黑色素细胞中黑色素含量的测定和酪氨酸酶活性的测定

细胞酪氨酸酶（tyrosinase）活性比色法定量检测试剂盒产品说明书（中文版）主要用途细胞酪氨酸酶（tyrosinase）活性比色法定量检测试剂是一种旨在通过酪氨酸酶反应系统中底物酪氨酸氧化后，产生多巴色素，呈现吸光峰值的变化，即采用比色法来测定细胞裂解悬液样品中酶活性的权威而经典的技术方法。

该技术经过精心研制、成功实验证明的。

其适用于各种人体和动物细胞，尤其是黑色素细胞和黑色素瘤细胞裂解悬液样品中酪氨酸酶的活性检测，以及抑制剂筛选。

产品严格无菌，即到即用，操作简捷，性能稳定。

技术背景酪氨酸酶（tyrosinase；EC1.14.18.1）是一种单酚单加氧酶（monophenol monooxygenase），又称为单酚酶（monophenolase）或单酚二羟基苯丙氨酸：O2氧化还原酶（monophenol dihydroxyphenylalanin：O2 oxidoreductase），具有双功能的含铜糖蛋白，广泛存在于植物、酵母和动物组织中，其蛋白结构、大小、糖基化方式、激活特征等都不同。

人体酪氨酸酶是一个跨膜蛋白，而催化结构域在黑色素细胞（melanocyte）或色素细胞的黑色素器（melanosome）里。

酪氨酸酶通过催化L-酪氨酸（L-tyrosine）等的o-羟基化（o-hydroxylation）反应变成L-多巴（L-dopamine），进而氧化产生黑色素底物多巴醌（dopaquinone），从而由多巴色素（dopachrome）转化为黑色素（melanin）中的真黑素（eumelanin），以及其它色素，包括毛发和眼睛色素。

紫外线可以激活酪氨酸酶活性，严重时，会引起色素沉着（hyperpigmentation）。

酪氨酸酶基因突变将导致I型眼皮肤白化病（oculocutaneous albinism）。

酪氨酸酶抑制剂成为增白化妆品的重要元素。

基于底物酪氨酸，在酪氨酸酶的催化下，产生多巴色素，通过其吸光值的变化（475nm波长），来定量测定酪氨酸酶的活性。

黑色素细胞体外培养技术的研究及应用维普资讯 ////0>.堑堡匿堂生箜墨鲞黑色素细胞体外培养技术的研究及应用新疆维吾尔自治区人民医院刘熔李红健综述一、体外培养技术的建立表皮培养而设计的培养液,钙离子浓度为.。

.概述:黑素细胞起源于外胚层的神经嵴,其数量与部但对一些要求较高的基础性研究来说,由于血清位、年龄有关与肤色、人种、性别等无关。

几乎所成分非常复杂,常使研究结果影响较大,同时血清中有组织内均有黑素细胞,但以表皮、毛囊、黏膜、视网也含有一定的细胞:莓性物质和抑制剂,对细胞有去分膜色素上皮等处为多。

位于表皮的基底层,与化作用,影响某些细胞功能的表达。

因此现在的研究者都在寻找不含血清或其他天然培养基成分的培养表皮细胞共同组成表皮黑素单位,其主要功能是产生液。

黑素小体保护皮肤免受损害。

最近研究表明,在某些不良条件刺激下,黑色素细胞可发生分裂、增殖、黑素无血清培养基一般包括基础培养液及辅加成分形成、移行等一系列行为参与机体代谢。

两部分。

无血清培养基的基础培养液一般采用人工应用细胞培养技术建立的体外纯培养,是研合成培养基,常用 /、培养液等。

究皮肤色素障碍性疾病的重要手段。

因此体外培养 .影响黑色素细胞生长的因子:人是人们十分关注的一项研究课题。

黑色素细年等在培养基中加入和胞的培养可以追溯到年代,但是直到年代才真首次成功地培养出了黑素细胞。

其机理是除正建立了可靠的黑色素细胞培养方法。

年了可以刺激黑素细胞自分泌特定的生长因子外,在化学结构上与二酯酰甘油类似,可以取代首先报告体外培养人黑色素细胞以来,由于培养技术没有突破,而未能培养出大量纯化的人活化蛋白激酶,是以为代表黑素细胞。

随着细胞生物技术的迅速发展,年的佛波酯类化合物的主要受体和靶点,通过信号转导美国和在含有辅助致癌因子途径/刺激黑素细胞增殖。

也有人认为培一一一一,或 ? 养成功的关键是对角质形成细胞有毒性作用,一一一 , 、霍舌毒可以抑制成纤维细胞生长,从而避免角质形成细素 ,和 %胎牛血清的培养液中, 胞和成纤维细胞污染,使黑素细胞快速生长。

化妆品中美白活性成分检测1美白活性成分作用机制白种人肤色较浅，是因为他们皮肤中含有较多的褐黑色素，而黄种人皮肤中含有较多的真黑色素，使得黄种人看起来更黑一些。

因此，阻断褐黑色素以及真黑色素的形成就成为美白祛斑的有效途径。

1.1 黑色素的形成[1-5]紫外线、活性氧等环境甚至是某些食物都可能导致皮肤变黑，这是因为这些直接或间接地参与了黑色素生成。

黑色素的形成依赖于三种酶：酪氨酸酶、多巴色素互变酶以及DHICA氧化酶。

酪氨酸在酪氨酸酶的作用下生成多巴，多巴继续在酪氨酸酶的作用下生成多巴酿，多巴醍是形成褐黑素的前体；同时多巴醍在酪氨酸酶催化作用下生成多巴色素，接着多巴色素在多巴色素互变酶作用下，大部分生成二羟基口引噪(DHI),小部分生成二羟基口引口朵竣酸(DH1CA),二者在DHICA 氧化酶作用下，生成DH1-黑色素和DHICA-黑色素两种真黑色素。

从黑色素的形成过程中可以看到，酪氨酸是形成黑色素的主要原料，减少酪氨酸的摄入，有利于从源头上减少黑色素的形成，同时可以发现，酪氨酸酶是形成黑色素的决定性酶，可以说没有酪氨酸酶，黑色素将难以形成。

因此，体内的酪氨酸酶活力越高，含量越高，形成黑色素的可能越大。

1.2 美白祛斑机制美白祛斑类化妆品的作用机制是干扰甚至阻断黑色素的形成。

目前有4种原理可用来解释美白活性成分的作用机制[6]o1.2.1 还原作用美白活性成分直接作用于黑色素）各具有明显黑色的黑色素加以还原，变为无色的还原型色素。

1.2.2 凝结作用一般而言，蛋白质发生凝结，会失去其本身的活性。

而酪氨酸酶是一种蛋白质，通过凝结作用，促使酪氨酸酶失活，失去催化的活性，从而使得色素细胞不能生成黑色素。

酚类化合物对蛋白质有凝结作用，苯酚、对苯二酚（氢酿）、熊果甘、曲酸及其衍生物等采用的就是这种原理。

1.2.3 嵌合作用酪氨酸酶是一种含铜离子的酶，其中铜离子作为辅酶。

嵌合作用就是作用于酪氨酸酶中的铜离子，使其发生络合作用，从而使酶的活性降低甚至失去催化功能□o1.2.4 破坏作用通过破坏自由基，从而导致黑色素小体结构改变，最后造成黑色素的细胞破坏。

几种复配美白剂的美白功效研究文/罗婷婷 孙祥灵 赵昆 段国梅 李土桂 何秋星本文通过对7种不同美白剂原料进行复配研究，采用单因素试验、正交试验以及综合评价，优选最佳美白复配组合，通过B16细胞试验评价美白复配组合的美白功效，以此探讨美白复配原料对皮肤色素沉着的改善作用，旨在为今后美白复配化妆品的开发提供依据。

关键词美白剂；复配；黑色素瘤细胞中国素有“一白遮三丑”的审美观念，追求“肤如雪，凝如脂”历来是中国女性关注的热点话题。

目前，大多数美白剂的美白作用机制主要通过抑制酪氨酸酶活性、阻断黑色素合成过程、加速黑色素角质细胞脱落、抑制黑色素向角质细胞迁移、清除自由基等方面来改善皮肤黑色素沉着、肤色暗沉等问题[1-2]。

因此，美白型护肤品在化妆品领域的研究中具有较为广阔的市场和发展潜力。

实验部分01试剂与仪器烟酰胺、熊果苷、维生素C、凝血酸、光甘草啶、谷胱甘肽、白藜芦醇，广州品赫生物技术有限公司；FeSO4、水杨酸，天津市福晨化学试剂厂；三羟甲基氨基甲烷，上海润捷化学试剂有限公司；DPPH、邻苯三酚、酪氨酸酶，上海宝曼生物科技有限公司；B16小鼠黑色素瘤细胞株，北京北纳创联生物技术研究院；DMEM培养基、0.25%胰蛋白酶、胎牛血清，美国Gbico公司；DMSO、MTT、TritonX-100，美国MP Biomedicals公司。

UV-2600，CH紫外分光光度计，岛津公司；SC-3610低速离心机，安徽中科中佳科学仪器有限公司；二氧化碳细胞培养箱，上海圣科仪器设备有限公司；Infinite M200 PRO酶标仪，帝肯贸易有限公司。

