CN117618273A - 用于皮肤表层修复的鼠李糖脂精华液及其制备方法 - Google Patents
用于皮肤表层修复的鼠李糖脂精华液及其制备方法 Download PDFInfo
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- CN117618273A CN117618273A CN202311660771.9A CN202311660771A CN117618273A CN 117618273 A CN117618273 A CN 117618273A CN 202311660771 A CN202311660771 A CN 202311660771A CN 117618273 A CN117618273 A CN 117618273A
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- rhamnolipid
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Abstract
本发明公开用于皮肤表层修复的鼠李糖脂精华液及其制备方法，属于化妆品技术领域。所述鼠李糖脂精华液按重量份记包括以下组分：鼠李糖脂10～20份、载药纳米脂质体10～15份、脂肪干细胞外泌体0.2～2份、桦树汁3～5份、尿囊素0.5～1份、羟乙基纤维素0.2～1份、甘油5～10份和去离子水66～81.5份。本发明的皮肤表层修复的鼠李糖脂精华液通过鼠李糖脂与脂肪干细胞外泌体的结合来提高包裹大分子的脂肪干细胞外泌体的渗透性，解决了术后修复部位出现的不同程度的红肿、发炎、疼痛等症状的皮肤表层修复的渗透性不高的技术问题。
Description
技术领域
本发明涉及一种用于皮肤表层修复的鼠李糖脂精华液及其制备方法，属于化妆品技术领域。
背景技术
皮肤及其附属器官在形态或功能上的增龄变化是人们最容易察觉到的老化现象。皮肤老化的组织学变化是表皮棘层呈空泡变形，真皮乳头变低，网状纤维消退，弹性纤维逐渐失去弹性，甚至断裂成片。胶原纤维更新变慢，细胞间质内透明质酸减少，故真皮质量降低，进而出现松弛、褶皱、色素沉着、肤色不均等。少量、细小的面部皱纹可通过针灸、理疗、激光等方法促进面部血液循环和新陈代谢进行修复，让面部重现青春活力。
水光针美容是一种新型注射类护肤疗法，将美容针剂如破尿酸、胶原蛋白注射到紧贴表皮下的真皮层，注射后能让皮肤变得水润、光亮而得名，具有保湿补水、改善肤色、收缩毛孔、祛除皱纹的功效。但使用水光针等注射疗法进行皮肤修复后会产生疼痛感，注射针口部位容易产生疤痕，术后修复部位还会出现不同程度的红肿、发炎、疼痛、过敏，需要三天到一周的时间才能慢慢恢复。常规方法一般采用冰敷进行缓解，但并不能加快修复部位的恢复。
鼠李糖脂是由假单胞菌或伯克氏菌类产生的一种生物代谢性质的生物表面活性剂，同时也是一种研究时间最长、应用技术最为成熟的一种生物表面活性剂，无毒性，能100％生物降解。
发明内容
本发明的目的在于，克服现有技术存在的技术缺陷，解决术后修复部位出现的不同程度的红肿、发炎、疼痛等症状的皮肤表层修复的渗透性不高的技术问题，提出一种用于皮肤表层修复的鼠李糖脂精华液及其制备方法。
本发明具体采用如下技术方案：用于皮肤表层修复的鼠李糖脂精华液，所述鼠李糖脂精华液按重量份记包括以下组分：鼠李糖脂10～20份、载药纳米脂质体10～15份、脂肪干细胞外泌体0.2～2份、桦树汁3～5份、尿囊素0.5～1份、羟乙基纤维素0.2～1份、甘油5～10份和去离子水66～81.5份；
所述载药纳米脂质体由以下组分按质量比组成：质量比为65～70份：20份：10～15份的蛋黄磷脂酰胆碱、胆固醇和药物组合物，所述药物组合物由如下组分组成：木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油；所述药物组合物中木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油的质量比为3～5：1～3：1～3：1～5：1～3。
作为一种较佳的实施例，所述脂肪干细胞外泌体的制备方法包括如下步骤：
步骤1、获得脂肪干细胞：取脂肪组织用PBS缓冲液冲洗3次后剪碎获得1mm3的碎块，加入0.1％胶原蛋白酶I和0.25％胰酶消化30～45min，然后静置分层后吸取上层脂肪细胞液，将所得脂肪细胞液加入等体积DMEM-F12培养基，10％胎牛血清终止消化，300×g离心10min，弃上清，在细胞沉淀中加入20mL无血清细胞培养基，37℃，5％CO2恒温培养48h后换液，之后每5天换液，观察到梭形细胞长出，细胞融合度达到70～80％时即可冻存得到脂肪干细胞；
