CN116687788A - 一种添加活性肽促进皮肤微生态平衡的复合物及其制备方法和应用 - Google Patents
一种添加活性肽促进皮肤微生态平衡的复合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种添加活性肽促进皮肤微生态平衡的复合物及其制备方法和应用，重点开发出多种秋葵活性肽，并配合了植物提取物，力求提供一种纯天然无添加，可修复肌肤，保持水油平衡，同时保湿作用效果好，保持肌肤水润，抗氧化抗衰的复合物。
Description
技术领域
本发明涉及化妆品技术领域，具体涉及一种添加活性肽促进皮肤微生态平衡的复合物及其制备方法和应用。
背景技术
化妆品是指以涂抹、喷洒或者其他类似方法，散布于人体表面的任何部位，如皮肤、毛发、指趾甲、唇齿等，以达到清洁、保养、美容、修饰和改变外观，或者修正人体气味，保持良好状态为目的的化学工业品或精细化工产品。
随着生活水平的不断提高，人们对于肌肤护理的要求也不断提高。人体在新陈代谢过程中，形成了各种各样的自由基。其中，95％的自由基都是对我们有害的氧自由基。它们攻击细胞，产生氧化作用，伤害细胞功能，使人体活力下降，脸部出现皱纹、色斑等衰老现象，皮肤变得灰暗。而且在夏季气温高的时候，肌肤水分流失严重，肌肤会大量分泌油脂，阻止水分的进一步散失。脸上出油很多时，会导致毛孔粗大、皮肤油腻光亮，存在油腻黏糊的感觉，容易使人产生烦躁郁闷的心理。并且，现今城市污染比较严重，长期存在出油过大，容易堵塞毛孔，也容易粘附灰尘和污物，引起皮肤的感染与痤疮等。
维持肌肤健康，首先要维持皮肤的水油平衡，皮肤的水油平衡就是指肌肤处在不油不干的最健康理想的状态，pH值在5-5.6之间，汗腺和皮脂腺的分泌量都很适中，处于不油腻也不干燥的中和状态，这样的皮肤红润细腻，对外界的刺激也不是很敏感。
秋葵含氨基酸、蛋白质、微量元素、多糖化合物、多酚类成分、黄酮类化合物、油脂和维生素等多种营养成分，具有促进皮肤微循环，促进皮肤对营养成分的吸收和代谢物的排出，具有减轻皮肤松弛、去除皱纹的效果，通过调研国内黄秋葵提取的非专利文献发现，目前关于秋葵提取的研究多集中于食品、饮料方面，在化妆品中应用的研究较少，但国际品牌如科颜氏、悦木之源、馥蕾诗等均有含秋葵提取物的产品，主打修复、抗衰、保湿等功效，黄秋葵提取物作为一种天然、无毒、多功效的植物提取物，在化妆品方面的应用应当重视(刘苏亭,王秀娟,王萌,闫鑫.黄秋葵提取技术专利战略分析[J].化学研究,2023,34(02))。
因此，本发明重点分析了秋葵中的功能多肽，通过生物信息学进行计算机模拟等当代生物学手段，有针对有目的的开发其中的有益成分，尤其是小分子肽，在研究中也得到多种多糖化合物、多酚类成分、黄酮类化合物，后续将对上述化合物的化妆品应用前景进行分析。
发明内容
本发明的目的在于提供一种添加活性肽促进皮肤微生态平衡的复合物，重点开发出多种秋葵活性肽，并配合了植物提取物，力求提供一种纯天然无添加，可修复肌肤，保持水油平衡，同时保湿作用效果好，保持肌肤水润，抗氧化抗衰的复合物。
为实现以上目的，本发明通过以下技术方案予以实现：
本发明一方面提供了一种添加活性肽促进皮肤微生态平衡的复合物，包括活性物和化妆品基础物质，所述活性物质是化妆品中添加了活性肽，所述活性肽是秋葵的植物乳杆菌和酵母发酵提取物经过酶解提取分离提纯得到的。
所述酶解得到活性肽的酶包括胃蛋白酶、胰蛋白酶、羧肽酶、氨肽酶、中性蛋白酶、碱性蛋白酶和酸性蛋白酶。
所述活性肽包括MIGDHSFPQSSLIA，NTVEFVRSDDQY，RFFDIIPTVDS。
