CN116284211A - 胎盘蛋白的制备方法及其在化妆品中的应用 - Google Patents
胎盘蛋白的制备方法及其在化妆品中的应用 Download PDFInfo
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Abstract
本发明属于医用、牙科用或化妆用的配实施例领域，具体涉及一种胎盘蛋白的制备方法及其在化妆品中的应用。所述胎盘蛋白使用在化妆品中时，包括组分甘油、透明质酸钠、胎盘蛋白、卡波姆、芽孢杆菌/大豆发酵产物提取物、三肽‑1、六肽‑9、棕榈酰三肽‑5、棕榈酰五肽‑4、十肽‑4、糖蛋白、棕榈酰四肽‑7。本发明的提供的护肤组合物具有祛皱、抗衰和保湿和抗氧化的效果。本发明的成分胎盘蛋白具有生物活性，天然无毒性，有增强成纤维细胞活性，促进胶原蛋白、弹力蛋白、粘多糖合成功效。本发明中采用的重要原料为动物胎盘，是扔掉的医疗废物，提高了经济效益。
Description
技术领域
本发明属于医用、牙科用或化妆用的配实施例领域，具体涉及一种胎盘蛋白的制备方法及其在化妆品中的应用。
背景技术
随着社会的快速发展，人民生活水平的不断提高，各种护肤品在人们生活中扮演的角色越发重要，人们对其的追求也越来越高，也让护肤品的发展日新月异，功能和种类越来越多，面膜是其中重要的一类，面膜类型可分为撕剥型面膜、冻胶型面膜、乳霜型面膜、精油面膜、棉布式面膜、功效型面膜、膜粉型面膜、淡斑面膜、除皱面膜等。面膜的作用原理：就是在面部造成一个封闭的环境，使面部温度升高增加面部细胞活跃，毛细血管运输加快，毛孔扩张，使皮肤内的废物和有害物质排出，面膜精华液中的保湿因子，营养物质进入皮肤中，面膜中的水分渗入表皮的角质层，皮肤变得柔软，肌肤自然光亮而又弹性。
胎盘是母体与胎儿间进行物质交换的器官，是胚胎与母体组织的结合体，由羊膜、叶状绒毛膜和底蜕膜构成。胎盘又叫胞衣、衣胞、紫河车、胎衣、胎膜。胎盘中含有多种促进机体组织细胞代谢和修复的有效成份，随着科学发展来生物制药技术的越来越先进，现已经能制得较纯的胎盘内的蛋白。胎盘蛋白具有抗氧化、延缓衰老、保湿、促进生长等作用，但鉴于人们对于对应原料具有较大的抵触心理，故现在市场上利用胎盘提取的胎盘蛋白用在护肤品上还很少。
发明内容
本发明的目的在于提供一种胎盘蛋白的制备方法及其在化妆品中的应用，该提取物具有修复皮肤，抗皱嫩肤的作用。
胎盘蛋白的制备方法，包括以下步骤：
(1)选取新鲜的猪胎盘，去除胎膜和大血管，-40至-20℃速冻；
(2)然后将其切碎，放入打浆机中打浆至80-100目；
(3)称重然后按每100g组织加入浓度为0.15mol/L的硫酸铵溶液200ml，搅拌成浆；
(4)用无机酸调节匀浆液的pH至4.0-5.0，-8至5℃下搅拌1-3h；
(5)以6000-8000rpm离心45-75min，除去沉淀，取上清液，
(6)上清液用无机碱调节pH至5.0-7.0，搅拌中加入硫酸铵，盐析至不再出现沉淀为止；