所述脂肪组织取自健康人体抽脂术后的脂肪组织；
步骤2、脂肪干细胞的培养：配制诱导培养基，所述诱导培养基以低糖DMEM培养基为基础，添加了如下含量的诱导剂：重组白蛋白50μg/mL、重组表皮生长因子20μg/mL、维生素C 20μg/mL、尼克酰胺8μg/mL、丙酮酸钠12μg/mL、成纤维细胞生长因子30μg/mL、IBMX 0.5μg/mL、维甲酸0.5μg/mL；
将步骤1获得的脂肪干细胞接种于诱导培养基中进行培养，接种量为50％，培养条件为37℃，5％CO2恒温培养48h，吸除培养基，加入低糖DMEM培养基培养48h后收集上清液；
步骤3、收集脂肪干细胞外泌体：将步骤2收集得到的上清液300×g离心10min，收集上清，将所得上清10000×g高速离心30min，收集上清，将所得上清14000×g超速离心90min，收集沉淀得到外泌体，用PBS缓冲液洗涤沉淀并重悬，14000×g超速离心90min，收集沉淀，生理盐水重悬沉淀，冻存于-80℃备用。
作为一种较佳的实施例，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂10份、脂肪干细胞外泌体0.2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
作为一种较佳的实施例，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂15份、脂肪干细胞外泌体1份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
作为一种较佳的实施例，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂20份、脂肪干细胞外泌体2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
作为一种较佳的实施例，所述脂肪干细胞外泌体粒径为20～200nm。
本发明还提出一种用于皮肤表层修复的鼠李糖脂精华液的制备方法，包括如下步骤：
步骤一、制备脂肪干细胞外泌体：
步骤二、制备载药纳米脂质体：根据配方将蛋黄磷脂酰胆碱、胆固醇、木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油溶于无水乙醇，所有组分完全溶解后减压旋干获得脂膜，将所得脂膜再次用无水乙醇溶解并再次旋干得到二次脂膜，将所得二次脂膜用DEPC生理盐水溶解水化，对水化体系进行超声处理，使脂质体充分均匀分散在水化体系中，然后用聚碳酸酯膜对水化体系进行过膜挤出得到载药纳米脂质体；
步骤三、制备鼠李糖脂精华液：将步骤一提取得到的脂肪干细胞外泌体、步骤二制备的载药纳米脂质体、尿囊素与甘油混合均匀得到体系1，将羟乙基纤维素和鼠李糖脂溶于去离子水中得到体系2，再将体系1、体系2与桦树汁混合均匀得到鼠李糖脂精华液。
作为一种较佳的实施例，步骤二所述超声处理条件为300W超声处理15min。
作为一种较佳的实施例，步骤二所述用聚碳酸酯膜对水化体系进行过膜挤出是用孔径为300nm的聚碳酸酯膜对水化体系进行过膜挤出，反复挤出3～5次后，再用孔径为100nm的聚碳酸酯膜对体系进行过膜挤出，反复挤出2～3次后，得到粒径为100～300nm的载药纳米脂质体。
本发明所达到的有益效果：第一，本发明针对如何解决术后修复部位出现的不同程度的红肿、发炎、疼痛等症状的皮肤表层修复的渗透性不高的技术需求，提出用于皮肤表层修复的鼠李糖脂精华液及其制备方法，鼠李糖脂精华液的作用机理是通过鼠李糖脂与脂肪干细胞外泌体的结合来提高包裹大分子的脂肪干细胞外泌体的渗透性，一方面在脂肪干细胞外泌体作用下促进皮肤快速吸收、利用皮肤修复注入的营养物质，起到消肿作用；另一方面在载药纳米脂质体所载药物作用下起到消炎镇痛、抗敏抑菌的功效，能够快速缓解患者术后皮肤红肿疼痛、发炎过敏的症状。同时，面膜精华液中还复配了桦树汁、尿囊素等成分，辅以保湿补水、促进细胞生长、加速伤口愈合的作用，从整体上解决了术后修复部位出现的不同程度的红肿、发炎、疼痛等症状的皮肤表层修复的渗透性不高的技术需求。第二，本发明的用于皮肤表层修复的鼠李糖脂精华液各组分相复配，起到协同增效的作用，能使皮肤表层快速恢复，尽早实现皮肤修复效果，同时各组分温和无刺激，安全无毒副作用。第三，本发明制备方法得到的脂肪干细胞外泌体和载药纳米脂质体均为纳米级粒径，鼠李糖脂精华液在皮肤表层修复过程中易于被细胞吸收利用，其中脂肪干细胞外泌体纯度高，活性状态稳定，经冻干再水化后仍保持膜完整性，外泌体蛋白测得量恒定，通过鼠李糖脂与脂肪干细胞外泌体的结合来提高包裹大分子的脂肪干细胞外泌体的渗透性。
