CN113171411A - 一种具有美白功能的含有脂肪干细胞外泌体的组合物 - Google Patents
一种具有美白功能的含有脂肪干细胞外泌体的组合物 Download PDFInfo
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Abstract
本发明提供了一种具有美白功能的含有脂肪干细胞外泌体的组合物,该组合物由包括如下重量份的原料制成:脂肪干细胞外泌体0.01‑0.5份,月柿提取物1‑20份,烟酰胺1‑10份,茶多酚1‑10份,α‑熊果苷1‑10份。本发明所述的一种具有美白功能的含有脂肪干细胞外泌体的组合物含有月柿提取物与脂肪干细胞外泌体,结合烟酰胺、茶多酚与α‑熊果苷,可有效的作用于皮肤起到美白的作用。
Description
技术领域
本发明属于生物领域,尤其是涉及一种具有美白功能的含有脂肪干细胞外泌体的组合物。
背景技术
外泌体最早发现于体外培养的绵羊红细胞上清液中,是细胞主动分泌的大小均一,直径为40-100nm,密度1.10-1.18g/ml的囊泡样小体。1996年有学者发现B细胞可以通过释放能表达主要组织相容性复合体分子的外泌体促进T细胞增殖和抑制肿瘤生长。外泌体包含多种蛋白质和遗传物质,表明蛋白质等大分子物质可以装载到外泌体上。外泌体可广泛分布于不同体液,在体内有较长的半衰期。此外,外泌体可以穿透细胞膜释放mRNA到靶细胞,使受体细胞翻译转染的mRNA。目前,外泌体已经开始应用于临床治疗。
皮肤衰老是人体衰老固有的生理机制和外界风吹日晒、环境污染等损害共同作用的结果。皮肤衰老通常体现在皮肤弹性降低和皮肤皱纹的产生,据报道,胶原蛋白和弹性蛋白的减少将导致皮肤弹性降低和皮肤皱纹产生,胶原蛋白和弹性蛋白受胶原蛋白酶和弹性蛋白酶调控,胶原蛋白酶和弹性蛋白酶能够引起胶原蛋白和弹性蛋白降解,从而导致胶原蛋白和弹性蛋白减少,导致皱纹产生,皮肤弹性减低。目前尚未有对脂肪来源间充质干细胞外泌体在皮肤抗衰老方面的相关研究。
发明内容
有鉴于此,本发明旨在克服现有技术中的缺陷,提出一种具有美白功能的含有脂肪干细胞外泌体的组合物。
为达到上述目的,本发明的技术方案是这样实现的:
一种具有美白功能的含有脂肪干细胞外泌体的组合物,该组合物由包括如下重量份的原料制成:
优选的,所述的组合物由包括如下重量份的原料制成:
优选的,所述的组合物由包括如下重量份的原料制成:
进一步,所述的月柿提取物由包括如下步骤的方法制成:将月柿的汁液取出,将汁液加入至10-20倍量的浓度为30-60%的乙醇中进行超声提取,提取后过滤,将滤液浓缩后即得。
进一步,所述的超声步骤的功率为200-500W;提取步骤的温度为50-60℃。
进一步,所述的脂肪干细胞外泌体由包括如下步骤的方法制成:将脂肪冲洗后加入胶原酶消化,消化完成后加入胎牛血清培养基终止消化,离心后去除上层油脂及中层清液,然后离心去除上清液,加入胎牛血清重悬,筛网过滤后,移入培养皿行原代培养,每隔两三天换液,细胞浓度融合生长至60%-90%时,用胰蛋白酶消化后传代培养,将传代培养后的细胞置于培养皿中,当细胞融合达60-90%,清洗细胞,更换无外泌体血清的培养基,继续培养,收集细胞上清,离心去除细胞与细胞碎片,提取得到外泌体。
所述的具有美白功能的含有脂肪干细胞外泌体的组合物的制备方法,包括如下步骤:将烟酰胺、茶多酚与α-熊果苷加入到月柿提取物中,进行一次超声搅拌1-3小时,然后向其中加入脂肪干细胞外泌体,二次超声搅拌1-3小时后即成。
进一步,所述的一次超声步骤的温度为40-60℃,功率为300-600W;所述的二次超声步骤的温度为50-65℃,功率为400-600W。
所述的具有美白功能的含有脂肪干细胞外泌体的组合物的应用,所述的组合物在皮肤美白中的应用。
