献3)，所以是制造Gly-Pro-Hyp的特别高效的方法。本发明中使用的Gly-Pro-Hyp也可使用化学合成后的产物。
本发明中使用的Gly-His-Lys，可使用将α2前胶原蛋白通过胶原酶等的胶原蛋白分解酶分解的产物，也可使用化学合成后的产物。
本发明中使用的Lys-Thr-Thr-Lys-Ser，可使用将α1前胶原蛋白通过胶原酶等的胶原蛋白分解酶分解的产物，也可使用化学合成后的产物。
本发明中使用的Gly-Glu-Pro-Arg，可使用将免疫球蛋白G(immunoglobulin G)通过胰蛋白酶等的分解酶分解的产物，也可使用化学合成后的产物。
本发明的用于促进I型胶原蛋白产生的组合物，可促进构成真皮的细胞外基质的主要蛋白质I型胶原蛋白的产生，提高皮肤的张力、弹性，预防、防止、改善由增龄、紫外线照射等引起的皱纹、松弛的形成，使皮肤保持年轻。
本发明的用于促进I型胶原蛋白产生的组合物，可作为化妆料等的皮肤外用剂、经口用食品来制造。
化妆料，可将水飞蓟宾、作为水飞蓟宾供给源的水飞蓟素、含有水飞蓟宾的植物体或其植物提取物的任一种以及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser、Gly-Glu-Pro-Arg中选择的肽直接或添加于小麦胚芽油或橄榄油等中，作为化妆料成分使用，以制造化妆料。
食品，可将水飞蓟宾、作为水飞蓟宾供给源的水飞蓟素、含有水飞蓟宾的植物体或其植物提取物的任一种以及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser、Gly-Glu-Pro-Arg中选择的肽直接或添加各种营养成分后作为食品使用，也可配合在所需的食品中。例如，可添加淀粉、乳糖、麦芽糖、植物油脂粉末、可可脂粉末、硬脂酸等的适当的辅助剂后，使用常用的方法制成适于食用的形态，例如颗粒状、粒状、片剂、胶囊、糊状等作为健康补助食品、保健功能食品等。且可添加于各种食品，例如火腿、香肠等的肉食加工食品，鱼糕、圆筒状鱼糕等的水产加工食品，面包、点心、黄油、奶粉、发酵乳制品中，也可添加于水、果汁、牛乳、清涼饮料等的饮料中使用。
本发明的用于促进I型胶原蛋白产生的组合物中，水飞蓟宾与各种肽的配合比率(摩尔比)，可根据各种成分的配制法、制剂的形态等适当选择决定，无特别限定，水飞蓟宾为1时，优选将各种肽以2～6的比例配合。
本发明的向用于促进I型胶原蛋白产生的组合物、化妆料、食品中添加的水飞蓟宾及促进胶原蛋白产生的肽的有效配合量，根据各种成分的配制法、制剂的形态等适当选择决定，无特别限定，但作为皮肤外用剂使用时，优选使水飞蓟宾及促进胶原蛋白产生的肽分别含有0.01～2重量％。另外，作为片剂、饮料等、作为食品使用时，优选含有0.1～10重量％。
本发明的用于促进I型胶原蛋白产生的组合物、化妆料、食品的有效适用量，可通过适用途径、适用日程、制剂形态等适当決定。例如，可在每天0.01g～10g的范围适当调节，分1次或数次使用。
作为食品，可直接或添加各种营养成分后使用。例如，可添加淀粉、乳糖、麦芽糖、植物油脂粉末、可可脂粉末、硬脂酸等适当的辅助剂后使用常用的手段，制成适合食用的形态，例如颗粒状、粒状、片剂、胶囊、糊状等，作为健康补助食品、保健功能食品等用于食用，或也可添加于各种食品，例如火腿、香肠等的肉食加工食品，鱼糕、圆筒状鱼糕等的水产加工食品，面包、点心、黄油、奶粉、发酵乳制品中使用，也可添加于水、果汁、牛乳、清涼饮料等的饮料中使用。上述制剂、食品，可通过通常采用的制剂化技术制造。
作为化妆料，可直接或添加于小麦胚芽油或橄榄油等中作为化妆料的成分使用，使用这些物质可制造化妆料。
