CN117264078A - 一种透皮增强肽与谷胱甘肽的偶联多肽及其应用 - Google Patents
一种透皮增强肽与谷胱甘肽的偶联多肽及其应用 Download PDFInfo
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Abstract
本发明涉及一种透皮增强肽与谷胱甘肽的偶联多肽及其应用,属于生物活性肽技术领域。发明提出了一种将透皮增强肽TD1氮端与谷胱甘肽C端通过寡聚甘氨酸链直链连接的偶联多肽,结构为ACSSSPSKHCG‑GGGGG‑ECG,具有较强的透皮能力及黑色素抑制能力,具有良好的美白淡斑功效,同时具有良好的安全性和稳定性。本发明的偶联多肽不仅可以直接抑制B16F10细胞中黑色素的生成,还可以通过调节HaCaT细胞(人表皮角质形成细胞)IL‑6、IFN‑γ、α‑MSH三种细胞因子的分泌量,以HaCaT细胞的旁分泌作用间接抑制黑色素的生成。
Description
技术领域
本发明涉及一种透皮增强肽与谷胱甘肽的偶联多肽及其应用,属于生物活性肽技术领域。
背景技术
黑色素,也称为黑素,可分为真黑素和褐黑素,是一种高分子生物色素,在皮肤基底层中黑色素细胞内产生并分泌。黑色素本质上是一种蛋白质,通常以聚合形式存在于人体皮肤或者毛发中,其含量和分布会直接影响皮肤、头发和眼睛的颜色。而黑色素过度分泌会造成色素沉着异常,引起黄褐斑、雀斑、老年斑等,影响人们的生活质量。黑色素的合成必须有酪氨酸、酪氨酸酶以及氧元素这三种物质参与:酪氨酸在酪氨酸酶的催化作用下,羟化变成多巴(DOPA),多巴也是酪氨酸酶的底物,它在酪氨酸酶的催化作用下被催化生成多巴醌,多巴醌自动氧化生成多巴色素,多巴色素的反应产物5,6-二羟基吲哚和5,6-二羟基吲哚羧酸,经一系列的氧化反应生成真黑素,其为皮肤黑色素的主要组分;在半胱氨酸存在的条件下,多巴醌还会转变成半胱氨酰多巴,最后生成褐黑素。
目前,已经有许多产品被报道具有美白功效,例如谷胱甘肽、熊果苷、曲酸和某些黄酮类化合物等等。谷胱甘肽(Glutataione,GSH)是一种解除毒素的亲水性特效物质,是由三个氨基酸组成的小分子肽,是体内重要的抗氧化剂和自由基清除剂,可与自由基、重金属等结合,将机体内有害的毒物转化为无害的物质并排出体外。谷胱甘肽在美容行业也因其局部皮肤美白和/或亮白效果而闻名。如中国专利文献CN105919828A公开了一种美白淡斑组合物,包括:有效成分和辅助成分,所述有效成分包括1~25%谷胱甘肽、0.1~2%VC、0.1~2%熊果苷、0.01~0.2%生物素,余量为辅助成分;主要是采用谷胱甘肽、VC、熊果苷与生物素复合使用,增效效果非常明显。中国专利文献CN107397697A公开了一种美白祛斑组合物,包括以下重量份的组分:胶原蛋白肽40-50份,L-半胱氨酸5-10份,谷胱甘肽5-10份,针叶樱桃提取物5-10份,柑橘提取物5-10份,黑胡椒提取物1-5份,蓝莓花青素1-10份;该美白祛斑组合物通过抗皱补水、抑制酪氨酸酶活性、抗氧化、改善皮肤微循环等全方位达到美白皮肤的效果。