所述复合物中还包括铜三肽、水杨酸、二裂酵母发酵产物滤液、乳酸菌/绿豆籽提取物、水解透明质酸钠、白藜芦醇。
所述复合物中还包括植物提取物，植物提取物包括槲寄生提取物、马齿苋提取物、洋甘菊提取物、茅根提取物、高山蒿草提取物、雪松叶提取物、小球藻提取物、牡丹提取物、薄荷提取物、酸橙树提取物、甘草提取物、莲叶提取物、菊苣茎提取物、白芨提取物。
优选的所述复合物中各组分含量，活性肽10-20份，植物提取物各1-10份(槲寄生提取物、马齿苋提取物、洋甘菊提取物、茅根提取物、高山蒿草提取物、雪松叶提取物、小球藻提取物、牡丹提取物、薄荷提取物、酸橙树提取物、甘草提取物、莲叶提取物、菊苣茎提取物、白芨提取物)，0.01-0.05份铜三肽、0.01-0.05份水杨酸、1-5份二裂酵母发酵产物滤液、1-10份乳酸菌/绿豆籽提取物发酵产物滤液、1-5份水解透明质酸钠、1-5份白藜芦醇，基本基质是1-10份1,3-丙二醇、1-10份丁二醇、1-5份泛醇、其余为水。
本发明另一方面提供了所述添加活性肽促进皮肤微生态平衡的复合物的制备方法，所述秋葵的植物乳杆菌和酵母发酵提取物由以下方法制备得到：以秋葵为原料，在添加有葡萄糖、NaCl、尿素的培养基中，取植物乳杆菌和酵母的种子液体，加入发酵培养基发酵培养，设置发酵温度，按照发酵情况，调整pH、接种量、摇床转速、发酵装液量，发酵时间48h-72h；得到发酵液，离心取上清过滤纯化，得到蛋白提取液，真空冷冻干燥冻干，再以上述蛋白冻干粉为原料，加水，搅拌均匀，分阶段加入胃蛋白酶、胰蛋白酶、羧肽酶、氨肽酶、中性蛋白酶、碱性蛋白酶和酸性蛋白酶，每加入一种酶都需充分搅拌，充分酶解后，升温灭菌灭酶，卧锣离心机离心过滤去除酶解残渣，过滤纯化，去除不溶物，离心液经滤膜过滤截留10000道尔顿以下的小分子肽，将产物送至华大基因测序，并采用蛋白同源建模及分子对接方法分析(与皮肤屏障功能有关的基因OCLN、KLK7、COL17A1、SPRR1B，再进行生物信息学分析，得到经过排名靠前的活性肽MIGDHSFPQSSLIA，NTVEFVRSDDQY，RFFDIIPTVDS。
本发明第三方面提供上述复合物的应用，可用于促进皮肤平衡、修复肌肤、抗衰老抗炎抗氧化产品的应用，其特征在于，复合物可制备成水、乳、油、膏状，可以添加佛手柑和天竺葵等天然防腐剂。复合物可制备的产品包括爽肤水、洁面乳、乳液、修复膏。
技术效果
本发明提供了一种添加活性肽促进皮肤微生态平衡的复合物及其制备方法和应用，得到了多种秋葵活性肽，并配合了植物提取物，力求提供一种纯天然无添加，可修复肌肤，保持水油平衡，同时保湿作用效果好，保持肌肤水润，抗氧化抗炎抗衰的化妆品。弥补了国内市场的不足，通过现代生物学手段精准靶向皮肤平衡修复肌肤的领域，深入渗透达到基因修复层面，一改化妆品领域盲目添加，国外占据市场的局面。
本发明提供的活性肽还可以单独使用，无细胞刺激性，具有抗氧化，抗菌，抗炎，促进胶原生物合成，促进细胞修复，缓解刺激等多方面的作用。
白藜芦醇具有很强的抗氧化、抗自由基作用，抗晒老效果好，同时对于维持皮肤的水油平衡效果好；植物提取物中槲寄生提取物富含黄酮类化合物，具有清除自由基，增强机体免疫功能，抑制黑色素生成，具有抗氧化、延缓衰老和美白的作用；马齿苋提取物含有酚性物质、大量钾离子、氨基酸、含氮物质、生物碱、香豆素、黄酮、强心苷、蒽醌苷。马齿苋提取物、洋甘菊提取物、甘草提取物、茅根提取物、高山蒿草提取物、雪松叶提取物、小球藻提取物、牡丹提取物具有清洁，水油平衡，舒缓的作用，还能缓解皮肤泛红，抑制皮肤炎症，抗过敏。薄荷、酸橙树提取物皮肤疗效可以调理不洁、堵塞的肌肤，能收缩微血管、缓解发痒、发炎和灼伤等症状。莲叶提取物具有防护净化抗氧化的作用；白芨提取物具有美白、去斑。菊苣茎提取物是化妆品中的气味抑制剂。