适合非经口用的组合物，例如可适用水溶液、油剂、乳液、悬浊液等的液剂，凝胶、霜剂等的半固体剂，粉末、颗粒、胶囊、微胶囊、固体等的固体剂的形态。用以往的公知方法配制成这些形态，可制成洗液、乳剂、凝胶剂、霜剂、软膏、硬膏、巴布剂、气雾剂、坐剂、注射剂、粉末剂等的各种剂型。上述制剂可适用于向身体涂布、粘贴、喷雾等。特别是这些剂型中，洗液、乳剂、霜剂、软膏剂、硬膏剂、巴布剂、气雾剂等可适用于皮肤外用剂。化妆料，可制成化妆水、乳液、面霜、面膜等的皮肤化妆料、化妆用基础化妆水、化妆用面霜、乳液状或奶油状或软膏型的粉底、口红、眼影、胭脂类的化妆用化妆料、护手霜、美腿霜、润肤液等的身体用化妆料等。
制成与增量剂混合的组合物的状态可便于使用。增量剂可使用葡萄糖、乳糖、麦芽糖、蔗糖等的糖类、山梨糖醇等的糖醇、糊精、环状糊精等的加工淀粉、小麦淀粉、玉米淀粉等的淀粉类、酪蛋白、大豆蛋白质等的蛋白质、阿拉伯胶、海藻酸钠、酪蛋白钠、明胶、果胶、粉末纤维素、羧甲基纤维素等的高分子稳定剂、卵磷脂、蔗糖脂肪酸酯、丙二醇脂肪酸酯、甘油脂肪酸酯等的乳化剂、钙粉末等。
本发明的用于促进I型胶原蛋白产生的组合物、化妆料、食品中可含有具有抗氧化作用的化合物。具有抗氧化作用的化合物无特别限定，例如各种维生素类、芦丁等的各种多酚类、生育三烯酚、補酶Q10及含有这些物质的天然成分等。
本发明的用于促进I型胶原蛋白产生的组合物、化妆料、食品中可含有促进除去生物体内异常蛋白质积累的成分。促进除去生物体内異常蛋白质积累的成分，无特别限定，例如可列举为大豆皂角苷、羽衣甘蓝提取物、白芨提取物、水菖蒲提取物等。
其他可根据用途、剂型添加以下物质。
油脂类，例如可为山茶油、月见草油、澳洲胡桃油、橄榄油、菜籽油、玉米油、芝麻油、荷荷巴油、胚芽油、小麦胚芽油、三辛酸甘油酯等的液体油脂，可可脂、椰子油、氢化椰子油、棕榈油、棕榈核油、木蜡、木蜡核油、氢化油、氢化蓖麻油等的固体油脂，蜂蜡、小烛树蜡、棉蜡、糠蜡、羊毛脂、醋酸羊毛脂、液状羊毛脂、蔗蜡等的蜡类。
烃类，例如可为液体石蜡、角鲨烯、角鲨烷、微晶蜡等。
高级脂肪酸，例如可为月桂酸、肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸、二十二碳六烯酸(DHA)、二十碳五烯酸(EPA)等。
高级醇，例如可为月桂醇、硬脂醇、十六烷醇、十八十六醇等的直链醇，十八烷基甘油醚、羊毛醇、胆固醇、植物甾醇、辛基十二烷醇等的支链醇等。
硅酮，例如可为链状聚硅氧烷的二甲基聚硅氧烷、甲基苯基聚硅氧烷等，环状聚硅氧烷的十甲基环五硅氧烷等。
阴离子表面活性剂，例如可为月桂酸钠等的脂肪酸盐、月桂基硫酸钠等的高级烷基硫酸酯盐、POE月桂基硫酸三乙醇胺等的烷基醚硫酸酯盐、N-酰基肌氨酸、磺基琥珀酸盐、N-酰基氨基酸盐等。
阳离子表面活性剂，例如可为十八烷基三甲基氯化铵等的烷基三甲基铵盐、氯化苯甲烃铵、苄索氯铵等。
两性表面活性剂，例如可为烷基甜菜碱、酰胺甜菜碱等的甜菜碱系表面活性剂等。
非离子表面活性剂，例如可为失水山梨醇单油酸酯等的失水山梨醇脂肪酸酯类、氢化蓖麻油衍生物等。
防腐剂，例如可为对羟基苯甲酸甲酯、对羟基苯甲酸乙酯等。
金属离子封锁剂，例如可为乙二胺四乙酸二钠、乙二胺四乙酸、乙二胺四乙酸钠盐等的乙二胺四乙酸盐等。
高分子，例如可为阿拉伯胶、西黄蓍胶、半乳聚糖、瓜尔豆胶、卡拉胶、果胶、琼脂、榅桲子、葡聚糖、普鲁兰多糖、羧甲基淀粉、胶原蛋白、酪蛋白、明胶、甲基纤维素、羟丙基甲基纤维素、羟乙基纤维素、羧甲基纤维素钠(CMC)、海藻酸钠、聚羧乙烯(Carboxyvinylpolymer)(CARBOPOL等)等的乙烯类高分子等。
增粘剂，例如可为卡拉胶、西黄蓍胶、榅桲子、酪蛋白、糊精、明胶、羧甲基纤维素钠(CMC)、羟乙基纤维素、羟丙基纤维素、聚羧乙烯、瓜尔豆胶、黄原胶、膨润土等。
粉末成分，例如可为滑石粉、高岭土、云母、二氧化硅、沸石、聚乙烯粉末、聚苯乙烯粉末、纤维素粉末、无机白色颜料、无机红色系颜料、云母钛珠光颜料、氧化钛包覆的滑石粉、着色型云母钛珠光颜料等的珠光颜料、红色201号、红色202号等的有机颜料等。
