CN113599320A - 多潜能细胞培养的组合物及制备方法和应用 - Google Patents
多潜能细胞培养的组合物及制备方法和应用 Download PDFInfo
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Abstract
本发明属于细胞培养技术领域，公开了多潜能细胞培养的组合物及制备方法和应用。该组合物包括雪莲提取物、银杏叶提取物、金盏花提取物。该组合物含有雪莲提取物、银杏叶提取物、金盏花提取物，该三种物质的使用，具有协同增效作用，使得所述组合物显著提高皮肤类细胞的增殖能力，进一步有助于皮肤细胞的修复更新。该组合物应用到化妆品或医用产品中，可显著提高皮肤的抗衰老能力。
Description
技术领域
本发明属于细胞培养技术领域，特别涉及多潜能细胞培养的组合物及制备方法和应用。
背景技术
干细胞是一种具有自我复制能力的多潜能细胞，在一定条件下，它可以分化成多种功能的细胞。典型的干细胞培养基中包括必需氨基酸、盐类、维生素、矿物质、痕量金属、糖、脂质和核苷。最常规的培养干细胞培养基是添加有胎牛血清的培养基，然而，采用这种培养基培养的干细胞，不仅生长比较缓慢，而且传递次数超过5代之后其分化成功能细胞的潜能大大降低。现有技术中的细胞培养基对皮肤类的细胞的增殖作用不明显，这不利于皮肤细胞的修复更新。
另外，皮肤细胞的修复更新有助于进一步减缓皮肤衰老。
因此，亟需提供一种细胞培养的组合物，该组合物不仅能有助于皮肤类的细胞的增殖，进一步的，有助于提高皮肤的抗衰老能力，是十分有必要的。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出多潜能细胞培养的组合物及制备方法和应用，所述组合物有助于皮肤类细胞的增殖，有利于皮肤细胞的修复更新。所述组合物也有助于干细胞的培养分化。将所述组合物应用到化妆品或医用产品中，有助于提高皮肤的抗衰老能力。
本发明的发明构思：本发明所述组合物含有雪莲提取物、银杏叶提取物、金盏花提取物，该三种物质的使用，具有协同增效作用，使得所述组合物显著提高皮肤类细胞的增殖能力，进一步有助于皮肤细胞的修复更新。该组合物同时具备抗氧化性能，减缓皮肤衰老。将所述组合物应用到化妆品或医用产品中，可显著提高皮肤的抗衰老能力。
本发明的第一方面提供一种多潜能细胞培养的组合物。
具体的，多潜能细胞培养的组合物，包括雪莲提取物、银杏叶提取物、金盏花提取物。
进一步优选的，所述组合物还包括山药提取物。山药提取物的加入，有助于进一步提升组合物促进皮肤类细胞的增殖能力。
优选的，所述组合物还包括氨基酸、盐类、维生素、矿物质、糖、脂肪酸、溶剂中的至少一种。这些组分属于细胞培养基常规组分。
优选的，所述氨基酸包括精氨酸、赖氨酸、亮氨酸、异亮氨酸、苏氨酸、缬氨酸、苯丙氨酸、组氨酸、丝氨酸、甘氨酸、半胱氨酸、脯氨酸、蛋氨酸、色氨酸、谷氨酸、天冬氨酸或丙氨酸的至少三种。
优选的，所述盐类包括钠盐、钾盐、钙盐、镁盐、锌盐、铜盐。
进一步优选的，所述钠盐包括氯化钠。
进一步优选的，所述钾盐包括氯化钾。
进一步优选的，所述钙盐包括氯化钙、碳酸钙、泛酸钙中的至少一种。
进一步优选的，所述镁盐包括硫酸镁和/或氯化镁。
进一步优选的，所述锌盐包括硫酸锌和/或氯化锌。
进一步优选的，所述铜盐包括硫酸铜。
优选的，所述组合物还包括磷酸盐缓冲盐溶液。所述组合物的pH优选7.2-7.4，磷酸盐缓冲盐溶液可根据pH进行添加。
优选的，所述维生素包括维生素A、维生素B、维生素C、维生素E。
优选的，所述糖包括葡萄糖。
优选的，所述所述脂肪酸为亚油酸和亚麻酸。
优选的，所述矿物质包括硫酸铁和硫酸硒。
优选的，所述溶剂包括水和/或甘油。
优选的，所述组合物还包括肌肽和5,7-二羟基黄酮。肌肽和5,7-二羟基黄酮的同时加入，配合雪莲提取物、银杏叶提取物、金盏花提取物，可显著提高组合物的抗皮肤衰老能力。
优选的，所述组合物，按重量份数计，包括雪莲提取物5-18份、银杏叶提取物1-6份、金盏花提取物3-16份。
