CN115350115B - 包含在细胞培养基中的白蛋白、透明质酸或胶原蛋白作为活性成分的化妆品组合物 - Google Patents
包含在细胞培养基中的白蛋白、透明质酸或胶原蛋白作为活性成分的化妆品组合物 Download PDFInfo
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Abstract
本发明涉及包含细胞培养基和白蛋白、透明质酸或胶原蛋白作为有效成分的化妆品组合物或药物组合物。根据本发明的组合物可用作化妆品组合物或药物组合物,以用于通过增加细胞的恢复能力来缓解、预防或治疗皮肤创伤、皮肤病或皮肤状况。
Description
技术领域
本发明涉及包含在细胞培养基中的白蛋白、透明质酸或胶原蛋白作为活性成分的组合物。
本发明还涉及包含上述组合物作为活性成分的化妆品组合物,以及使用该化妆品组合物改善皮肤状况的方法。
本发明还涉及包含上述组合物作为活性成分的用于预防或治疗皮肤病的药物组合物,以及使用该药物组合物预防或治疗皮肤病的方法。
背景技术
由于工业化和城市化加剧导致的饮食和居住生活的变化,以及由于环境污染引起的化学或生物有害物质的暴露,过敏性疾病在各个年龄群中显著增加。人体对无害外部环境的敏感反应表现为呼吸障碍(例如哮喘或鼻炎)或皮肤病(例如接触性皮炎、特应性皮炎)等。
具体地,皮肤通过经空气、食物等与外部过敏原直接接触而显示出过敏性免疫反应。这种过敏性皮肤病是由于持续暴露于外部抗原(例如食物、细菌、螨虫、气候和环境因素等)而引起的炎性疾病,并且对各种环境因素的过敏反应的表现根据每个人的遗传特征因人而异。
在此,过敏是指由免疫失衡引起的一种超敏反应,并且是对被称为过敏原的特定抗原的反应。过敏反应通过过敏原与肥大细胞或嗜碱性粒细胞结合而引发,在所述肥大细胞或嗜碱性粒细胞中以颗粒形式储存由L-组氨酸脱羧酶使组氨酸脱羧而形成的组胺,然后细胞脱颗粒而分泌组胺和β-己糖胺酶。
特应性皮炎是一种过敏性疾病,其是一种慢性和复发性皮肤病,主要发生于婴儿期或儿童期,其患病率在世界范围内不断增加。特应性皮炎的病因尚未明确,但环境因素、遗传易感性、免疫化学反应、皮肤屏障异常等被认为是该疾病的主要原因。它表现出各种症状,例如瘙痒、皮肤干燥、湿疹等。皮肤病变的分布和反应模式还根据患者的年龄而变化。
目前,广泛用于治疗特应性皮炎的药物通常包括局部类固醇、局部免疫调节剂、全身类固醇、全身免疫抑制剂、抗组胺药等。但是,这些药物的给药或口服摄入可能存在各种副作用的风险,因此需要开发一种安全并且对皮肤干燥和皮肤屏障异常具有改善作用的活性成分。
发明内容
技术问题
因此,本发明人致力于研究和开发用于改善皮肤状况和治疗皮肤病的物质,并且发现了与特定物质混合的组合物增加了细胞的创伤愈合能力。
问题的解决方案
根据本发明的一个目的,提供了一种化妆品组合物,其包含细胞培养基以及作为活性成分的白蛋白、透明质酸和胶原蛋白中的任何一种。
根据本发明的另一个目的,提供了一种改善皮肤状况的方法,包括将上述化妆品组合物施用于有此需要的对象的皮肤。
根据本发明的又另一个目的,提供了一种药物组合物,其包含细胞培养基以及作为活性成分的白蛋白、透明质酸和胶原蛋白中的任何一种。
根据本发明的又另一个目的,提供了一种预防或治疗皮肤病的方法,包括将上述药物组合物施用于有此需要的对象的皮肤上。
发明的有益效果
包含细胞培养基以及作为活性成分的白蛋白、透明质酸和胶原蛋白中的任何一种的化妆品或药物组合物提高了细胞的恢复能力,因此它可以有效地用作改善、预防或治疗由于外部损伤引起的皮肤创伤、皮肤病或皮肤状况的化妆品组合物或药物组合物,并且可用作新的化妆品或治疗剂。而且,它可以用于目前使用的皮肤软膏和功能性化妆品中,因此它可以容易地被开发为添加剂。