02实验方法2.1 单因素试验将维生素C(0.10、0.12、0.14、0.16、0.18mg/mL)、白藜芦醇(0.04、0.08、0.12、0.15、0.20mg/mL)、谷胱甘肽(0.03、0.05、0.08、0.10、0.12mg/mL)、光甘草叮(0.05、0.08、0.10、0.12、0.15mg/mL)、凝血酸(0.02、0.05、0.08、0.12、0.14mg/ mL)、熊果苷(0.25、0.30、0.35、0.40、0.45mg/mL)和烟酰胺(0.14、0.16、0.18、0.20、0.25mg/mL)分别以DPPH 自由基清除率为指标，考察7种美白原料的浓度对DPPH 自由基的抗氧化作用，根据其结果筛选出对DPPH自由基清除效果最好的3种美白原料，其为组合1。

第34卷第1期 海南师范大学学报（自然科学版）V〇l.34N〇.l 2021 年3 月Journal of Hainan Normal University(Natural Science)Mar.2021Doi ： 10.12051/j.issn. 1674-4942.2021.01.010黑木耳黑色素的研究综述陈雅，徐苗，王欣宜，单欣荷，季琳凯，张拥军*(中国计量大学生命科学学院，浙江杭州310018)摘要：黑木耳是中国一种分布极为广泛且具有独特的营养价值与保健功能的食用菌，含有多 种生物活性化合物，其中黑色素是主要生物活性成分之一，且具有极高的安全性，可进一步开发具 备保健作用的功能色素，该方面的研究已初见成效且应用前景广阔。

文章综述了黑木耳黑色素的 理化性质、分子组成与结构表征、分离制备手段以及其清除自由基、抑菌抗病毒、抗辐射、改善肝损 伤、保护D N A等生物活性的国内外研究现状，分析了限制黑木耳黑色素开发应用的实际问题并提 出展望，并为今后黑木耳黑色素生物活性作用机制的研究及其在食品、药品、化妆品等领域的应用 提供参考。

关键词：黑木耳；黑色素；分离提取；生物合成；分子结构；生物活性中图分类号:0629.1 文献标志码:A文章编号：1674-4942(2021)01-0063-07Summary of Melanin of A u ricu la ria au ricu laCHEN Ya, XU Miao, WANG Xinyi, SHAN Xinhe, Jl Linkai, ZHANG Yongjun' (College of L ife Science,China Jiliang University,Hangzhou 310018, China) Abstract：Auricularia auricula is an edible fungus with a unique nutritional value and healthy function and is widely dis­tributed in China. It contains a variety of bioactive compounds. Melanin is one of the main bioactive components with high safety. It can be further developed into func tional pigments with health care function. The research in this field has broad application prospects. In this paper, the physicochemical properties, molecular composition, structural characterization, sep­aration and prej)aration methods of Auricularia auricula melanin, as well as its biological activities such as scavenging free radicals, antibacterial and antiviral, antiradiation, improving liver injury, protecting DNA and so on, were reviewed. The problems limiting the development and application of Auricularia auricula melanin were analyzed, and the prospects were put forward, which would be helpful for the study of the biological mechanism of Auricularia auricula melanin and its appli­cation in food, medicine, cosmetics and other fields.K ey w〇r d s：/lz/m*z//rtrirt ai/rzcw/a;melanin;separation and extraction;biosynthesis;molecular stnicture;biological activity黑木耳(4w r/c a/a r i Y/aur/cu/fl)隶属担子菌亚门（Basi(liomycotina)层菌纲（Hymenomycetes)木耳目（Auricu- I a r i a les)木耳科(A u r i c u1a riaceae)木耳属(yWfcw/rmVz )，为中国珍贵的药用和食用菌，早在19世纪，黑木耳就被 用于民间医药，用于治疗咽喉痛、眼痛、黄疸等病症，并作为收敛剂"_21。

负载酪氨酸酶抑制肽的牛奶外泌体抑制酪氨酸酶活性及黑色素生成研究栗瑞斌;王倩;张雷杰;荆韧威;尹海芳【期刊名称】《天津医科大学学报》【年(卷),期】2022(28)4【摘要】目的:探讨酪氨酸酶抑制肽(YRS)功能化的牛奶外泌体(mEXOYRS),对酪氨酸酶活性及黑色素生成的抑制作用。

方法:通过超速离心法获取牛奶外泌体(mEXO);利用外泌体特异锚定肽CP05将YRS修饰在mEXO表面,获得mEXOYRS,随后通过流式细胞仪分析YRS的负载效率;在黑色素瘤细胞B16-F10中,检测细胞对mEXOYRS的摄取效率以及YRS与酪氨酸酶的共定位效率,评估mEXOYRS对B16-F10黑色素瘤细胞酪氨酸酶活性的抑制效果及对黑色素合成的抑制作用;进一步将mEXOYRS均匀涂抹于Nude BALB/c小鼠黑色素瘤细胞B16-F10所成皮下瘤模型上,评估其在体内对黑色素合成的抑制作用;通过对C57BL/6小鼠表面皮肤涂抹mEXOYRS,评估其对小鼠毛囊的着色程度及小鼠皮肤的美白效果。

结果:YRS通过外泌体特异锚定肽CP05高效负载至mEXO,并且不影响mEXO的形态结构及标志性蛋白的表达;在B16-F10黑色素瘤细胞摄取实验中,mEXO能够增强YRS与细胞内酪氨酸酶共定位的效率,在1周、2周及6周时均显著抑制细胞产生黑色素(F=56.117、48.954、560.006,均P<0.05);在Nude BALB/c小鼠黑色素皮下瘤模型中,与YRS组相比,mEXOYRS更好的抑制了黑色素的产生;在C57BL/6小鼠模型中,mEXOYRS降低了黑色素的产生,并显著降低了黑色素含量(F=173.083,P<0.05)及酪氨酸酶活性指标(F=34.156,P<0.05),取得了美白效果。

结论:YRS修饰的mEXO可通过内体转运途径,靶向运输到酪氨酸酶处,降低酪氨酸酶活性,并有效抑制黑色素生成,提高YRS的美白效果。

酪氨酸酶活性测定
一．实验材料
（一）仪器：倒置显微镜、96孔板、微量移液器（枪及枪头）、恒温培养箱、喷壶、脱脂棉、封口膜、培养瓶100ml、点滴瓶(250ml、500ml)、烧杯（500ml、50ml）、酒精灯、移液管、吸球、吸管架及瓶架、镊子、标签贴、超净台、酶标仪、超低温冰箱或液氮冷冻罐
（二）试剂：胎牛血清、DMEM培养基、胰蛋白酶、PBS液、75%酒精、左旋多巴、TritonX-100液
二．实验方法
采用多巴速率氧化法。

取对数期B16黑素瘤细胞，弃去培养液，用PBS液清洗2次。

调
节细胞浓度为0.5×104
—1×10
4
/孔相当于干细胞悬液5×10
4
—10×10
4
个（ml
-1
）,加入96
孔板中，每孔加入100ul。

过夜后（12h），细胞贴壁，使用同MTT法同浓度同体积的含药培养基加入，并设置对照组（细胞+培养基）和空白对照组（培养基），每个浓度组设置5个复
孔，培养基中均含5%胎牛血清，于37摄氏度5%CO2恒温培养箱中培养56h后，吸去培养液
后，用PH=7.4的PBS液洗涤2次，然后每孔加入1%TritonX-100液50ul，迅速与-80摄氏度冷冻30min,室温下融化，由于速冻过程是细胞破裂，加入0.5%左旋多巴20ul，37摄氏度下反应4h,酶标仪490nm处测吸光度A，以A表示酪氨酸酶活性。

