CN116751257B - 一种蔷薇多肽及其用于美白保湿的药品或者化妆品中的用途 - Google Patents
一种蔷薇多肽及其用于美白保湿的药品或者化妆品中的用途 Download PDFInfo
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Abstract
本发明涉及一种蔷薇多肽及其用于美白保湿的药品或者化妆品中的用途。本发明从蔷薇中分离鉴定获得了具有较好的抗酪氨酸酶特性的多肽，所述多肽能够抑制黑色素的合成进而具有美白的特性。同时所述多肽还有较好的抗氧化和保湿的特性，所述多肽可以制备成为相应的抑制黑色素合成的药物或者美白的化妆品。
Description
技术领域
本申请涉及生物领域和化妆品领域，具体的涉及一种蔷薇多肽及其用于美白保湿的药品或者化妆品中的用途。
背景技术
在寒冷的冬季尤其是在干燥、少雨的北方城市，皮肤瘙痒、干燥、粗糙成为普遍困扰人们生活的一大皮肤问题。引起皮肤问题的关键是皮肤角质层缺水，所以如何保持皮肤适宜的水分成为保持皮肤光泽性、滋润有弹性、防止皮肤老化的关键，因此保湿类化妆品在人们的日常生活中显得尤为重要。
化妆品中的保湿成分可以被分成天然来源保湿成分和化学合成保湿成分。近年来，人们开始将目光转向含有天然保湿成分的化妆品，寻求来源于中草药的安全保湿成分。据悉，仅我国目前研制的化妆品中使用的中草药就达到500余种，化妆品广告也均打出“取自天然精华”的王牌。常规的化学合成小分子保湿剂能给皮肤带来即时的补水，但却不能达到持久保湿。而中草药保湿成分中的物质如多糖类，由于结构中含有大量的羟基，能够以氢键的形式与水结合，锁住水分，因而具有良好的保湿性。同时一些相对分子质量较大的多糖类物质如银耳多糖，它的相对分子质量能够达到100万至130万，具有较好的成膜性，减少水分流失，形成致密的“锁水膜”，达到持久保湿效果。此外，中草药保湿成分中的油脂类如山茶花油、橄榄油等，因其自有的成膜特性也能够在皮肤表层迅速形成一层保护膜以起到长效保湿作用。
化妆品领域中主要应用植物黄酮及多酚的抗氧化性作为抗氧化剂，但由于该类化合物还可以通过抑制透明质酸酶的活性，减少透明质酸的分解，能够达到深层的保湿效果，故也应用在化妆品保湿剂方面。有研究发现金盏花总黄酮提取液具有一定保湿性。橙皮素是从植物总黄酮中分离出的一种天然保湿成分，能够促进转谷氨酰胺酶的活性，催化酰基转移所形成的空间网络结构，容纳大量水分，达到保水保湿的目的。
植物多肽是由植物蛋白质水解得到的一类相对分子质量在1000-30000Da的小分子物质，是一种分子结构较简单的生物活性肽，能够通过皮肤屏障，为人体所吸收。该类化合物具有很强的亲水性，可保持大量水分，故可用在化妆品中作为保湿剂。研究发现在相对湿度81%时，麦麸多肽的吸湿率持续上升，达到80%以上。还有实验表明，藜麦多肽具有很好的成膜性，对皮肤有一定的亲合力。燕麦多肽为小分子活性肽，并含有大量的亲水基团，能够通过抑制水解胶原蛋白的分解来保持皮肤弹性和维持皮肤水分含量。植物中也含有能够应用于化妆品的游离氨基酸，如鹿药属植物中的精氨酸、甘氨酸、赖氨酸等都具有保湿效果。含有鹿药氨基酸的润肤霜，在温度25℃、相对湿度87%的环境下，其保湿性能优于空白对照样品，并只略低于含透明质酸的润肤霜。目前市场上已经有知名品牌在销售含有各种植物多肽的化妆品。