附图说明
图1示出了包含制备的细胞培养基组合物和白蛋白或胶原蛋白I的组合物对人皮肤成纤维细胞的创伤愈合作用。
图2是对包含制备的细胞培养基组合物和白蛋白或胶原蛋白I的组合物对人皮肤成纤维细胞的创伤愈合作用进行定量的图。
图3示出了包含制备的细胞培养基组合物、白蛋白、透明质酸或胶原蛋白III的组合物对人皮肤成纤维细胞的创伤愈合作用。
图4是对包含制备的细胞培养基组合物、白蛋白、透明质酸或胶原蛋白III的组合物对人皮肤成纤维细胞的创伤愈合作用进行定量的图。
图5示出了细胞培养基和白蛋白的混合组合物的特应症改善效果。
图6示出了细胞培养基和白蛋白的混合组合物的特应症改善效果。
图7显示了细胞培养基和白蛋白的混合组合物的角膜再生效果。
具体实施方式
在一个方面,本发明提供了一种组合物,包含细胞培养基;以及选自由白蛋白、透明质酸和胶原蛋白组成的组中的任何一种。
如本文所用的术语“细胞培养基”含有细胞在体外生长和存活所需的组分,或有助于细胞生长和存活的组分。具体地,该组分可以是维生素、必需或非必需氨基酸和微量元素。该培养基可以是用于培养细胞的培养基,该细胞优选为真核细胞,更优选为动物细胞。
细胞培养基的已知实例包括DMEM/F-12(杜氏改良伊格尔培养基/营养混合物F-12)、DMEM、MEMα(最低必需培养基α)、BME(伊格尔基础培养基)、GMEM(格拉斯哥最低必需培养基)、RPMI-1640培养基,但不限于此。
此外,本发明的细胞培养基是无血清的。无血清培养基是指未补充来自包括人的动物的血清(例如动物来源的血清)的培养基。虽然动物来源的血清为要培养的细胞的生长提供了通用的营养素,但是其具有由于含有未鉴定的微量成分而难以分析的问题,难以建立可重复的试验和生产过程,并且在人体内的稳定性无法得到保证。然而,根据本发明的细胞培养基是无血清培养基,其确保在人体内的稳定性并且能够建立可重复的测试和生产过程。
另外,所述细胞培养基是指培养细胞前的细胞培养基。由于它是培养细胞前的培养基,所以细胞培养基的组成没有变化,因此它可以在商业上以均匀的品质使用。
根据本发明的细胞培养基由氨基酸组分、维生素组分、无机盐组分、其他组分和纯净水组成,其中:
a)氨基酸组分是选自由甘氨酸、L-丙氨酸、L-缬氨酸、L-亮氨酸、L-异亮氨酸、L-苏氨酸、L-丝氨酸、L-半胱氨酸、L-蛋氨酸、L-天冬氨酸、L-天冬酰胺、L-谷氨酸、L-谷氨酰胺、L-赖氨酸、L-精氨酸、L-组氨酸、L-苯丙氨酸、L-酪氨酸、L-色氨酸、L-脯氨酸、β-丙氨酸、γ-氨基丁酸、鸟氨酸、瓜氨酸、高丝氨酸、三碘酪氨酸、甲状腺素和二氧苯丙氨酸组成的组中的至少一种氨基酸,或其组合,并且优选地,它是选自由甘氨酸、L-丙氨酸、L-精氨酸、L-半胱氨酸、L-谷氨酰胺、L-组氨酸、L-赖氨酸、L-蛋氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸和L-缬氨酸组成的组中的至少一种,或其组合,
b)维生素组分是选自由生物素、D-泛酸钙、叶酸、烟酰胺、盐酸吡哆醇、核黄素、盐酸硫胺素、维生素B12、氯化胆碱、i-肌醇和抗坏血酸组成的组中的至少一种维生素,或其组合,优选地,它是选自由i-肌醇、盐酸硫胺素、烟酰胺和盐酸吡哆醇组成的组中的至少一种,或其组合,
c)该无机盐成分是选自由氯化钙(CaCl2)(无水)、五水合硫酸铜(CuSO4-5H2O)、七水合硫酸铁(FeSO4-7H2O)、氯化镁(无水)、硫酸镁(MgSO4)(无水)、氯化钾(KCl)、氯化钠(NaCl)、磷酸氢二钠(Na2HPO4)、一水合磷酸氢钠(NaH2PO4-H2O)、七水硫酸锌(ZnSO4-7H2O)、硝酸铁九水合物((Fe(NO3)3·9H2O)和碳酸氢钠(NaHCO3)的至少一种无机盐,或其组合,优选地,它是选自由氯化钠(NaCl)、碳酸氢钠(NaHCO3)、氯化钾(KCl)、氯化钙(CaCl2)(无水)和一水合磷酸氢钠(NaH2PO4-H2O)组成的组中的至少一种无机盐,或其组合,