紫外线吸收剂，例如可为对氨基苯甲酸、水杨酸苯酯、对甲氧基肉桂酸异丙酯、对甲氧基肉桂酸辛酯、2，4-二羟基二苯甲酮等。
紫外线屏蔽剂，例如可为氧化钛、滑石粉、胭脂红、膨润土、高岭土、氧化锌等。
保湿剂，例如可为聚乙二醇、丙二醇、二丙二醇、1，3-丁二醇、1，2-戊二醇、甘油、双甘油、聚甘油、木糖醇、麦芽糖醇、麦芽糖、山梨糖醇、葡萄糖、果糖、硫酸软骨素钠、透明质酸钠、乳酸钠、吡咯烷酮羧酸、环糊精等。
药效成分，例如可为维生素A油、视黄醇等的维生素A类，核黄素等的维生素B2类、吡哆醇盐酸盐等的B6类，L-抗坏血酸、L-抗坏血酸磷酸酯、L-抗坏血酸单棕榈酸酯、L-抗坏血酸二棕榈酸酯、L-抗坏血酸-2-葡萄糖苷等的维生素C类，泛酸钙等的泛酸类，维生素D2、维生素D3等的维生素D类；α-维生素E、醋酸维生素E、DL-α-维生素E烟酸酯等的维生素E类等的维生素类等。
且可为胎盘提取物、谷胱甘肽、虎耳草提取物等的美白剂，蜂王浆、山毛榉提取物等的皮肤活化剂，辣椒素、姜酮、斑蝥素、鱼石脂、咖啡因、单宁酸、γ-谷维素等的血流促进剂，甘草酸衍生物、甘草次酸衍生物、甘菊环等的消炎剂，精氨酸、丝氨酸、亮氨酸、色氨酸等的氨基酸类，常驻菌控制剂的麦芽糖蔗糖缩合物、氯化溶菌酶等。
并且，还可为洋甘菊提取物、荷兰芹提取物、山毛榉提取物、葡萄酒酵母提取物、葡萄柚提取物、金银花提取物、米提取物、葡萄提取物、啤酒花提取物、米糠提取物、枇杷提取物、黄檗提取物、薏米仁提取物、当药提取物、草木樨提取物、桦树提取物、甘草提取物、芍药提取物、肥皂草提取物、丝瓜提取物、辣椒提取物、柠檬提取物、龙胆提取物、紫苏提取物、芦荟提取物、迷迭香提取物、鼠尾草提取物、铺地百里香提取物、茶提取物、海藻提取物、黄瓜提取物、丁香提取物、胡萝卜提取物、欧洲七叶树提取物、金缕梅提取物、桑树提取物等的各种提取物。
以下可列举试验例、实施例更详细地说明本发明，但本发明的效果不限于这些试验例、实施例。
[药剂的配制]
在培养液中适量添加将水飞蓟宾(Silibin；Sigma Aldrich)通过生物化学用试剂级别的二甲亚砜(DMSO；和光纯药)溶解的溶液，对三维皮肤模型进行处理。
在培养液中适量添加将水飞蓟素(Sigma Aldrich)通过生物化学用试剂级别的二甲亚砜(DMSO；和光纯药)溶解的溶液，对三维皮肤模型进行处理。
水飞蓟宁、水飞蓟亭用如下方法进行分离。将2g水飞蓟素(SigmaAldrich)溶解到10ml乙醇中，在200ml ODS色谱柱(FUJISILYSIACHEMICAL DM1020T)中，用水比甲醇的比率为1比1的溶剂条件分离，得到水飞蓟宁粗画分、水飞蓟亭粗画分。
水飞蓟宁粗画分、水飞蓟亭粗画分分别在200ml硅胶色谱中，用氯仿比甲醇的比率为95比5的溶剂条件分离，得到17mg水飞蓟亭精制物、34mg水飞蓟宁精制物。在培养液中适量添加将水飞蓟亭精制物、水飞蓟宁精制物通过生物化学用试剂级别的二甲亚砜(DMSO；和光纯药)溶解的溶液，对三维皮肤模型进行处理。
GLY-PRO-HYP使用了市售品(销售厂家；和光纯药，制造厂家；BachemAG，Switzerland)。
Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg委托Nikka Techno Service Co.，LTD.，[日语原名：「日化テクノサ一ビス(株)」]使用通常方法合成。合成后通过反相HPLC精制。证实通过根据氨基酸分析及反相HPLC的纯度分析，合成了90％以上纯度的目的肽。在培养液中适量添加将这些肽通过生物化学用试剂级别的二甲亚砜(DMSO；和光纯药)溶解的溶液，对三维皮肤模型进行处理。
[人体皮肤三维模型]
人体皮肤三维模型是人体皮肤的仿真模型，广泛用于安全性评价、有效性评价。人体皮肤三维模型使用人造皮肤(TESTSKIN)(LSE-high)(東洋纺绩)。