二裂酵母发酵产物滤液、乳酸菌/绿豆籽提取物发酵产物滤液中含有含有丰富的氨基酸、多肽、矿物质以及各种维生素，可有效改善肌肤条件，修复肌肤效果好，对于维持皮肤的水油平衡具有一定的作用。本发明进一步的还添加了铜三肽、水杨酸，以达到协助伤口愈合，抗痘的作用。
综上，本发明由多种植物提取物组合而成，不添加色素、矿物油、人工香料、表面活性剂等对皮肤不利的成份，扩大了秋葵在化妆品中的用途，能够改善肌肤新陈代谢延缓衰老，无细胞刺激性，具有抗氧化，抗菌，抗炎，促进胶原生物合成，促进细胞修复，缓解刺激等多方面的作用。提供了一种纯天然无添加，可修复肌肤，保持水油平衡，同时保湿作用效果好，保持肌肤水润，抗氧化抗炎抗衰的化妆品。
具体实施方式
下面结合实施例对本发明的实施方式作更进一步的说明。
本发明将实验分为5组，第一组空白组基本基质，第二组基本基质+活性肽(10g，下同)，第三组基本基质+植物提取物(槲寄生提取物、马齿苋提取物、洋甘菊提取物、茅根提取物、高山蒿草提取物、雪松叶提取物、小球藻提取物、牡丹提取物、薄荷提取物、酸橙树提取物、甘草提取物、莲叶提取物、菊苣茎提取物、白芨提取物，上述物质分别为1g)，第四组基本基质+活性肽+植物提取物，第五组基本基质+活性肽+植物提取物+添加物(0.01g铜三肽、0.01g水杨酸、1g二裂酵母发酵产物滤液、10g乳酸菌/绿豆籽提取物发酵产物滤液、1g水解透明质酸钠、1g白藜芦醇)，部分实验结果记为整数。基本基质是10g1,3-丙二醇、10g丁二醇、5g泛醇、其余为水(使得每组都为100g)等常用的化妆品基本基质。如无特殊说明实施例中验证发酵物功能的实验中发酵物在培养基中使用的浓度为5％。
实施例1细胞刺激性
为了确认实验各组对细胞的刺激性，发明人参考常见的细胞刺激性试验方法，以角质形成细胞(HaCat)为对象进行了细胞毒性实验。
将角质形成细胞(HaCat)。48小时后，在DMEM培养基中混合10％EZ-Cytox以测量细胞活力和增殖，在培养基加入各组实验物。培养2小时后，用酶标仪在450nm波长下测量吸光度。
细胞生存率(％)＝(实验组2-5的吸光度/实验组1的吸光度)×100结果：实验组2的显示出2.25％的细胞毒性，实验组3的显示出4.25％的细胞毒性，实验组4的显示出3.85％的细胞毒性，实验组5显示出4.15％的细胞毒性。上述组别均具有5.0％以下的低细胞毒性，上述组别可以用作化妆品安全的材料。
实施例2DNA修复系统增强作用
参考常见的DNA修复测试方法，UV照射后会生成胸腺嘧啶二聚体，其可被人体中的DNA修复系统修复，并且当DNA修复系统有效地工作时，胸腺嘧啶摄取增加。因此，可以通过测量溴脱氧尿苷(BrdU)(一种胸苷类似物)的摄取来评估修复DNA的能力。
取Wistar雌性大鼠乳房皮肤，用HEV蓝光持续照射2小时，测量溴脱氧尿苷(BrdU)的含量，结果见表1。
表1溴脱氧尿苷(BrdU)的含量测定结果。
| 样本 | 溴脱氧尿苷(BrdU)的含量 |
| 实验组1 | 0.12±0.02 |
| 实验组2 | 0.20±0.05 |
| 实验组3 | 0.18±0.02 |
| 实验组4 | 0.25±0.04 |
| 实验组5 | 0.25±0.06 |
结果：实验组2-实验组5显示出显著的DNA修复作用。
实施例3细胞刺激的缓解作用