d)其他成分是选自由D-葡萄糖(右旋糖)、丙酮酸钠、次黄嘌呤钠、胸苷、亚油酸、硫辛酸、腺苷、胞苷、鸟苷、尿苷、2'-脱氧腺苷、2'-脱氧胞苷HCl、2'-脱氧鸟苷组成的组中的至少一种,或其组合,优选地,它是丙酮酸钠,
e)纯化水用于溶解上述氨基酸、维生素、无机盐和其它组分,该纯化水可以是通过至少单次蒸馏的蒸馏或通过经过滤器纯化而获得的水。
根据本发明的实施方式,细胞培养基优选由甘氨酸、L-丙氨酸、L-精氨酸盐酸盐、L-半胱氨酸盐酸盐一水合物、L-谷氨酰胺、L-组氨酸盐酸盐一水合物、L-赖氨酸盐酸盐、L-蛋氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-缬氨酸、i-肌醇、盐酸硫胺素、烟酰胺、盐酸吡哆醇、氯化钠(NaCl)、碳酸氢钠(NaHCO3)、氯化钾(KCl)、氯化钙(CaCl2)(无水)、一水合磷酸氢钠(NaH2PO4-H2O)和丙酮酸钠组成。
氨基酸组分用于细胞生长以作为蛋白质合成的原材料。基于细胞培养基中包含1重量份的氨基酸丝氨酸,氨基酸组分可包含0.2至1.2重量份的甘氨酸、0.08至0.3重量份的L-丙氨酸、2至7重量份的L-精氨酸盐酸盐、0.2至0.9重量份的L-半胱氨酸盐酸盐一水合物、10至18重量份L-谷氨酰胺、0.8至2.0重量份L-组氨酸盐酸盐一水合物、1.0至5.0重量份L-赖氨酸盐酸盐、0.2至1.2重量份L-蛋氨酸、0.2至1.2重量份的L-脯氨酸、1.0至5.0重量份的L-苏氨酸和1.0至5.0重量份的L-缬氨酸。
根据本发明的细胞培养基中氨基酸组分和含量的一个具体实例可以是0.5至1.0重量份的甘氨酸、0.10至0.2重量份的L-丙氨酸、5.0至6.0重量份的L-盐酸精氨酸盐、0.4至0.7重量份的L-半胱氨酸盐酸盐一水合物、12至15重量份的L-谷氨酰胺、1.0至1.5重量份的L-组氨酸盐酸盐一水合物、2.0至4.0重量份的L-盐酸赖氨酸、0.4至0.8重量份的L-甲硫氨酸、0.4至0.8重量份的L-脯氨酸、1.5至2.5重量份的L-苏氨酸和1.5至2.5重量份的L-缬氨酸。通过根据上述含量和范围包含氨基酸组分,可以有助于细胞的生长并改善制剂的制剂稳定性。
在细胞培养基中,维生素组分起到维持细胞活性的作用,并且基于1重量份的氨基酸丝氨酸,可包含0.2至0.9重量份的i-肌醇、0.04至0.8重量份的盐酸硫胺素、0.04至0.8重量份的烟酰胺,和0.04至0.8重量份的盐酸吡哆醇。
根据本发明的细胞培养基中维生素组分和含量的一个具体实例可以是0.4至0.7重量份的i-肌醇,0.06至0.2重量份的盐酸硫胺素,0.06至0.2重量份的烟酰胺和0.06至0.2重量份盐酸吡哆醇。通过根据上述含量和范围包含维生素成分,可以有助于维持细胞活性。
无机盐组分起到控制细胞功能表达的作用,并且基于1重量份氨基酸丝氨酸,可包含50至200重量份的氯化钠(NaCl)、50至120重量份的碳酸氢钠(NaHCO3)、2.0至20重量份氯化钾(KCl)、1.0至10重量份的氯化钙(CaCl2)(无水)和0.5至5.0重量份的一水合磷酸氢钠(NaH2PO4-H2O)。
基于1重量份的氨基酸丝氨酸,根据本发明的细胞培养基中无机盐组分和含量的一个具体实例可以是100至150重量份的氯化钠(NaCl)、80至100重量份的碳酸氢钠(NaHCO3)、8至15重量份氯化钾(KCl)、2.0至8.0重量份氯化钙(CaCl2)(无水)和1.0至4.0重量份一水合磷酸氢钠(NaH2PO4-H2O))。
