CN113750030A - 一种含外泌体的抗衰精华液及其制备方法 - Google Patents
一种含外泌体的抗衰精华液及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种含外泌体的抗衰精华液及其制备方法,该含外泌体的抗衰精华液以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.05‑0.1份、玫瑰纯露20‑30份、甘油8‑10份、二裂酵母发酵产物溶胞物4‑5份、透明质酸2‑4份、银耳提取物3‑6份、寡肽‑1 2‑3份、水解胶原蛋白1‑2份、肌肽0.3‑0.7份、卡波U20 0.1‑0.2份、EDTA‑2Na 0.2‑0.8份、抗菌剂0.1‑0.3份、三乙醇胺0.1‑0.2份,其余为无菌水。本发明的含外泌体精华液所用组分精简,为无油配方,使用感更清爽,无需乳化,具有长效保湿、防止皮肤老化、使皱纹舒展的功效。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种含外泌体的抗衰精华液及其制备方法。
背景技术
皮肤是人体表面积最大的器官,覆盖于人体表面,但随着年龄增长和各种外界环境的侵害,面部皮肤会出现皱纹、下垂、暗黄等衰老问题。使用各种抗衰老护肤品来护理皮肤已经成为消费者的日常。随着护肤品市场的发展,消费者对护肤品的需求早已不满足于简单的水、乳、霜,越来越多的人需求一种高机能的营养丰富的护肤品品类----精华液。
外泌体是由活细胞分泌的40-200nm的小囊泡,携带各种蛋白质、mRNA、micro-RNA和脂质类物质,可以促进成纤维细胞增殖和胶原合成、角质化细胞再生,促进给皮肤细胞生长供给营养的血管生成,从而激活皮肤的自我修护系统,对皮肤问题进行自我修护,从皮肤细胞内部解决多种皮肤问题。现在市面上的精华液品类繁多,但添加外泌体的精华液市场还比较空白,急需一款将干细胞外泌体应用到精华液中的产品,满足消费者需求。
发明内容
针对上述现有技术,本发明提供一种含外泌体的抗衰精华液及其制备方法,用以解决现有精华液抗衰抗皱效果不佳的问题。
为了达到上述目的,本发明所采用的技术方案是:提供一种含外泌体的抗衰精华液,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.05-0.1%、玫瑰纯露20-30%、甘油8-10%、二裂酵母发酵产物溶胞物4-5%、透明质酸2-4%、银耳提取物3-6%、寡肽-1 2-3%、水解胶原蛋白1-2%、肌肽0.3-0.7%、卡波U20 0.1-0.2%、EDTA-2Na 0.2-0.8%、抗菌剂0.1-0.3%、三乙醇胺0.1-0.2%,其余为无菌水。
甘油和透明质酸分别为吸湿剂和仿生剂类保湿剂,两者同时使用既可以做到吸收环境中水分又可以结合皮肤中的水分,双重保湿;银耳提取物里富含α-甘露聚糖,具有强大锁水性和成膜性,防止皮肤水分蒸发,达到长效保湿效果。三者配合使用保湿效果更好更长久。
水解胶原蛋白可以被吸收并填充在皮肤基质间,使皮肤丰满、皱纹舒展,使皮肤柔软富有弹性;肌肽具有抗氧化特性,是一种天然抗氧化剂,可清除在氧化应激过程中使细胞膜的脂肪酸过度氧化而形成的活性氧自由基(ROS)以及α-β不饱和醛;外泌体可以促进成纤维细胞增殖和胶原合成。三种抗衰老原料配合使用能更好地解决皮肤衰老问题。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,包含以下质量份组份:脐带干细胞外泌体0.05-0.1份、玫瑰纯露20-30份、甘油8-10份、二裂酵母发酵产物溶胞物4-5份、透明质酸2-4份、银耳提取物3-6份、寡肽-12-3份、水解胶原蛋白1-2份、肌肽0.3-0.7份、卡波U20 0.1-0.2份、EDTA-2Na 0.2-0.8份、抗菌剂0.1-0.3份、三乙醇胺0.1-0.2份,其余为无菌水。
进一步,脐带干细胞外泌体由以下步骤制得:
(1)新鲜脐带用含青霉素或链霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎至0.5-1.5mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中培养,当细胞融合达到70-80%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清离心去除活细胞后,再次离心去除死细胞;
(4)取离心后的上清过滤,收集滤液;
(5)滤液超速离心去掉上清,再用PBS重悬,然后再次超速离心去掉上清,所得滤液用450-550ul PBS重悬,即得。
