CN112198143B - 一种评价化妆品和保健食品抗光老化、糖基化功效的方法 - Google Patents
一种评价化妆品和保健食品抗光老化、糖基化功效的方法 Download PDFInfo
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Abstract
本发明涉及保健食品功效评价技术领域,尤其涉及一种评价化妆品和保健食品抗光老化、糖基化功效的方法,包括以下步骤:(1)选取斑马鱼,加入待测试样,UVB照射,建立试样组;(2)选取斑马鱼,UVB照射,建立模型对照组;(3)分析尾鳍面积(A),评价皮肤损伤修复功效;(4)测定糖化血红蛋白含量,评价抗糖基化功效;(5)测定斑马鱼β‑半乳糖苷酶染色强度,评价β‑半乳糖苷酶抑制功效;(6)测定斑马鱼卵黄囊荧光强度,评价抗氧化功效;(7)测定斑马鱼20min内的总运动距离,评价运动改善功效。本发明实验周期短,检测抗糖基化功效的同时可展示皮肤损伤修复功效、β‑半乳糖苷酶抑制功效、抗氧化功效和运动能力改善功效。
Description
技术领域
本发明涉及保健食品功效评价技术领域,尤其涉及一种评价化妆品和保健食品抗光老化、糖基化功效的方法。
背景技术
在分子水平上老化的一个显著特征是经过非酶修饰的蛋白质的逐渐积累,其中最常见的是糖基化。还原糖与蛋白质(或其他分子)上的游离氨基发生反应,产生可逆的反应中间体,最终生成不可逆的晚期糖基化终产物(AGEs)。AGEs储存在组织中并与其受体连接,损伤组织功能。能够清除AGEs的物质提示具有抗老化作用。
紫外线通常分为三类:UVA(400–315nm),UVB(315–280nm)和UVC(280–100nm)。过去,化妆品抗光老化功效评价方法主要是UVA、UVB对脱毛大鼠进行照射,通过皮肤外观评价、皮损病理组织观察、生化检测氧化应激相关因子的变化。由于欧盟、英国、巴西、挪威、土耳其、韩国、印度、新西兰和以色列等国家和地区禁止用动物评价化妆品的功效,现有化妆品抗光老化、糖基化功效评价主要用体外的替代方法。1.人成纤维细胞抗老化实验:用UVA照射人原代成纤维细胞,检测细胞活性、β-半乳糖苷酶含量、氧自由基含量和总胶原蛋白含量。2.体外重建皮肤模型抗光老化实验:用UVB照射表皮模型,检测基质金属酶活性。3.AGEs清除实验:体外人血白蛋白与葡萄糖反应,检测荧光AGEs含量(《化妆品评价替代方法标准实施指南》)。现行的防晒功效评价方法,主要用人体评价防晒系数。
临床研究发现正常老年人体内的糖化血红蛋白含量比正常年轻明显升高,而患有白内障的老年人(未患有糖尿病)体内的糖化血红蛋白含量比正常老年人明显升高。除单糖外,美拉德反应、糖的自氧化作用和其他代谢途径(如糖酵解)产生的二羰基化合物也会产生AGEs,这可以解释非糖尿病白内障患者血清AGEs的增加。由于体内不含任何能够降解AGEs的酶,所以糖化血红蛋白随着年龄的增长不断积累,即血清AGEs是与年龄呈线性关系。说明糖化血红蛋白不仅仅是长期血糖水平衡量的“金标准”,更是评价老化的指标之一。流行病学数据表明AGEs与正常血糖患者的慢性疾病风险存在正相关关系,如动脉粥样硬化、阿尔茨海默症、肾衰竭和皮肤老化(包括促进黑色素生成)。