其他组分可包括维持细胞培养基等的pH的组分,并且基于1重量份的氨基酸丝氨酸,可包含0.5至5.0重量份的丙酮酸钠。
基于1重量份的氨基酸丝氨酸,根据本发明的组合物中的其他组分和含量的一个具体实例可以是1.0至4.0重量份的丙酮酸钠。通过根据上述含量和范围包含其他组分,可以有助于维持pH并因此改善制剂的稳定性。
构成本发明组合物的白蛋白可以是人血清白蛋白或牛血清白蛋白。
人血清白蛋白是人血液中发现的血清白蛋白,并且是人血浆中存在的最丰富的蛋白质。在本发明中,人血清白蛋白可以特别是重组人血清白蛋白。此外,牛血清白蛋白是在牛中发现的血清白蛋白,并且通常在实验中用作蛋白质浓度标准。
白蛋白的含量可以为10μg/ml、50μg/ml、100μg/ml、200μg/ml、300μg/ml、500μg/ml或1000μg/ml的浓度。可供选择地,白蛋白的含量可以为10至1000μg/ml、50至500μg/ml或100至250μg/ml的浓度。
另外,构成本发明组合物的透明质酸是大量存在于动物皮肤等中的生物合成的天然物质,其是亲水性物质,并且在动物等的皮肤中起到保湿作用。
透明质酸的浓度可以为0.01mg/ml、0.1mg/ml、0.5mg/ml、1mg/ml、5mg/ml或10mg/ml。透明质酸的浓度可以为0.01至1mg/ml、0.05至0.5mg/ml、或0.01至0.1mg/ml。
同时,对于构成根据本发明的组合物的胶原蛋白,可以使用胶原蛋白I至胶原蛋白IV,并且在具体实例中,可以使用胶原蛋白I和胶原蛋白III。最优选地,胶原蛋白可以是胶原蛋白III。
人体内约有20种胶原蛋白。胶原蛋白I是人体中发现的最丰富的胶原蛋白,它占皮肤、头发、指甲、器官、骨骼和韧带的约80%至85%。胶原蛋白III是人体内第二丰富的胶原蛋白。它构成皮肤、骨骼、软骨、肌腱和其他结缔组织的约10%至15%,并且也被称为胎盘胶原蛋白。
胶原蛋白在组合物中的浓度可以为1μg/ml、5μg/ml、10μg/ml、20μg/ml、50μg/ml、100μg/ml、150μg/ml或200μg/ml。胶原蛋白的浓度可以为1至200μg/ml、2至100μg/ml或10至80μg/ml。
在此,组合物中的胶原蛋白I的浓度可以为1至100μg/ml、5至50μg/ml、或10至25μg/ml,优选为10μg/ml。另外,胶原蛋白III的浓度可以为2至200μg/ml、10至120μg/ml和20至70μg/ml,优选为20μg/ml。
本发明的一种实施方式可以是包含细胞培养基和白蛋白的组合物。它也可以是包含细胞培养基和透明质酸的组合物。它也可以是包含细胞培养基和胶原蛋白的组合物。
另外,该组合物可以是包含细胞培养基、白蛋白和胶原蛋白的组合物。该组合物还可以是包含细胞培养基、白蛋白和透明质酸的组合物。另外,该组合物可以是包含细胞培养基、胶原蛋白和透明质酸的组合物。该组合物还可以是包含细胞培养基、白蛋白、胶原蛋白和透明质酸的组合物。
作为实验的结果,发现与单独治疗每种组分相比,当将组分混合并用于治疗时,人皮肤成纤维细胞的恢复能力进一步提高。此外,在本发明的实施方式中,与已知具有优异的皮肤改善效果的包含细胞培养基和EGF的组合物相比,包含全部细胞培养基、白蛋白和胶原蛋白I,或细胞培养基、白蛋白、胶原蛋白III和透明质酸的组合物显示出更为优异的创伤愈合效果。(参见图2和图4)。
本发明还提供包含上述组合物的化妆品组合物。该组合物具有改善皮肤状况的作用。“改善皮肤状况”是指保护皮肤免于皮肤细胞功能的下降或丧失,或改善皮肤状况,或预防或治疗皮肤病的作用。皮肤状况改善选自由抑制皱纹发展、抑制皮肤老化、改善皮肤弹性、皮肤再生、损伤或创伤愈合、角膜再生、缓解皮肤刺激或其组合。皮肤病可选自特应性皮炎、过敏、皮肤湿疹、痤疮、牛皮癣、瘙痒及其组合。