目前，从蔷薇中分离多肽类来制备美白保湿化妆品的研究还不多，提供可供商业化形式的化妆品还没有，急需开发研究。
发明内容
本发明基于现有技术的不足，开发了一种特异性提取自蔷薇的活性美白保湿肽。
所述多肽的具有抗酪氨酸酶活性以及较好的抗氧化的特性，并且具有较好的安全性。
本发明的多肽其疏水性氨基酸含量较多，而疏水氨基酸对体外抗氧化有着很好的效果。
具体的，所述的美白保湿肽为LASY-3D，其氨基酸序列如SEQ ID NO：1所示。
具体的，本发明还提供一种抗氧化特性的多肽LASY-3D，其氨基酸序列如SEQ IDNO：1所示。
具体的，本发明还提供一种抑制黑色素合成的多肽LASY-3D，其氨基酸序列如SEQID NO：1所示。
进一步的，本发明还提供了一种具有美白、保湿的化妆品，所述化妆品中含有本发明的多肽LASY-3D，其氨基酸序列如SEQ ID NO：1所示。
具体的，本发明的化妆品还包含一种或两种以上的油剂。作为油剂，也可使用通常的化妆料中使用的固体、半固体、液状的任意油剂。
作为本发明中使用的油剂的硅油，可列举出二甲基聚硅氧烷、辛酰基聚甲基硅氧烷、苯基聚三甲基硅氧烷、甲基苯基聚硅氧烷、甲基己基聚硅氧烷、甲基氢聚硅氧烷、二甲基硅氧烷·甲基苯基硅氧烷共聚物等低粘度至高粘度的直链或支链状的有机聚硅氧烷、八甲基环四硅氧烷、十甲基环五硅氧烷、十二甲基环六硅氧烷、四甲基四氢环四硅氧烷、四甲基四苯基环四硅氧烷等环状有机聚硅氧烷、甲基三(三甲基硅氧烷基)硅烷、四(三甲基硅氧基)硅烷等支链状有机聚硅氧烷、氨基改性有机聚硅氧烷、高聚合度胶状二甲基聚硅氧烷、胶状氨基改性有机聚硅氧烷、胶状的二甲基硅氧烷·甲基苯基硅氧烷共聚物等有机硅橡胶、及有机硅橡胶或橡胶的环状硅氧烷溶液、三甲基硅烷氧基硅酸、三甲基硅烷氧基硅酸的环状有机聚硅氧烷溶液、硬脂氧基有机硅等高级烷氧基改性有机聚硅氧烷、高级脂肪酸改性有机聚硅氧烷、烷基改性有机聚硅氧烷、长链烷基改性有机聚硅氧烷、氟改性有机聚硅氧烷、有机硅树脂及硅树脂的溶解物等。硅油的掺合量为1～60质量％，优选为3～40质量％，进一步优选为5～30质量％。也可同时使用容许掺合于化妆品的其他油剂。
此外，本发明的乳化化妆品可包含表面活性剂。本发明的乳化化妆品通过根据使用目的而掺合表面活性剂，由此成为使用性更优异的化妆品。作为表面活性剂，有阴离子性、阳离子性、非离子性及两性的表面活性剂，但本发明的乳化化妆品中包含的除多肽成分以外的表面活性剂没有特别限制，只要为可用于一般的化妆品的表面活性剂，则可使用任意的表面活性剂。
本发明的化妆品中也可以包含粉体。作为粉体，只要是使用于一般的化妆品的粉体，则不论其形状(球状、针状、板状等)或粒径(烟雾状、微粒、颜料级等)、颗粒结构(多孔质、无孔质等)如何，均可使用。例如可列举出无机粉体、有机粉体、表面活性剂金属盐粉体、有色颜料、珠光颜料、金属粉末颜料、焦油色素、天然色素、染料等着色剂。
本发明的有益效果：本发明从蔷薇中分离鉴定获得了具有较好的抗酪氨酸酶特性的多肽，所述多肽能够抑制黑色素的合成进而具有美白的特性。同时所述多肽还有较好的抗氧化和保湿的特性，所述多肽可以制备成为相应的抑制黑色素合成或者美白的化妆品。
附图说明
图1 不同分级的多肽的抑制酪氨酸酶活性结果图
图2 LASY-3D多肽不同浓度对细胞内黑色素含量的影响结果图
实施方式
下面将参照附图更详细地描述本发明的具体实施例。虽然附图中显示了 本发明的具体实施例，然而应当理解，可以以各种形式实现本发明而不应被 这里阐述的实施例所限制。相反，提供这些实施例是为了能够更透彻地理解 本发明，并且能够将本发明的范围完整的传达给本领域的技术人员。