细胞实验显示:在紫外的照射下,血红蛋白发生过氧化形成高铁血红蛋白,这种反应也发生在皮肤。紫外辐射造成的红细胞损伤可能通过抑制膜结合ATP酶、乙酰胆碱酯酶(AchE)和G6PD、改变GSH含量和增强脂质过氧化作用破坏了负责维持细胞膜正常结构和功能的红细胞膜成分。有证据表明,氧化损伤和糖基化损伤呈正相关。紫外照射能诱发氧化损伤。AGEs通过产生涉及·O2-,H2O2和·OH的活性氧来促进皮肤光老化。AGEs的积累不但随着年龄的增长而增加,而且通过紫外线照射而被放大。
现有抗光老测试均为体外方法,缺乏吸收、分布、代谢和排泄的过程,无法准确预测抗光老化的供试品在人体是否真正有效,也无法预测在体内代谢后代谢产品具有抗光老化功效的供试品。体外检测不具备可视化的优势,无法观察到在抗光老化、糖基化的同时皮肤损伤修复的变化,不利于化妆品和保健食品的宣传推广。可用于替代实验的光老化动物模型尚未建立。化妆品防晒功效评价方法主要用于化妆品,无法评价保健食品的抗光老化、糖基化功效。
由于受精后5天(5dpf)内的斑马鱼幼鱼不需要进食,不属于动物,被欧盟认为在伦理学方面是可以接受,便被用于化妆品功效和安全性评价的替代实验。紫外照射能诱导斑马鱼氧自由基反应,但是否能诱导斑马鱼糖基化反应尚未见报道。我们迫切需要开发一种简便、快速检测的AGEs作为斑马鱼糖基化反应的指标。
发明内容
本发明为了克服上述现有技术中存在的问题,提供了一种实验周期短、检测抗糖基化功效的同时可展示皮肤损伤修复的效果的评价化妆品和保健食品抗光老化、糖基化功效的方法。
为了实现上述目的,本发明采用以下技术方案:
一种评价化妆品和保健食品抗光老化、糖基化功效的方法,包括以下步骤:
(1)随机选取若干尾斑马鱼,加入待测试样,UVB照射,建立试样组;
(2)随机选取与步骤(1)中数目相同的斑马鱼,UVB照射,建立模型对照组;
(3)在解剖显微镜下,分别采集试样组和模型对照组中斑马鱼的尾鳍照片,用尼康NIS-Elements D 4.30.00高级图像处理软件分析尾鳍面积(A),按照以下公式评价皮肤损伤修复功效:
(4)分别采集试样组和模型对照组中的斑马鱼,用生理盐水制备成匀浆液,测定所述匀浆液的糖化血红蛋白OD值,根据糖化血红蛋白OD值换算糖化血红蛋白含量(H),按照以下公式评价抗糖基化功效:
(5)分别将试样组和模型对照组中的斑马鱼用4%多聚甲醛固定过夜,用β-半乳糖苷酶染色试剂盒进行染色,测定斑马鱼β-半乳糖苷酶染色强度(S,蓝色代表β-半乳糖苷酶的含量),按照以下公式评价β半乳糖苷酶抑制功效:
(7)分别测定试样组和模型对照组中斑马鱼20min内的总运动距离(L),按照以下公式评价运动改善功效:
斑马鱼是脊椎动物,与人基因相似度高达87%。斑马鱼皮肤结构与功能与人类高度相似。含有基底层、棘层、颗粒层、透明层和表皮角质细胞层;另外尚有与人皮肤结构相同的固有层、半桥粒、黑色素细胞、血管和皮下脂肪细胞等。斑马鱼皮肤间质结缔组织、胶原及其临近的纤维母细胞及皮肤基因表达与人类皮肤相似。斑马鱼已获得广泛认可研究皮肤病变包括遗传病、色素异常、炎症、牛皮鲜、损伤修复、黑色素瘤等。因此,斑马鱼模型评价人用化妆品功效也具有较好的预测性。
斑马鱼的消化系统由肝脏、胆囊和肠组成,肝和肠细胞的分化与人类高度保守;食欲调节(如血清素)和胰岛素调节功能保守;消化和营养物质的吸收以及运输与人类高度相似;拥有新陈代谢的关键器官(如下丘脑回路、胰腺和胰岛素反应组织,白色脂肪组织、肝脏和肌肉)。因此,斑马鱼服用保健食品后评价其功效,具有较高的预测性。