根据本发明的化妆品组合物可用于制备用于改善皮肤状况的化妆品组合物。
可以将化妆品组合物配制成本领域常规制备的化妆品制剂。化妆品组合物可以配制成例如溶液剂、悬液剂、乳剂、糊剂、凝胶剂、霜剂、洗剂、粉剂、肥皂、含表面活性剂的清洁剂、油、粉底、乳液粉底、蜡粉底、喷雾剂等。但是,本发明不限于此。更具体地,化妆品组合物可以配制成柔软化妆水、营养化妆水、营养霜、按摩霜、精华素、眼霜、清洁霜、清洁泡沫、清洁水、面膜、喷雾或粉末。
当本发明的化妆品组合物的制剂是糊剂、霜剂或凝胶剂时,它可以包含选自由动物油、植物油、蜡、石蜡、淀粉、黄蓍胶、纤维素衍生物、聚乙二醇、硅氧烷、膨润土、二氧化硅、滑石、氧化锌及其混合物组成的组的载体组分。
当本发明的化妆品组合物的制剂是溶液剂或乳剂时,其可含有选自溶剂、溶剂化物、乳化剂及其混合物的载体组分。该载体组分的实例包括水、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、脱水山梨糖醇脂肪酸酯及其混合物。
当本发明的化妆品组合物的制剂是悬液剂时,它可以包含选自液体稀释剂(例如水、乙醇或丙二醇)、悬浮剂(例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糖醇酯)、微晶纤维素、偏氢氧化铝、膨润土、琼脂、黄蓍胶及其混合物。
本发明还提供一种使用该化妆品组合物改善皮肤状况的方法。
改善皮肤状况的方法可包括将根据本发明的化妆品组合物施用于有需要的对象的皮肤。对象可以是哺乳动物,特别是人。
将组合物施用于皮肤的步骤可包括根据组合物的形式将根据本发明的化妆品组合物直接施用或喷雾到皮肤上。在此,化妆品组合物的施用量和日常使用次数可以根据使用者的年龄、性别、目的、症状的严重程度等适当调整。例如,可以将适量的化妆品组合物施用于皮肤每天一到六次。
本发明还提供用于预防或治疗皮肤病的药物组合物,其包含根据本发明的组合物作为活性成分。
本发明的药物组合物具有与上述化妆品组合物相同的活性成分。
皮肤病可选自特应性皮炎、过敏、皮肤湿疹、痤疮、牛皮癣、瘙痒及其组合。
药物组合物可进一步包含药学上可接受的载体。
基于本发明的药物组合物的总重量,载体的含量可以为约1重量%至约99.99重量%,优选为约90重量%至约99.99重量%。
本发明的药物组合物可以施用于皮肤。
本发明的药物组合物的制剂可以是皮肤外用制剂的形式,例如软膏剂、溶液剂、霜剂、喷雾剂、贴剂等。
考虑到各种因素,例如制剂方法、给药方法、患者年龄、体重、性别、病理状况、食物、给药时间、给药途径、排泄率、反应敏感性等,确定本发明的药物组合物的合适剂量,因此上述剂量不应被解释为在任何方面限制本发明的范围。
本发明提供使用上述药物组合物预防或治疗皮肤病的方法。
本发明还提供了该药物组合物在制备用于预防或治疗皮肤病的药物中的用途。
用于预防或治疗皮肤病的方法可包括将根据本发明的药物组合物施用于有需要的对象的皮肤。可以施用药物组合物的对象可以是哺乳动物,特别是人。
将组合物施用于皮肤的步骤可包括根据组合物的形式将根据本发明的化妆品组合物直接施用或喷雾到皮肤上。在此,化妆品组合物的施用量和日常使用次数可以根据使用者的年龄、性别、目的、症状的严重程度等适当调整。例如,可以将适量的化妆品组合物应用于皮肤每天一到六次。
本发明的方式
在下文中,通过实施例详细说明本发明。以下实施例旨在进一步说明本发明而不限制其范围。
制备例1.细胞培养基的制备
将表1中描述的每种组分彻底溶解在超纯水中,确认所有组分均被溶解,然后通过0.2μm过滤器过滤。下表1中列出的所有组分均购自美国Sigma-Aldrich。制备细胞培养基,其是含有下列22种组分的混合组合物,并且在下文中称为BG。