[各药剂处理的人体皮肤三维模型提取样品的制备]
在人造皮肤(TESTSKIN)(LSE-high)的外侧孔中添加培养基，培养24小时。之后在更换培养液的同时，将100μl各药剂添加在检验环(assayring)内的组织上，培养36小时。回收组织，加入组织提取用溶液{50mMTris-HCl(pH7.5)、0.5％(Octylphenoxy)polyethoxyethanol(Sigma-Aldrich)}，用氟碳树脂制均质器均质。10,000×G、30分钟离心除去组织片后，在蒸馏水中4℃透析一夜。其后通过冷冻干燥除去水分。加入组织提取用溶液至20倍浓缩，三维皮肤模型提取样品用于Western印迹法。
[通过Western印迹法的促进I型胶原蛋白产生的作用的评价]
用人体皮肤三维模型提取样品，每泳道使用(apply)10μg蛋白质，用SDS-PAGE分离后，转移至硝酸纤维素膜。将转移后的硝酸纤维素膜浸入封闭溶液(用含有0.1％聚氧乙烯(20)失水山梨醇单月桂酸酯的PBS溶解脱脂奶，使脱脂奶为5％的浓度的溶液)，4℃下封闭一昼夜。用洗涤液{含有0.1％的聚氧乙烯(20)失水山梨醇单月桂酸酯的PBS}洗涤后，浸入一次抗体{用洗涤液配制的500ng/ml抗I型胶原蛋白的多克隆抗体(Rockland)}，室温反应1小时。洗涤后浸入二次抗体(用洗涤液配制成250ng/ml的辣根过氧化物酶标记抗兔免疫球蛋白G)，室温反应1小时。洗涤后，使用ECL Plus Western印迹法检测试剂(Amersham Biosciences公司)检测。且检测出的条带使用图像扫描仪(Image Scanner)(AmershamBiosciences公司)输入后，通过ImageMaster软件(Amersham Biosciences公司)分析并数值化。
实施例
实施例1
有关水飞蓟素中含有的促进I型胶原蛋白产生的有效成分的评价
用10μg/ml水飞蓟素、分别用10μM浓度的水飞蓟宾、水飞蓟亭、水飞蓟宁对人体皮肤三维模型进行处理，通过Western印迹法测定I型胶原蛋白的量。检测出的条带如图1所示，将检测出的条带数值化后的图如图2所示。
用10μg/ml水飞蓟素、10μM水飞蓟素中所含有的成分水飞蓟宾处理时，与无处理对照相比，I型胶原蛋白的产生被促进，分别为2.3倍及3.2倍(图1及图2)。另外，用10μM水飞蓟素中所含有的成分水飞蓟亭、水飞蓟宁处理时，与无处理对照相比，I型胶原蛋白的产生分别为0.95倍及0.9倍(图1及图2)。因此可表明，水飞蓟素中所含有的有关促进I型胶原蛋白产生的有效成分是水飞蓟宾。
实施例2
单独使用Gly-Pro-Hyp及并用Gly-Pro-Hyp与水飞蓟宾时对促进I型
胶原蛋白产生作用的评价
用5、10、30μM浓度的Gly-Pro-Hyp对人体皮肤三维模型进行处理，通过Western印迹法测定I型胶原蛋白的量。检测出的条带如图3表示，将检测出的条带数值化的图如图4所示。同样，单独使用30μMGly-Pro-Hyp、单独使用5μM水飞蓟宾、并用30μM Gly-Pro-Hyp和5μM水飞蓟宾进行测定。所得到的条带如图5所示，其数值化的图如图6所示。
用5、10、30μM Gly-Pro-Hyp处理时，与无处理对照相比，I型胶原蛋白的产生分别为1.05倍、1.15倍及1.25倍(图3及图4)。因此可表明，即使单独使用Gly-Pro-Hyp也具有促进I型胶原蛋白产生的作用，但其作用不太强。
单独使用30μM Gly-Pro-Hyp、单独使用5μM水飞蓟宾、并用30μMGly-Pro-Hyp和5μM水飞蓟宾时，与无处理对照相比，I型胶原蛋白的产生分别为1.25倍、1.85倍及4.25倍(图5及图6)。因此可表明，与各自单独使用相比，并用Gly-Pro-Hyp及水飞蓟宾能协同促进I型胶原蛋白产生。