但是由于谷胱甘肽是一种亲水性多肽,很难透过皮肤角质层屏障,从而无法发挥更加有效的美白效果。对此,为增强药物分子穿透皮肤的能力,须使用适当的物理和/或化学方法来克服这些问题。例如,二甲基亚砜(DMSO)、氮酮、油酸、丙二醇、萜烯和萜类化合物能够破坏角质形成细胞紧密的排列结构,增大细胞间距,促进吸收。另外,离子电渗疗法、电穿孔法、超声波空化作用的超声导入疗法以及产生新的渗透途径的微针等物理促透方法可在角质层表面产生微孔一次提高皮肤渗透性。然而,化学和物理增强剂都会引起刺激,造成损伤,并降低皮肤屏障功能。蛋白质转导系统是一种被广泛接受的方法,通过与聚精氨酸等细胞穿透肽(CPP)和TAT的蛋白质转导结构域融合,将蛋白质、肽、siRNA和生物活性化合物穿过细胞膜。TD1是一种透皮增强肽,具有优秀的皮肤促透效果及穿透细胞膜的能力,能够渗透至表皮的基底层。对此,本发明设计将GSH作为功效分子修饰TD1,利用TD1优秀的透皮能力将GSH“拉”入皮肤表皮,保持皮肤屏障功能正常的同时作用于黑色素细胞及角质形成细胞,实现调节黑色素过度产生,最终达到美白祛斑效果。
发明内容
针对现有技术的不足,本发明提供了一种透皮增强肽与谷胱甘肽的偶联多肽及其应用。
本发明的技术方案如下:
一种透皮增强肽与谷胱甘肽的偶联多肽,氨基酸序列如SEQ ID NO.1所示,具体为ACSSSPSKHCG-GGGGG-ECG。GGGGG作为直链连接肽,能够保证偶联多肽的正常折叠结构,满足大多数融合蛋白的需求。
上述偶联多肽在制备美白产品中的应用。
根据本发明优选的,所述美白产品包括护肤品、化妆品、药品。
上述偶联多肽在制备淡斑产品中的应用。
根据本发明优选的,所述淡斑产品包括护肤品、化妆品、药品。
一种美容制剂,包括上述透皮增强肽与谷胱甘肽的偶联多肽。
根据本发明优选的,所述美容制剂还包括制剂学上可接受的助剂或载体。
一种药物组合物,包括上述透皮增强肽与谷胱甘肽的偶联多肽。
根据本发明优选的,所述药物组合物还包括药剂学上可接受的助剂或载体。
有益效果:
1、本发明提出了一种将透皮增强肽TD1氮端与谷胱甘肽C端通过寡聚甘氨酸链直链连接的偶联多肽,组分合理,具有较强的透皮能力及黑色素抑制能力,具有良好的美白淡斑功效,同时具有良好的安全性和稳定性。
2、本发明的偶联多肽不仅可以直接抑制B16F10细胞中黑色素的生成,还可以通过调节HaCaT细胞(人表皮角质形成细胞)IL-6、IFN-γ、α-MSH三种细胞因子的分泌量,以HaCaT细胞的旁分泌作用,间接抑制黑色素的生成。
3、偶联多肽能够作为抑制剂抑制酪氨酸酶活性,能够抑制黑色素瘤细胞内酪氨酸酶活性和黑色素的合成,应用效果通过试验验证,具有很好的美白祛斑的作用。
附图说明
图1为不同多肽浓度下HaCaT细胞和B16F10细胞的细胞活力曲线图。
图2为不同多肽组B16F10细胞内和上清液中黑色素含量柱状图
图3为不同多肽组的HaCaT细胞培养上清液中的细胞因子浓度柱状图。
图4为不同药物组B16F10细胞内酪氨酸酶的相对活性柱状图
图5为不同多肽组豚鼠皮肤中总谷胱甘肽含量柱状图。
图6为豚鼠背部皮肤组织黑色素沉着情况。
具体实施方式
下面结合实施例对本发明的技术方案做进一步详细说明,但是本发明的保护范围并不仅限于此。实施例中涉及的材料及试剂,若无特殊说明,均为普通市售产品。
实施例1:透皮增强肽与谷胱甘肽的混合与偶联
谷胱甘肽(GSH):是由谷氨酸(Glu,E)、半胱氨酸(Cys,C)和甘氨酸(Gly,G)结合而成,氨基酸序列为ECG。
透皮增强肽(TD1):氨基酸序列为ACSSSPSKHCG。
谷胱甘肽与透皮增强肽的物理混合多肽(GSH+TD1):将谷胱甘肽和透皮增强肽按照摩尔比1:1混合。
谷胱甘肽与透皮增强肽直链连接的偶联多肽(TD1-linker-GSH):氨基酸序列为ACSSSPSKHCG-GGGGG-ECG。GGGGG作为连接肽,其不影响蛋白的折叠,能满足大多数融合蛋白的需求。
