CN113813263A - 苦茶碱在制备抗皮肤组织光损伤的产品中的应用 - Google Patents
苦茶碱在制备抗皮肤组织光损伤的产品中的应用 Download PDFInfo
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Abstract
本发明公开了苦茶碱在制备抗皮肤组织光损伤的产品中的应用,所述皮肤组织光损伤是由紫外光线照射导致的。本发明发现苦茶碱能对抗紫外光线导致的皮肤组织光损伤,且浓度越高效果越明显。能够有效的避免紫外光线导致的皮肤组织光损伤,减少其造成的皮肤红斑、水肿,点状血管结构和毛细血管扩张,皮纹增粗,表面多发脱屑;有效干预角质层增厚,减少晒伤细胞产生,胞浆红染、核固缩、核溶、核碎、棘层细胞气球变性以及真皮层弥漫性炎症细胞浸润等皮肤细胞晒伤现象。较于其他天然化合物,苦茶碱取材容易,稳定性强,价格经济,且我国具有原产地优势和出口优势,有很好的经济前景,是值得深度研究开发的预防及治疗光损伤的原材料。
Description
技术领域
本发明涉及天然化合物苦茶碱技术领域,具体地,涉及苦茶碱在制备抗皮肤组织光损伤的产品中的应用。
背景技术
皮肤是人体最外层的器官,反复暴露在阳光下容易受到环境损伤。紫外线是日光引起人皮肤损伤的主要成分,它能够穿过表皮到达真皮的上皮层,导致DNA损伤和氧化应激增加,启动细胞信号转导通路,诱导特定基因的表达,最终引起多种细胞内蛋白激酶、各种细胞因子和基质金属蛋白酶的表达与活化,引起皮肤急性损伤(晒黑、日晒伤)及慢性累积性损伤(海员皮肤、光线性角化病、光老化、皮肤癌)。由于臭氧层的破坏,地球上UVB辐射增加,这加重了环境对皮肤的损害风险并带来长期后果,如光老化、光免疫抑制和光致癌等。此外,随着人口老龄化速度加快,皮肤光损伤对老年人皮肤健康的影响愈发明显,光相关性皮肤肿瘤的发病率也逐年增加。
目前对抗紫外线的产品主要是局部应用防晒产品,局部应用的防晒产品可以通过吸收或反射皮肤表面的UV辐射来保护皮肤。防晒剂基于其保护机制分为两大类:无机化合物和有机化合物。无机化合物是能反射UVA和UVB辐射的惰性粒子,主要包括氧化锌和二氧化钛,这些化合物不会引起皮肤过敏反应,但是在皮肤表面是可见的,所以从美观方面考虑无机防晒霜并不受欢迎。有机防晒霜一般是羰基共轭的芳香族化合物,共轭化合物可以吸收UV和释放能量较低的射线,从而防止皮肤受UV的辐射。有机防晒剂涂在皮肤表面不易发现,具有更多美容性,但是很多成分如氧苯酮、舒利苯酮被UV活化后产生光敏性产物,随后与皮肤相互作用引起不良的皮肤反应。因此天然化合物纳入防晒配方变得越来越重要。在氧化应激介导的光损伤中,皮肤的内源性抗氧化能力是一个主要的决定因素。而大部分天然化合物具有抗氧化作用,食用或外用这些天然化合物不仅安全性强,还能够防止和减少紫外线照射引起的皮肤疾病的发生发展。
天然化合物是自然界发现的生物体产生的次生代谢产物。局部应用或食用天然化合物能预防皮肤光损伤。很多天然有机物暴露在强烈辐射下可以演变形成具有多种光适应机制的产物,包括产生的抗氧化剂和吸收紫外线的次生代谢物。
其中,苦茶碱(1,3,7,9-四甲基尿酸,theacrine)是一类甲基黄嘌呤类生物碱,可溶于氯仿、乙醇,微溶于水,并具有良好的热稳定性。主要存在于苦茶中,具有一定的镇静、催眠、消炎、镇痛作用。CN101543498A公开了苦茶碱在制备消炎镇痛药物中的应用,具体地公开了(1)苦茶碱具有一定的对抗外周神经痛的作用:苦茶碱高浓度时可在给药后30min时产生镇痛作用,而苦茶碱低浓度时的镇痛作用时间呈现延长,可能与其对中枢神经系统的抑制作用有关;(2)苦茶碱高浓度时具有一定的抗炎作用。