柿果营养价值很高,具有抑菌抗病毒、抗过敏、抗氧化和延缓衰老等作用。柿子中还含有大量的多酚类物质,此类物质具有众多的生物活性,尤其是在清除自由基能力方面表现了很好的抗氧化性,对慢性疾病起到很好的预防作用,同时可以使皮肤达到抗老化、美白、防晒和保湿等目的。
相对于现有技术,本发明具有以下优势:
本发明所述的一种具有美白功能的含有脂肪干细胞外泌体的组合物含有月柿提取物与脂肪干细胞外泌体,结合烟酰胺、茶多酚与α-熊果苷,可有效的作用于皮肤起到美白的作用。
具体实施方式
除有定义外,以下实施例中所用的技术术语具有与本发明所属领域技术人员普遍理解的相同含义。以下实施例中所用的试验试剂,如无特殊说明,均为常规生化试剂;所述实验方法,如无特殊说明,均为常规方法。
下面结合实施例来详细说明本发明。
实施例1组分制备
称取500g月柿,将月柿的汁液取出,将汁液加入至20倍量的浓度为40%的乙醇中进行300W、55℃下超声提取2小时,提取后过滤,将滤液浓缩至50ml后得到月柿提取物。
将脂肪50ml冲洗2遍后加入胶原酶消化30分钟,消化完成后加入胎牛血清培养基终止消化,离心后去除上层油脂及中层清液,然后离心20分钟去除上清液,加入胎牛血清重悬,筛网过滤后,移入培养皿行原代培养,每隔两三天换液,细胞浓度融合生长至80%-90%时,用胰蛋白酶消化后传代培养,将传代培养后的细胞置于培养皿中,当细胞融合达80-90%,清洗细胞,更换无外泌体血清的培养基,继续培养,收集细胞上清,离心去除细胞与细胞碎片,提取得到脂肪干细胞外泌体。
称量2mg烟酰胺、2mg茶多酚与5mgα-熊果苷加入到10mg月柿提取物中,进行400W、45℃下一次超声搅拌2小时,然后向其中加入0.5mg脂肪干细胞外泌体,600W、55℃下二次超声搅拌2小时后得到组合物A。
称量2mg烟酰胺、2mg茶多酚与5mgα-熊果苷加入到10mg月柿提取物中,进行400W、45℃下一次超声搅拌2小时,然后向其中加入0.01mg脂肪干细胞外泌体,600W、55℃下二次超声搅拌2小时后得到组合物B。
称量2mg烟酰胺、2mg茶多酚与5mgα-熊果苷混合,进行400W、45℃下一次超声搅拌2小时,然后向其中加入0.5mg脂肪干细胞外泌体,600W、55℃下二次超声搅拌2小时后得到组合物C。
称量2mg烟酰胺、2mg茶多酚与5mgα-熊果苷加入到10mg月柿提取物中,进行400W、45℃下一次超声搅拌2小时后得到组合物D。
称量10mg月柿提取物与0.5mg脂肪干细胞外泌体进行混合,600W、55℃下二次超声搅拌2小时后得到组合物E。
各组合物的具体配比如表1所示。
表1各组合物的成分配比
实施例2皮肤黑色素实验
选取70名20-30岁的健康、无化妆品过敏史的受试者,选择受试者左、右手臂内侧距手掌基部5cm处为试验部位,试验面积为5×5cm2。将受试者分为6组,前五组分别涂抹组合物A-E,第6组作为空白组,第7组为修正牌烟酰胺精华液作为对照组,试验前,受试者清洁双手前臂内侧,测试受试者试验区域的起始值。然后在8周内每天早晚在同一测试区域内涂抹相应等量,在第7天、14天、21天、28天、60天测试皮肤中黑色素含量,计算平均值,将试验结果列于表2中。
表2皮肤黑色素含量测试结果
注:仪器的测量范围是0-999,测量数值越高,说明皮肤中黑色素的含量越高。
由表2可知,与空白组相比,使用组合物A与组合物B涂抹于受试者手臂皮肤后皮肤中的黑色素含量显著降低,与对照组相比,也有显著的优势,说明烟酰胺、茶多酚、α-熊果苷、月柿提取物与脂肪干细胞外泌体的组合对皮肤有突出的美白作用,组合物C、D与E中缺少任一组分组无法达到理想的效果,而其中缺少脂肪干细胞外泌体对组合物的美白效果影响尤为明显。
实施例3体外酪氨酸酶抑制能力和抗氧能力检测实验