本发明的检测原理为:UVB对斑马鱼的毒性要比对UVA的毒性大,LD 50值之间的差异超过350倍。为了便于诱导损伤,选择UVB作为光源。(1)UVB照射后,鱼鳍会出现缺失和减小。(2)在皮肤老化的过程中,斑马鱼体内形成AGEs。糖化血红蛋白是AGEs的一种,不仅反应血糖的水平,也能反应光老化、糖基化的严重程度。将预先包被的糖化血红蛋白(HbA1c)抗体包被在微孔板中,依次加入斑马鱼匀浆上清液、标准品、HRP标记的检测抗体,经过温预并彻底洗涤。用底物TMB显色,TMB在过氧化物酶的催化下转化成蓝色,并在酸的作用下转化成最终的黄色。颜色的深浅和斑马鱼体内HbA1c呈正相关。(3)以X-Gal为底物,在衰老特异性β-半乳糖苷酶催化下细胞或组织会生成深蓝色产物。该染色仅染衰老细胞,不染衰老前、静止期细胞、永生细胞或肿瘤细胞。(4)绿色试剂是一种DNA染料,具有细胞通透性,原本无荧光或者在还原状态荧光非常弱,氧化后与DNA结合,发出荧光信号。它的信号主要定位在细胞核和线粒体中。由于/>在有机溶剂(如二甲基亚砜)中溶解性较好,斑马鱼卵黄囊的成分为脂肪,因此,/>在卵黄囊的通透性较强,该部位染色明显。氧化应激试剂产生的荧光可通过传统荧光显微镜、高含量成像和分析、微板荧光测定法或流式细胞仪检测。(5)行为分析仪通过斑马鱼身体上的黑色素作为信号捕捉斑马鱼的运动轨迹。斑马鱼鱼鳍受损,运动能力下降,鱼鳍损伤修复,运动能力改善。
作为优选,步骤(1)中,所述待测试样为化妆品,所述斑马鱼为受精后2天的斑马鱼。化妆品给药方法:2dpf野生型AB品系斑马鱼,每组30尾,6孔板定容3mL,UVB照射前,在6孔板中加入化妆品相应的含量。UVB照射建立斑马鱼保健食品抗光老化、糖基化模型。化妆品的总给药时长维持48小时。实验终点为4dpf。平行4次实验,1次用于皮肤损伤修复的数据采集,3次用于抗糖基化的数据采集。
作为优选,步骤(1)中,所述待测试样为保健食品,所述斑马鱼为受精后3天的斑马鱼。保健食品给药方法:3dpf野生型AB品系斑马鱼,每组30尾,6孔板定容3mL,UVB照射建立斑马鱼保健食品抗光老化、糖基化模型。照射结束,在6孔板中加入保健食品相应的含量,保健食品的给药时长维持48小时。实验终点为5dpf。平行4次实验,1次用于皮肤损伤修复的数据采集,3次用于抗糖基化的数据采集。在4~5dpf收集斑马鱼糖基化反应比较明显。
上述不同待测试样所选取斑马鱼的发育阶段不同,受精后3天(3dpf)和3dpf前斑马鱼化合物吸收只经过皮肤;4dpf之后化合物吸收通过皮肤和肠道。
作为优选,步骤(4)中,所述匀浆液的糖化血红蛋白OD值的测定方法为:将所述匀浆液离心取上清液,加入到包被了HbA1c抗体的微孔板中,再加入辣根过氧化物酶(HRP)标记的检测抗体100μL,37℃避光孵育1h;洗涤后,分别加入底物A、B各50μL,37℃避光孵育15min;每孔加入终止液50μL,15min内在450nm处检测OD值,做标准曲线,测定所述匀浆液的糖化血红蛋白OD值。
作为优选,步骤(5)中,步骤(5)中,所述β-半乳糖苷酶染色强度(S)的测定方法为:将斑马鱼用PBS清洗3遍,4%多剧甲醛4℃固定过夜;用PBS洗3遍,弃PBS,加入β-半乳糖苷酶染色工作液1mL,37℃避光孵育过夜;用PBS洗1遍,在解剖显微镜下拍照。
作为优选,步骤(6)中,所述卵黄囊荧光强度(F)的测定方法为:将斑马鱼用PBS洗脱2遍,70rpm,洗脱结束,用终浓度为10μM的避光28℃染色0.5小时;染色结束,再用PBS洗脱2遍,70rpm,避光,在荧光显微镜下拍照。