谷胱甘肽与透皮增强肽支链连接的偶联多肽(TD1-GSH):氨基酸序列为ACSSSPSK{gamma-GCE}HCG,其中,谷胱甘肽的甘氨酸(Gly,G)链接在TD1第八个氨基酸赖氨酸(Lys,K)的侧链氨基上。
上述偶联多肽TD1-linker-GSH和TD1-GSH由江苏金斯瑞生物科技有限公司合成。
实施例2:细胞增殖评价多肽的细胞毒性
以人角质形成细胞(HaCaT细胞)和小鼠黑色素瘤细胞(B16F10细胞)为研究对象,设计一系列浓度的多肽溶液,通过CCK8细胞毒性试验,测试多肽的细胞毒性,确定其安全剂量范围。
具体地,将HaCaT细胞和B16F10细胞以每孔1×105个/mL的细胞密度接种在96孔板中,每孔添加100μL细胞液,并将其置于37℃、5%CO2培养箱中培养24h使细胞贴壁;24小时后HaCaT细胞使用高糖DMEM维持培养基(含2%胎牛血清、1%青链霉素双抗、97%基础DMEM培养基)、B16F10细胞使用RPMI-1640维持培养基(含2%胎牛血清、1%青链霉素双抗、97%基础RPMI-1640培养基)配制的多肽溶液替换原培养基,多肽的浓度分别为:1μM、5μM、10μM、50μM、100μM、200μM、400μM、800μM,每个浓度设置3个复孔;不同浓度的多肽溶液与细胞共培养48h;同时以不含任何药物的维持培养基培养组为阴性对照组。以不含任何细胞的培养组为空白组。培养结束后,分别加入10μL CCK8溶液到96孔板的孔中,并将96孔板置于培养箱中培养2h。培养结束后使用酶标仪测试96孔板每孔中的溶液在450nm处吸光值(OD),并按照如下公式计算细胞活力。
细胞活力(%)=(ODs-ODb)/(ODc-ODb)×100%
如上述公式所示,ODs、ODb和ODc分别代表样本组、空白组和阴性对照组的吸光值。
细胞活力结果如图1所示,分析可知,对于HaCaT细胞,GSH具有一定的细胞毒害作用,且浓度越高,细胞毒害作用越强;但与TD1物理混合或偶联后,在小于400μM浓度下,细胞毒害作用基本消失,还具有促进HaCaT细胞增殖的能力,而且偶联多肽的促增殖效果优于物理混合多肽。另外,TD1在50~100μM浓度范围下的促增殖效果最佳,细胞活力可以达到120%左右。对于B16F10细胞,除了偶联多肽TD1-GSH之外,在小于400μM浓度下,各多肽对于细胞生长的影响不大,细胞活性无显著变化,基本不会产生明显的细胞毒性;但是TD1-GSH在200~400μM浓度下,可以促进B16F10细胞的增殖,细胞活力可以达到180%以上,B16F10细胞活力越高,越有利于黑色素的生成。综合各多肽的表现,以下实验选择的多肽剂量范围为1~400μM。
实施例3:B16F10细胞黑色素生成检测
通过对B16F10细胞上清液中黑色素含量的测定及B16F10细胞内黑色素含量的测定,确定多肽对黑色素生成的影响。
(1)B16F10细胞上清液中黑色素含量测量
在6孔板中,每孔加入细胞密度5×105个/mL的B16F10细胞悬液,于37℃、5%CO2培养箱中培养。待细胞贴壁后,弃去原培养基,加入用无酚红RPMI-1640维持培养基配制的不同浓度的多肽溶液,多肽溶液的浓度分别为10μM、50μM、100μM、200μM、400μM,以不加任何药物的无酚红RPMI-1640维持培养基为阴性对照组(Control),以无酚红RPMI-1640维持培养基配制的50μM的苯硫脲(PTU)溶液为阳性对照组,每组处理都设置3个重复孔。培养48h后,吸取上清液到新的96孔板,用酶标仪在490nm处测量上清液的吸光值(OD)。