发明内容
本发明的目的是为了克服现有技术的上述不足,提供苦茶碱在制备抗皮肤组织光损伤的产品中的应用。
本发明的目的是提供苦茶碱在制备抗皮肤组织光损伤的产品中的应用。
为了实现上述目的,本发明是通过以下方案予以实现的:
发明人发现,UV可以引起小鼠皮肤光损伤模型的皮肤细胞凋亡,而苦茶碱可以对抗这种凋亡现象,且随着苦茶碱剂量的增加,皮肤细胞的凋亡水平逐渐减少。同时还可以减少炎症反应、氧化应激和影响多种信号通路来保护皮肤免受紫外线辐射引起的损伤。
因此本发明要求保护苦茶碱在制备抗皮肤组织光损伤的产品中的应用,其特征在于,所述皮肤组织光损伤是由紫外光线照射导致的。
优选地,所述产品为药物和/或保健品。所述药物和/或保健品为预防和/或治疗皮肤光损伤的保护剂。
在此所述的产品除了含有有效剂量的苦茶碱,还含有制备成不同剂型所需的辅料。
优选地,所述皮肤组织光损伤为细胞凋亡、炎症反应、和/或氧化应激。苦茶碱可以抑制皮肤组织光损伤产生的细胞凋亡、炎症反应、和/或氧化应激。
优选地,所述皮肤组织光损伤为皮肤肿胀、紫癜、和/或增厚。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为皮肤毛细血管扩张、皮肤血管结构改变、皮肤血管成片分布形成红斑、皮肤表面脱屑增多、皮肤表面毛发减少、皮肤纹理增粗、和/或皮肤纹理排列紊乱。苦茶碱可以显著抑制以上症状。
更优选地,所述皮肤表面脱屑为皮肤表面白色细碎脱屑。苦茶碱可以显著抑制以上症状。
更优选地,所述血管结构改变为点状、线状、和/或分支状血管结构改变。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为角质层明显增厚、晒伤细胞增多、和/或真皮层炎症细胞增多。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为皮肤角质细胞胞浆红染、细胞核固缩、细胞核溶、或细胞核碎的一种或几种。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为棘层细胞气球变性。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为真皮层弥漫性炎症细胞浸润。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为凋亡相关蛋白Bcl-2与Bax表达量的比值升高。苦茶碱可以显著抑制以上症状。
优选地,所述皮肤组织光损伤为凋亡相关蛋白caspase3活化成Cleaved-caspase-3。苦茶碱可以显著抑制以上症状。
苦茶碱可以显著抑制皮肤组织光损伤的各种症状,比且随着剂量增加,抑制效果更好。
与现有技术相比,本发明具有以下有益效果:
本发明发现苦茶碱能对抗紫外光线导致的皮肤组织光损伤,且浓度越高效果越明显。能够有效的避免紫外光线导致的皮肤组织光损伤造成的皮肤呈红斑、水肿,可见毛细血管扩张、点状血管结构和毛细血管扩张,表面多发脱屑,皮纹增粗;有效干预角质层明显增厚,减少晒伤细胞产生,胞浆红染、核固缩,甚至核溶、核碎,棘层细胞气球变性,以及真皮层弥漫性炎症细胞浸润等皮肤细胞晒伤现象。较于其他天然化合物,苦茶碱取材容易,稳定性强,价格经济,且我国具有原产地优势和出口优势,有很好的经济前景,是值得深度研究开发的预防及治疗光损伤的原材料。