（酪氨酸酶活性率=（A含药组-A空白组）/（A对照组-A空白组）×100%）。

酪氨酸酶与黑色素的形成过程一、酪氨酸的结构与性质酪氨酸是一种含有芳香环的氨基酸，其化学式为C9H11NO3。

它在生物体内广泛存在于蛋白质中，其侧链上含有一个羟基和一个苯环，使得它具有良好的水溶性和稳定性。

同时，酪氨酸还是合成黑色素的重要前体之一。

二、黑色素的形成过程1. 酪氨酸的氧化反应在人类和动物体内，黑色素主要由皮肤中的黑色素细胞合成。

这些细胞含有一种特殊的酶——酪氨酸酶(Tyrosinase)，它能够催化酪氨酸分子中的羟基被氧化为半胱氨酸，并且将半胱氨酸转化为多巴(DOPA)。

这个反应是合成黑色素过程中最关键的一步。

2. 多巴的自身聚合反应多巴分子具有两个邻位间亲电性较强的羟基，因此它们可以发生自身聚合反应，形成多巴二聚体(DOPAquinone)。

这个反应是黑色素形成的第二步。

3. 多巴二聚体的聚合反应多巴二聚体可以通过氧化还原反应被还原为多巴分子，并与另一个多巴二聚体结合，形成黑色素前体——5,6-二羟基间苯二酮(DHI)。

这个反应是黑色素形成的第三步。

4. DHI的氧化反应DHI分子中含有两个邻位间亲电性较强的羟基，因此它们可以发生自身聚合反应，形成DHI三聚体。

同时，DHI也可以被氧化为5,6-间苯醌(DHQ)，这个反应是黑色素形成的最后一步。

5. DHQ和DHI三聚体的交替聚合DHQ和DHI三聚体可以通过交替聚合反应形成多种不同类型的黑色素分子，包括橙褐色、棕褐色和黑色等。

三、影响黑色素形成的因素1. 酪氨酸酶活性：酪氨酸酶是合成黑色素过程中最关键的一种酶，其活性越高，则黑色素合成速度越快。

2. 皮肤类型：不同肤色的人体内酪氨酸和黑色素的含量也不同，因此黑色素形成的速度也不同。

3. 紫外线辐射：紫外线可以抑制黑色素细胞的活性，从而降低黑色素的合成速度。

4. 饮食习惯：含有大量酪氨酸和维生素C等营养物质的食物可以促进黑色素合成。

四、应用1. 化妆品：黑色素是皮肤颜色的主要调节因子之一，因此在化妆品中添加具有促进或抑制黑色素合成作用的成分，可以实现美白或增加肤色深度等效果。

酪氨酸酶活性检测试剂盒说明书微量法注意：正式测定之前选择2-3个预期差异大的样本做预测定。

货号：BC4055规格：100T/96S产品内容：提取液：液体125mL×1瓶，4℃保存。

试剂一：粉剂×3瓶，4℃保存。

临用前每瓶加入7mL提取液充分溶解待用。

现配现用。

产品说明：酪氨酸酶（tyrosinase：EC1.14.18.1）是一种单酚单加氧酶，是具有双功能的含铜糖蛋白，广泛存在于植物、酵母和动物组织中。

酪氨酸酶是生物体合成黑色素的关键酶，也是引起果蔬酶促褐变的主要因素，同时也对昆虫的免疫及生长有重要影响酪氨酸酶催化L-多巴生成多巴色素，其在475nm下有特征吸收峰，进而测定出酪氨酸酶的活性。

自备实验用品及仪器：可见分光光度计/酶标仪、低温离心机、水浴锅、可调式移液器、微量玻璃比色皿/96孔板、研钵/匀浆器、冰、蒸馏水。

操作步骤：一、样本处理：（1）组织：称取约0.1g组织，加入1mL提取液进行冰浴匀浆。

12000g，4℃离心20min，取上清，置冰上待测。

（2）细胞或微生物样品的制备：先收集细胞或微生物样品到离心管内，弃上清，按照每500万细胞或微生物加入1mL提取液，超声波破碎细菌或细胞（功率20％，超声3s，间隔10s，重复30次）。

12000g，4℃离心20min，取上清，置冰上待测。

（3）血清（浆）：直接检测。

二、测定步骤：（1）分光光度计/酶标仪预热30min，波长调至475nm。

蒸馏水调零。

（2）加样表：在微量玻璃比色皿/96孔板中分别加入试剂名称（μL）测定管试剂一180样品20充分混匀后立即测定10s时在475nm下的吸光度，记为A1，之后迅速将其放入37℃（哺乳动物）或25℃（其他物种）水浴或培养箱中3min（若酶标仪自带控温功能，将温度调至37℃或25℃）。

然后迅速拿出擦净后测定190s时的吸光度，记为A2。

计算ΔA=A2-A1。

三、酪氨酸酶活力计算：1、按微量玻璃比色皿计算：(1)按蛋白浓度计算：单位的定义：每mg组织蛋白每分钟催化生成1nmol多巴色素的酶量定义为一个酶活性单位。

酪氨酸酶活性与黑素生成关系的基础及临床研究作者：闻人庆等来源：《中国美容医学》2014年第23期黑素细胞是产生黑素的高度特异性细胞，细胞内的高尔基体可合成黑素小体，为黑素合成的场所。

树突是黑素细胞形态学的重要特征性标志，在黑素转运中起着至关重要的作用。

它形成树枝状分叉，朝邻近的角质形成细胞生长，促使黑素细胞将黑素输送给它们，增加机体色素沉着 [1]。

黑素是一种化学本质为蛋白衍生物的无定形小颗粒，广泛存在于人的皮肤、毛发和眼球组织中，决定着相应部位的颜色。

并吸收阳光中的紫外线，保护皮肤不受阳光照射的伤害，因此，又称作光防护色素。

可分为真黑素（eumelanin）和褐黑素（pheomelanin）两类，前者在黑色毛发中大量存在，后者在褐色及红色毛发中比率相对较高。

黑素生成异常与黄褐斑、白化病等色素障碍性疾病关系密切，伴随着人们对自身形象越来越高的追求，对黑素的相关研究日益成为医学美容界关注的热点问题。

本文就酪氨酸酶的活性与黑素生成关系的基础及研究综述如下。

1 酪氨酸酶基因家族、酪氨酸酶及黑素的生成1.1酪氨酸酶基因家族与酪氨酸酶：酪氨酸酶基因家族成员包括酪氨酸酶基因（TYR基因）、酪氨酸酶相关蛋白1基因（TYRP1基因）和酪氨酸酶相关蛋白2基因（TYRP2基因）等。