(2)B16F10细胞内黑色素含量测量
按照上述操作吸取上清液后,贴壁的细胞用PBS缓冲液清洗2次,每孔加入100μL1M NaOH溶液,80℃的水浴锅中溶解2小时,设定酶标仪波长为490nm,检测该波长下溶液的吸光值(OD)。
黑色素能够吸收490nm波长的光,酶标仪所测的吸光度值越高,说明黑色素含量越高。其中黑色素相对含量按照如下公式计算得到:
黑色素含量(%)=OD给药组/OD阴性对照组×100%
式中,OD给药组为加入了多肽的细胞处理组的平均吸光值;OD阴性对照组为阴性对照组的平均吸光值。
黑色素含量结果如图2所示,分析可知:TD1对B16F10细胞黑色素生成的抑制效果最弱,其他多肽对B16F10细胞黑色素的抑制情况呈现出浓度依赖性,均能够在一定程度上抑制B16F10细胞黑色素的生成与分泌。其中,GSH与GSH+TD1和TD1-GSH对B16F10细胞内黑色素的抑制效果相当;与GSH+TD1相比,在10μM的浓度下,TD1-linker-GSH和GSH+TD1对B16F10细胞培养上清液中黑色素的抑制效果相当,在50~400μM的浓度下,TD1-linker-GSH对B16F10细胞培养上清液中黑色素的抑制效果更佳;在10~100μM的浓度下,TD1-linker-GSH和GSH+TD1对B16F10细胞内黑色素的抑制效果相当,在200~400μM的浓度下,TD1-linker-GSH对B16F10细胞内黑色素的抑制效果更佳。
其中,当不同多肽的浓度为400μM时,黑色素抑制效果最佳,B16F10细胞内和上清液中黑色素相对含量具体如表1所示。
表1.药物浓度为400μM时B16F10细胞内/上清液中黑色素相对含量
实验组 | 阴性对照 | PTU | GSH | TD1 | GSH+TD1 | TD1-linker-GSH | TD1-GSH |
细胞内 | 100% | 80% | 75% | 83% | 78% | 55% | 75% |
上清液 | 100% | 79% | 52% | 90% | 54% | 33% | 53% |
由表1可知,除TD1以外,其他多肽对B16F10细胞内和上清液中黑色素抑制效果均优于阳性对照组(PTU);GSH+TD1浓度为400μM(是指GSH和TD1的浓度均为400μM)时,对B16F10细胞内和上清液中黑色素抑制效果与GSH相差不大,说明在物理混合状态时,TD1对GSH的黑色素抑制能力作用不大;但是相对于GSH、TD1、GSH+TD1和TD1-GSH,偶联多肽TD1-linker-GSH可以显著降低B16F10细胞内和上清液中黑色素的含量,说明在直链偶联状态时,TD1和GSH产生了协同增效的作用。
实施例4:HaCaT旁分泌对B16F10细胞黑色素生成的影响
通过对HaCaT细胞培养上清液中分泌的细胞因子含量的测定,确定偶联多肽通过HaCaT旁分泌对B16F10细胞黑色素生成的影响。
在6孔板中,每孔加入细胞密度5×105个/mL的HaCaT细胞悬液,于37℃、5%CO2培养箱中培养,待细胞贴壁后,弃去原培养基,加入用无酚红DMEM维持培养基配制的多肽溶液,多肽溶液的浓度为400μM,以不加任何药物的无酚红DMEM维持培养基为阴性对照组(Control),加药培养48h。
(1)HaCaT上清液中人白细胞介素6(IL-6)含量的测量