实施例3
分别单独使用Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu
-Pro-Arg及将Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-
Pro-Arg分别与水飞蓟宾并用时对促进I型胶原蛋白产生作用的评价
分别用10及30μM浓度的Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg对人体皮肤三维模型进行处理，通过Western印迹法测定I型胶原蛋白的量。检测出的条带如图7所示，将检测出的条带数值化后的图如图8所示。同样，分别单独使用10μM Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg，单独使用5μM水飞蓟宾，将10μM Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg分别与5μM水飞蓟宾并用，对人体皮肤三维模型进行处理，测定I型胶原蛋白产生量。所得到的条带如图9所示，将其数值化的图如图10所示。
分别用10及30μM Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg处理时，与无处理对照相比，I型胶原蛋白产生在用10及30μM的Lys-Thr-Thr-Lys-Ser处理时为1.80倍、2.45倍，用10及30μM的Gly-His-Lys处理时为1.75倍、2.70倍，用10及30μM的Gly-Glu-Pro-Arg处理时为2.15倍及3.55倍(图7及图8)。因此可表明，Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg促进I型胶原蛋白产生的效果，分别为浓度依赖型促进效果。
且分别单独用10μM Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg及单独用5μM水飞蓟宾处理时，与无处理对照相比，I型胶原蛋白的产生分别为1.80倍、1.75倍、2.10倍及1.95倍(图9及图10)，而分别用10μM Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg与5μM水飞蓟宾并用时，与无处理对照相比，I型胶原蛋白产生分别为5.75倍、5.35倍、4.95倍(图9及图10)。因此可表明，与各自单独使用相比，通过使Lys-Thr-Thr-Lys-Ser、Gly-His-Lys、Gly-Glu-Pro-Arg分别与水飞蓟宾并用能协同促进I型胶原蛋白产生。
以下表示本发明的处方例，但本发明不限于这些处方例。
处方例1 胶囊剂的制造
混合下述成分，充填到混合有明胶及甘油的胶囊基质中，得到软胶囊。
(组成) (配合量；mg)
水飞蓟提取物(含有35％水飞蓟宾) 25
大豆提取物(含有80％大豆皂角苷) 25
Gly-Pro-Hyp(胶原蛋白分解精制物) 25
生育三烯烃 30
蜂蜡 10
葡萄籽油 110
处方例2 片剂的制造
混合下述成分，压片，得到片剂。
(组成) (配合量；mg)
水飞蓟提取物(含有35％水飞蓟宾) 25
大豆提取物(含有80％大豆皂角苷) 25
Gly-Pro-Hyp(胶原蛋白分解精制物) 25
纤维素 40
淀粉 20
蔗糖脂肪酸酯 2
处方例3 片剂的制造
混合下述成分，压片，得到片剂。
(组成) (配合量；mg)
水飞蓟提取物(含有35％水飞蓟宾) 25
大豆提取物(含有80％大豆皂角苷) 25
Lys-Thr-Thr-Lys-Ser(肽) 25
纤维素 40
淀粉 20
蔗糖脂肪酸酯 2
处方例4 片剂的制造
混合下述成分，压片，得到片剂。
(组成) (配合量；mg)