它们的序列具有高度一致性，共同作用于黑素细胞，调节黑素生成的种类和数量。

核心成员TYR 基因在人类定位于11q14-q21，长度超过65kb，包括5个外显子和4个内含子，是眼皮肤白化病Ⅰ型（OCA1）的致病基因。

其第一外显子最长，几乎占了全部编码序列的一半。

基因上游存在着TATA、CAAT盒等调控序列。

在11p11至着丝粒区域，存在着一个TYR基因的相关序列，是第四和第五外显子的同源序列，同源性高达97%[2]。

其编码的酪氨酸酶是由529个氨基酸残基组成的黑素小体跨膜酶蛋白，分子量为55KD，糖基化后分子量为65～75KD。

包括CuA和CuB这两个铜原子结合位点、由18个氨基酸残基组成的信号肽及C末端疏水的跨膜区[3]。

化妆品中的美白效果评价方法研究近年来，随着人们对美白肌肤的需求不断增长，化妆品市场上涌现出众多美白产品。

然而，如何准确评价化妆品的美白效果成为了广大消费者关注的焦点。

本文将从不同角度研究化妆品中的美白效果评价方法，为消费者提供参考，以便选择适合自己的产品。

以下是几个重要的评价指标：一、肤色改善度评价法肤色改善度是评价美白产品效果的重要指标之一。

目前，常见的评价方法包括使用色度仪测量皮肤颜色变化、使用专业的肤色分析仪、化妆品临床试验和消费者自评等。

1. 色度仪测量皮肤颜色变化：色度仪是一种用于测量色彩的仪器，通过比较使用美白产品前后的肤色变化，可以评估产品的美白效果。

通常在不同时间点测量肌肤的颜色，并进行数据分析，以便获取肤色改善的度量值。

2. 肤色分析仪：肤色分析仪是专业化妆工具，通过拍摄和分析肌肤照片，可以评估肤色的均匀度、亮度和红润度等参数。

通过分析这些参数的变化，可以得出美白产品的效果。

3. 化妆品临床试验：化妆品临床试验是用一定数量的参与者进行的实验，通过评估其肌肤颜色的变化，以确定美白产品的效果。

这种方法能够更真实地反映出产品对肌肤的影响。

4. 消费者自评：消费者自评是在使用美白产品一段时间后，让用户根据自身感受评估产品的效果。

这种方法有助于了解产品在日常使用中的实际效果。

二、黑色素含量评价法黑色素是肌肤暗沉、肤色不均匀的主要原因之一。

因此，评价化妆品的美白效果时，测量黑色素含量是一种常见的方法。

以下是几种常用的黑色素含量评价方法：1. 高效液相色谱法（HPLC）：HPLC是一种用于分离、定量和分析化合物的方法。

通过采集肌肤样本，提取其中的黑色素，并利用HPLC 技术分析黑色素的含量，以评估化妆品的美白效果。

2. 酪氨酸酶酶联免疫吸附测定法（TyrELISA）：TyrELISA是一种测定黑色素含量的方法。

它通过酪氨酸酶特异性地结合黑色素，利用酶标仪测定黑色素含量，从而评估美白产品的效果。

卵黄高磷蛋白在抑制黑素原形成过程中的功能特性关键词：卵黄高磷蛋白黑素原形成酪氨酸酶小眼畸形相关转录因子摘要：卵黄高磷蛋白是鸡蛋蛋黄中的磷糖蛋白。

卵黄高磷蛋白分子中一多半氨基酸为丝氨酸，其中90%以上被磷酸化。

因此，卵黄高磷蛋白具有较强的金属键能。

本文旨在研究卵黄高磷蛋白在黑素瘤细胞中对黑素原形成的抑制作用。

结果显示，卵黄高磷蛋白能够抑制蘑菇中酪氨酸酶的活性。

与空白相比，当卵黄高磷蛋白的浓度增加到50μg/ml 时，B16F10黑素瘤细胞中酪氨酸酶的活性下降了约42%，黑色素形成活性下降17%。

卵黄高磷蛋白抑制了B16F10黑素瘤细胞中酪氨酸酶、酪氨酸相关蛋白1（TRP-1）、TRP-2和小眼畸形相关转录因子（MITF）的表达。

另外，卵黄高磷蛋白降低了B16F10黑素瘤细胞中cAMP的浓度。

这些结果说明卵黄高磷蛋白有能力抑制食物和化妆品工业中黑素原的形成。

2012年艾斯维尔股份有限公司版权所有。

1、介绍黑色素是影响皮肤和头发颜色的重要因素。

黑色素的合成场所为上皮最内层的生黑色素细胞，可保护皮肤免受紫外辐射的损伤（Gilchrest & Eller, 1999）。

然而，黑色素过多积累易导致色素沉着过度而引起黄褐斑、老年斑等美学问题，也增加了患恶性黑色素瘤的风险(Kim & Uyama, 2005)。

因此，鉴于在医学和化妆品工业中蛋白质能够显著调节或抑制黑素原的形成，找到能够调节黑素原形成的天然化合物对医学和化妆品领域的发展意义重大。

酪氨酸酶是真黑素和褐黑素合成过程中的关键酶。

真黑素是生物黑色素的典型形式，为黑褐色色素，而褐黑素则为橙色。

其他酶如酪氨酸酶相关蛋白1和2（TRP1&2）可调节真黑色素生成(Kobayashiet al., 1995)。

为了找到在人和动物皮肤生黑色素细胞中可以控制过量黑色素合成的黑素原生成抑制剂，多项研究已经展开。

因此，几种来源于天然或者加工的黑素原生成抑制剂已经鉴定并被使用，如对苯二酚、曲酸、熊果苷和乙基-（4-羟苯基）草氨酸钠(Cho & Shin,2011)。

Synthesized quercetin derivatives stimulate melanogenesis in B16melanoma cells by influencing the expression of melanin biosynthesis proteins MITF and p38MAPKKosei Yamauchi,Tohru Mitsunaga ⇑,Mizuho Inagaki,Tohru SuzukiThe United Graduate School of Agricultural Science,Gifu University,1-1Yanagido,501-1193Gifu,Japana r t i c l e i n f o Article history:Received 26March 2014Revised 23April 2014Accepted 25April 2014Available online 5May 2014Keywords:B16melanoma cells Tyrosinase TRP-1Quercetina b s t r a c tIn order to understand the effect of structure–activity relationships on melanogenesis using B16mela-noma cells,19quercetin derivatives were synthesized.Among the synthesized compounds,3-O -meth-ylquercetin (11)and 30,40,7-O -trimethylquercetin (14)increased melanin content more potently than the positive control theophylline,while exhibiting low pound 11exhibited less melano-genesis-stimulating activity than compound 14.However,11increased the expression of tyrosinase and tyrosinase-related protein 1(TRP-1)to a greater extent than 14,thereby suggesting that melanogenesis in melanoma cells does not depend solely on the expression of the enzymes catalyzing melanin biosyn-thesis.Furthermore,14also stimulated the expression of the microphthalmia-associated transcription factor (MITF)and p-p38mitogen activated protein kinase (MAPK),while they were not increased by 11.These results suggest that 11may enhance the expression of tyrosinase and TRP-1by regulating the proteasomal degradation of melanogenic enzymes and/or by activating other transcriptional factors regulating enzyme expression.Ó2014Elsevier Ltd.All rights reserved.1.IntroductionMelanin,a pigment present in several tissues in the human body,plays an important role in preventing skin cancer caused by ultraviolet (UV)rays.1,2The overall level of melanogenesis is changed by aging,stress,and damage caused by the UV rays,resulting in the appearance of gray hairs,sunburn,and mottlingof the skin.Therefore,controlling melanogenesis is important for maintaining the good health and cosmetic appearance of the human body.A number of studies have investigated novel pharma-cological and herbal agents aimed at controlling melanogenesis.3–7Tyrosinase,tyrosinase-related protein (TRP)-1,and TRP-2are key enzymes involved in melanin biosynthesis.Tyrosinase,an enzyme containing copper ions,catalyzes two separate reactions within the melanin biosynthetic pathway.8Tyrosinase catalyzes the first step in melanin biosynthesis that is,the hydroxylation of L -tyrosine to L -3,4-dihydroxyphenylalanine (L -DOPA),as well as the subsequent oxidation of L -DOPA to L -DOPA quinone.Two types of melanin are ultimately biosynthesized,reddish-orange and blackish-brown pigments called pheomelanin and eumelanin,respectively,with the enzymes TRP-1and TRP-2playing a key role in the biosynthe-sis of eumelanin.9Melanin is biosynthesized in the melanosome in the perinuclear region of the melanocyte and transported in the mature melano-some to the periphery of the cell.10–12The melanosome is further transported to the hair matrix or the keratinocyte present above the melanocyte.The keratinocyte that receives the melanin under-goes cornification,resulting in skin pigmentation.Similarly,hair pigmentation occurs due to melanin released on the outside of the melanocyte.Despite the importance of the extracellular/10.1016/j.bmc.2014.04.0530968-0896/Ó2014Elsevier Ltd.All rights reserved.Abbreviations:a -MSH,a -melanocyte-stimulating hormone;BSA,bovine serum albumin;cAMP,cyclic adenosine monophosphate;DMEM,Dulbecco’s modified Eagle medium;DMF,dimethylformamide;L -DOPA,L -3,4-dihydroxyphenylalanine;ECL,enhanced chemiluminescence;EtOAc,ethyl acetate;EtOH,ethanol;GAPDH,glyceraldehyde-3-phosphate dehydrogenase;HMBC,heteronuclear multiple-bond correlations;HRP,horseradish peroxidase;IR,infrared;JNK,c-Jun N-terminal kinase;MAPK,mitogen-activated protein kinase;MALDI,matrix-assisted laser desorption/ionization;MeOH,methanol;MS,mass spectroscopy;MITF,microph-thalmia-associated transcription factor;MTT,microculture tetrazolium technique;NMR,nuclear magnetic resonance;PBS,phosphate-buffered saline;Pd/C,palladium on carbon;p-HPLC,preparative high-performance liquid chromatography;PVDF,polyvinylidene difluoride;SDS–PAGE,sodium dodecyl sulfate–polyacrylamide gel electrophoresis;RIPA,radioimmunoprecipitation assay;TBST,tris-buffered saline Tween20;THF,tetrahydrofuran;TRP,tyrosinase-related protein;UPLC–TOFMS,ultra performance liquid chromatography time-of-flight mass spectrometry;UV,ultraviolet.⇑Corresponding author.Tel./fax:+81582932920.E-mail address:mitunaga@gifu-u.ac.jp (T.Mitsunaga).pigment stores,an evaluation of melanogenesis activity using mel-anin-controlling agents has only been performed on intracellular melanin in B16melanoma cells thus far.13–15The present study focused on evaluating extracellular melanin released from B16 melanoma cells and investigated the levels of melanin released from the B16melanoma cells in addition to measuring the intracel-lular melanin content.Quercetin is aflavonoid present as a glycoside in various fruits and vegetables.16–18A number of studies have demonstrated that quercetin exhibits a variety of pharmacological effects,including antioxidant and anti-cancer activities,19,20while some reports relate to effectiveness in controlling melanogenesis.Quercetin is recog-nized as a potent inhibitor of tyrosinase activity and melanogenesis, as evidenced by the studies performed in the mouse B16melanoma cells.21However,quercetin has been reported to elicit the opposite effect and accelerate melanogenesis in human melanoma cells.22 Furthermore,it was reported that the direction of its melanogene-sis-regulating activity depends on the concentration of quercetin used.23A small number of studies have shown that quercetin deriv-atives can control melanogenesis.Quercetin-3-O-b-D-glucoside enhances melanogenesis by stimulating the expression of TRP-1 and TRP-2.24In our previous study,we evaluated the effect on mela-nogenesis of two quercetin