[表1]
组分 | 浓度(g/L) |
甘氨酸 | 0.01875 |
L-丙氨酸 | 0.00445 |
L-精氨酸盐酸盐 | 0.1475 |
L-半胱氨酸盐酸盐一水合物 | 0.01756 |
L-谷氨酰胺 | 0.365 |
L-组氨酸盐酸盐一水合物 | 0.03148 |
L-赖氨酸盐酸盐 | 0.09125 |
L-蛋氨酸 | 0.01724 |
L-脯氨酸 | 0.01725 |
L-丝氨酸 | 0.02625 |
L-苏氨酸 | 0.05345 |
L-缬氨酸 | 0.05285 |
i-肌醇 | 0.0126 |
盐酸硫胺素 | 0.00217 |
烟酰胺 | 0.00202 |
盐酸吡哆醇 | 0.002 |
氯化钠(NaCl) | 3.49775 |
碳酸氢钠(NaHCO3) | 2.438 |
氯化钾(KCl) | 0.3118 |
氯化钙(CaCl2)(无水) | 0.1166 |
一水合磷酸二氢钠(NaH2PO4-H2O) | 0.0625 |
丙酮酸钠 | 0.055 |
实施例1至9.制备包含细胞培养基和白蛋白、胶原蛋白或透明质酸的组合物
根据表2中所示的含量,通过将白蛋白、胶原蛋白或透明质酸与制备例1中制备的BG混合来制备组合物。
[表2]
实施例10至16.包含细胞培养基和白蛋白、胶原蛋白或透明质酸的组合物的制备
根据表3中所示的含量,通过将MEMα(Sigma-Aldrich,USA)与白蛋白、胶原蛋白或透明质酸混合来制备组合物。
[表3]
实验例1.评估细胞的恢复能力
使用补充有10%FBS(胎牛血清)和1%P/S(青霉素-链霉素)的细胞培养基DMEM(1X),在保持在5%CO2下的37℃恒温恒湿器中培养人皮肤成纤维细胞系(Hs27)。将ibidi培养插件附接到6孔板上,在其中以每个插件孔中5.2×104个细胞培养人皮肤成纤维细胞。在上述条件下培养人皮肤成纤维细胞24小时后,将细胞在无血清DMEM(1X)培养液中再培养18小时,保持无血清状态直至本实验。然后,移除ibidi培养插件。
1.1.检验含有BG和白蛋白或胶原蛋白I的组合物的细胞恢复能力
用BG(对照组)、BG和100μg/ml白蛋白的混合物组合物(实施例1)、BG和10μg/ml胶原蛋白I的混合物组合物(实施例2)或100μg/ml白蛋白和10μg/ml胶原蛋白I的混合物组合物(实施例3)处理制备的细胞培养溶液。
处理后9小时,使用显微镜拍摄细胞,使用TScratch程序测量由损伤引起的间隙的距离,并与对照组进行比较。结果如图1和图2所示。
如图1和图2所示,发现与对照组相比,当用包含BG和白蛋白或胶原蛋白I中的至少一种的组合物处理细胞时,人皮肤成纤维细胞的增殖和迁移增加。
具体地,与用白蛋白或胶原蛋白I单独处理的细胞培养溶液相比,当用白蛋白和胶原蛋白I组合处理含有BG的细胞培养溶液时,人皮肤成纤维细胞的增殖和迁移增加,证实了混合处理对创伤愈合有效。也就是说,发现与用白蛋白或胶原蛋白I单独处理相比,当用白蛋白和胶原蛋白I组合处理含有BG的细胞培养溶液时,人皮肤成纤维细胞的恢复能力进一步增加。
1.2.检验含有细胞培养基和白蛋白、透明质酸或胶原蛋白III的组合物的细胞恢
复能力
用BG(对照组)、BG和100μg/ml白蛋白的混合物组合物(实施例1)、BG和0.1mg/ml透明质酸的混合物组合物(实施例4)、BG和20μg/ml胶原蛋白III的混合物组合物(实施例5)、或BG、100μg/ml白蛋白、0.1mg/ml透明质酸和20μg/ml胶原蛋白III的混合物组合物(实施例9)处理制备的细胞培养溶液。在对照组中,单独地添加BG、100μg/ml白蛋白、0.1mg/ml透明质酸或20μg/ml胶原蛋白III。