(7)静置20-30h，以6000-8000rpm离心45-75min，除去沉淀，取上清液加入硫酸铵，盐析至不再出现沉淀为止，以6000-8000rpm离心45-75min，除去上清液，取沉淀即为胎盘蛋白。
本发明还提供前述的胎盘蛋白在化妆品领域中的应用。
具体例子为：
一种护肤组合物，按质量份计算包括以下原料：
特别地，按质量份计算还包括以下原料：
所述醇类保湿剂为丁二醇、1，3-丙二醇和1，2-己二醇中的至少一种。
所述调节剂为精氨酸、EDTA二钠和磷酸氢二钠中的至少一种。
所述舒缓剂为甘草酸二钾、尿囊素、叶酸和甘油辛酸酯中的至少一种。
所述防腐剂为对羟基苯乙酮和辛酰羟肟酸中的至少一种。
前述护肤组合物的制备方法，包括以下步骤：
(1)无菌条件下，在反应容器中加入水，加热后，加入卡波姆，均质，制成混合物A；
(2)将醇类保湿剂在另一反应器中加热，然后将其加入到步骤(1)所述的反应容器中，均质，与所述混合物A制成混合物B，
(3)将步骤(2)所述的混合物B降温后，向反应容器中加入调节剂、防腐剂、透明质酸和甘油，搅拌均质，与所述混合物B制成混合物C，
(4)将步骤(3)所述的混合物C降温后，向反应容器中加入舒缓剂、胎盘蛋白、芽孢杆菌/大豆发酵产物提取物、三肽-1、六肽-9、棕榈酰三肽-5、棕榈酰五肽-4、十肽-4、糖蛋白、棕榈酰四肽-7，搅拌均匀，与所述混合物C制成混合物D，所述混合物D降温至常温，即为成品。
所述步骤(1)中加热的温度为80℃-90℃；
所述步骤(2)中所述加热的温度为75℃-90℃；
所述步骤(3)中所述继降温是降至65℃-80℃；
所述步骤(4)中所述继降温是降至40℃-60℃。
胎盘蛋白：可以清除超氧阴离子自由基、羟基自由基、DPPH自由基，从而维持自由基平衡、保护细胞和DNA免受自由基攻击，防止脂质过氧化和炎症反应，延缓皮肤衰老。胎盘蛋白中的活性成分，能控制和调节皮肤老化进程，保护受损皮肤，延缓皮肤衰老。在皮肤整体水平上，胎盘蛋白能促进表皮和真皮细胞增殖分化，使皮肤增厚、修复皮肤损伤，逐渐恢复正常结构和生理功能，而且还能增强成纤维细胞活性，促进胶原蛋白、弹力蛋白、粘多糖等物质合成，增厚真皮，恢复皮肤弹性、改善皮肤萎缩缺水状态，抚平细纹、保持皮肤年轻。
三肽-1：三肽具抗氧性,对胶原蛋白、弹性蛋白、透明质酸等的生成有促进作用,有抗衰抗皱活肤功能；三肽-1对粘连蛋白生成的促进,可作为组织重塑的激活剂,促进伤疤外部大量胶原蛋白集聚物的降解,具有伤口修复功能。
六肽-9：主要作用是皮肤调理剂，有效舒缓并抑制额头抬头纹，眼睛鱼尾细纹及周边肌肉的收缩与活动，帮助肌肉放松，使肌肤弹性组织恢复柔顺平滑的线条。能够减能缓肌肉收缩的力量，让肌肉放松，减少动态纹的发生与消除细纹；有效重新组织胶原弹力，可以增加弹力蛋白的活性，使脸部线条放松，皱纹抚平改善松弛。
榈酰三肽-5：促进肌肤细胞生长，抑制氧自由基和羟基自由基，促进基质蛋白尤其是胶原蛋白的合成，同时还可能增加弹性蛋白、透明质酸、糖胺聚糖和纤维连接蛋白的生成。该类胜肽通过增加基质细胞活动促进胶原蛋白的合成，使得皮肤看起来更显弹性和年轻。.