glycosides,40-O-b-D-glucopyranosyl-quercetin-3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranoside(3) and40-O-b-D-glucopyranosyl-(1?2)-b-D-glucopyranosyl-querce-tin-3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranoside isolated from Helminthostachys zeylanica root extract.25While compound3 was found to stimulate melanogenesis,the latter compound had no effect,suggesting that the structure and positions of the sugars in the quercetin glycosides may affect their melanogenesis-modu-lating activity in B16melanoma cells.Furthermore,in order to investigate the structure–activity relationship of quercetin glyco-sides,ten structurally distinct quercetin glycosides(Fig.1)were synthesized,and their effects on intracellular melanogenesis were determined.Quercetin-3-O-b-D-glucopyranoside(1),quercetin-3-O-b-D-glucopyranosyl-(1?4)-b-D-glucopyranoside(2),and3 stimulated intracellular melanogenesis in our previous report,26 suggesting the potency of the quercetin derivatives as stimulators of melanogenesis.In this study,9quercetin derivatives were newly synthesized,and their melanogenesis-stimulating activities deter-mined by measuring both intra-and extracellular melanin levels in order to clarify the structure–activity relationships of the querce-tin derivatives.Furthermore,the expression of proteins related to melanin biosynthesis were determined by western blot analysis in B16melanoma cells treated with the synthesized quercetin derivatives for understanding the mechanisms underlying the observed activity.2.Materials and methods2.1.General experimental procedures1H and13C NMR spectra were recorded in methanol-d4,acetone-d6or dimethylsulfoxide-d6using JEOL EC600M Hz NMR(JEOL, Tokyo,Japan).Coupling constants were expressed in Hz,and the chemical shifts were expressed on a d(ppm)scale.Ultra perfor-mance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS;WatersÒXevo™QT for MS)was performed using a C18column(2.1mm uÂ100mm length;Waters,Milford,MA, USA).UPLC–TOFMS data were collected in the negative ionization mode.The capillary voltage was3.0kV.Cone and desolvation gas flow rates were set at50L/h and1000L/h,respectively,and the source and desolvation temperatures were150°C and500°C, respectively.Matrix-assisted laser desorption/ionization TOFMS (MALDI-TOFMS)spectra were measured on a Shimadzu AXIMA-Resonance spectrometer(Kyoto,Japan)equipped with a nitrogen laser(k=337nm).Samples were mixed with the matrix(2,3-dihy-droxybenzoic acid in30%acetonitrile,10mg/mL)and loaded onto a384-well MALDI sample plate.Preparative HPLC(SHIMAZU LC-6AD)was performed using an Inertsil ODS-3column(20mm uÂ250mm;GL Sciences,Tokyo,Japan)with gradient elution. Microculture tetrazolium technique(MTT)assay kit and bovine serum albumin(BSA)was purchased from Sigma–Aldrich(St. Louis,MO,USA).Antibodies against tyrosinase(H-109),TRP-1 (H-90),TRP-2(H-150),p38MAPK(H-147),and p-p38MAPK (D-8)were purchased from Santa Cruz Bio technology(Santa Cruz, CA,USA).Horseradish peroxidase(HRP)-conjugated anti-rabbit IgG donkey antibody(NA934)and HRP-conjugated anti-mouse IgG sheep antibody(NA931)were purchased from GE Healthcare(Pis-catawawy,NJ,USA),while MITF-specific antibody(EPR9731)and glyceraldehyde-3-phosphate dehydrogenase(GAPDH)-specific antibody(GTX100118)were purchased from Abcam(Cambridge, MA,USA)and GeneTex(Irvine,CA,USA)respectively.Radioimmu-noprecipitation assay(RIPA)buffer(ab156034)and protease inhib-itors cocktail(539134)were purchased from Abcam(Cambridge, MA,USA)and Merck Millipore(Billerica,MA,USA).Other commer-cially available products were purchased from Wako Chemicals (Richmond,VA,USA).IR spectra were recorded on a Perkin-Elmer Spectrum100FT-IR system(Waltham,MA,USA).UV spectra were recorded on a Shimadzu SPD-M20A diode array detector.Optical3332K.Yamauchi et al./Bioorg.Med.Chem.22(2014)3331–3340rotations were measured using a JASCO P-2300system(Easton, MD,USA).2.2.Synthesis of quercetin derivatives2.2.1.Synthesis of compounds1–10Compounds1–10were synthesized using methods presented in our previous publication.262.2.2.Synthesis of3-O-methylquercetin(11)Rutin(5.00g,8.19mmol),K2CO3(9.04g,65.52mmol),and BnBr (7.79mL,65.52mmol)were added to60mL of dimethylformam-ide(DMF),and the mixture was stirred for10h under argon at room temperature.The resulting mixture was diluted with 150mL of ethyl acetate(EtOAc)and washed with water (2Â150mL).The residue obtained after evaporation of the solvent was dissolved in100mL of1N HCl,and the mixture was refluxed at80°C for2h.The precipitate was allowed to cool andfiltered.Next,1.0g of the precipitate,K2CO3(22.5g,163mmol),and dimethylsulfate (17.5mL,184mmol)were added to26mL of DMF,and the mixture was stirred for6h at65°C.The reaction mixture was diluted with 150mL of EtOAc and washed with water(2Â150mL).After the EtOAc phase was dried using Na2SO4,the reactant was obtained by evaporating the solvent.The reactant was dissolved in40mL of ethanol(EtOH)/tetrahy-drofuran(THF)(1:1,v/v),and200mg of10%Pd/C was added.The mixture was stirred for1h at room temperature under0.05MPa hydrogen.The Pd/C wasfiltered off.After the solvent was evapo-rated,11was isolated by preparative HPLC with an ODS-3column (20mm uÂ250mm).Elution was performed with a linear gradi-ent of MeOH/0.05%aqueous solution TFA(0min,50/50;50min, 100/0;60min,100/0)to obtain11as a yellowish powder with 41.1%yield.The structure of the synthesized methylquercetin11 was confirmed using NMR,UPLC–TOFMS,UV,and IR spectra,and by measuring the specific optical rotation:UV k max204,255, 357nm;[a]25DÀ3.75°(c0.75,MeOH);IR(KBr)m max3402,1655, 1607,1505,1440,1361cmÀ1;1H NMR(CD3OD,600MHz)d H3.75 (3H,s,3-OCH3), 6.16(1H,d,J=2.04Hz,H-6), 6.36(1H,d, J=2.04Hz,H-8), 6.88(1H,d,J=8.28Hz,H-50),7.50(1H,dd, J=8.22,2.04Hz,H-60),7.59(1H,d,J=2.04Hz,H-20);13C NMR (CD3OD,150MHz)d C59.2(3-OCH3),93.4(C-8),98.4(C-6),104.5 (C-4a),115.1(C-20,50),121.0(C-60),121.6(C-10),138.2(C-3), 145.1(C-30),148.6(C-40),156.6(C-2),157.1(C-8a),161.7(C-5), 164.5(C-7),178.7(C-4);UPLC–TOFMS m/z315.049[M–H]À.2.2.3.Synthesis of3,40-O-dimethylquercetin(12),3,7-O-dimethylquercetin(13)and3,40,7-O-trimethylquercetin(14) Methylquercetin11(100mg,0.316mmol),K2CO3(43.7mg, 0.316mmol),and dimethylsulfate(35.1l L,0.316mmol)were added to10mL of DMF.The mixture was stirred for6h at65°C. The resulting mixture was diluted with30mL of EtOAc and washed with water(2Â30mL).After evaporating the solvent, compounds12,13,and14were obtained by preparative HPLC with an ODS-3column(20mm uÂ250mm).Compounds were eluted with the linear gradient MeOH/0.05%aqueous solution TFA (0min,50/50;50min,100/0;60min,100/0)to obtain12,13and 14as yellowish powders with yields of11.9%,9.1%,and9.1%, respectively.The structures of synthesized methylquercetins were confirmed using NMR,UPLC–TOFMS,UV,and IR spectra,and by measuring the specific optical rotation.Compound12:UV k max208,254,355nm;[a]25DÀ0.51°(c0.90, MeOH);IR(KBr)m max3402,1654,1609,1507,1441,1364cmÀ1;1H NMR(CD3OD,600MHz)d H3.76(3H,s,3-OCH3),3.91(3H,s,40-OCH3), 6.16(1H,dr s,H-6), 6.36(1H,br s,H-8),7.02(1H,d, J=8.22Hz,H-50),7.57(1H,br s,H-20),7.60(1H,br d,J=8.28Hz,H-60);13C NMR(CD3OD,150MHz)d C55.0(40-OCH3),59.2(3-OCH3),93.4(C-8),98.4(C-6),104.6(C-4a),114.8(C-20,50),120.8 (C-60),122.8(C-10),138.5(C-3),146.3(C-30),150.3(C-40),156.2 (C-2),157.1(C-8a),161.8(C-5),164.6(C-7),178.7(C-4);UPLC–TOFMS m/z329.0645[MÀH]À.Compound13:UV k max209,256,357nm;[a]25DÀ19.6°(c0.10, THF);IR(KBr)m max3415,1657,1595,1498,1441,1350cmÀ1;1H NMR(Acetone-d6,600MHz)d H3.84(3H,s,3-OCH3),3.89(3H,s, 7-OCH3),6.28(1H,dr s,H-6),6.62(1H,br s,H-8),6.97(1H,d, J=8.22Hz,H-50),7.57(1H,br d,J=8.22Hz,H-60),7.70(1H,br s, H-20);13C NMR(Acetone-d6,150MHz)d C55.6(7-OCH3),59.3 (3-OCH3),91.9(C-8),97.6(C-6),105.7(C-4a),115.4(C-20),115.6 (C-50),121.3(C-60),122.1(C-10),138.6(C-3),145.0(C-30),148.3 (C-40),156.1(C-2),156.9(C-8a),162.0(C-5),165.7(C-7),178.8 (C-4);UPLC–TOFMS m/z329.0653[MÀH]À.Compound14:UV k max209,255,354nm;[a]25DÀ26.1°(c0.52, THF);IR(KBr)m max3420,1656,1595,1501,1441,1333cmÀ1;1H NMR(dimethylsulfoxide-d6,600MHz)d H 3.76(3H,s,3-OCH3), 3.83(6H,s,40-OCH3,7-OCH3),6.33(1H,dr s,H-6),6.69(1H,br s, H-8),7.07(1H,d,J=8.22Hz,H-50),7.54(2H,br s,H-60,20);13C NMR(dimethylsulfoxide-d6,150MHz)d