优选的，所述组合物，按重量份数计，包括雪莲提取物5-15份、银杏叶提取物1-5份、金盏花提取物3-15份。
优选的，所述组合物，按重量份数计，还包括山药提取物10-25份；进一步优选的，所述组合物，按重量份数计，还包括山药提取物10-20份。
优选的，所述组合物，按重量份数计，还包括氨基酸5-20份、盐类5-15份、维生素5-25份、矿物质0.01-0.5份、糖20-50份、脂肪酸20-70份、溶剂80-300份。
进一步优选的，所述组合物，按重量份数计，还包括氨基酸8-15份、盐类8-12份、维生素10-20份、矿物质0.01-0.5份、糖40-50份、脂肪酸40-70份、水100-200份。
优选的，所述组合物，按重量份数计，还包括肌肽0.1-2份和5,7-二羟基黄酮0.1-3份；进一步优选的，所述组合物，按重量份数计，还包括肌肽0.5-1份和5,7-二羟基黄酮0.5-2份。
本发明的第二方面提供一种多潜能细胞培养的组合物的制备方法。
具体的，一种多潜能细胞培养的组合物的制备方法，包括以下步骤：
取各原料组分，搅拌混合，制得所述组合物。
优选的，上述组合物的制备方法，包括以下步骤：
将雪莲提取物、银杏叶提取物、金盏花提取物与部分溶剂混合，制得混合物A，然后将氨基酸、盐类、维生素、矿物质、糖、脂肪酸与剩余溶剂混合，制得混合物B，将混合物A、混合物B以及剩余组分混合，制得所述组合物。
本发明的第三方面提供上述多潜能细胞培养的组合物的应用。
上述多潜能细胞培养的组合物在制备化妆品或医用品中的应用。
一种化妆品，包括上述组合物和辅料。所述辅料为化妆品常用物质，例如防腐剂。
优选的，所述化妆品包括洁面乳、化妆水、喷雾、精华液、面膜、凝胶、美容冻干粉、溶媒、乳液和膏霜。
一种医用品，包括上述组合物和医用辅料。医用辅料为本领域常规物质。
所述医用产品包括医用冻干粉、溶媒、敷料、软膏和冷敷贴。
相对于现有技术，本发明的有益效果如下：
(1)本发明所述组合物含有雪莲提取物、银杏叶提取物、金盏花提取物，该三种物质的使用，具有协同增效作用，使得所述组合物显著提高皮肤类细胞的增殖能力，进一步有助于皮肤细胞的修复更新。该组合物同时具备抗氧化性能，减缓皮肤衰老。
(2)所述组合物还包括山药提取物。山药提取物的加入，有助于进一步提升组合物促进皮肤类细胞的增殖能力。所述组合物还包括肌肽和5,7-二羟基黄酮。肌肽和5,7-二羟基黄酮的同时加入，配合雪莲提取物、银杏叶提取物、金盏花提取物，可显著提高组合物的抗皮肤衰老能力。
(3)将所述组合物应用到化妆品或医用产品中，可显著提高皮肤的抗衰老能力。
具体实施方式
为了让本领域技术人员更加清楚明白本发明所述技术方案，现列举以下实施例进行说明。需要指出的是，以下实施例对本发明要求的保护范围不构成限制作用。
以下实施例中所用的原料、试剂或装置如无特殊说明，均可从常规商业途径得到，或者可以通过现有已知方法得到。
实施例1：组合物的制备
多潜能细胞培养的组合物，按重量份数计，包括雪莲提取物6份、银杏叶提取物2份、金盏花提取物4份、氨基酸6份(精氨酸0.5份、赖氨酸0.5份、亮氨酸0.5份、异亮氨酸0.5份、苏氨酸0.5份、缬氨酸0.5份、苯丙氨酸0.5份、组氨酸0.5份、丝氨酸0.5份、甘氨酸0.5份、半胱氨酸0.5份、天冬氨酸0.5份)、盐类5份(氯化钠3.35份、氯化钾0.5份、泛酸钙0.5份、硫酸镁0.5份、硫酸锌0.1份、硫酸铜0.05份)、维生素5份(维生素A 2份、维生素B1份、维生素C1份、维生素E1份)、矿物质0.11份(硫酸铁0.1份、硫酸硒0.01份)、葡萄糖40份、脂肪酸60份(亚油酸30份、亚麻酸30份)、溶剂150份(水130份、甘油20份)。
上述多潜能细胞培养的组合物的制备方法，包括以下步骤：
将雪莲提取物、银杏叶提取物、金盏花提取物与部分溶剂(60份水、10份甘油)混合，制得混合物A，然后将氨基酸、盐类、维生素、矿物质、葡萄糖、脂肪酸与剩余溶剂混合，制得混合物B，将混合物A、混合物B混合，制得组合物。
实施例2：组合物的制备