处理9小时后,使用显微镜拍摄细胞,使用TScratch程序测定由损伤引起的间隙的距离,并与实验例进行比较。结果如图3和图4所示。
如图3和图4所示,发现与用BG、白蛋白、透明质酸或胶原蛋白III单独治疗相比,当混合组分后用BG、白蛋白、透明质酸和胶原蛋白III的组合处理细胞时,人皮肤成纤维细胞的增殖和迁移进一步增加。也就是说,发现与用BG、白蛋白、透明质酸或胶原蛋白III单独处理相比,当用BG、白蛋白、透明质酸和胶原蛋白III组合处理细胞培养溶液时,发现人皮肤成纤维细胞的恢复能力进一步增加。
1.3.检验包含MEMα和白蛋白、透明质酸或胶原蛋白III的细胞组合物的细胞恢复
能力
通过与实验例1.2相同的方法进行实验。检测实施例10至16的细胞恢复能力。作为结果,发现与用每种成分单独处理相比,当混合组分后用白蛋白、透明质酸和胶原蛋白III的组合处理细胞培养基时,细胞的恢复能力是优异的。此外,当组合添加上面列出的所有三种物质时,观察到最佳的恢复能力。
实验例2.检验包含细胞培养基和白蛋白的组合物的特应症改善能力
检验包含制备例1中制备的细胞培养基和500μg/ml白蛋白的组合物是否对作为一种皮肤病的特应症有效。
将包含细胞培养基和白蛋白的组合物每天两次施用于6月龄的男性特应症患者的受影响区域(头部),持续2天。施用结果如图5所示。此外,将包含细胞培养基和白蛋白的组合物每天两次施用于24岁女性特应症患者的受影响区域(颈部),持续10天。结果如图6所示。
如图5和图6所示,在施用两天后,6月龄男孩的头部上由特应症诱发的创伤显著减少,并且施用10天后,24岁女性女性患者的颈部上的由特应症诱发的创伤显著减少。这意味着包含细胞培养基和白蛋白的混合组合物可有效改善特应症。
实验例3.评价包含细胞培养基和白蛋白的组合物的角膜再生效果
检验包含制备例1中制备的细胞培养基(BG)和100μg/ml白蛋白的组合物是否对角膜糜烂有效。
使用将包含细胞培养基和白蛋白的组合物对患有难治性角膜糜烂的32岁韩国女性患者的角膜(眼睛)每天两次进行滴眼,持续2周。滴眼的结果如图7所示。
如图7所示,发现患有难治性角膜糜烂的患者的损伤角膜在滴眼2周后被治愈。这意味着包含细胞培养基和白蛋白的混合培养基对角膜再生有效。
Claims (19)
1.一种用于改善皮肤状况的化妆品组合物,包括:
细胞培养基;以及
选自由白蛋白、透明质酸和胶原蛋白组成的组中的任何一种,
其中,所述细胞培养基由氨基酸组分、维生素组分、无机盐组分和其他组分组成;
其中,所述氨基酸组分包含甘氨酸、L-丙氨酸、L-精氨酸盐酸盐、L-半胱氨酸盐酸盐一水合物、L-谷氨酰胺、L-组氨酸盐酸盐一水合物、L-赖氨酸盐酸盐、L-蛋氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-缬氨酸;
其中,所述维生素组分包含i-肌醇、盐酸硫胺素、烟酰胺和盐酸吡哆醇;
其中,基于1重量份的L-丝氨酸,所述无机盐组分包含50至200重量份的氯化钠(NaCl)、50至120重量份的碳酸氢钠(NaHCO3)、2.0至20重量份氯化钾(KCl)和1.0至10重量份的氯化钙(CaCl2)(无水);以及
其中,基于1重量份的L-丝氨酸,所述其他组分包含0.5至5.0重量份的丙酮酸钠。
2.权利要求1所述的组合物,其特征在于,所述细胞培养基是动物细胞培养基。
3.权利要求1所述的组合物,其特征在于,所述细胞培养基由甘氨酸、L-丙氨酸、L-精氨酸盐酸盐、L-半胱氨酸盐酸盐一水合物、L-谷氨酰胺、L-组氨酸盐酸盐一水合物、L-赖氨酸盐酸盐、L-蛋氨酸、L-脯氨酸、L-丝氨酸、L-苏氨酸、L-缬氨酸、i-肌醇、盐酸硫胺素、烟酰胺、盐酸吡哆醇、氯化钠(NaCl)、碳酸氢钠(NaHCO3)、氯化钾(KCl)、氯化钙(CaCl2)(无水)、一水合磷酸氢钠(NaH2PO4-H2O)和丙酮酸钠组成。