作为优选,步骤(7)中,所述总运动距离(L)的测定方法为:将斑马鱼从6孔板转移至96孔板,每孔1尾鱼,定容200μL,采用行为分析仪检测黑暗环境下20min内的总运动距离。
作为优选,步骤(1)和(2)中,UVB照射的条件为:照射强度为9Mw/cm2;照射10~20min/次,共3~5次,间隔30min。根据公式照射时间[s]=照射剂量[mJ/cm2]/辐照强度[Mw/cm2]计算可得照射总剂量为27~41J/cm2。
本发明必须严格控制UVB的照射条件:UVB的照射时间较短,斑马鱼未出现光老化症状;UVB单次照射时间过长,斑马鱼无法耐受;UVB单次照射时间过短,则损伤不够严重;任一条件的变化,均会导致该模型无法进行抗光老化、抗糖基化效果的检测。
作为优选,步骤(3)~(7)中,两组间用T检验,多组间用ANOVA分析,以P<0.05为差异有显著性。
作为优选,步骤(1)~(7)中,所述斑马鱼为野生型AB品系斑马鱼。
因此,本发明具有如下有益效果:首次利用斑马鱼抗光老化、糖基化功效评价模型,实验周期短,仅需2天,可筛选抗糖基化的化妆品和保健食品配方;检测抗糖基化功效的同时可展示皮肤损伤修复的效果。
附图说明
图1是UVB照射5次不同时长尾鳍损伤表型图。
图2是UVB照射15min不同次数尾鳍损伤表型图。
图3是鼠尾藻多酚处理后斑马鱼尾鳍表型图(虚线为数据分析区域)。
图4是茶多酚处理后斑马鱼尾鳍表型图(虚线为数据分析区域)。
图5是茶多酚处理后斑马鱼全身β-半乳糖苷酶含量表型图。
图6是松珍胶囊处理后斑马鱼氧自由基含量表型图。
图7是紫草提取物处理后斑马鱼尾鳍凋亡细胞表型图。
图8是超氧化物歧化酶(SOD)处理后斑马鱼氧自由基含量表型图。
图9是SOD处理后斑马鱼运动轨迹图。
具体实施方式
下面通过具体实施例,并结合附图,对本发明的技术方案作进一步具体的说明。
在本发明中,若非特指,所有设备和原料均可从市场购得或是本行业常用的,下述实施例中的方法,如无特别说明,均为本领域常规方法。
主要仪器和试剂:
紫外线光疗仪(KN-4006,徐州市科诺医学仪器设备有限公司);解剖显微镜(SZX7,OLYMPUS,Japan);CCD相机(TK-C1481EC,Japan);电动聚焦连续变倍荧光显微镜(AZ100,Nikon,Japan);精密电子天平(CP214,OHAUS,America);多功能酶标仪(Tecan,Austria);行为分析仪(V3,ViewPoint Life Sciences,France);6孔板(Nest Biotech,China);96孔板(Nest Biotech,China);糖化血红蛋白ELISA检测试剂盒(批号202004,台湾力钧生物,China);细胞衰老β-半乳糖苷酶染色试剂盒(货号C0602,碧云天生物,China);CellROX(货号C10444,Thermofisher,America)。
本发明以下实施例均采用以下方法:
用鱼阶段:受精后3天(3dpf)和3dpf前斑马鱼化合物吸收只经过皮肤;4dpf之后化合物吸收通过皮肤和肠道。因此,化妆品功效评价的造模阶段为2dpf,实验终点在4dpf。保健食品功效评价的造模阶段为3dpf,实验终点在5dpf。
化妆品给药方法:2dpf野生型AB品系斑马鱼,每组30尾,6孔板定容3mL,UVB照射前,在6孔板中加入化妆品相应的含量。UVB照射建立斑马鱼保健食品抗光老化、糖基化模型。化妆品的总给药时长维持48小时。实验终点为4dpf。平行4次实验,1次用于皮肤损伤修复的数据采集,3次用于抗糖基化的数据采集。