多潜能细胞培养的组合物，按重量份数计，包括雪莲提取物12份、银杏叶提取物4份、金盏花提取物3份、氨基酸10份(精氨酸1份、赖氨酸1份、亮氨酸1份、异亮氨酸1份、苏氨酸1份、缬氨酸0.5份、苯丙氨酸0.5份、组氨酸0.5份、丝氨酸0.5份、甘氨酸0.5份、半胱氨酸0.5份、天冬氨酸0.5份、脯氨酸0.5份、蛋氨酸0.5份、色氨酸0.5份)、盐类6.65份(氯化钠3份、氯化钾1份、泛酸钙2份、硫酸镁0.5份、硫酸锌0.1份、硫酸铜0.05份)、维生素8份(维生素A 3份、维生素B 2份、维生素C 2份、维生素E1份)、矿物质0.11份(硫酸铁0.1份、硫酸硒0.01份)、葡萄糖40份、脂肪酸50份(亚油酸20份、亚麻酸30份)、溶剂200份(水180份、甘油20份)。
上述多潜能细胞培养的组合物的制备方法，包括以下步骤：
将雪莲提取物、银杏叶提取物、金盏花提取物与部分溶剂(80份水、10份甘油)混合，制得混合物A，然后将氨基酸、盐类、维生素、矿物质、葡萄糖、脂肪酸与剩余溶剂混合，制得混合物B，将混合物A、混合物B混合，制得组合物。
实施例3：组合物的制备
与实施例1相比，实施例3的区别在于，组合物中还含有山药提取物15份，其余组分和制备方法与实施例1相同。
实施例4：组合物的制备
与实施例2相比，实施例4的区别在于，组合物中还含有肌肽0.5份和5,7-二羟基黄酮1份，其余组分和制备方法与实施例2相同。
实施例5：组合物的制备
与实施例4相比，实施例5的区别在于，组合物中含有肌肽1份，但不含5,7-二羟基黄酮，其余组分和制备方法与实施例4相同。
实施例6：组合物的制备
与实施例4相比，实施例6的区别在于，组合物中含有5,7-二羟基黄酮1.5份，但不含肌肽，其余组分和制备方法与实施例4相同。
实施例7：组合物的制备
与实施例1相比，实施例7的区别在于，组合物中雪莲提取物为4份、银杏叶提取物7份、金盏花提取物1份，其余组分和制备方法与实施例1相同。
对比例1
与实施例1相比，对比例1的区别在于用金黄洋甘菊提取物替代金盏花提取物，其余组分和制备方法与实施例1相同。
对比例2
与实施例2相比，对比例2的区别在于组合物中不含金盏花提取物和雪莲提取物，其余组分和制备方法与实施例2相同。
对比例3
与实施例2相比，对比例3的区别在于组合物中不含雪莲提取物和银杏叶提取物，其余组分和制备方法与实施例2相同。
对比例4
与实施例2相比，对比例4的区别在于组合物中不含金盏花提取物和银杏叶提取物，其余组分和制备方法与实施例2相同。
产品效果测试
1.皮肤细胞增殖效果测试
将处于对数生长期的HaCaT细胞以5×104个/mL的密度接种于96孔板，于37℃，CO2培养箱培养24小时(培养用的培养基为常规DMEM培养基)，分别加入等量实施例1、2、3、7和对比例1-4制得的组合物(称为待测样品)进行细胞处理24小时、48小时，然后采用CCK-8(应用于细胞增殖和细胞毒性的检测试剂)方法检测待测样品对细胞增殖的影响。另外，在相同条件下，以重组牛碱性成纤维生长因子bFGF(重组牛碱性成纤维生长因子bFGF是已知的对皮肤细胞增殖有促进作用)作为阳性对照组。HaCaT细胞存活率结果如表1所示。
表1：细胞存活率结果
| 24小时细胞存活率(％) | 48小时小时细胞存活率(％) | |
| 实施例1 | 130 | 139 |
| 实施例2 | 131 | 140 |
| 实施例3 | 135 | 148 |
| 实施例7 | 128 | 136 |
| 对比例1 | 118 | 123 |
| 对比例2 | 101 | 106 |
| 对比例3 | 104 | 110 |
| 对比例4 | 106 | 113 |
| 阳性对照组 | 101 | 103 |
从表1可以看出，实施例1、2、3、7制得的组合物对HaCaT细胞的增殖具有明显的促进作用，促进作用远大于对比例1-4和阳性对照组。特别是从对比例2-4与实施例2的结果可以看出，本发明组合物中雪莲提取物为、银杏叶提取物、金盏花提取物的同时使用，具有明显促进HaCaT细胞的增殖的作用，即雪莲提取物为、银杏叶提取物、金盏花提取物具有协同增效的作用。