进一步,步骤(5)中超速离心条件为4℃、100000xg下离心70min。
进一步,银耳提取物经过以下步骤制得:
(1)将银耳于70~80℃下烘干2~3小时,再碾磨至粒度为40~50目;
(2)将所得银耳粉末用初始银耳重量2.5~3.5倍的80%的乙醇浸泡10~20小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8~9倍的可可碱水溶液中,在90~100℃下浸提2~3小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置10~20小时,离心分离取沉淀物即得。
本发明还提供了该种含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)将卡波U20分散在无菌水中,然后静置20-30h,再用三乙醇胺调节pH至6-7;
(2)将除脐带干细胞外泌体外的其余组份加入到步骤(1)所得溶液中,搅拌均匀后加入脐带干细胞外泌体,继续搅拌10~20min,即可得到含外泌体精华液。
进一步,步骤(1)中静置时间为24h。
本发明的有益效果是:
1.本发明的含外泌体精华液,通过添加脐带间充质干细胞外泌体,配合水解胶原蛋白和肌肽,可促进成纤维细胞增殖和胶原合成,激活皮肤的自我修护系统,防止皮肤老化,使皮肤柔软富有弹性、使皱纹舒展。
2.本发明通过将甘油、透明质酸、银耳提取物三种物质配合使用,从而拥有良好保湿作用,能够长效保湿。
3.本发明配方为无油配方,所用组分精简,使用感更清爽,无需乳化,减少皮肤负担的同时兼顾产品功效。
具体实施方式
下面结合实施例对本发明的具体实施方式做详细的说明。
实施例1
一种含外泌体的抗衰精华液,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.05份、玫瑰纯露20份、甘油8份、二裂酵母发酵产物溶胞物3份、透明质酸2份、银耳提取物3份、寡肽-1 2份、水解胶原蛋白1.5份、肌肽0.5份、卡波U20 0.1份、EDTA-2Na 0.2份、抗菌剂0.1份、三乙醇胺0.1份,其余为无菌水。
其中,脐带干细胞外泌体经过以下步骤制得:
(1)新鲜脐带用含青霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎成1mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中于37℃、5%CO2的饱和湿度下培养,当细胞融合达到70%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清于离心管中在4℃、300xg下离心10min去除活细胞后,再次在4℃、2000xg下离心10min去除死细胞;
(4)取离心后的上清用0.22μm的过滤器过滤,收集滤液;
(5)滤液于离心管中在4℃、100000xg下离心70min去掉上清,再用PBS重悬,然后再次于离心管中在4℃、100000xg下离心70min去掉上清,所得滤液用500ul PBS重悬收集即可得到脐带干细胞外泌体。
其中,银耳提取物经过以下步骤制得:
(1)将银耳于75℃下烘干2.5小时,再碾磨至粒度为45目;
(2)将所得银耳粉末用初始银耳重量3倍的80%的乙醇浸泡15小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8.5倍的可可碱水溶液中,在95℃下浸提2.5小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置15小时,离心分离取沉淀物即得。
上述含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)卡波U20撒入无菌水中,搅拌使其均匀分散在无菌水中,然后静置24h,其后用三乙醇胺调节pH至6;
(2)向所得溶液中加入除脐带干细胞外泌体的其余组份,搅拌均匀,当各组分混匀后,加入脐带干细胞外泌体,继续搅拌15min,即可得到含外泌体精华液。
实施例2
一种含外泌体的抗衰精华液,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.1份、玫瑰纯露30份、甘油10份、二裂酵母发酵产物溶胞物4份、透明质酸4份、银耳提取物2份、寡肽-1 3份、水解胶原蛋白1份、肌肽0.3份、卡波U20 0.2份、EDTA-2Na0.8份、抗菌剂0.3份、三乙醇胺0.2份,其余为无菌水。
其中,脐带干细胞外泌体经过以下步骤制得:
(1)新鲜脐带用含链霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎成1mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中于37℃、5%CO2的饱和湿度下培养,当细胞融合达到80%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清于离心管中在4℃、300xg下离心10min去除活细胞后,再次在4℃、2000xg下离心10min去除死细胞;
(4)取离心后的上清用0.22μm的过滤器过滤,收集滤液;
(5)滤液于离心管中在4℃、100000xg下离心70min去掉上清,再用PBS重悬,然后再次于离心管中在4℃、100000xg下离心70min去掉上清,所得滤液用450ul PBS重悬收集即可得到脐带干细胞外泌体。
其中,银耳提取物经过以下步骤制得:
(1)将银耳于70℃下烘干3小时,再碾磨至粒度为40目;
(2)将所得银耳粉末用初始银耳重量2.5倍的80%的乙醇浸泡10小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8倍的可可碱水溶液中,在90℃下浸提3小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置10小时,离心分离取沉淀物即得。
上述含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)卡波U20撒入无菌水中,搅拌使其均匀分散在无菌水中,然后静置20h,其后用三乙醇胺调节pH至7;
(2)向所得溶液中加入除脐带干细胞外泌体的其余组份,搅拌均匀,当各组分混匀后,加入脐带干细胞外泌体,继续搅拌10min,即可得到含外泌体精华液。
实施例3
一种含外泌体的抗衰精华液,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.07份、玫瑰纯露25份、甘油9份、二裂酵母发酵产物溶胞物4份、透明质酸3份、银耳提取物5份、寡肽-1 2份、水解胶原蛋白2份、肌肽0.7份、卡波U20 0.2份、EDTA-2Na0.5份、抗菌剂0.2份、三乙醇胺0.1份,其余为无菌水。
脐带干细胞外泌体经过以下步骤制得:
(1)新鲜脐带用含青霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎成1mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中于37℃、5%CO2的饱和湿度下培养,当细胞融合达到75%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清于离心管中在4℃、300xg下离心10min去除活细胞后,再次在4℃、2000xg下离心10min去除死细胞;
(4)取离心后的上清用0.22μm的过滤器过滤,收集滤液;
(5)滤液于离心管中在4℃、100000xg下离心70min去掉上清,再用PBS重悬,然后再次于离心管中在4℃、100000xg下离心70min去掉上清,所得滤液用550ul PBS重悬收集即可得到脐带干细胞外泌体。
其中,银耳提取物经过以下步骤制得:
(1)将银耳于80℃下烘干2小时,再碾磨至粒度为50目;
(2)将所得银耳粉末用初始银耳重量3.5倍的80%的乙醇浸泡20小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量9倍的可可碱水溶液中,在100℃下浸提2小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置20小时,离心分离取沉淀物即得。
上述含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)卡波U20撒入无菌水中,搅拌使其均匀分散在无菌水中,然后静置30h,其后用三乙醇胺调节pH至6.5;
(2)向所得溶液中加入除脐带干细胞外泌体的其余组份,搅拌均匀,当各组分混匀后,加入脐带干细胞外泌体,继续搅拌20min,即可得到含外泌体精华液。
对比例1
一种的抗衰精华液,以总质量份为100份计,包含以下质量份组份:玫瑰纯露20份、甘油8份、二裂酵母发酵产物溶胞物3份、透明质酸2份、银耳提取物3份、寡肽-1 2份、水解胶原蛋白1.5份、肌肽0.5份、卡波U20 0.1份、EDTA-2Na 0.2份、抗菌剂0.1份、三乙醇胺0.1份,其余为无菌水。
其中,银耳提取物经过以下步骤制得:
(1)将银耳于75℃下烘干2.5小时,再碾磨至粒度为45目;