保健食品给药方法:3dpf野生型AB品系斑马鱼,每组30尾,6孔板定容3mL,UVB照射建立斑马鱼保健食品抗光老化、糖基化模型。照射结束,在6孔板中加入保健食品相应的含量,保健食品的给药时长维持48小时。实验终点为5dpf。平行4次实验,1次用于皮肤损伤修复的数据采集,3次用于抗糖基化的数据采集。
照射方法:UVB照射15-20min/次,共3-5次,间隔30min。照射强度:9Mw/cm2,根据公式照射时间[s]=照射剂量[mJ/cm2]/辐照强度[Mw/cm2]计算可得照射总剂量为27-41J/cm2。
检测方法:
1、在解剖显微镜下,采集斑马鱼尾鳍照片。
数据分析:1、用尼康NIS-Elements D 4.30.00高级图像处理软件分析尾鳍面积(A),用于评价皮肤损伤修复功效。评价公式如下:
2、将每孔斑马鱼收集,用生理盐水制备成匀浆液。将斑马鱼匀浆液在4℃,3000rpm离心20min,取上清。将上清液10μL稀释到50μL,加入到包被了HbA1c抗体的微孔板中,再加入辣根过氧化物酶(HRP)标记的检测抗体100μL,37℃避光孵育1小时。洗涤后,分别加入底物A、B各50μL,37避光孵育15min。每孔加入终止液50μL,15min内在450nm处检测OD值。
做标准曲线,根据糖化血红蛋白OD值换算糖化血红蛋白含量(H),用于评价抗糖基化功效。评价公式如下:
3、将斑马鱼用PBS清洗3遍,4%多剧甲醛4℃固定过夜;用PBS洗3遍,70rpm,弃PBS,加入β-半乳糖苷酶染色工作液1mL,37℃避光孵育过夜;用PBS洗1遍。每个实验组随机选取10尾斑马鱼置于解剖显微镜下观察、拍照并保存图片,用尼康NIS-Elements D 4.30.00高级图像处理软件进行图像分析,计算斑马鱼β-半乳糖苷酶染色强度(S,蓝色代表β-半乳糖苷酶的含量)。评价公式如下:
4、将斑马鱼用PBS洗脱2遍,70rpm。洗脱结束,用终浓度为10μM的避光28℃染色0.5小时。染色结束,再用PBS洗脱2遍,70rpm。每个实验组随机选取10尾斑马鱼置于解剖显微镜下观察、拍照并保存图片,用尼康NIS-Elements D 4.30.00高级图像处理软件进行图像分析,计算斑马鱼卵黄囊荧光强度(F,绿色荧光代表氧自由基含量)。评价公式如下:
5、将斑马鱼从6孔板转移至96孔板,每孔1尾鱼,定容200μL,用行为分析仪检测20min内的总运动距离(L),用于评价运动改善功效。评价公式如下:
统计学分析方法:两组间用T检验,多组间用ANOVA分析,以P<0.05为差异有显著性。
模型确定依据:
3dpf野生型AB品系斑马鱼用UVB分别照射11s、22s和33s,每次间隔30min,共照射6次。照射结束,观察斑马鱼死亡情况。
表1.第一次UVB照射条件摸索(n=30)
结果表明:UVB照射时间较短,斑马鱼未出现光老化症状,需延长照射时间。
3dpf野生型AB品系斑马鱼用UVB分别照射5、15和30min,仅照射1次。照射结束,观察斑马鱼死亡情况。
表2.第二次UVB照射条件摸索(n=30)
结果表明:UVB单次照射时间过长,斑马鱼无法耐受,需要控制单次照射时间。
3dpf野生型AB品系斑马鱼用UVB分别照射1、3和5min,每次间隔30min,共照射6次。照射结束,观察斑马鱼尾鳍损伤情况。