水飞蓟提取物(含有35％水飞蓟宾) 25
大豆提取物(含有80％大豆皂角苷) 25
Gly-His-Lys(肽) 25
纤维素 40
淀粉 20
蔗糖脂肪酸酯 2
处方例5 片剂的制造
混合下述成分，压片，得到片剂。
(组成) (配合量；mg)
水飞蓟提取物(含有35％水飞蓟宾) 25
大豆提取物(含有80％大豆皂角苷) 25
Gly-Glu-Pro-Arg(肽) 25
纤维素 40
淀粉 20
蔗糖脂肪酸酯 2
处方例6 果汁的制造
(组成) (配合；质量％)
果糖葡萄糖液糖 5.00
柠檬酸 10.40
L-抗坏血酸 0.20
香料 0.02
色素 0.10
水飞蓟提取物(含有35％水飞蓟宾) 1.00
Gly-Pro-Hyp(胶原蛋白分解精制物) 1.00
大豆提取物(含有85％大豆皂角苷) 1.00
水 81.28
处方例7 霜剂的制造
将下述成分(1)～(12)在80℃下加热溶解成为油相。将成分(13)～(15)在70℃下加热溶解成为水相。在油相中缓缓加入水相进行乳化，搅拌的同时冷却到40℃，进一步搅拌冷却到30℃后得到霜剂。
(组成) (配合；质量％)
(1)硬脂醇 6.0
(2)硬脂酸 2.0
(3)氢化羊毛脂 4.0
(4)角鲨烷 9.0
(5)辛基十二烷醇 10.0
(6)POE(25)十六烷醇醚 3.0
(7)单硬脂酸甘油酯 2.0
(8)白芨提取物 0.1
(9)水飞蓟提取物(含有85％水飞蓟宾) 0.7
(10)Gly-Pro-Hyp(胶原蛋白分解精制物) 0.1
(11)防腐剂 适量
(12)香料 适量
(13)1，3丁二醇 6.0
(14)PEG 1500 4.0
(15)精制水 剩余
处方例8 霜剂的制造
将下述成分(1)～(12)在80℃下加热溶解成为油相。将成分(13)～(15)在70℃下加热溶解成为水相。在油相中缓缓加入水相进行乳化，搅拌的同时冷却到40℃，进一步搅拌冷却到30℃后得到霜剂。
(组成) (配合；质量％)
(1)硬脂醇 6.0
(2)硬脂酸 2.0
(3)氢化羊毛脂 4.0
(4)角鲨烷 9.0
(5)辛基十二烷醇 10.0
(6)POE(25)十六烷醇醚 3.0
(7)单硬脂酸甘油酯 2.0
(8)白芨提取物 0.1
(9)水飞蓟提取物(含有85％水飞蓟宾) 0.7
(10)Lys-Thr-Thr-Lys-Ser(肽) 0.1
(11)防腐剂 适量
(12)香料 适量
(13)1，3丁二醇 6.0
(14)PEG 1500 4.0
(15)精制水 剩余
处方例9 霜剂的制造
将下述成分(1)～(12)在80℃下加热溶解成为油相。将成分(13)～(15)在70℃下加热溶解成为水相。在油相中缓缓加入水相进行乳化，搅拌的同时冷却到40℃，进一步搅拌冷却到30℃后得到霜剂。
(组成) (配合；质量％)
(1)硬脂醇 6.0
(2)硬脂酸 2.0
(3)氢化羊毛脂 4.0
(4)角鲨烷 9.0
(5)辛基十二烷醇 10.0
(6)POE(25)十六烷醇醚 3.0
(7)单硬脂酸甘油酯 2.0
(8)白芨提取物 0.1
(9)水飞蓟提取物(含有85％水飞蓟宾) 0.7
(10)Gly-His-Lys(肽) 0.1
(11)防腐剂 适量
(12)香料 适量
(13)1，3丁二醇 6.0
(14)PEG 1500 4.0
(15)精制水 剩余
处方例10 霜剂的制造
将下述成分(1)～(12)在80℃下加热溶解成为油相。将成分(13)～(15)在70℃下加热溶解成为水相。在油相中缓缓加入水相进行乳化，搅拌的同时冷却到40℃，进一步搅拌冷却到30℃后得到霜剂。
(组成) (配合；质量％)
(1)硬脂醇 6.0
(2)硬脂酸 2.0
(3)氢化羊毛脂 4.0
(4)角鲨烷 9.0
(5)辛基十二烷醇 10.0
(6)POE(25)十六烷醇醚 3.0
(7)单硬脂酸甘油酯 2.0
(8)白芨提取物 0.1