C56.2(40-OCH3),56.6(7-OCH3),60.2(3-OCH3),92.8(C-8),98.3(C-6),105.7(C-4a),115.6 (C-20,50),121.0(C-60),122.7(C-10),138.7(C-3),146.9(C-30), 150.8(C-40),156.1(C-2),156.8(C-8a),161.4(C-5),165.7(C-7), 178.6(C-4);UPLC–TOFMS m/z343.0825[MÀH]À.2.2.4.Synthesis of40-O-methylquercetin(15),7-O-methylquercetin(16)and40,7-O-dimethylquercetin(17) Rutin(200mg,0.328mmol),K2CO3(90.6mg,0.656mmol),and dimethylsulfate(62.2l L,0.656mmol)were added to10mL of DMF,and the mixture was stirred for6h at65°C.The resulting mixture was added to50mL of2.5N HCl and refluxed at80°C for2h.The reaction mixture was diluted with150mL of EtOAc and washed with water(2Â150mL).The reaction mixture was obtained by drying the EtOAc phase using Na2SO4.After evaporat-ing the solvent,compounds15,16,and17were obtained by pre-parative HPLC with an ODS-3column(20mm uÂ250mm). Compounds were eluted with the linear gradient MeOH/0.05% aqueous solution of TFA(0min,60/40;50min,100/0;60min, 100/0)to obtain15,16and17as yellowish powders with yields of4.1%,10.0%,and7.7%,respectively.The structures of the synthe-sized compounds15,16,and17were confirmed using NMR, MALDI-TOFMS,UV and IR spectra,and by the measurements of specific optical rotation.Compound15:UV k max207,254,368nm;[a]25DÀ26.9°(c0.20, MeOH);IR(KBr)m max3430,1655,1617,1499,1456cmÀ1;1H NMR (dimethylsulfoxide-d6,600MHz)d H3.81(3H,s,40-OCH3),6.15(1H, d,J=2.04Hz,H-6), 6.39(1H,d,J=2.04Hz,H-8),7.05(1H,d, J=8.22Hz,H-50),7.62(1H,dd,J=8.22,2.76Hz,H-60),7.63(1H, d,J=2.10Hz,H-20);13C NMR(dimethylsulfoxide-d6,150MHz)d C 56.1(40-OCH3),93.9(C-8),98.7(C-6),103.6(C-4a),112.4(C-50), 115.1(C-20),120.3(C-60),123.9(C-10),136.7(C-3),146.7(C-30), 146.8(C-2),149.9(C-40),156.7(C-8a),161.3(C-5),164.5(C-7), 176.5(C-4);MALDI-TOFMS m/z317.0494[M+H]+.Compound16:UV k max206,255,371nm;[a]25DÀ17.3°(c0.20, THF);IR(KBr)m max3402,1656,1593,1502,1442,1324cmÀ1;1H NMR(dimethylsulfoxide-d6,600MHz)d H 3.82(3H,s,7-OCH3), 6.31(1H,d,J=2.04Hz,H-6),6.60(1H,d,J=1.38Hz,H-8),6.86 (1H,d,J=8.22Hz,H-50),7.56(1H,dd,J=8.22,2.04Hz,H-60),7.69 (1H,d,J=2.04Hz,H-20);13C NMR(dimethylsulfoxide-d6,150MHz)d C56.5(7-OCH3),92.4(C-8),98.0(C-6),104.5(C-4a), 115.6(C-20),116.1(C-50),120.5(C-60),122.4(C-10),136.6(C-3), 145.6(C-30),147.8(C-2),148.4(C-40),156.6(C-8a),160.9(C-5), 165.4(C-7),176.5(C-4);MALDI-TOFMS m/z317.0274[M+H]+.Compound17:UV k max206,254,368nm;[a]25DÀ3.72°(c0.23, THF);IR(KBr)m max3420,1656,1594,1501,1441,1332cmÀ1;1HK.Yamauchi et al./Bioorg.Med.Chem.22(2014)3331–33403333NMR(dimethylsulfoxide-d6,600MHz)d H 3.82(3H,s,7-OCH3), 3.83(3H,s,40-OCH3),6.32(1H,d,J=2.10Hz,H-6),6.68(1H,d, J=2.04Hz,H-8),7.05(1H,d,J=8.94Hz,H-50),7.65(1H,dd, J=8.94,2.04Hz,H-60),7.69(1H,d,J=2.04Hz,H-20);13C NMR (dimethylsulfoxide-d6,150MHz)d C56.2(40-OCH3),56.6(7-OCH3), 92.5(C-8),98.0(C-6),104.6(C-4a),115.3(C-20),112.3(C-50), 120.3(C-60),123.8(C-10),137.0(C-3),146.7(C-30),147.3(C-2), 150.0(C-40),156.7(C-8a),160.9(C-5),165.5(C-7),176.6(C-4); MALDI-TOFMS m/z331.0608[M+H]+.2.2.5.Synthesis of3-O-acetylquercetin(18)Rutin(5.00g,8.19mmol),K2CO3(9.04g,65.52mmol),and BnBr (7.79mL,65.52mmol)were added to60mL of DMF,and the mix-ture was stirred for10h under argon at room temperature.The resulting mixture was diluted with150mL of EtOAc and washed with water(2Â150mL).The residue obtained by evaporating the solvent was dissolved in100mL of1N HCl and refluxed at 80°C for2h.The obtained precipitate wasfiltered after cooling, and100l g was acetylated overnight using acetic anhydride (1.0mL,10.6mmol)in1.0mL of pyridine at room temperature. The reaction mixture was diluted with50mL of EtOAc and washed with water(2Â50mL).After the EtOAc phase was dried using Na2-SO4,the reactant was obtained by evaporating the solvent.The reaction mixture was dissolved in40mL of EtOH/THF(1:1, v/v),and100mg of10%Pd/C was added.The mixture was stirred for1h at room temperature under0.05MPa hydrogen.After the Pd/C was removed byfiltration,the solvent was evaporated to obtain18as a yellowish powder with39.0%yield from original rutin.It was purified using preparative HPLC with an ODS-3col-umn(20mm uÂ250mm L).Compound18was eluted using a lin-ear gradient of MeOH/0.05%aqueous solution of TFA(0min,50/50; 50min,100/0;60min,100/0).The structure of the synthesized18 was confirmed using NMR,UPLC–TOFMS,UV,and IR spectra,and the measurement of specific optical rotation:UV k max204,255, 350nm;[a]25DÀ2.67°(c0.55,MeOH);IR(KBr)m max3402,1656, 1604,1507,1444,1367cmÀ1;1H NMR(CD3OD,600MHz)d H2.30 (3H,s,3-OAc), 6.20(1H,d,J=2.10Hz,H-6), 6.40(1H,d, J=2.04Hz,H-8), 6.88(1H,d,J=8.94Hz,H-50),7.27(1H,dd, J=8.94,2.04Hz,H-60),7.32(1H,d,J=2.10Hz,H-20);13C NMR (CD3OD,150MHz)d C19.1(3-OAc),93.7(C-8),98.8(C-6),103.9 (C-4a),114.8(C-20),115.2(C-50),120.8(C-60),120.7(C-10),130.1 (C-3),145.3(C-30),149.1(C-40),157.0(C-2),157.3(C-8a),161.7 (C-5),164.9(C-7),168.5(3-OAc),175.9(C-4);UPLC–TOFMS m/z 343.043[MÀH]À.2.2.6.Synthesis of quercetin-3-O-tetra-O-acetyl-b-D-glucopyranoside(19)Rutin(5.00g,8.19mmol),K2CO3(9.04g,65.52mmol),and BnBr (7.79mL,65.52mmol)were added to60mL of DMF,and the mix-ture was stirred for10h under argon at room temperature.The resulting mixture was diluted with150mL of EtOAc and washed with water(2Â150mL).The residue obtained after evaporation of the solvent was dissolved in100mL of1N HCl and refluxed at 80°C for2h.After the mixture was cooled and the precipitatefiltered, 200mg of the precipitate,K2CO3(83.4mg,0.604mmol),and acet-obromoglucose(250mg,0.604mmol)were added to 2.0mL of DMF,and the mixture was stirred for6h at room temperature under argon.The reaction mixture was diluted with30mL of EtOAc and washed with water(2Â30mL).After the EtOAc phase was dried using Na2SO4,the reactant was obtained by evaporating the solvent.The reaction mixture was dissolved in40mL of EtOH/THF(1:1, v/v).Following addition of100mg of10%Pd/C,the mixture was stirred for2h at room temperature under0.05MPa hydrogen. Pd/C wasfiltered,and,after the solvent was evaporated,19was obtained by preparative HPLC with an ODS-3column(20mm uÂ250mm).Fractions were eluted with the linear gradient MeOH/0.05%TFA aqueous solution(0min,50/50;50min,100/0; 60min,100/0)to obtain19as a yellowish powders with a yield of25.2%.The structure of the synthesized19was confirmed using NMR,UPLC–TOFMS,UV,and IR spectra,and the measurement of specific optical rotation:UV k max205,255,354nm;[a]25DÀ68.1°(c0.86,MeOH);IR(KBr)m max3436,1747,1656,1609,1498, 1442,1368cmÀ1;1H NMR(CD3OD,600MHz)d H1.89(3H,s,A2-OAc),1.93(3H,s,A4-OAc),1.96(3H,s,A3-OAc),2.14(3H,s,A6-OAc),3.92(1H,m,H-A5),3.94(1H,m,H-A6),4.03(1H,m,H-A6), 5.01(1H,m,H-A4),5.21(1H,dd,J=9.66,8.28Hz,H-A2),5.35 (1H,t,J=9.66Hz,H-A3),5.56(1H,d,J=8.28Hz,H-A1),6.02(1H, d,J=2.04Hz,H-6), 6.24(1H,d,J=2.10Hz,H-8), 6.85(1H,d, J=8.22Hz,H-50),7.48(1H,dd,J=8.28,2.10Hz,H-60),7.56(1H, d,J=2.04Hz,H-20);13C NMR(CD3OD,150MHz)d C19.2(A2-OAc, A4-OAc),19.3(A3-OAc),19.7(A6-OAc),61.3(C-A6),68.6(C-A4), 71.4(C-A5),71.7(C-A2),72.8(C-A3),93.5(C-8),98.4(C-6),99.5 (C-A1),104.4(C-4a),114.6(C-20),116.2(C-50),121.9(C-10),122.0 (C-60),133.3(C-3),144.5(C-30),148.3(C-40),157.0(C-2),158.3 (C-8a),161.5(C-5),164.4(C-7),169.9(A4-OAc),170.3(A6-OAc), 170.6(A3-OAc),171.1(A2-OAc),177.4(C-4);UPLC–TOFMS m/z 631.126[MÀH]À.2.3.Tyrosinase activity assayMeasurements of tyrosinase activity were performed using a technique modified from previous reports.27,28Briefly,60l L ali-quots of the sample were added to a96-well plate.Following addi-tion of30l L of mushroom tyrosinase(333U/mL in phosphate buffer,50mM,pH6.5),and110l L of substrates(2mM L-tyrosine or2mM L-DOPA),samples were incubated at37°C for30min. Absorbance was measured at510nm using a microplate reader. Each experiment was repeated twice.Tyrosinase activity was expressed as a percentage of the activity measured in control cells incubated with vehicle(solvent DMSO/water)without sample materials.2.4.Cell cultureMurine melanoma B16-F0cells(DS Pharma Biomedical,Osaka, Japan)were grown in Dulbecco’s modified Eagle medium(DMEM) supplemented with10%fetal bovine serum,100,000unit/L penicil-lin,and100mg/L streptomycin.Cells were cultured at37°C in humidified atmosphere of5%CO2.2.5.Measurement of cellular melanin contentBriefly,confluent cultures of B16melanoma cells were rinsed in