表3.第三次UVB照射条件摸索(n=30)
实验中各组均未见死亡。结果表明:UVB照射5min,6次,损伤不够严重,仍需延长照射时间。
3dpf野生型AB品系斑马鱼用UVB分别照射10、15和20min,每次间隔30min,共照射5次。照射结束,观察斑马鱼尾鳍损伤情况。
表4.第四次UVB照射条件摸索(n=30)
图1为UVB照射不同时长尾鳍损伤表型,实验中各组均未见死亡。结果表明:UVB照射10-20min,5次,可建立光老化模型。
2dpf野生型AB品系斑马鱼用UVB均照射15min,每次间隔30min,分别照射3、4、5次。照射结束,观察斑马鱼尾鳍损伤情况。
表5.第五次UVB照射15min不同次数(n=30)
图2是UVB照射15min不同次数尾鳍损伤表型对比图,实验中各组均未见死亡。结果表明:UVB照射15min,最少3次即可建立光老化模型。
综上所述,在照射强度为9Mw/cm2的UVB灯光照射下,照射10-20min/次,共3-5次,间隔30min,可建立光老化模型。
6、3dpf野生型AB品系斑马鱼用UVB均照射10、15和20min,每次间隔30min,共照射5次。照射结束,在4dpf收集斑马鱼,进行糖化血红蛋白检测。
表6.UVB不同照射时长的糖基化反应(n=3)
与正常对照组比较,*p<0.05,**p<0.01
结果表明:在照射强度为9Mw/cm2的UVB灯光照射下,照射15-20min/次,共5次,间隔30min,可诱发糖基化。
7、3dpf野生型AB品系斑马鱼用UVB均照射15min,每次间隔30min,共照射5次。照射结束,分别于4、5、6dpf收集斑马鱼,进行糖化血红蛋白检测。
表6.UVB不同照射时长的糖基化反应(n=3)
组别 | 正常对照组 | 模型对照组 |
4dpf | 86±5.3 | 115±10.3* |
5dpf | 76±11.3 | 111±7.3* |
6dpf | 72±12.0 | 57±5.3 |
与正常对照组比较,*p<0.05
结果表明:在照射强度为9Mw/cm2的UVB灯光照射下,照射15min/次,共5次,间隔30min,在4-5dpf收集斑马鱼糖基化反应比较明显。
实施例1:鼠尾藻多酚抗光老化、糖基化功效评价实验
正常对照组:标准稀释水处理
模型对照组:UVB处理
鼠尾藻多酚组1:0.8μg/mL
鼠尾藻多酚组2:1.2μg/mL
鼠尾藻多酚组3:1.6μg/mL
如图3所示,模型对照组尾鳍与正常对照组比较明显粗糙、皱缩、糖化血红蛋白含量升高,表明UVB照射诱发光老化、糖基化。服用鼠尾藻多酚2天后,斑马鱼尾鳍明显再生、糖化血红蛋白含量下降。表明鼠尾藻多酚具有明显的抗光老化、糖基化功效。
表7.鼠尾藻多酚处理后斑马鱼尾鳍面积(n=10)
与模型对照组比较,***p<0.001
表8.鼠尾藻多酚处理后斑马鱼糖化血红蛋白含量(n=3)
与模型对照组比较,*p<0.05,**p<0.01
实施例2:茶多酚抗光老化、糖基化功效评价实验
正常对照组:标准稀释水处理
模型对照组:UVB处理
茶多酚组1:10μg/mL
茶多酚组2:20μg/mL
茶多酚组3:30μg/mL;
如图4、图5所示,模型对照组尾鳍与正常对照组比较明显粗糙、皱缩、糖化血红蛋白、β-半乳糖苷酶含量较高,表明UVB照射诱发光老化、糖基化。服用茶多酚2天后,斑马鱼尾鳍明显再生、糖化血红蛋白、β-半乳糖苷酶含量下降。表明茶多酚具有明显的抗光老化、糖基化功效。