(9)水飞蓟提取物(含有85％水飞蓟宾) 0.7
(10)Gly-Glu-Pro-Arg(肽) 0.1
(11)防腐剂 适量
(12)香料 适量
(13)1，3丁二醇 6.0
(14)PEG 1500 4.0
(15)精制水 剩余
序列表
<110>株式会社芳珂(FANCL Corporation)
<120>用于促进I型胶原蛋白产生的组合物
(Composition for Promoting Production of Type 1 Collagen)
<130>PC26-023
<160>2
<210>1
<211>5
<212>PRT
<213>人工序列(Artificial Sequence)
<400>
Lys Thr Thr Lys Ser
1 5
<210>2
<211>4
<212>PRT
<213>人工序列(Artificial Sequence)
<400>
Gly Glu Pro Arg
1
Claims (6)
1.用于促进I型胶原蛋白产生的组合物，其特征在于，含有水飞蓟宾及促进胶原蛋白产生的肽。
2.权利要求1中所述的组合物，其中，促进胶原蛋白产生的肽是从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上。
3.权利要求1～2中任一项所述的组合物，其用于预防及/或改善皱纹、松弛、暗淡、斑点。
4.化妆料，其特征在于，含有水飞蓟宾及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上。
5.食品，其特征在于，含有水飞蓟宾及从Gly-Pro-Hyp、Gly-His-Lys、Lys-Thr-Thr-Lys-Ser或Gly-Glu-Pro-Arg的氨基酸序列所组成的肽中选择的1种或2种以上。
6.权利要求1或2中所述的组合物，其用于动物。
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- 2006-07-20 US US12/088,655 patent/US8022038B2/en not_active Expired - Fee Related
- 2006-07-20 WO PCT/JP2006/314393 patent/WO2007037060A1/ja active Application Filing
- 2006-07-28 TW TW101113938A patent/TWI438002B/zh active
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Also Published As
| Publication number | Publication date |
|---|---|
| HK1117080A1 (en) | 2009-01-09 |
| JP4033877B2 (ja) | 2008-01-16 |
| TWI438002B (zh) | 2014-05-21 |
| US8022038B2 (en) | 2011-09-20 |
| TW200740453A (en) | 2007-11-01 |
| JP2007091637A (ja) | 2007-04-12 |
| US20090325885A1 (en) | 2009-12-31 |
| CN101277713B (zh) | 2012-03-21 |
| TW201249455A (en) | 2012-12-16 |
| TWI373342B (zh) | 2012-10-01 |
| WO2007037060A1 (ja) | 2007-04-05 |
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