phosphate-buffered saline(PBS)and removed using0.25%trypsin/ EDTA.The cells were loaded into a24-well plate(5.0Â104cells/ well)and allowed to adhere at37°C for24h.Sample compounds prepared at200–10l M for1–10and50–6.25l M for11–19were added and the cells incubated for72h.Following incubation,cell medium was collected and200l L aliquots were loaded into a 96-well plate.The absorbance of the medium was measured at 510nm by using a microplate reader and used as a measure of extracellular melanin contents.The cells were washed with PBS following lysis in600l L of1M NaOH by heating at100°C for 30min to solubilize the melanin.A portion of the resulting lysate (250l L)was loaded into a96-well microplate,and the absorbance was measured at405nm using a microplate reader.Measured absorbance was used as an index of intracellular melanin contents. Each experiment was repeated twice.The melanin-producing activities were expressed as a percentage of the activity measured in the control cells treated with DMSO without sample materials.3334K.Yamauchi et al./Bioorg.Med.Chem.22(2014)3331–33402.6.Cell viabilityMeasurement of cell viability was performed according to a pre-viously described method,29using the microculture tetrazolium technique(MTT).Cultures were initiated in24-well plates at 5.0Â104cells per well.Following incubation with compounds prepared the same concentrations as described in measurement of cellular melanin content for72h,50l L of MTT reagent(5mg/ mL of3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide in PBS)was added to each well.The plates were incubated in a humidified atmosphere of5%CO2at37°C for4h.After the medium was removed, 1.0mL of isopropyl alcohol(containing 0.04N HCl)was added to each well,and a150l L sample were withdrawn and transferred to a96-well plate.Absorbance was measured at590nm by using a microplate reader.Each experi-ment was repeated twice.Cell viability was expressed as a percent-age of the viability measured in control cells treated with solvent DMSO without sample materials.2.7.Western blot analysisB16melanoma cells treated with the samples at12.5–0l M for 72h were lysed with radioimmunoprecipitation assay(RIPA) buffer containing protease inhibitor cocktail at0°C for30min.Pro-tein concentrations were determined using a Bradford protein assay kit(Thermo)and a BSA solution as a standard.Cell lysates were loaded at10l g of protein per lane and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis(SDS–PAGE)on 10%polyacrylamide gel.Proteins were subsequently transferred onto a polyvinylidene difluoride(PVDF)membrane(Millipore, Billerica,MA,USA)using a semi-dry transfer system(Atto)run at 150mA for30min.The membrane was blocked with2%BSA in tris-buffered saline Tween20(TBST)at4°C overnight.After wash-ing,the membranes were incubated with dilutions of rabbit mono-clonal anti-MITF(1:1000),rabbit polyclonal anti-tyrosinase (1:200),rabbit polyclonal anti-TRP-1(1:200),rabbit polyclonal anti-TRP-2(1:100),rabbit polyclonal anti-p38MAPK(1:100),or mouse monoclonal anti-p-p38MAPK(1:200)antibodies.Following incubation for2h,the membranes were washed and incubated with1:5000diluted HRP-conjugated secondary antibody for2h. Following addition of the Luminata™Forte(Millipore),protein density was visualized using enhanced chemiluminescence(ECL) detection system(LAS-3000,Fujifilm,Tokyo,Japan)and quantified using the Multi Gauge V3.0quantification system(Fujifilm).2.8.Statistical analysisAll data were expressed as means±SD values.Statistical signif-icance of differences were evaluated using the Student’s t-test. 3.Results and discussion3.1.Synthesis of quercetin derivativesQuercetin derivatives1–19were synthesized using commer-cially available rutin as the starting material.The syntheses of quercetin glycosides1–10were performed according to the previously described methods.26The methylquercetin derivatives 11–14were synthesized according to the synthesis route shown in Scheme1.Thefirst step in this synthetic approach involved append the methyl group at the C-3hydroxyl group of quercetin. All phenolic hydroxyl groups of rutin were protected by benzyla-tion.After removing rutinose by acid hydrolysis,the free C-3 hydroxyl group was methylated using dimethylsulfate.Debenzyla-tion was subsequently performed by hydrogenation using palladium on carbon(Pd/C),yielding3-O-methylquercetin11.Fur-ther methylation of11was performed to obtain compounds12–14.Compounds15–17were synthesized according to the route shown in Scheme2.Following methylation of rutin by dimethyl-sulfate,rutinose was removed by acid hydrolysis to obtain15–17.Compounds18and19were synthesized according to the route shown in Scheme3.In order to specifically append the acetyl group or acetoglucose at the C-3hydroxyl group,the phenolic hydroxyl group of rutin was protected by benzylation using the method described above.Following acid hydrolysis,the C-3hydroxyl group was acetylated or glucosylated by acetic acid anhydride or acetob-romoglucose to yield18or19,respectively.All compounds(1–19)were purified using preparative high-performance liquid chromatography(p-HPLC).Structural analysis of compounds11–19was performed by nuclear magnetic reso-nance(NMR),mass spectrometry(MS),infrared(IR)spectroscopy, UV spectroscopy,and the measurement of specific optical rotation. The protons existing in the methoxyl group of compound11were correlated with C-3,as shown in Figure2,indicating that the meth-oxyl group is bonded to the C-3position.The key heteronuclear multiple-bond correlations(HMBC)within compound14are also presented in Figure2,showing the binding position of the meth-oxyl group.The anomeric proton of acetoglucose of19was corre-lated with C-3,as shown in Figure2,indicating that acetoglucose binds to the C-3position as a glycoside.Similarly,HMBC correla-tions indicated the exact position of the substituent groups in the other synthesized quercetin derivatives(not shown).Comparing the data with the spectrum of quercetin itself,30,31the position of the acetyl group in18is likely to be at C-3,as suggested by the observed chemical shift of the C-3carbon(130.1ppm).3.2.Melanogenesis-stimulating activities of quercetin derivativesThe melanogenesis-stimulating activities of synthesized quer-cetin glycosides were determined by measuring both intra-and extracellular melanin content in B16melanoma cells.The effects of compounds1–3and11–19on cell viability and melanogenesis are shown in Table1.In our previous study,we evaluated the mod-ulation of intracellular melanin levels by1–10with theophylline as a positive control.26The current experiment investigated the effects of quercetin derivatives on extracellular melanin release and melanogenesis.On measuring the melanogenesis activity assay for each compounds,we adopted a concentration which was not shown strong cytotoxicity of the B16melanoma cells.As shown in Table1,quercetin glycosides1,2,and3stimulated intra-cellular melanogenesis in a dose-dependent manner.However, none of the synthesized quercetin glycosides increased the extra-cellular levels of melanin.On the other hand,quercetin methyle-thers11–14increased both intra-and extracellular melanin content(Table1),demonstrating higher melanogenesis-stimula-tion than theophylline,a positive control.Significant effects were observed on the extracellular melanin paring the activities of compounds11–14,medium of cells incubated with 50l M of compound11showed224.9%higher extracellular mela-nin levels compared to controls.The increases of melanin levels following incubation with compounds12–14were higher than 220%,even at6.25l M,indicating the most potent stimulation of extracellular melanin levels in this study.Furthermore,the 3-hydroxyl quercetin methylethers such as15–17,3-O-acethylqu-ercetin(18),and quercetin-3-O-b-D-2,3,4,6-tetra-O-acetoglucopy-lanoside(19)showed no stimulatory effect on the extracellular melanin levels,suggesting that the3-methoxyl group of com-pounds11–14is an essential moiety for stimulation activity.Addi-tionally the40and/or7-methoxyl group may further increase the melanogenesis-stimulating pounds12–14showedK.Yamauchi et al./Bioorg.Med.Chem.22(2014)3331–33403335。