表9.茶多酚处理后斑马鱼尾鳍面积(n=10)
与模型对照组比较,*p<0.05
表10.茶多酚处理后斑马鱼糖化血红蛋白含量(n=3)
与模型对照组比较,**p<0.01,***p<0.001
表11.茶多酚处理后斑马鱼β-半乳糖苷酶含量(n=10)
与模型对照组比较,***p<0.001
实施例3:松珍胶囊抗糖基化、抗氧化功效评价实验
正常对照组:标准稀释水处理
模型对照组:UVB处理
松珍胶囊组1:25μg/mL
松珍胶囊组2:50μg/mL
松珍胶囊组3:100μg/mL;
如图6卵黄囊荧光越亮,说明氧自由基含量越多。模型对照组与正常对照组比较糖化血红蛋白含量和氧自由基含量明显增强,表明模型对照组出现糖基化和过氧化。服用松珍胶囊2天后,斑马鱼糖化血红蛋白含量和氧自由基含量明显减少。表明松珍胶囊具有明显的抗糖基化和过氧化功效。
表11.松珍胶囊处理后斑马鱼糖化血红蛋白含量(n=3)
与模型对照组比较,***p<0.001
表12.松珍胶囊处理后斑马鱼氧自由基含量(n=10)
与模型对照组比较,***p<0.001
实施例4:紫草叶提取物抗凋亡、糖基化功效评价实验
正常对照组:标准稀释水处理
模型对照组:UVB处理
紫草叶提取物组1:50μg/mL
紫草叶提取物组2:75μg/mL
紫草叶提取物组3:100μg/mL
如图7所示:尾鳍荧光亮点越多,凋亡细胞越多,模型对照组尾部凋亡细胞与正常对照组比较明显增多,呈绿色荧光小点,糖化血红蛋白含量升高,表明模型对照组出现细胞凋亡和糖基化反应。涂抹紫草提取物2天后,斑马鱼细胞凋亡明显减少、糖化血红蛋白含量明显下降。表明紫草提取物具有明显的抗糖基化、抗凋亡功效。
表13.紫草提取物处理后斑马鱼尾部凋亡细胞数量(n=10)
与模型对照组比较,*p<0.05,***p<0.001
表14.紫草提取物处理后斑马鱼糖化血红蛋白含量(n=3)
与模型对照组比较,***p<0.001
实施例5:超氧化物歧化酶(SOD)抗氧化、抗糖基化、运动能力改善功效评价实验
正常对照组:标准稀释水处理
模型对照组:UVB处理
SOD组1:33μg/mL
SOD组2:100μg/mL
SOD组3:300μg/mL;
如图8所示:卵黄囊荧光越亮,氧自由基越多;如图9所示:运动轨迹图,线条越少运动能力越弱。模型对照组卵黄囊荧光强度与正常对照组比较明显增强、糖化血红蛋白增多、运动能力下降,表明存在大量的氧自由基并诱发糖基化反应,鱼鳍损伤,运动能力受到影响。服用SOD 2天后,斑马鱼卵黄囊荧光强度明显减弱、糖化血红蛋白含量下降、鱼鳍再生从而运动能力改善。表明西SOD具有明显的抗氧化、抗糖基化、运动能力改善功效。
表15.SOD处理后斑马鱼氧自由基含量(n=10)
与模型对照组比较,*p<0.05,**p<0.01,***p<0.001
表16.SOD处理后斑马鱼糖化血红蛋白含量(n=3)
在本实验含量条件下,鼠尾藻多酚、茶多酚、松珍胶囊、紫草提取物和SOD有明显抗光老化、糖基化功效。
以上所述仅为本发明的较佳实施例,并非对本发明作任何形式上的限制,在不超出记载的技术方案的前提下还有其它的变体及改型。
Claims (7)
1.一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,包括以下步骤:
(1)随机选取若干尾斑马鱼,加入待测试样,UVB照射,建立试样组;
(2)随机选取与步骤(1)中数目相同的斑马鱼,UVB照射,建立模型对照组;