参考常见的细胞刺激缓解测试方法，将人成纤维细胞在培养基中培养，至细胞汇合达80％。将细胞以5×104个细胞/孔的密度转移到96孔板中，培养48小时。培养后，在显微镜下观察细胞贴壁和生长状态。使用乳酸(0.2％)作为皮肤刺激物。加入MTT溶液3mg/ml，然后使用ELISA读数器(测量570nm处的吸光度)，比较各组细胞存活率。以实验组1细胞存活率为100％。
结果：实验组2的细胞存活率为175％，实验组3细胞存活率为127％，实验组4为190％，实验组5为225％。镜检细胞状态处于良好至活跃等不同等级，各组细胞存活良好且处于增殖状态，不受细胞刺激的影响。
实施例4促进胶原生物合成作用
参考常见的胶原生物合成测试方法，将人成纤维细胞接种在含有DMEM的48孔培养板中，1×106个细胞/孔，培养24小时。然后用无血清DMEM培养基替换之前的培养基，培养24小时。培养后，收集每孔上清，并使用试剂盒(Takara)测量人I型前胶原羧基端肽PICP水平以计算新合成的胶原的量。以实验组1的PICP含量定义为100％。使用抗坏血酸作为阳性对照。
结果：实验组2的PICP含量提高至156％，实验组3的PICP含量为115％，实验组4为174％，实验组5为175％，抗坏血酸的PICP含量提高至123％。
实施例5抗氧化作用
采用实施例4的细胞培养方法，根据试剂盒说明书检测细胞中的羟基和超氧阴离子自由基清除率，结果见表2。以每组细胞初始状态的羟基和超氧阴离子自由基清除率为0％进行计算。
表2羟基和超氧阴离子自由基清除率测定结果。
| 样本 | 羟基自由基清除率 | 超氧阴离子自由基清除率 |
| 实验组1 | 54％ | 46％ |
| 实验组2 | 71％ | 73％ |
| 实验组3 | 67％ | 65％ |
| 实验组4 | 75％ | 81％ |
| 实验组5 | 78％ | 80％ |
结果：实验2-5组都具有羟基和超氧阴离子自由基清除作用。
实施例6促进有益菌的合成和抑制有害菌的形成
采用实施例4的细胞培养方法，观察金黄色葡萄球菌抑菌圈大小，对10CFU/ML肠膜明串珠菌进行计数，结果见表3。抑菌圈大小测定结果。
表3抑菌圈大小测定结果
| 样本 | S.aureus(mm) | Leuconostocmesenteorides(mm) |
| 实验组1 | 20.05±0.15 | 3.05x107 |
| 实验组2 | 8.05±0.20 | 3.65x107 |
| 实验组3 | 12.10±0.55 | 3.74x107 |
| 实验组4 | 7.55±0.45 | 4.08x107 |
| 实验组5 | 8.05±0.35 | 4.51x107 |
实施例7性能测试：
选取200名20-45岁的志愿者(不分男女)作为试用对象。试用对象采用公司的皮肤检测仪检测，鉴定为皮肤健康，无皮肤过敏史为一组100名，皮肤受损缺水需要修复为一组100名；再随机将每组分为5个小组，一组20人。使用方法为：试用对象清洁脸部后将复合物爽肤水均匀涂抹于脸部，轻轻拍打至全部吸收，每日早晚各使用一次，连续使用50天。然后对复合物对于肌肤的清爽性、抗敏感性、抗皱性、水润光滑性进行评分。各项使用效果总分为5分。以下为各项平均得分。结果如表4所示。
表4皮肤健康，无皮肤过敏史组的清爽性、敏感性、抗皱性、水润光滑性评分结果。
| 样本 | 清爽性 | 抗敏感性 | 抗皱性 | 水润光滑性 |
| 实验组1 | 4.7 | 4.5 | 4.3 | 4.3 |
| 实验组2 | 5 | 4.7 | 4.6 | 4.9 |
| 实验组3 | 5 | 4.5 | 4.4 | 4.5 |