采用Human IL-6ELISA KIT试剂盒(货号SEKH-0013,北京索莱宝科技有限公司)检测IL-6含量。将细胞加药培养48h后,加入100μL标准样品或HaCaT细胞培养上清液于IL-6抗体预包被酶标板中,室温孵育2h后加入100μL生物素化抗体工作液至反应孔中,室温孵育1h。孵育结束后洗板,加入100μL酶结合物工作液室温孵育30min。洗板5次并拍干板内溶液,加入100μL显色底物室温避光孵育20min后加入50μL终止液,立即用酶标仪450nm波长下测量吸光度。绘制的标准曲线:y=101.47x-9.2593,R2=0.9961。根据标准曲线和样品吸光度计算HaCaT细胞培养上清液中IL-6细胞因子浓度。
(2)HaCaT上清液中人干扰素γ(IFN-γ)含量的测量
采用Human IFN-γELISA KIT试剂盒(货号SEKH-0046,北京索莱宝科技有限公司)检测IFN-γ含量。将细胞加药培养48h后,加入100μL标准样品或HaCaT细胞培养上清液于IFN-γ抗体预包被酶标板中,室温孵育2h后加入100μL预先与酶结合物偶联的生物素化抗体工作液至反应孔中,室温孵育1h。孵育结束后洗板,加入100μL酶结合物工作液室温孵育30min。洗板5次并拍干板内溶液,加入100μL显色底物室温避光孵育20min后加入50μL终止液,立即用酶标仪450nm波长下测量吸光度。绘制的标准曲线:y=148.56x-20.091,R2=0.9986。根据标准曲线和样品吸光度计算HaCaT细胞培养上清液中IFN-γ细胞因子浓度。
(3)HaCaT上清液中人α黑色素细胞刺激素(α-MSH)含量的测量
采用Humanα-MSH ELISA KIT试剂盒(货号JL14434,上海江莱生物科技有限公司)检测α-MSH含量。将细胞加药培养48h后,加入100μL标准样品或HaCaT细胞培养上清液于α-MSH抗体预包被酶标板中,每孔加入50μL HRP-抗原工作液后37℃温育1小时。洗板5次并拍干板内溶液,加入100μL显色底物室温避光孵育15min后加入50μL终止液,立即用酶标仪450nm波长下测量吸光度。绘制的标准曲线:y=-94.109x+381.59,R2=0.9914。根据标准曲线和样品吸光度计算HaCaT细胞培养上清液中α-MSH细胞因子浓度。
上述细胞因子的检测结果如图3和表2所示,IL-6、IFN-γ、α-MSH三种细胞因子是由HaCaT细胞分泌,是三种重要的与黑色素生成有关的细胞因子。
IL-6和IFN-γ因子含量越高,抑制黑色素生成效果越好。检测结果发现,GSH和GSH+TD1组IL-6含量相差不大,而TD1-linker-GSH组IL-6含量约为GSH+TD1组的2.5倍,约为TD1-GSH组的1.5倍。对于IFN-γ因子而言,TD1-GSH组表现出降低趋势,GSH、TD1、GSH+TD1和TD1-linker-GSH组均表现出不同程度的增加,其中TD1-linker-GSH组IFN-γ细胞因子含量高达48.5pg/mL。
α-MSH因子是一种非常重要的促黑因子,α-MSH含量越高,促进黑色素生成效果越好。研究结果表明,TD1、GSH+TD1对α-MSH含量几乎没有影响。相比较于对照组,GSH抑制约10%的α-MSH细胞因子分泌,而TD1-GSH促进约6%的α-MSH细胞因子分泌,TD1-linker-GSH则表现出了约25%的α-MSH分泌抑制率。