尽管本文使用了特定的术语，但它们仅用于一般性和描述性的意义，而 不是为了限制的目的。除非另有定义，否则本文使用的所有技术和科学术语 具有与本公开描述的主题所属领域的普通技术人员通常理解的相同的含义。
实施例1 蔷薇抗酪氨酸酶活性组分的鉴定
取新鲜蔷薇地上叶部分，液氮冷冻，研磨，在4℃条件下，用超纯水浸提12h，去渣取汁，4℃条件下5000r/min离心10min，分离出沉淀留取上清液。
上清液采用硫酸铵沉淀，静置6h，4000r/min 离心15min，去上清，取沉淀，室温减压干燥，得到冻干蛋白。
将冻干蛋白配成2.0%的蛋白悬浮液,按照1:1的配比加入风味蛋白酶和碱性蛋白酶进行酶解反应。其中总的酶添加量为10000U/g,温度设置为50℃,pH8.0下酶解5h。酶解液95℃下灭酶10min后,恢复室温,过滤,将酶解物依次通过6ku和3ku的超滤膜，将酶解物分级为＜3、3～6、＞6ku，将分级后的多肽水溶液进行冷冻干燥，测其对酪氨酸酶抑制活性抑制效果，选择抗酪氨酸酶效果最好的组分。
酪氨酸酶活性测定实验：将黑素细胞（PCLH0118-RT，毕合生物）及共培养细胞按1×104/孔接种于96孔板，在37℃及5％CO2中孵育24h，去上清液，每孔加人含100μg/mL的各组分的黑素细胞培养基100μl，对照组只加培养基，空白组不加细胞，阳性对照组为熊果苷（100μg/mL），每组重复3次，每隔1d换1次培养基，继续孵育6d后用不含Ca2+和Mg2+的PBS洗1次，然后每孔加入0.5％Triton-X溶液100μl，超声振荡30min后．每孔加人10mM左旋多巴溶液50μl，于37℃下孵育3h，在酶标仪490nm波长（参考波长620nm）下测量各孔吸光度值，酪氨酸酶相对活性＝［（待筛组分平均吸光度-空白组平均吸光度）/（对照组平均吸光度-空白组平均吸光度）×100％。
从图1可以看出，分级为＜3Ku的多肽水溶液具有最强的抑制酪氨酸酶活性的效果。将＜3Ku的多肽水溶液进一步采用Sephadex G-25柱进行层析，将具有抑制酪氨酸酶活性的最强的组分继续进行DEAE-cellulose分离以及RP-HPLC分离，最终鉴定并获得了具有最强的抑制酪氨酸酶活性的多肽LASY-3D，其氨基酸序列如SEQ ID NO：1所示。所述多肽序列委托合成，鉴定纯度为99.96%后备用。
实施例2 LASY-3D多肽对细胞内黑色素含量的影响
为了验证所述的多肽具有广谱的抑制酪氨酸酶的特性，选择B16细胞作为实验对象，检验多肽对其中黑色素含量的影响。
取对数生长期的B16细胞,经0.25%胰酶消化,以RPMI1640完全培养基配成密度为5×105/mL的细胞悬液,接种于6孔培养板中,每孔2mL,经24h贴壁培养,分别加入含不同LASY-3D多肽浓度(浓度分别设为1,10,50,100μg/mL)的完全培养液,阴性对照组仅加同体积的完全培养基，阳性对照组为加入熊果苷（100μg/mL）的完全培养基。于37℃、5%CO2的培养箱内培养48h。分别计数、洗涤、收集细胞。每一样本加入1mLNaOH液(1moL/L,10%DMSO配制),超声波处理30min,90℃水浴2h,冷却后于酶标仪450nm测吸光度值。黑色素合成抑制率(%)=[1-(受试物孔吸光度值÷受试物孔细胞密度)÷(对照孔吸光度值÷对照孔细胞密度)]×100%。