| 实验组4 | 5 | 4.7 | 4.6 | 5 |
| 实验组5 | 5 | 4.8 | 4.6 | 4.9 |
表5皮肤受损缺水需要修复组的清爽性、敏感性、抗皱性、水润光滑性评分结果。
| 样本 | 清爽性 | 抗敏感性 | 抗皱性 | 水润光滑性 |
| 实验组1 | 4.4 | 3.0 | 4.0 | 3.9 |
| 实验组2 | 4.7 | 4.3 | 4.1 | 4.3 |
| 实验组3 | 4.5 | 3.5 | 3.9 | 4.0 |
| 实验组4 | 4.7 | 4.4 | 4.5 | 5 |
| 实验组5 | 4.6 | 4.7 | 4.7 | 4.9 |
以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (10)
1.一种添加活性肽促进皮肤微生态平衡的复合物，其特征在于，复合物中添加了活性肽，所述活性肽是秋葵的植物乳杆菌和酵母发酵提取物经过酶解提取分离提纯得到的。
2.根据权利要求1所述的一种添加活性肽促进皮肤微生态平衡的复合物，所述酶解得到活性肽的酶包括胃蛋白酶、胰蛋白酶、羧肽酶、氨肽酶、中性蛋白酶、碱性蛋白酶和酸性蛋白酶。
3.根据权利要求1所述的一种添加活性肽促进皮肤微生态平衡的复合物，所述活性肽包括MIGDHSFPQSSLIA，NTVEFVRSDDQY，RFFDIIPTVDS。
4.根据权利要求1所述的一种添加活性肽促进皮肤微生态平衡的复合物，其特征在于，所述复合物中还包括铜三肽、水杨酸、二裂酵母发酵产物滤液、乳酸菌/绿豆籽提取物发酵产物滤液、水解透明质酸钠、白藜芦醇。
5.根据权利要求1所述的一种添加活性肽促进皮肤微生态平衡的复合物，其特征在于，所述复合物中还包括植物提取物，植物提取物包括槲寄生提取物、马齿苋提取物、洋甘菊提取物、茅根提取物、高山蒿草提取物、雪松叶提取物、小球藻提取物、牡丹提取物、薄荷提取物、酸橙树提取物、甘草提取物、莲叶提取物、菊苣茎提取物、白芨提取物。
6.一种添加如权利要求1所述活性肽促进皮肤平衡修复肌肤用复合物的制备方法，所述秋葵的植物乳杆菌和酵母发酵提取物由以下方法制备得到：以秋葵为原料，在添加有葡萄糖、NaCl、尿素的培养基中，取植物乳杆菌和酵母的种子液体，加入发酵培养基发酵培养，设置发酵温度，按照发酵情况，调整pH、接种量、摇床转速、发酵装液量，发酵时间48h-72h；得到发酵液，离心取上清过滤纯化，得到蛋白提取液，真空冷冻干燥冻干。
7.根据权利要求6所述的活性肽促进皮肤平衡修复肌肤用复合物的制备方法，其特征在于，所述活性肽的制备方法包括：以秋葵蛋白冻干粉为原料，加水，搅拌均匀，分阶段加入胃蛋白酶、胰蛋白酶、羧肽酶、氨肽酶、中性蛋白酶、碱性蛋白酶和酸性蛋白酶，每加入一种酶都需充分搅拌，充分酶解后，升温灭菌灭酶，卧锣离心机离心过滤去除酶解残渣，过滤纯化，去除不溶物，离心液经滤膜过滤截留10000道尔顿以下的小分子肽，进行测序生物信息学分析以及计算机模拟，得到经过功能预测的活性肽。
8.根据权利要求1-5所述的活性肽促进皮肤平衡修复肌肤用复合物用于促进皮肤平衡修复肌肤的应用，其特征在于，复合物可制备成水、乳、油、膏状。
9.根据权利要求1-5所述的活性肽促进皮肤平衡修复肌肤用复合物用于抗衰老抗炎抗氧化产品的应用，其特征在于，复合物可制备成水、乳、油、膏状。
10.根据权利要求9所述的活性肽促进皮肤平衡修复肌肤用复合物，其特征在于，复合物可制备的产品包括爽肤水、洁面乳、乳液、修复膏。
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