附图说明
图1是本发明的用于皮肤表层修复的鼠李糖脂精华液的制备方法的流程图。
具体实施方式
下面结合附图对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例1：本发明提出用于皮肤表层修复的鼠李糖脂精华液，所述鼠李糖脂精华液按重量份记包括以下组分：鼠李糖脂10份、脂肪干细胞外泌体0.2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份；
所述载药纳米脂质体由以下组分按质量比组成：质量比为65～70份：20份：10～15份的蛋黄磷脂酰胆碱、胆固醇和药物组合物，所述药物组合物由如下组分组成：木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油；所述药物组合物中木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油的质量比为3～5：1～3：1～3：1～5：1～3。
所述脂肪干细胞外泌体的制备方法包括如下步骤：
步骤1、获得脂肪干细胞：取脂肪组织用PBS缓冲液冲洗3次后剪碎获得1mm3的碎块，加入0.1％胶原蛋白酶I和0.25％胰酶消化30～45min，然后静置分层后吸取上层脂肪细胞液，将所得脂肪细胞液加入等体积DMEM-F12培养基，10％胎牛血清终止消化，300×g离心10min，弃上清，在细胞沉淀中加入20mL无血清细胞培养基，37℃，5％CO2恒温培养48h后换液，之后每5天换液，观察到梭形细胞长出，细胞融合度达到70～80％时即可冻存得到脂肪干细胞；所述脂肪干细胞外泌体粒径为20～200nm；
所述脂肪组织取自健康人体抽脂术后的脂肪组织；
步骤2、脂肪干细胞的培养：配制诱导培养基，所述诱导培养基以低糖DMEM培养基为基础，添加了如下含量的诱导剂：重组白蛋白50μg/mL、重组表皮生长因子20μg/mL、维生素C 20μg/mL、尼克酰胺8μg/mL、丙酮酸钠12μg/mL、成纤维细胞生长因子30μg/mL、IBMX 0.5μg/mL、维甲酸0.5μg/mL；
将步骤1获得的脂肪干细胞接种于诱导培养基中进行培养，接种量为50％，培养条件为37℃，5％CO2恒温培养48h，吸除培养基，加入低糖DMEM培养基培养48h后收集上清液；
步骤3、收集脂肪干细胞外泌体：将步骤2收集得到的上清液300×g离心10min，收集上清，将所得上清10000×g高速离心30min，收集上清，将所得上清14000×g超速离心90min，收集沉淀得到外泌体，用PBS缓冲液洗涤沉淀并重悬，14000×g超速离心90min，收集沉淀，生理盐水重悬沉淀，冻存于-80℃备用。
实施例2：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂10份、脂肪干细胞外泌体1份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例3：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂10份、脂肪干细胞外泌体2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例4：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂15份、脂肪干细胞外泌体0.2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例5：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂15份、脂肪干细胞外泌体1份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例6：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂15份、脂肪干细胞外泌体2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例7：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂20份、脂肪干细胞外泌体0.2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例8：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂20份、脂肪干细胞外泌体1份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
实施例9：所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂20份、脂肪干细胞外泌体2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
本发明的鼠李糖脂的制备方法如下：(1)将鼠李糖脂发酵液过滤，收集滤液，离心，取上清液；(2)用酸调节步骤(1)上清液的pH，使鼠李糖脂析出，静置，离心，收集沉淀；(3)用0.1mol/L的碳酸氢钠溶液重悬步骤(2)的沉淀，自然沉降，收集上清液；(4)用酸调节步骤(3)上清液的pH，使鼠李糖脂析出，静置，离心，收集沉淀；(5)将步骤(4)的沉淀加入有机溶剂中进行萃取，收集有机相，将有机相与磷酸盐缓冲溶液混匀，静置分层，使有机相中的鼠李糖脂反萃取到水相中，下层水相即为纯化后的鼠李糖脂溶液；步骤(5)所述磷酸盐缓冲溶液的pH为7～8；磷酸盐缓冲溶液的体积为有机相体积的1～3倍；所述磷酸盐缓冲溶液的浓度为0.1～0.2mol/L；步骤(5)所述有机溶剂为氯仿和乙酸乙酯中的至少一种；步骤(5)所述有机溶剂与步骤(4)沉淀的质量比为10：1～20：1。
本发明还提出一种用于皮肤表层修复的鼠李糖脂精华液的制备方法，包括如下步骤：
步骤一、制备脂肪干细胞外泌体：
步骤二、制备载药纳米脂质体：根据配方将蛋黄磷脂酰胆碱、胆固醇、木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油溶于无水乙醇，所有组分完全溶解后减压旋干获得脂膜，将所得脂膜再次用无水乙醇溶解并再次旋干得到二次脂膜，将所得二次脂膜用DEPC生理盐水溶解水化，对水化体系进行超声处理，使脂质体充分均匀分散在水化体系中，然后用聚碳酸酯膜对水化体系进行过膜挤出得到载药纳米脂质体；
步骤三、制备鼠李糖脂精华液：将步骤一提取得到的脂肪干细胞外泌体、步骤二制备的载药纳米脂质体、尿囊素与甘油混合均匀得到体系1，将羟乙基纤维素和鼠李糖脂溶于去离子水中得到体系2，再将体系1、体系2与桦树汁混合均匀得到鼠李糖脂精华液。
可选的，步骤二所述超声处理条件为300W超声处理15min。
可选的，步骤二所述用聚碳酸酯膜对水化体系进行过膜挤出是用孔径为300nm的聚碳酸酯膜对水化体系进行过膜挤出，反复挤出3～5次后，再用孔径为100nm的聚碳酸酯膜对体系进行过膜挤出，反复挤出2～3次后，得到粒径为100～300nm的载药纳米脂质体。
最后应当说明的是：以上实施例仅用以说明本发明的技术方案而非对其限制，尽管参照上述实施例对本发明进行了详细的说明，所属领域的普通技术人员应当理解：依然可以对本发明的具体实施方式进行修改或者等同替换，而未脱离本发明精神和范围的任何修改或者等同替换，其均应涵盖在本发明的权利要求保护范围之内。
Claims (9)
1.用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述鼠李糖脂精华液按重量份记包括以下组分：鼠李糖脂10～20份、载药纳米脂质体10～15份、脂肪干细胞外泌体0.2～2份、桦树汁3～5份、尿囊素0.5～1份、羟乙基纤维素0.2～1份、甘油5～10份和去离子水66～81.5份；
所述载药纳米脂质体由以下组分按质量比组成：质量比为65～70份：20份：10～15份的蛋黄磷脂酰胆碱、胆固醇和药物组合物，所述药物组合物由如下组分组成：木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油；所述药物组合物中木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油的质量比为3～5：1～3：1～3：1～5：1～3。