酪氨酸酶和黑色素的关系酪氨酸酶啊，那可是黑色素世界里的超级魔法师。

你可以把它想象成一个住在我们皮肤细胞里的小工匠，整天忙忙碌碌地制造黑色素这种神奇的“黑颜料”。

黑色素就像是皮肤的小盾牌，保护着我们的皮肤细胞免受紫外线这个大坏蛋的伤害。

而酪氨酸酶呢，它就像是制造这些小盾牌的超级工厂。

这个工厂开工的时候可有趣啦，它就像一个超级大厨，把酪氨酸这种原料放进它的大锅里，然后添加上各种神奇的“调料”（酶促反应所需的条件），经过一番神奇的操作，就像变魔术一样，黑色素就诞生了。

如果把我们的皮肤比作一个大画布，黑色素就是画布上的黑色颜料。

酪氨酸酶这个调皮的小画家，有时候会画得太多啦，就像小朋友画画太兴奋，把一整瓶黑色颜料都倒在画布的某个地方，于是就有了黑斑或者雀斑，哎呀，这就有点不太美观啦。

有时候呢，酪氨酸酶这个小工匠可能会偷懒，就像工人罢工一样。

那可不得了，黑色素制造不出来，皮肤就像没有穿铠甲的小士兵，紫外线这个敌人一来，皮肤就很容易受伤，然后就变得红红的，就像一个害羞的西红柿。

你再看那些皮肤特别白的人，他们的酪氨酸酶就像是一个慢性子的小老头，不慌不忙地制造着黑色素，产量特别少。

而那些皮肤黝黑的人呢，酪氨酸酶就像是一个精力旺盛的大力士，不停地生产黑色素，让皮肤黑得像夜晚的天空。

不过，黑色素也不总是因为酪氨酸酶才出现的。

有时候它也会像一个不请自来的小客人，可能是因为我们身体内部出了点小状况，就像家里突然闯进了一个迷路的小动物。

要是我们能和酪氨酸酶对话就好了，我们就可以跟它说：“小酶啊，你可悠着点，别把黑色素制造得太多或者太少啦，不然我们的皮肤可就遭罪了。

”它可能会像一个知错的小孩子一样点点头，然后调整自己的工作节奏。

酪氨酸酶和黑色素就像是一对欢喜冤家，一个负责制造，一个负责发挥作用。

它们在我们的皮肤里演绎着一场场奇妙的故事，虽然有时候会给我们带来一些小烦恼，但总体来说，它们可是我们皮肤健康的重要守护者呢。

要是没有它们，我们的皮肤在这个充满紫外线和各种伤害的世界里，可就像没有城墙保护的城堡一样脆弱啦。

・45•黑龙江医药科学2021年2月第44卷第1期丁香精油对黑色素细胞抗氧化特性及黑色素生成的影响①刘玉荣1,曹雅菲1,雒洋洋1,高怡1,刘景源1,刘君星2（12!木斯大学临床&学-，黑龙江住木斯154067；2.!木斯大学基铀&学-，黑龙江住木斯150929）摘要：目的：考察丁香精油对体外培养的黑色素细胞抗氧化特性的变化及对黑色素生成抑制作用，为精油在食V、化妆品等领域的应用提供理论支撑。

方法：通过测定精油作用后细胞增殖活力，选择合适的药物浓度。

然后用不同浓度精油作用细胞，测定细胞内T-SOD、ROS的含量、黑色素含量和酪氨酸酶相对活性。

结果：MTT法可知丁香精油最大无毒浓度为0.084my/mL,用于后续试验，可显著抑制黑色素细胞生长，抑制率与药物浓度正相关。

细胞经药物作用48h后，细胞内T-SOD增加，ROS下降，黑色素含量和酪氨酸酶活性下降。

结论:丁香精油具有抗氧化性及抑制黑色素细胞黑色素生成的功效。

关键词：丁香精油；黑色素细胞;抗氧化；黑色素生成中图分类号:R285.7文献标识码:A文章编号：108-014（2021）01-0048-02丁香（FlosCaryophyl/）系桃金娘科蕃樱桃属植物丁香（Euaecia Camo/hyllata Thunb.6的干燥花蕾，又称公丁香。

实验结果表明丁香精油具有良好的抗氧化活性和抑菌4~5］。

目前国内外对丁香的研究与应用主要集中在食品的抗氧化抑菌、化妆品和医药卫生方面+7］。

自科学家们开始对精油的研究，精油的各种功效逐渐显露，但是精油对皮肤美白的作用研究的时间并不久［7］。

随着提取工艺的不断提升,各种提取方法之间的对比,我们可以得到含抗氧化物质更多的精油4］。

植物精油作为一种天然的酪氨酸酶抑制剂，具有显著的促透皮吸收能力和酪氨酸酶抑制活性,而且具有独特的芳香气味,应用前景广阔54］。

本实验基于黑色素细胞评价体系探索了丁香精油对黑色素细胞活力的影响、抗氧化损伤作用及对其生成黑色素的抑制作用，为进一步扩大丁香精油在食品、药品和化妆品等领域的应用提供数据支撑。

黑色素细胞中黑色素含量的测定和酪氨酸酶活性的测定
黑色素细胞中黑色素含量的测定
和酪氨酸酶活性的测定方法
一、黑色素含量的测定
1.B16小鼠黑色素瘤细胞的准备
离心收集约107个细胞，再用PBS洗2次，离心条件为1000rpm、5min，细胞沉淀备用。

2.除杂
向细胞沉淀中加入200µL纯水重悬细胞，再加入1mL乙醇/乙醚混合液（体积比为1:1），充分摇匀并于室温下静置15min。

时间到后，该混合物于3000rpm，5min条件下继续离心，弃上清液，沉淀备用。

3.黑色素颗粒溶解与检测
向所得细胞沉淀中加入1mL10%的DMSO（用1MNaOH配制），充分振荡后于80℃条件下封口放置30min。

时间到后于470nm波长处检测吸光度值。

黑色素含量计算：
黑色素含量=A/细胞数
二、酪氨酸酶活性的测定
1.B16黑色素瘤细胞的准备
离心收集约107个细胞，再用PBS洗2次，离心条件为1000rpm、5min，细胞沉淀备用。

2.细胞裂解
想细胞沉淀中加入1mL0.5%脱氧胆酸钠，于0℃条件下放置15min。

3.酪氨酸酶活性的测定
时间到后，向以上混合物中加入3mL0.1%L-dopa（用0.1MpH=6.8的磷酸缓冲液配制，该反应混合液应在配制后2h内使用），反应温度37℃，于475nm波长处检测反应0min和10min时的吸光度值。

酪氨酸酶活性计算：
酪氨酸酶活性=（A0-A10）/细胞数。

酪氨酸酶与⿊⾊素酪氨酸酶与⿊⾊素 (2006-12-01 01:31:46)转载▼分类：好⾊之旅酪氨酸酶的本质：⼀种含铜的⾦属酶。

酪氨酸酶的来源：由位于⼈的表⽪基层细胞间的⿊⾊素细胞合成，结合于亚细胞颗粒——⿊⾊素体内。

酪氨酸酶的功能：⿊⾊素细胞活化酶。

⿊⾊素代谢中⽬前唯⼀已知的酶，控制⿊⾊素细胞活性的关键，决定了⿊素合成的速率。

酪氨酸酶的催化作⽤：酪氨酸酶与⾎液中的酪氨酸反应，⽣成⼀种叫“多巴”的物质。

多巴其实就是⿊⾊素的前⾝，经酪氨酸氧化⽽成，释放出⿊⾊素。

⿊⾊素⼜经由细胞代谢的层层移动，到了肌肤表⽪层形成雀斑、晒斑、⿊斑等形状了。

⿊⾊素蛋⽩（Melanin）的来龙去脉：⿊⾊素细胞（Melanocytes）以10：1的⽐例存在于⽪肤基底（Stratum Basale）细胞内，具有树枝样的外形，穿梭在表⽪细胞间。

当热辐射或太阳光照射⽪肤时，可激发并活化⿊⾊素细胞。

⿊⾊素细胞中含有⾊素颗粒，在粗内质⽹合成⿊⾊素体（Melanosomes），分泌⼀种麦拉宁的褐⾊⾊素，当紫外线(B波、A波)照射到⽪肤上(B 波即UVB作⽤于⽪肤基底层，⽽A波更厉害，作⽤于⽪肤的真⽪层)，肌肤就会处于“⾃我防护”的状态，藉由紫外线刺激麦拉宁⾊素，激活位于⾼尔基体的酪氨酸酶（Tyrosinase）的活性，来保护⽪肤细胞。

麦拉宁⾊素在完成保护任务后，变成污垢剥落。

酪氨酸酶结合于亚细胞颗粒－－⿊⾊素体内，催化酪氨酸（Tyrosine）氧化⽣成“多巴”，经过⼀系列反应释放出⿊⾊素，⽣成⿊⾊素蛋⽩。

同时酪氨酸酶将失去活性，并担任运输⼯作，将⿊⾊素蛋⽩经过细胞代谢的层层移动，转移⾄⾓质细胞。

当⾝体新陈代谢⽋佳时，部分⾊素会留在⽪肤表层，形成雀斑、晒斑、⿊斑等形状，⿊⾊素蛋⽩转移⼊⾓质细胞越多，肤⾊越深。

形成⿊斑。

在正常情况下，⿊素的代谢过程需要28天，分泌产⽣的⿊素能够保护⽪肤免受紫外线的伤害。

另外，因妊娠⽽造成的内分泌失调、氧化或作息时间不稳定，都会造成⾊斑。