在各组合物中加入500倍量的PBS进行稀释,作为最后的样品进行检测。
酪氨酸酶抑制能力测试方法:将100μL不同浓度样品溶液和50μL的200U/mL酪氨酸酶依次加入到96孔板中,37℃孵育10min,之后加入50μL的质量分数0.05%L-酪氨酸,混匀,在37℃温育30min,在490nm处测量其吸光度。以样品溶剂作为空白对照,进行10次平行实验。
酪氨酸酶抑制率(%)=(A空白对照-A样品)/A空白对照×100%
DPPH自由基清除能力测试方法:将0.25mM DPPH溶液100μL和各样品100μL依次加入到96孔板中,暗处静置30min,490nm处测定其吸光度,以样品溶剂作为空白对照,进行10次平行实验。
自由基清除率(%)=(A空白对照-A样品)/A空白对照×100%
表3酪氨酸酶抑制率与自由基清除率测试结果
各样品 | 酪氨酸酶抑制率(%) | 自由基清除率(%) |
样品A(组合物A) | 73.41±3.54 | 43.85±2.96 |
样品B(组合物B) | 58.41±0.92 | 38.45±1.48 |
样品C(组合物C) | 64.52±6.52 | 18.14±4.10 |
样品D(组合物D) | 30.25±2.46 | 41.75±5.13 |
样品E(组合物E) | 65.37±5.41 | 26.71±1.81 |
空白对照 | 3.49±0.34 | 6.74±1.25 |
由表3可知,烟酰胺、茶多酚、α-熊果苷、月柿提取物与脂肪干细胞外泌体的组合可显著的提高酪氨酸酶抑制率与自由基清除率,表明其对皮肤具有美白效果,由样品C、样品D、样品E的结果可知,组合物中缺少任一组分均会降低其美白效果。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (9)
4.根据权利要求1所述的具有美白功能的含有脂肪干细胞外泌体的组合物,其特征在于:所述的月柿提取物由包括如下步骤的方法制成:将月柿的汁液取出,将汁液加入至10-20倍量的浓度为30-60%的乙醇中进行超声提取,提取后过滤,将滤液浓缩后即得。
5.根据权利要求4所述的具有美白功能的含有脂肪干细胞外泌体的组合物,其特征在于:所述的超声步骤的功率为200-500W;提取步骤的温度为50-60℃。
6.根据权利要求1述的具有美白功能的含有脂肪干细胞外泌体的组合物,其特征在于:所述的脂肪干细胞外泌体由包括如下步骤的方法制成:将脂肪冲洗后加入胶原酶消化,消化完成后加入胎牛血清培养基终止消化,离心后去除上层油脂及中层清液,然后离心去除上清液,加入胎牛血清重悬,筛网过滤后,移入培养皿行原代培养,每隔两三天换液,细胞浓度融合生长至60%-90%时,用胰蛋白酶消化后传代培养,将传代培养后的细胞置于培养皿中,当细胞融合达60-90%,清洗细胞,更换无外泌体血清的培养基,继续培养,收集细胞上清,离心去除细胞与细胞碎片,提取得到外泌体。
7.权利要求1-6中任一项所述的具有美白功能的含有脂肪干细胞外泌体的组合物的制备方法,其特征在于:包括如下步骤:将烟酰胺、茶多酚与α-熊果苷加入到月柿提取物中,进行一次超声搅拌1-3小时,然后向其中加入脂肪干细胞外泌体,二次超声搅拌1-3小时后即成。
8.根据权利要求7所述的具有美白功能的含有脂肪干细胞外泌体的组合物的制备方法,其特征在于:所述的一次超声步骤的温度为40-60℃,功率为300-600W;所述的二次超声步骤的温度为50-65℃,功率为400-600W。
9.权利要求1-6中任一项所述的具有美白功能的含有脂肪干细胞外泌体的组合物的应用,其特征在于:所述的组合物在皮肤美白中的应用。
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