以上结果表明,TD1-linker-GSH可以通过调节HaCaT细胞IL-6、IFN-γ、α-MSH三种细胞因子的分泌量,以HaCaT细胞旁分泌的形式影响B16F10细胞黑色素的生成。
表2.不同多肽组的HaCaT细胞培养上清液中细胞因子的浓度
浓度(pg/mL) | 对照组 | GSH | TD1 | GSH+TD1 | TD1-linker-GSH | TD1-GSH |
IL-6 | 5.606 | 6.976 | 5.251 | 6.418 | 16.007 | 10.578 |
IFN-γ | 18.124 | 21.078 | 31.972 | 37.510 | 48.496 | 14.339 |
α-MSH | 257.743 | 230.169 | 257.617 | 255.170 | 193.142 | 273.647 |
实施例5:B16F10细胞内酪氨酸酶活力检测
在24孔板中,每孔加入细胞密度1×105个/mL的B16F10细胞悬液,于37℃、5%CO2培养箱中培养,待细胞贴壁后,弃去原培养基,加入用无酚红RPMI-1640维持培养基配制的不同浓度多肽溶液,多肽溶液的浓度分别为10μM、50μM、100μM、200μM、400μM,以不加任何药物的无酚红RPMI-1640维持培养基为阴性对照(Control),以无酚红RPMI-1640维持培养基配制的50μM的苯硫脲(PTU)溶液为阳性对照,每个浓度都设置3个重复孔。细胞培养48h后,弃去培养基,PBS缓冲液清洗细胞2次;每孔加200μL 1%TritonX-100,将24孔板置于4℃冰箱30min裂解;将裂解液转移至EP管,12000转离心20min,取100μL上清液于96孔板中;每孔再加50μL浓度为4mM的L-DOPA,37℃培养箱培养10h;酶标仪490nm处检验吸光值(OD),按照如下公式计算细胞内酪氨酸酶活性。
酪氨酸酶活性(%)=OD给药组/OD阳性对照组×100%
式中,OD给药组为加入了多肽的细胞处理组的平均吸光值;OD阴性对照组为阴性对照组的平均吸光值。
酪氨酸酶的相对活性如图4所示,分析可知:所有的多肽都可以抑制B16F10细胞内酪氨酸酶的活性,其中,GSH+TD1对酪氨酸酶的抑制效果较差,说明在物理混合状态下,两者未产生协同增效的作用效果,但是在400μM时,TD1-linker-GSH对酪氨酸酶的抑制效果优于其他四组多肽。
其中,当多肽浓度为400μM时,B16F10细胞内酪氨酸酶相对活性如表3所示。
表3.B16F10细胞内酪氨酸酶相对活性
400μM | 阴性对照 | 阳性对照 | GSH | TD1 | GSH+TD1 | TD1-Linker-GSH | GSH-TD1 |
直接作用 | 100% | 52% | 47% | 54% | 69% | 42% | 66% |
由表3数据可知,相对于GSH、TD1、GSH+TD1和GSH-TD1,偶联多肽TD1-linker-GSH可以更显著降低B16F10细胞内酪氨酸酶活性,说明在直链偶联状态时,TD1和GSH对于抑制酪氨酸酶活性产生了协同增效的作用。
实施例6:谷胱甘肽透皮能力检测
为验证偶联多肽的透皮能力,采用ELISA试剂盒检验在给药之后的不同时间段内皮肤组织中谷胱甘肽的含量。共设计四组药物组,分别为GSH组、TD1+GSH组、TD1-linker-GSH组,每组受试豚鼠数量为3只,使用PBS缓冲液配制,浓度为400μM,涂药量为100μL/4cm2,分别在涂药后0、1、2、4、8h时对豚鼠进行麻醉并取下4cm2的皮肤组织(含真皮层),分别与PBS缓冲液和无水乙醇中清洗三次,去除残留脂肪后用液氮速冻,然后研成粉末。每20毫克研碎的组织粉末,使用碧云天总谷胱甘肽试剂盒(货号S0052)检验皮肤组织中总谷胱甘肽含量。