棕榈酰五肽-4：在化妆品、护肤品里主要作用是保湿剂，抗氧化剂，属于抗衰老成分，棕榈酰五肽-4可以刺激皮肤基质的关键部件的合成如胶原蛋白等。
十肽-4，由精氨酸、天门冬氨酸、半胱氨酸、谷氨酸、亮氨酸、蛋氨酸和酪氨酸组成的合成肽，具有皮肤调理的作用。
棕榈酰四肽-7：通过刺激层黏连蛋白(LamininV)IV,VII型胶原蛋白使皮肤紧实，可减少白细胞介素的生成，消除炎症。防损伤，阻止形成皱纹、松弛和肤色不均。
与现有技术相比，本发明的提供的护肤组合物具有祛皱、抗衰和保湿和抗氧化的效果。
本发明的成分胎盘蛋白具有生物活性，天然无毒性，有增强成纤维细胞活性，促进胶原蛋白、弹力蛋白、粘多糖合成功效。
本发明中采用的重要原料为动物胎盘，是扔掉的医疗废物，提高了经济效益。
具体实施方式
下面结合具体实施例，对本发明作进一步详细的阐述，下述实施例不用于限制本发明，仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明，实施例中未注明具体条件的实验方法，通常按照常规条件，下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。
在具体实施例中，没有详细说明的步骤、材料选择、数值参数均为现有技术中的常规选择，或者任何现有公开的现有技术。
胎盘蛋白的制备与测试
1.一种胎盘蛋白的制备方法：
(1)选取新鲜的猪胎盘，去除胎膜和大血管，-40至-20℃速冻；
(2)然后将其切碎，放入打浆机中打浆至80-100目；
(3)称重然后按每100g组织加入浓度为0.15mol/L的硫酸铵溶液200ml，搅拌成浆；
(4)用无机酸调节匀浆液的pH至4.0-5.0，-8至5℃下搅拌1-3h；
(5)以6000-8000rpm离心45-75min，除去沉淀，取上清液，
(6)上清液用无机碱调节pH至5.0-7.0，搅拌中加入硫酸铵，盐析至不再出现沉淀为止；
(7)静置20-30h，以6000-8000rpm离心45-75min，除去沉淀，取上清液加入硫酸铵，盐析至不再出现沉淀为止，以6000-8000rpm离心45-75min，除去上清液，取沉淀即为胎盘蛋白。
2.测试胎盘蛋白对细胞的毒性
(1)细胞接种：按2.2*104个/孔的接种密度接种细胞至96孔板，放置培养箱孵育过夜(37℃、5％CO2)。
(2)实验分组：实验设置空白对照组与实验组，另设3个无细胞的孔做调零孔，实验组中样品以前述制备的胎盘蛋白为基础，设置5个浓度梯度，每个梯度浓度下设置3个复孔。
(3)配液：按实验设计，用基础培养基配置不同浓度的受试物。
表1测试浓度设定表
(4)给药：24h后取出96孔板，废除旧培养基，空白对照组每孔加100μL培养液,样品组每孔加入100μL含有相应浓度样品的培养液；调零组无细胞接种，仅加入100μL细胞培养液。给药完成后放回培养箱继续培养。
(5)检测：细胞培养24h后，弃掉上清，加入MTT工作液。37℃避光孵育4h，孵育结束后，弃掉上清，每孔加100μL DMSO，在490nm处读取OD值。
(6)细胞相对活力计算：根据公式计算；
(7)利用SPSS 19.0统计软件包进行统计学分析，对测试区域的测量值进行描述性统计。计算分析数值的变化，对照组与样品组之间的差异。如测试数据为正态分布，则采用独立T检验方法进行统计分析；如测试数据为非正态分布，则采用秩和检验方法进行统计分析。统计方法均采用双尾检验，检验水准α＝0.05。
结果如表2所示。