由图2可见,不同浓度的多肽对B16黑素瘤细胞黑色素的生成均有一定的抑制作用,抑制作用具有剂量依赖性，与空白对照组相比，差异及其显著（P<0.01）。与阳性对照组比较，100μg/mL 浓度的本发明的多肽具有更强的黑色素生成抑制率，其达到了（23.12±1.25）%。
实施例3 LASY-3D多肽抗氧化特性的鉴定
清除DPPH·能力的测定：取不同浓度的多肽样品（1,10,50,100μg/mL）1mL，加入1×10-4mol/L DPPH·无水乙醇溶液1mL，混均后在室温避光保存30min，在517nm波长条件下测定吸光度，空白组以等体积无水乙醇溶液代替DPPH·溶液，阴性对照组以等体积蒸馏水代替样品溶液，阳性对照组为采用100μg/mL Vc代替样品溶液，并以等体积蒸馏水和无水乙醇混合液空白调零，所有测定值均为3次结果的平均值，清除率计算公式如下：DPPH·清除率/%=(1－（Ai－Aj）/ A0)×100%；A0式中：A0为对照组吸光度；Ai为样品组吸光度；Aj为空白组吸光度。结果如表1所示。
表1 不同浓度多肽对DPPH·清除率的影响
由表1可知，不同浓度的多肽，其清除DPPH·的能力有所不同，随着多肽浓度的增加，其清除DPPH·的能力逐步提高。这与该多肽的疏水性氨基酸含量较多有关，疏水氨基酸对体外抗氧化有着很好的效果。
实施例4 多肽的皮肤刺激实验
选择健康、成年、皮肤无损的白色家兔5只,试验前24h用8%硫化钠将试验动物背部脊柱两侧背毛去除，去除面积左右各约3cmx3cm，将0.5mL50%(W/V) LASY-3D多肽溶液涂抹于一侧,另一侧作为空白对照,每天涂抹一次，涂抹后清洗观察皮肤状态，连续涂抹观察14天。第二天开始,每次涂抹前剪毛，用水清除残留受试物。涂抹1h后观察结果，根据2015年版《化妆品卫生规范》中皮肤刺激性反应评分标准对实验结果进行评分，计算给药动物积分均值,并对照皮肤刺激强度评分表判定皮肤刺激强度。结果如下表2所示。
表2 多肽皮肤刺激性测定
从表2可以看出，涂抹多肽液14天后,5只受皮肤兔，每天每只动物的积分为0，因此涂抹50%多肽溶液对皮肤无刺激性，具有较好的安全性。
实施例5 多肽保湿性实验
取本发明多肽1g以及对照的甘油，分别加入2mL蒸馏水充分溶解后置于干燥器内，在RH40%条件下放置10h，称量并计算出保湿率，计算公式为：保湿率=（Hn/H0）×100%，其中H0为放置前样品质量（g），Hn为放置后样品质量（g）。结果如表3所示。
表3 保湿率结果（%）
从表3可以看出，本发明的LASY-3D多肽具有较好的保湿特性，虽然比已知的保湿产品甘油的保湿率有所差距，但是效果已较为显著。
尽管以上结合附图对本发明的实施方案进行了描述，但本发明并不局限 于上述的具体实施方案和应用领域，上述的具体实施方案仅仅是示意性的、 指导性的，而不是限制性的。本领域的普通技术人员在本说明书的启示下和在不脱离本发明权利要求所保护的范围的情况下，还可以做出很多种的形式，这些均属于本发明保护之列。
Claims (2)
1.一种具有抑制酪氨酸酶活性、抗氧化特性的多肽，命名为LASY-3D，其氨基酸序列如SEQ ID NO：1所示。
2.权利要求1所述的多肽在制备用于美白、保湿的化妆品中的用途。
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