附图说明
图1为苦茶碱抑制UV对小鼠皮肤光损伤的大体影响。
图2为苦茶碱抑制UV对小鼠皮肤光损伤的皮肤镜影响(50×)。
图3为苦茶碱对UV照射损伤的小鼠皮肤的病理影响-HE染色(400×)。
图4为苦茶碱对UV皮肤光损伤凋亡相关蛋白Bcl-2、Bax表达的影响;Mean±SD,n>3.*P<0.05vs the Control;#P<0.05vs UV。
图5为苦茶碱对UV皮肤光损伤凋亡相关蛋白caspase3表达的影响;Mean±SD,n>3.*P<0.05vs the Control;#P<0.05vs UV。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
(1)实验材料:
ICR小鼠,6~8周龄,25~30g,SPF级。
(2)实验动物饲养:
实验动物为ICR小鼠,饲养于温度为24±2℃、相对湿度为50±10%的12h白昼/12h黑夜交替的普通级动物饲养房;普通颗粒饲料及清洁自来水自由取食;每笼10只。在正式开始试验前,于动物房中饲养以1周适应环境。
(3)苦茶碱配制:
苦茶碱用纯水溶解为浓度分别为0.4g/L、0.8g/L、1.6g/L的苦茶碱溶液。
实施例1
一、实验方法
1、实验分组
取60只小鼠,随机分为6组,每组10只,雌、雄各5只,分别为空白对照组(A组)、UV对照组(B组)、苦茶碱对照组(C组)、苦茶碱低量组(D组)、苦茶碱中量组(E组)、苦茶碱高量组(F组)。
建立UV光损伤模型前根据分组给药。苦茶碱组给予0.4g/L、0.8g/L、1.6g/L三个不同浓度剂量的苦茶碱溶液灌胃14天(分别作为D组、E组、F组),苦茶碱对照组(C组)予高浓度剂量的苦茶碱溶液灌胃14天,空白对照组(A组)及UV对照组(B组)给予纯水灌胃14天。
2、建立UV光损伤模型:
10%水合氯醛溶液(4ml/kg)腹腔麻醉实验小鼠,婴儿理发器粗剃小鼠背部毛后,使用脱毛膏脱毛;观察评估,拍照记录;UV灯开灯稳定光源2分钟,UV照度仪确认辐照强度后,将灌胃后的实验小鼠置于UV灯下30cm处,背部向上,摆正已麻醉的实验小鼠体位充分暴露皮肤避免阴影遮挡。
在黑暗环境中,UV对照组(B组)、苦茶碱低量组(D组)、苦茶碱中量组(E组)、苦茶碱高量组(F组)小鼠予1000mJ/cm2/d的UV照射,连续照射14天。空白对照组(A组)和苦茶碱对照组(C组)未予UV照射。
3、观察小鼠皮肤变化:
连续每日灌胃及UV照射14天后,予小鼠背部裸露皮肤行皮肤镜检查、病理活检(取相同部位)并安乐死,拍照记录。
4、Western blot法验证凋亡蛋白的影响:
观察小鼠皮肤变化的同时采集样本,采用Western blot免疫印迹法检测各组处理后小鼠皮肤光损伤模型凋亡相关蛋白Bcl-2和Bax表达的影响,以及凋亡相关蛋白caspase-3和Cleaved-caspase-3的表达。
(1)总蛋白提取
将皮肤组织置于冰上,加100μl裂解液,摇匀置于冰上20min,期间震荡2-3次。裂解完后,研磨,4℃下14000rpm离心5min。将离心后的上清分装转移倒干净的离心管中放于-80℃保存。
(2)蛋白浓度测定
稀释实验样品:每个样品取2μL,加38μL H2O稀释20倍。按顺序排列好。配制BCA试剂浓度测定工作液:取50体积A液,加入1体积B液,充分混匀,现配现用。准备96孔板,各取20μL稀释好的标准样品和实验样品于96孔板中。每孔加入200μL工作液,37℃避光孵育30min。于酶标仪中读取吸光值即OD值,波长为560nm。将提取好样品的蛋白溶液和5×上样缓冲液按4:1混合,煮沸5min,中间打开盖子1-2次,放于-20℃保存。