豚鼠皮肤组织中谷胱甘肽含量检测结果如图5所示,分析可知:偶联多肽在涂药后4小时皮肤中谷胱甘肽含量是未涂抹时的5.6倍,GSH+TD1是未涂抹时的2.4倍,而GSH几乎不被皮肤吸收。
实施例7:偶联多肽对体内豚鼠色沉模型的改善
(1)建立紫外线照射诱导色素沉着的豚鼠模型
选择健康的雌性未怀孕花色豚鼠(3只),清洁级。豚鼠使用刮刀及脱毛膏使豚鼠背部皮肤充分暴露。随后用亚甲基蓝在背部分区。用紫外线光疗仪(徐州市科诺医学设备有限责任公司)照射豚鼠裸露的背部皮肤,每次照射前背部实验区域皮肤剃毛,调整焦距,使用紫外线光疗仪照射,照射剂量为900mJ/cm2,每周照射1次,连续照射4周,照射总剂量为2700mJ/cm2,成功建立紫外线照射诱导色素沉着的豚鼠模型。
(2)药物配制
使用PBS缓冲液配制浓度为400μM的偶联多肽TD1-linker-GSH溶液。
(3)分组治疗
在紫外线照射诱导色素沉着的过程中,连续28天对豚鼠模型每天两次(早晚各一)涂抹偶联多肽溶液,用药量为10μL/cm2。
除各用药组外,还设置了阴性对照组:只光照,不涂PBS缓冲液,不用任何药物;溶剂对照组:光照的同时涂抹PBS缓冲液,不加任何药物;阳性对照组:光照的同时涂抹PBS缓冲液及一种公认的黑色素抑制剂苯硫脲(PTU)。
(4)黑色素沉着变化观察和标本切取
取材前通过拍摄豚鼠外观照片观察豚鼠背部色素沉着变化情况,然后取豚鼠背部皮肤组织标本放入4%多聚甲醛溶液中固定。其中4%多聚甲醛溶液中固定的豚鼠背部皮肤组织行黑色素颗粒Masson法染色。
经过治疗后,豚鼠背部皮肤组织黑色素沉着情况如图6所示。分析可知:照光前,豚鼠裸露的背部皮肤红润白皙,照光后皮肤明显变黑,其中,1号区域为阴性对照区,2号区域为溶剂对照区,3号区域为阳性对照区,4号区域为偶联多肽给药区。连续治疗28天后取下1,2,3,4号区域内皮肤组织的同时,取相同大小且未接受UVB光照处理的皮肤组织作为正常组进行黑色素颗粒贪色,结果说明偶联多肽TD1-linker-GSH对豚鼠UVB色沉模型具有阳性效果,能够改善豚鼠背部表皮组织色斑沉着情况。
Claims (9)
1.一种透皮增强肽与谷胱甘肽的偶联多肽,其特征在于,氨基酸序列如SEQ ID NO.1所示,具体为ACSSSPSKHCG-GGGGG-ECG。
2.权利要求1所述的偶联多肽在制备美白产品中的应用。
3.如权利要求2所述的应用,其特征在于,所述美白产品包括护肤品、化妆品、药品。
4.权利要求1所述的偶联多肽在制备淡斑产品中的应用。
5.如权利要求4所述的应用,其特征在于,所述淡斑产品包括护肤品、化妆品、药品。
6.一种美容制剂,其特征在于,包括权利要求1所述的透皮增强肽与谷胱甘肽的偶联多肽。
7.如权利要求6所述的美容制剂,其特征在于,所述美容制剂还包括制剂学上可接受的助剂或载体。
8.一种药物组合物,其特征在于,包括权利要求1所述的透皮增强肽与谷胱甘肽的偶联多肽。
9.如权利要求8所述的药物组合物,其特征在于,所述药物组合物还包括药剂学上可接受的助剂或载体。
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