(2)将所得银耳粉末用初始银耳重量3倍的80%的乙醇浸泡15小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8.5倍的可可碱水溶液中,在95℃下浸提2.5小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置15小时,离心分离取沉淀物即得。
上述含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)卡波U20撒入无菌水中,搅拌使其均匀分散在无菌水中,然后静置24h,其后用三乙醇胺调节pH至6;
(2)向所得溶液中加入其余组份,搅拌均匀,即可得到抗衰精华液。
对比例2
一种抗衰精华液,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.05份、玫瑰纯露20份、甘油8份、二裂酵母发酵产物溶胞物3份、透明质酸2份、银耳提取物3份、寡肽-1 2份、卡波U20 0.1份、EDTA-2Na 0.2份、抗菌剂0.1份、三乙醇胺0.1份,其余为无菌水。
其中,脐带干细胞外泌体经过以下步骤制得:
(1)新鲜脐带用含链霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎成1mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中于37℃、5%CO2的饱和湿度下培养,当细胞融合达到70%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清于离心管中在4℃、300xg下离心10min去除活细胞后,再次在4℃、2000xg下离心10min去除死细胞;
(4)取离心后的上清用0.22μm的过滤器过滤,收集滤液;
(5)滤液于离心管中在4℃、100000xg下离心70min去掉上清,再用PBS重悬,然后再次于离心管中在4℃、100000xg下离心70min去掉上清,所得滤液用500ul PBS重悬收集即可得到脐带干细胞外泌体。
其中,银耳提取物经过以下步骤制得:
(1)将银耳于75℃下烘干2.5小时,再碾磨至粒度为45目;
(2)将所得银耳粉末用初始银耳重量3倍的80%的乙醇浸泡15小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8.5倍的可可碱水溶液中,在95℃下浸提2.5小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置15小时,离心分离取沉淀物即得。
上述含外泌体的抗衰精华液的制备方法,包括以下步骤:
(1)卡波U20撒入无菌水中,搅拌使其均匀分散在无菌水中,然后静置24h,其后用三乙醇胺调节pH至6;
(2)向所得溶液中加入除脐带干细胞外泌体的其余组份,搅拌均匀,当各组分混匀后,加入脐带干细胞外泌体,继续搅拌15min,即可得到含外泌体精华液。
下面对实施例、对比例以及市面上在售的一款保湿抗皱精华液进行使用效果对比。选取受试者60名分为1-6组,1-6组分别使用实施例1-3、对比例1、2以及市售精华液十五天,接下来15天交换为1-6组分别使用市售精华液、对比例1、2以及实施例1-3中精华液。使用过程中对各受试者进行皮肤检测,检测指标包括:皮肤水分含量、皮肤弹性、皮肤皱纹、受试者自我评价。受试者使用前并不知道两款产品信息,使用频率和护肤步骤前后一致。检测部位统一为右边脸颊,检测环境22-25℃湿度50-60%。
表1受试者使用过程中皮肤检测指标统计如下:
从表1可以看出使用实施例1-3中精华液的1-3组前15天皮肤水分含量上升较快,在后15天交换产品使用后使用实施例1-3中精华液的4-6组皮肤水分含量上升较快。
表2 VISIA检测皮肤皱纹改善百分比
5天 | 10天 | 15天 | 20天 | 25天 | 30天 | |
1组 | 57% | 62% | 65% | 64% | 61% | 60% |
2组 | 58% | 63% | 66% | 64% | 62% | 59% |
3组 | 57% | 63% | 67% | 65% | 63% | 60% |
4组 | 55% | 57% | 58% | 60% | 63% | 63% |
5组 | 54% | 55% | 56% | 59% | 59% | 61% |
6组 | 54% | 56% | 56% | 60% | 62% | 62% |
从表2以看出使用实施例1-3中精华液的1-3组前15天皮肤皱纹改善百分比上升较快,在后15天交换产品使用后使用实施例1-3中精华液的4-6组皮肤水分含量上升较快。