(3)SDS电泳
向电泳槽中加入电泳缓冲液,使两块玻璃内侧电泳液高于上样孔,外侧电泳槽内电泳液浸没凝胶底部,玻璃板内侧液面高于外侧,然后依次上样,保证每孔蛋白总量在20ug,总上样量小于30uL。盖好电泳槽的盖子,注意正负极选择80V恒压电泳至溴酚蓝至分离胶后,120V恒压电泳至溴酚蓝刚出胶底部止。电泳时间大约2-3h。
(4)转膜
电泳结束后,取出凝胶,在转膜缓冲液中漂洗数秒,“三明治”模式打开电转印夹,每侧垫上一块专用的转膜液浸泡透的海绵,再各自放一块转膜液浸泡的NC膜平放在阴极测滤纸上,去除气泡,夹好电转印夹,转印槽加满转膜缓冲液,插入电转印夹,将转印槽放入冰箱内,确保NC膜靠近正极,带负点的氨基酸和蛋白质像正极移动。低温条件下,100V恒压根据蛋白分子量每1kDa转膜1分钟来进行转膜。取出杂交膜,TBST漂洗5min,5%脱脂奶粉溶液室温封闭1小时。TBST洗膜10min。
(5)免疫杂交
一抗敷育4℃过夜。洗膜液洗膜10min×4次。相应二抗稀释液37℃孵育1h。洗膜液洗膜10×4次。将杂交膜置于一透明塑料板上,注意不要让膜干燥。用一干净移液器将化学组化发光底物均匀地加到膜的表面,并使反应持续5min。用试剂盒提供的滤纸吸去膜表面多余的底物溶液,放至暗盒。
(6)显色
暗房内采用ECL化学发光液显色,以下所有操作均在暗房红灯下操作。将ECL发光液的A和B液按1:1比例配制在1.5mL EP管中。取出X光片夹,裁剪出适当大小的保鲜膜,铺平。用镊子从孵育盒中夹起PVDF膜,膜的一角先接触到滤纸上,吸去膜上的多余的TBST液。把膜放置在X光片夹的保鲜膜上,铺平后滴加配好的ECL发光液,把保鲜膜折叠并覆盖整个PVDF膜。取出X光片用剪刀裁剪适当宽度的X光片,放在保鲜膜覆盖的膜上,按下X光片夹,压片3min-10min。取出X光片,迅速放入洗片机。显影结束后,室温晾干胶片,扫描仪扫描后,用Image J软件分析结果。
二、实验结果
1、苦茶碱抑制UV对小鼠皮肤光损伤的总体影响
分别在连续1000mJ/d UV照射14天前和照射14天后2个时间点对各组小鼠背部皮肤进行观察。如图1所示,空白对照组(A组)小鼠背部皮肤色泽正常而光滑。UV对照组(B组)在连续照射14天后小鼠背部明显的皮肤肿胀,色泽红润、透亮,局部可见紫癜,触之质中,明显增厚。苦茶碱对照组(C组)小鼠背部皮肤色泽正常而光滑,与A组相似。苦茶碱低量组(D组)小鼠背部明显的皮肤红肿,
2、苦茶碱抑制UV对小鼠皮肤光损伤的皮肤镜影响
在连续1000mJ/d UV照射14天后对各组小鼠背部皮肤进行皮肤镜观察。如图2所示,空白对照组(A组)小鼠背部皮肤镜呈淡红色,表面光滑。苦茶碱对照组(C组)皮肤镜表现与A组相似,呈正常皮肤图像。UV对照组(B组)皮肤呈红色,可见多方毛细血管扩张,局部可见点状、线状、分支状血管结构改变,血管成片分布形成红斑,皮肤表面可见白色细碎脱屑,局部毛发减少,皮肤纹理增粗伴排列紊乱。苦茶碱低量组(D组)皮肤镜下皮肤呈红斑,可见点状血管结构,表面多发脱屑,皮纹增粗。苦茶碱中量组(E组)较D组红斑减淡,呈淡红色,皮纹增粗,表面可见白色脱屑。苦茶碱高量组(F组)皮肤镜下呈淡红色,皮纹稍增粗,表面无明显脱屑。
3、苦茶碱对UV照射损伤的小鼠皮肤的病理影响