2.皮肤抗衰老效果测试
2.1.自由基清除率测试
当外界因素(例如阳光、紫外线、污染的空气及化学试剂)刺激皮肤时，会使皮肤产生羟自由基，加速皮肤老化。
取本发明实施例1、2、4-7，对比例1-4制得的组合物，测试其抗衰老效果(抗衰老效果的强弱用DPPH自由基清除率来衡量，DPPH指1,1-二苯基-2-三硝基苯肼，DPPH自由基清除率的测试方法是本领域常规方法)，结果如表2所示。
表2：抗衰老效果
| DPPH自由基清除率(％) | |
| 实施例1 | 95.9 |
| 实施例2 | 96.1 |
| 实施例4 | 98.5 |
| 实施例5 | 96.9 |
| 实施例6 | 97.1 |
| 实施例7 | 95.1 |
| 对比例1 | 89.0 |
| 对比例2 | 72.2 |
| 对比例3 | 75.8 |
| 对比例4 | 78.9 |
从表2可以看出，本发明实施例制得的组合物的抗衰老效果明显优于对比例制得的组合物的抗衰老效果。而且从实施例1-2与实施例4的结果可以看出，肌肽和5,7-二羟基黄酮的加入，可显著提高组合物的抗衰老效果。
2.2.皮肤弹性测试
选存在皱纹较多的志愿者50名，年龄30-50岁，随机分为5组，每组10人。志愿者使用相同的皮肤清洁剂清理脸部之后，在脸部左右对称位置选6×6cm2作为实验区域；取实施例4和对比例1-4制得的组合物作为待测样品，每次涂抹0.2g，早晚各一次。在涂抹待测样品前，用Callegari soft plus皮肤测试仪检测脸部实验区域的皮肤弹性，每位志愿者的实验区域测试10次取平均值，进一步计算每组的平均值，记为a；连续使用待测样品3周后，用Callegari soft plus皮肤测试仪检测脸部实验区域的皮肤弹性，每位志愿者的实验区域测试10次取平均值，进一步计算每组的平均值，记为b；b除以a的结果记为弹性评级，弹性评级值越大，说明待测样品增加皮肤弹性的效果越好。弹性评级结果如表3所示。
表3：弹性评级结果
| 实施例4 | 对比例1 | 对比例2 | 对比例3 | 对比例4 | |
| 弹性评级值 | 1.50 | 1.41 | 1.12 | 1.25 | 1.31 |
从表3可以看出，本发明实施例4制得的组合物可显著增加皮肤弹性，具有皮肤抗衰作用。
另外，使用上述待测样品涂抹脸部，未见脸部出现红肿等过敏现象，表明本发明制得的组合物是安全可靠的。
应用例1
一种化妆品抗衰面霜，包括实施例4制得的组合物。
应用例2
一种抗衰医用敷料，包括实施例4制得的组合物。
本发明实施例制得的组合物培养干细胞，传递次数超过5代之后其分化成功能细胞的潜能较大。
Claims (10)
1.一种组合物，其特征在于，包括雪莲提取物、银杏叶提取物、金盏花提取物。
2.根据权利要求1所述的组合物，其特征在于，所述组合物还包括山药提取物。
3.根据权利要求1所述的组合物，其特征在于，所述组合物还包括氨基酸、盐类、维生素、矿物质、糖、脂肪酸、溶剂中的至少一种。
4.根据权利要求1所述的组合物，其特征在于，所述组合物还包括肌肽和5,7-二羟基黄酮。
5.根据权利要求1所述的组合物，其特征在于，所述组合物，按重量份数计，包括雪莲提取物5-18份、银杏叶提取物1-6份、金盏花提取物3-16份。
6.根据权利要求5所述的组合物，其特征在于，所述组合物，按重量份数计，还包括氨基酸5-20份、盐类5-15份、维生素5-25份、矿物质0.01-0.5份、糖20-50份、脂肪酸20-70份、溶剂80-300份。
7.权利要求1-6中任一项所述的组合物的制备方法，其特征在于，包括以下步骤：
取各原料组分，搅拌混合，制得所述组合物。
8.权利要求1-6中任一项所述的组合物在制备化妆品或医用品中的应用。
9.一种化妆品，其特征在于，包括权利要求1-6中任一项所述的组合物，以及辅料。
10.一种医用品，其特征在于，包括权利要求1-6中任一项所述的组合物，以及医用辅料。
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