表2MTT检测结果
护肤组合物的制备与测试
一种含有胎盘动物蛋白的面膜液制备方法，包括如下步骤：
(1)称取如下表3按重量百分比计的原料；无菌条件下，在反应容器中加入水，加热后，加入卡波姆，均质，制成混合物A；
(2)将醇类保湿剂在另一反应器中加热，然后将其加入到步骤(1)所述的反应容器中，均质，与所述混合物A制成混合物B，
(3)将步骤(2)所述的混合物B降温后，向反应容器中加入调节剂、防腐剂、透明质酸和甘油，搅拌均质，与所述混合物B制成混合物C，
(4)将步骤(3)所述的混合物C降温后，向反应容器中加入舒缓剂、胎盘蛋白、芽孢杆菌/大豆发酵产物提取物、三肽-1、六肽-9、棕榈酰三肽-5、棕榈酰五肽-4、十肽-4、糖蛋白、棕榈酰四肽-7，搅拌均匀，与所述混合物C制成混合物D，所述混合物D降温至常温，即为成品。
表3面膜液的原料配比
表3中，空白表示不添加对应成分。
稳定性测试
1、耐热测试：恒温培养箱调节到40℃，将前述制取的4个实施例，每个实施例取三支装于铝膜面膜袋中，装样量为30ml/袋，封口后置于恒温培养箱内，三个月后取出，恢复至室温，观察外观变化。
2、耐寒测试：恒温培养箱调节到-10℃，将前述制取的4个实施例，每个实施例取三支装于铝膜面膜袋中，装样量为30ml/袋，封口后置于恒温培养箱内，三个月后取出，恢复至室温，观察外观变化。
3、常温测试：将前述制取的4个实施例，每个实施例取三支装于铝膜面膜袋中，装样量为230ml/袋，封口后常温下放置6个月后，观察精华液的外观变化。
耐热测试、耐寒测试、常温测试均未观察到析出、沉淀等现象，保持原来外观。
成分测试
将进行稳定性测试后的样品，根据《2022化妆品安全技术规范》对以下指标进行测试，如表4所示。
表4检测结果
| 检测项目 | 指标 | 实施例1 | 实施例2 | 实施例3 |
| 汞 | ≤1mg/Kg | 合格 | 合格 | 合格 |
| 铅 | ≤40mg/Kg | 合格 | 合格 | 合格 |
| 砷 | ≤10mg/Kg | 合格 | 合格 | 合格 |
| PH | 4.5-8.5 | 6.9 | 6.8 | 6.8 |
| 菌落总数 | ≤1000CFU/g | <10 | <10 | <10 |
| 霉菌和酵母总数 | ≤100CFU/g | <10 | <10 | <10 |
| 粪大肠杆菌 | 不得检出 | 未检出 | 未检出 | 未检出 |
| 金黄色葡萄球菌 | 不得检出 | 未检出 | 未检出 | 未检出 |
| 铜绿假单胞菌 | 不得检出 | 未检出 | 未检出 | 未检出 |
人体皮肤斑贴测试
将获得的4组实施例参照《2022化妆品安全技术规范》中的人体皮肤斑贴实验。
分别于30min(待压痕消失后)、24h和48h按标准观察皮肤反应，并记录观察结果，见表5。
表5人体安全性测试结果
| 编号 | 30min | 24h | 48h |
| 实施例1 | 0级，10人 | 0级，10人 | 0级，10人 |
| 实施例2 | 0级，10人 | 0级，10人 | 0级，10人 |
| 实施例3 | 0级，10人 | 0级，10人 | 0级，10人 |
| 实施例4 | 0级，10人 | 0级，10人 | 0级，10人 |
人体主观感受测试
对获得的4组实施例，选取年龄为30-50岁的女性为实验对象进行临床试验，实验总人数为40人，连续1个月每晚使用，以面膜纸浸润实施例后，贴敷在使用者脸部，每次使用15min后清水洗涤残留物；在实验过程中通过实验对象对面部皮肤的弹性、皱纹减少数量和保湿度进行评价，实验结果如下表6。