如图3所示,空白对照组(A组)和苦茶碱对照组(C组)小鼠镜下皮肤均无明显异常;UV对照组(B组)和苦茶碱低量组(D组)可见角质层明显增厚,较多晒伤细胞,胞浆红染、核固缩,时可见核溶、核碎,偶见棘层细胞气球变性,以及真皮层炎症细胞增多,真皮层弥漫性炎症细胞浸润。苦茶碱中量组(E组)角质层增厚,较B组、D组增量少,未见明显晒伤细胞,真皮层炎症细胞增多。苦茶碱高量组(F组)镜下小鼠皮肤未见明显异常。
综上所述,苦茶碱能对抗UV导致的皮肤组织光损伤,且浓度越高效果越明显。
4、苦茶碱对凋亡相关蛋白的影响
如图4所示,采用Western blot免疫印迹法检测不同剂量苦茶碱对UV小鼠皮肤光损伤模型凋亡相关蛋白Bcl-2和Bax表达的影响。Bcl-2是抗凋亡蛋白,而Bax是促凋亡蛋白。如图所示:随着苦茶碱剂量的增加,抗凋亡蛋白Bcl-2与促凋亡蛋白Bax的比值逐渐增加,且与对照组相比均具有统计学意义(P<0.05)。结果提示UV可以引起小鼠皮肤光损伤模型的皮肤细胞凋亡,而苦茶碱可以对抗这种凋亡现象,且随着苦茶碱剂量的增加,皮肤细胞的凋亡水平逐渐减少。
如图5所示,检测苦茶碱对UV诱导小鼠皮肤损伤后凋亡相关蛋白caspase-3和Cleaved-caspase-3的表达。在凋亡过程中,caspase3可以活化成Cleaved-caspase-3。结果显示:苦茶碱能明显降低caspase3的活化,且苦茶碱剂量越大,其抑制活化作用越明显,结果均具有统计学意义(P<0.05)。因此苦茶碱能明显降低UV引起的皮肤细胞的凋亡。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
Claims (10)
1.苦茶碱在制备抗皮肤组织光损伤的产品中的应用,其特征在于,所述皮肤组织光损伤是由紫外光线照射导致的。
2.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为细胞凋亡、炎症反应、和/或氧化应激。
3.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为皮肤肿胀、紫癜、和/或增厚。
4.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为皮肤毛细血管扩张、皮肤血管结构改变、皮肤血管成片分布形成红斑、皮肤表面脱屑增多、皮肤表面毛发减少、皮肤纹理增粗、和/或皮肤纹理排列紊乱。
5.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为角质层明显增厚、晒伤细胞增多、和/或真皮层炎症细胞增多。
6.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为皮肤角质细胞胞浆红染、细胞核固缩、细胞核溶、或细胞核碎的一种或几种。
7.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为棘层细胞气球变性。
8.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为真皮层弥漫性炎症细胞浸润。
9.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为凋亡相关蛋白Bcl-2与Bax表达量的比值升高。
10.根据权利要求1所述的应用,其特征在于,所述皮肤组织光损伤为凋亡相关蛋白caspase3活化成Cleaved-caspase-3。
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