4.权利要求3所述的组合物,其特征在于,基于1重量份的L-丝氨酸,所述氨基酸组分包含0.2至1.2重量份的甘氨酸、0.08至0.3重量份的L-丙氨酸、2至7重量份的L-精氨酸盐酸盐、0.2至0.9重量份的L-半胱氨酸盐酸盐一水合物、10至18重量份L-谷氨酰胺、0.8至2.0重量份L-组氨酸盐酸盐一水合物、1.0至5.0重量份L-赖氨酸盐酸盐、0.2至1.2重量份L-蛋氨酸、0.2至1.2重量份的L-脯氨酸、1.0至5.0重量份的L-苏氨酸和1.0至5.0重量份的L-缬氨酸。
5.权利要求1所述的组合物,其特征在于,基于1重量份的L-丝氨酸,所述维生素组分包含0.2至0.9重量份的i-肌醇、0.04至0.8重量份的盐酸硫胺素、0.04至0.8重量份的烟酰胺和0.04至0.8重量份的盐酸吡哆醇。
6.权利要求1所述的组合物,其特征在于,所述细胞培养基是无血清培养基。
7.权利要求2所述的组合物,其特征在于,所述动物细胞培养基选自DMEM/F-12(杜氏改良伊格尔培养基/营养混合物F-12)、DMEM、MEMα(最低必需培养基α)、BME(伊格尔基础培养基)、GMEM(格拉斯哥最低必需培养基)、RPMI-1640培养基及其组合。
8.权利要求1所述的组合物,其特征在于,所述白蛋白是人血清白蛋白或牛血清白蛋白。
9.权利要求1所述的组合物,其特征在于,所述白蛋白的浓度为10至1000μg/ml。
10.权利要求1所述的组合物,其特征在于,所述透明质酸的浓度为0.01至1mg/ml。
11.权利要求1所述的组合物,其特征在于,所述胶原蛋白是胶原蛋白I或胶原蛋白III。
12.权利要求11所述的组合物,其特征在于,所述胶原蛋白I的浓度为1至100μg/ml。
13.权利要求11所述的组合物,其特征在于,所述胶原蛋白III的浓度为2至200μg/ml。
14.权利要求1所述的组合物,其特征在于,其保护皮肤免于皮肤细胞功能的下降或丧失,或改善皮肤状况,其中皮肤状况改善选自由抑制皱纹发展、抑制皮肤老化、改善皮肤弹性、皮肤再生、损伤或创伤愈合、角膜再生、缓解皮肤刺激或其组合。
15.一种用于预防或治疗皮肤病的药物组合物,包含权利要求1所述的组合物作为活性成分。
16.权利要求15所述的组合物,其特征在于,所述皮肤病选自过敏、瘙痒及其组合。
17.权利要求16所述的组合物,其特征在于,所述皮肤病选自特应性皮炎、皮肤湿疹、痤疮、牛皮癣及其组合。
18.权利要求1所述的化妆品组合物在制备用于改善皮肤状况的化妆品组合物中的用途。
19.权利要求15所述的药物组合物在制备用于预防或治疗皮肤病的药物中的用途。
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KR102506310B1 (ko) | 2020-09-14 | 2023-03-03 | 문외숙 | 칼슘아스코르베이트를 함유하는 콜라겐 히알루론산 매트릭스 및 그 제조방법 |
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CN113817046A (zh) * | 2021-09-28 | 2021-12-21 | 山东汉肽医美生物科技有限公司 | 一种重组人胶原蛋白、编码基因及其在制备修复敷料上的应用 |
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US20200297613A1 (en) | 2020-09-24 |
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