表6人体主观感受测试结果
人体仪器测试
通过仪器测定，科学的评价实施例的效果。
(1)实验前准备
1.受试者
纳入标准：
a)18岁～60岁健康志愿者；
b)面部敏感泛红且乳酸刺痛试验评分3分及以上者。
c)自愿参加并签署知情同意书；
d)能按测试要求完成规定内容者。
排除标准
a)近一周使用抗组胺药或近一个月内使用免疫抑制剂者；
b)近两个月内受试部位应用任何抗炎药物者；
c)受试者患有炎症性皮肤病临床未愈者；
d)胰岛素依赖性糖尿病患者；
e)正在接受治疗的哮喘或其他慢性呼吸系统疾病患者；
f)近六个月内接受抗癌化疗者；
g)免疫缺陷或自身免疫性疾病患者；
h)哺乳期或妊娠妇女；
i)双侧乳房切除及双侧腋下淋巴结切除者；
j)参加其他的临床试验研究者；
k)体质高度敏感者；
1)面部有大面积胎记、抓痕、白斑、色素痣、疤痕疙瘩等其他影响测试的皮肤表征；
m)非自愿参加者或不能按试验要求完成规定内容者。
受试人数设定
志愿者人数保证10例以上，受试者以室内活动为主,避免长期暴露在光照下及高温、高寒环境下。
(2)测试方案与测试方法
方案设计
通过筛选10名18-56岁，面部敏感泛红且乳酸刺痛试验评分3分及以上者的东亚人皮肤者，使用前述实施例1的制品4周，通过皮肤含水量测试仪、经皮失水率测试仪、皮肤黑色素和血红素测试探头、面部图像分析仪VISIA 7和乳酸刺痛测试，评价其保湿、舒缓、修护的功效。
试验环境
实验室温度21±1℃；相对湿度50％±10％。
评价方法
实验采用双盲法和自身前后对照法，测试周期为4周，皮肤数据采集次数为3次，分别为0W、2W、4W。按照要求筛选出合格的受试者，在志愿者第一次来访时，与其签订志愿者知情同意书、采集0周数据以及发放样品。志愿者每次来访时，用指定洁面清洗面部后，贴敷在使用者脸部，每次使用15min后清水洗涤残留物；开始采集皮肤图像和数据。
试验仪器
皮肤水分含量测试仪(Corneometer CM825，德国CK)：采用电容法测量人体皮肤角质层的水分含量。
经皮失水率测试仪(Tewameter TM300；德国CK)：通过两组温度、湿度不同的传感器测定表皮由角度层水分散失形成的不同点的水蒸气压梯度，测量经表皮蒸发的水分量。
皮肤黑色素和血红素测试探头(Mexameter MX18,德国C&K)：血红素含量越高，血管通透性增强，代表肤色越红；血红素含量越低，血管通透性减弱，代表肤色泛红减少。
皮肤观察评估
借助面部图像分析仪VISIA 7(美国Canfield)，该仪器采用3种不同的光源进行面部图像拍摄，能直观观察面部皮肤状态的变化。
测试指标
皮肤水分含量(MMV值)：MMV值表征皮肤水分，MMV值越大，角质层含水量越高。
经皮水分流失(TEWL值)：TEWL值用来评价皮肤屏障功能的重要参数，其数值越小皮肤经皮失水越少。
皮肤血红素含量(EI值)：血红素含量越高，血管通透性增强，代表肤色越红；血红素含量越低，血管通透性减弱，代表肤色泛红减少。
乳酸刺痛评分：表示10％乳酸对受试者的刺激程度，其数值越小，受试者对乳酸刺激的感受程度越低。
数据分析
1.计算公式
平均值
其中x＝个体参数测量值
2统计分析方法
用SPSS 19.0数据分析软件对数据进行分析。统计分析前对各参数进行正态分布和方差齐性检验，当数据呈现正态分布时，进行配对t检验分析。当数据呈现非正态分布时，进行配对秩和检验分析。统计方法采用双尾检验，检验水平为α＝0.05。