(3)分别采集试样组和模型对照组中斑马鱼的尾鳍照片,分析尾鳍面积A,按照以下公式评价皮肤损伤修复功效:
(4)分别采集试样组和模型对照组中的斑马鱼,用生理盐水制备成匀浆液,测定所述匀浆液的糖化血红蛋白OD值,根据糖化血红蛋白OD值换算糖化血红蛋白含量H,按照以下公式评价抗糖基化功效:
(5)分别将试样组和模型对照组中的斑马鱼用4%多聚甲醛固定过夜,用β-半乳糖苷酶染色试剂盒进行染色,测定斑马鱼β-半乳糖苷酶染色强度S,按照以下公式评价β半乳糖苷酶抑制功效:
(6)分别将试样组和模型对照组中的斑马鱼用CellROX®避光染色,测定斑马鱼卵黄囊荧光强度F,按照以下公式评价抗氧化功效:
(7)分别测定试样组和模型对照组中斑马鱼20 min内的总运动距离L,按照以下公式评价运动改善功效:
所述步骤(1)和(2)中,UVB照射的条件为:照射强度为9 mW/cm2;照射10~20 min/次,共3~5次,间隔30 min;
步骤(1)中,所述待测试样为化妆品,所述斑马鱼为受精后2天的斑马鱼;或
步骤(1)中,所述待测试样为保健食品,所述斑马鱼为受精后3天的斑马鱼。
2.根据权利要求1所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(4)中,所述匀浆液的糖化血红蛋白OD值的测定方法为:将所述匀浆液离心取上清液,加入到包被了HbA1c抗体的微孔板中,再加入辣根过氧化物酶标记的检测抗体100 μL,37℃避光孵育1h;洗涤后,分别加入底物A、B各50 μL,37℃避光孵育15 min;每孔加入终止液50 μL,15 min内在450 nm处检测OD值,做标准曲线,测定所述匀浆液的糖化血红蛋白OD值。
3.根据权利要求1所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(5)中,所述β-半乳糖苷酶染色强度S的测定方法为:将斑马鱼用PBS清洗3遍,4%多剧甲醛4℃固定过夜;用PBS洗3遍,弃PBS,加入β-半乳糖苷酶染色工作液1 mL,37℃避光孵育过夜;用PBS洗1遍,在解剖显微镜下拍照。
4.根据权利要求1所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(6)中,所述卵黄囊荧光强度F的测定方法为:将斑马鱼用PBS洗脱2遍,70rpm,洗脱结束,用终浓度为10 μM的CellROX®避光28℃染色0.5小时;染色结束,再用PBS洗脱2遍,70 rpm,避光,在荧光显微镜下拍照。
5.根据权利要求1所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(7)中,所述总运动距离L的测定方法为:将斑马鱼从6孔板转移至96孔板,每孔1尾鱼,定容200 μL,采用行为分析仪检测黑暗环境下20 min内的总运动距离。
6.根据权利要求1所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(3)~(7)中,两组间用T检验,多组间用ANOVA分析,以P < 0.05为差异有显著性。
7.根据权利要求1-6任一所述的一种评价化妆品和保健食品抗光老化、糖基化功效的方法,其特征在于,步骤(1)~(7)中,所述斑马鱼为野生型AB品系斑马鱼。
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