表3皮肤弹性测试仪检测皮肤弹性表征值
0天 | 5天 | 10天 | 15天 | 20天 | 25天 | 30天 | |
1组 | 0.32 | 0.59 | 0.68 | 0.77 | 0.73 | 0.70 | 0.69 |
2组 | 0.42 | 0.66 | 0.72 | 0.79 | 0.75 | 0.72 | 0.70 |
3组 | 0.35 | 0.62 | 0.70 | 0.78 | 0.75 | 0.72 | 0.68 |
4组 | 0.39 | 0.42 | 0.47 | 0.51 | 0.68 | 0.72 | 0.75 |
5组 | 0.37 | 0.39 | 0.45 | 0.49 | 0.56 | 0.62 | 0.69 |
6组 | 0.35 | 0.41 | 0.49 | 0.53 | 0.66 | 0.71 | 0.75 |
从表2以看出使用实施例1-3中精华液的1-3组前15天皮肤弹性表征值上升较快,在后15天交换产品使用后使用实施例1-3中精华液的4-6组皮肤弹性表征值上升较快。
表4受试者使用感描述
从4个统计表可以看出从保湿性、抗皱、皮肤弹性改善情况、受试者使用感和喜爱度四个方面来看,本发明产品均更受喜爱,且在保湿性、抗皱、皮肤弹性改善功效方面都有优异效果。
虽然结合实施例对本发明的具体实施方式进行了详细地描述,但不应理解为对本专利的保护范围的限定。在权利要求书所描述的范围内,本领域技术人员不经创造性劳动即可作出的各种修改和变形仍属本专利的保护范围。
Claims (7)
1.一种含外泌体的抗衰精华液,其特征在于,以总质量份为100份计,包含以下质量份组份:脐带干细胞外泌体0.05-0.1%、玫瑰纯露20-30%、甘油8-10%、二裂酵母发酵产物溶胞物4-5%、透明质酸2-4%、银耳提取物3-6%、寡肽-1 2-3%、水解胶原蛋白1-2%、肌肽0.3-0.7%、卡波U20 0.1-0.2%、EDTA-2Na 0.2-0.8%、抗菌剂0.1-0.3%、三乙醇胺0.1-0.2%,其余为无菌水。
2.根据权利要求1所述的含外泌体的抗衰精华液,其特征在于,包含以下质量份组份:脐带干细胞外泌体0.05-0.1份、玫瑰纯露20-30份、甘油8-10份、二裂酵母发酵产物溶胞物4-5份、透明质酸2-4份、银耳提取物3-6份、寡肽-1 2-3份、水解胶原蛋白1-2份、肌肽0.3-0.7份、卡波U20 0.1-0.2份、EDTA-2Na 0.2-0.8份、抗菌剂0.1-0.3份、三乙醇胺0.1-0.2份,其余为无菌水。
3.根据权利要求1或2所述的含外泌体的抗衰精华液,其特征在于,所述脐带干细胞外泌体由以下步骤制得:
(1)新鲜脐带用含青霉素或链霉素的生理盐水浸洗,排除残余血渍后,剔除脐带中的血管,然后剥离脐带中的华通氏胶,再将其剪碎至0.5-1.5mm3大小的碎片;
(2)将所得碎片接种于含DMEM培养基的培养瓶中培养,当细胞融合达到70-80%时进行细胞传代;
(3)收集传代至第4代时的培养液上清,转移上清离心去除活细胞后,再次离心去除死细胞;
(4)取离心后的上清过滤,收集滤液;
(5)滤液超速离心去掉上清,再用PBS重悬,然后再次超速离心去掉上清,所得滤液用450-550ulPBS重悬,即得。
4.根据权利要求3述的含外泌体的抗衰精华液,其特征在于:所述步骤(5)中超速离心条件为4℃、100000xg下离心70min。
5.根据权利要求1或2所述的含外泌体的抗衰精华液,其特征在于,所述银耳提取物经过以下步骤制得:
(1)将银耳于70~80℃下烘干2~3小时,再碾磨至粒度为40~50目;
(2)将所得银耳粉末用初始银耳重量2.5~3.5倍的80%的乙醇浸泡10~20小时,再过滤取不溶物;
(3)将所得不溶物加入到初始银耳重量8~9倍的可可碱水溶液中,在90~100℃下浸提2~3小时,再冷冻离心,得一次浸提液,再向不溶物中加水浸提,得二次浸提液;
(4)取两次浸提的上清液混合,再静置10~20小时,离心分离取沉淀物即得。
6.根据权利要求1~5所述的含外泌体的抗衰精华液的制备方法,其特征在于,包括以下步骤:
(1)将卡波U20分散在无菌水中,然后静置20-30h,再用三乙醇胺调节pH至6-7;
(2)将除脐带干细胞外泌体外的其余组份加入到步骤(1)所得溶液中,搅拌均匀后加入脐带干细胞外泌体,继续搅拌10~20min,即可得到含外泌体精华液。
7.根据权利要求6所述的含外泌体的抗衰精华液的制备方法,其特征在于:所述步骤(1)中静置时间为24h。
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