与初始值0W进行比较，“*”：P<0.05，表示有显著性差异，“**”：P<0.01、“***”：P<0.001，表示非常显著性差异。
(3)结果及分析
1.皮肤含水量MMV值结果，如表7所示。
表7
2.皮肤经皮失水TEWL值结果，如表8所示。
表8
3.皮肤血红素EI值结果，如表9所示。
表9
4.乳酸刺激评分结果，如表10所示。
表10
上述详细说明是针对本发明其中之一可行实施例的具体说明，该实施例并非用以限制本发明的专利范围，凡未脱离本发明所为的等效实施或变更，均应包含于本发明技术方案的范围内。
Claims (10)
1.胎盘蛋白的制备方法，其特征在于，包括以下步骤：
(1)选取新鲜的猪胎盘，去除胎膜和大血管，-40至-20℃速冻；
(2)然后将其切碎，放入打浆机中打浆至80-100目；
(3)称重然后按每100g组织加入浓度为0.15mol/L的硫酸铵溶液200ml，搅拌成浆；
(4)用无机酸调节匀浆液的pH至4.0-5.0，-8至5℃下搅拌1-3h；
(5)以6000-8000rpm离心45-75min，除去沉淀，取上清液，
(6)上清液用无机碱调节pH至5.0-7.0，搅拌中加入硫酸铵，盐析至不再出现沉淀为止；
(7)静置20-30h，以6000-8000rpm离心45-75min，除去沉淀，取上清液加入硫酸铵，盐析至不再出现沉淀为止，以6000-8000rpm离心45-75min，除去上清液，取沉淀即为胎盘蛋白。
2.权利要求1所述的胎盘蛋白在化妆品领域中的应用。
3.一种护肤组合物，其特征在于，按质量份计算包括以下原料：
4.根据权利要求3所述的护肤组合物，其特征在于，按质量份计算还包括以下原料：
5.根据权利要求4所述的护肤组合物，其特征在于，所述醇类保湿剂为丁二醇、1，3-丙二醇和1，2-己二醇中的至少一种。
6.根据权利要求4所述的护肤组合物，其特征在于，所述调节剂为精氨酸、EDTA二钠和磷酸氢二钠中的至少一种。
7.根据权利要求4所述的护肤组合物，其特征在于，所述舒缓剂为甘草酸二钾、尿囊素、叶酸和甘油辛酸酯中的至少一种。
8.根据权利要求4所述的护肤组合物，其特征在于，所述防腐剂为对羟基苯乙酮和辛酰羟肟酸中的至少一种。
9.权利要求4-8任一所述的护肤组合物的制备方法，其特征在于，包括以下步骤：
(1)无菌条件下，在反应容器中加入水，加热后，加入卡波姆，均质，制成混合物A；
(2)将醇类保湿剂在另一反应器中加热，然后将其加入到步骤(1)所述的反应容器中，均质，与所述混合物A制成混合物B，
(3)将步骤(2)所述的混合物B降温后，向反应容器中加入调节剂、防腐剂、透明质酸和甘油，搅拌均质，与所述混合物B制成混合物C，
(4)将步骤(3)所述的混合物C降温后，向反应容器中加入舒缓剂、胎盘蛋白、芽孢杆菌/大豆发酵产物提取物、三肽-1、六肽-9、棕榈酰三肽-5、棕榈酰五肽-4、十肽-4、糖蛋白、棕榈酰四肽-7，搅拌均匀，与所述混合物C制成混合物D，所述混合物D降温至常温，即为成品。
10.根据权利要求9所述的护肤组合物的制备方法，其特征在于，
所述步骤(1)中加热的温度为80℃-90℃；
所述步骤(2)中所述加热的温度为75℃-90℃；
所述步骤(3)中所述继降温是降至65℃-80℃；
所述步骤(4)中所述继降温是降至40℃-60℃。
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