2.根据权利要求1所述的用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述脂肪干细胞外泌体的制备方法包括如下步骤：
步骤1、获得脂肪干细胞：取脂肪组织用PBS缓冲液冲洗3次后剪碎获得1mm3的碎块，加入0.1％胶原蛋白酶I和0.25％胰酶消化30～45min，然后静置分层后吸取上层脂肪细胞液，将所得脂肪细胞液加入等体积DMEM-F12培养基，10％胎牛血清终止消化，300×g离心10min，弃上清，在细胞沉淀中加入20mL无血清细胞培养基，37℃，5％CO2恒温培养48h后换液，之后每5天换液，观察到梭形细胞长出，细胞融合度达到70～80％时即可冻存得到脂肪干细胞；
所述脂肪组织取自健康人体抽脂术后的脂肪组织；
步骤2、脂肪干细胞的培养：配制诱导培养基，所述诱导培养基以低糖DMEM培养基为基础，添加了如下含量的诱导剂：重组白蛋白50μg/mL、重组表皮生长因子20μg/mL、维生素C20μg/mL、尼克酰胺8μg/mL、丙酮酸钠12μg/mL、成纤维细胞生长因子30μg/mL、IBMX 0.5μg/mL、维甲酸0.5μg/mL；
将步骤1获得的脂肪干细胞接种于诱导培养基中进行培养，接种量为50％，培养条件为37℃，5％CO2恒温培养48h，吸除培养基，加入低糖DMEM培养基培养48h后收集上清液；
步骤3、收集脂肪干细胞外泌体：将步骤2收集得到的上清液300×g离心10min，收集上清，将所得上清10000×g高速离心30min，收集上清，将所得上清14000×g超速离心90min，收集沉淀得到外泌体，用PBS缓冲液洗涤沉淀并重悬，14000×g超速离心90min，收集沉淀，生理盐水重悬沉淀，冻存于-80℃备用。
3.根据权利要求1所述的用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂10份、脂肪干细胞外泌体0.2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
4.根据权利要求1所述的用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂15份、脂肪干细胞外泌体1份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
5.根据权利要求1所述的用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述鼠李糖脂精华液由以下组分组成，按重量份计为：鼠李糖脂20份、脂肪干细胞外泌体2份、载药纳米脂质体12份、桦树汁5份、尿囊素1份、羟乙基纤维素0.5份、甘油10份和去离子水70份。
6.根据权利要求1所述的用于皮肤表层修复的鼠李糖脂精华液，其特征在于，所述脂肪干细胞外泌体粒径为20～200nm。
7.一种如权利要求1-6任一项所述的用于皮肤表层修复的鼠李糖脂精华液的制备方法，其特征在于，包括如下步骤：
步骤一、制备脂肪干细胞外泌体：
步骤二、制备载药纳米脂质体：根据配方将蛋黄磷脂酰胆碱、胆固醇、木犀草素、羟基积雪草酸、红没药醇、邻羟基苯甲酸甲酯和薄荷油溶于无水乙醇，所有组分完全溶解后减压旋干获得脂膜，将所得脂膜再次用无水乙醇溶解并再次旋干得到二次脂膜，将所得二次脂膜用DEPC生理盐水溶解水化，对水化体系进行超声处理，使脂质体充分均匀分散在水化体系中，然后用聚碳酸酯膜对水化体系进行过膜挤出得到载药纳米脂质体；
步骤三、制备鼠李糖脂精华液：将步骤一提取得到的脂肪干细胞外泌体、步骤二制备的载药纳米脂质体、尿囊素与甘油混合均匀得到体系1，将羟乙基纤维素和鼠李糖脂溶于去离子水中得到体系2，再将体系1、体系2与桦树汁混合均匀得到鼠李糖脂精华液。
8.根据权利要求7所述的用于皮肤表层修复的鼠李糖脂精华液的制备方法，其特征在于，步骤二所述超声处理条件为300W超声处理15min。
9.根据权利要求8所述的用于皮肤表层修复的鼠李糖脂精华液的制备方法，其特征在于，步骤二所述用聚碳酸酯膜对水化体系进行过膜挤出是用孔径为300nm的聚碳酸酯膜对水化体系进行过膜挤出，反复挤出3～5次后，再用孔径为100nm的聚碳酸酯膜对体系进行过膜挤出，反复挤出2～3次后，得到粒径为100～300nm的载药纳米脂质体。
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