菟丝子体外和在体实验中对黑素代谢的作用评价
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摘要
研究背景
色素障碍性皮肤病(如白癜风、黄褐斑)是临床中常见的皮肤疾病,其对形象的损毁给患者带来巨大的心理压力,自古以来一直受到医界的重视,而在亚洲,普通人群对于皮肤美白等美容方面的要求也越来越高,从自然界寻找安全有效的美白剂早已是化妆品界的研究热点。随着分子生物学技术的迅速发展及不同模型的出现,黑素代谢的相关研究取得了极大的进步,中医药在黑素代谢中的作用也积累了丰富的研究成果。然而,由于所选模型不一致,或者药物提取方法不同,部分中医药对黑素代谢的作用时而出现不一致的结果,究其机制却鲜有深入的探讨。
本课题在结合古代和现代中医药对黑素代谢作用的基础上,选用菟丝子为实验药物。早在《神农本草经》中菟丝子就被记载有“汁去面皯”的作用,“皯”即黑斑、黧黑斑的意思。菟丝子的功能主治国家标准首次收载于1963年版《中国药典》,记载为：补肝肾,益精,明目。主治腰膝痠痛,遗精,目暗,尿频余沥。未提及菟丝子的外用功效,自1985年修订版开始,历经各次修订,各版均增加了“外治白癜风”的主治标准,2010年版则修订为“外用消风祛斑…外治白癜风”,但功效表述为“滋补肝肾,固精缩尿,安胎,明目,止泻”,未能为外治功效提供理论依据。文献检索提示,国外的实验研究认为菟丝子能抑制黑色素合成,可用于增色性皮肤病的治疗,未见菟丝子促进黑色素合成的报道；与之相对应的,近现代以来,国内对菟丝子治疗增色性皮肤病的研究较少,其应用于内服方或外治白癜风则更为常见。根据文献,在促进黑色素合成和抑制黑色素合成的研究中菟丝子均被认为发挥调节作用,但菟丝子的作用机理尚无进一步的揭示。菟丝子是否具有双向调节黑素代谢的作用?其调节机制如何?作为色素障碍性皮肤病临床治疗中应用广泛而且有效的一味中药,菟丝子常用于治疗表现完全相反的两类色素障碍性皮肤病,但其发挥作用的机制并不明确,这给临床用药带来了一定的混乱,我们认为对菟丝子的作用机制进一步深入研究具有非常重要的现实意义。
色素代谢的研究模型可以分为三类：非细胞模型测试系统,细胞模型测试系统,动物模型测试系统。蘑菇酪氨酸酶是研究受试药物对酶抑制机理最常用的非细胞测试模型,酶法测试的优点是无需细胞培养、简单快速,可对受试物的作用类型进行初步的判断,缺点是体系过于简单,单凭受试物对酪氨酸酶活性的抑制程度判定其作用效果显得片面；细胞测试系统可反应不同受体基因表达的变化,一定程度上弥补了蘑菇酪氨酸酶的缺陷。最常用的细胞是小鼠B16黑色素瘤细胞,其基因组成与人表皮黑素细胞有较高的相似性,较后者相比更容易获取、培养,是用于测试受试物对黑素细胞生物学功能影响的首选模型之一。动物测试系统主要包括C57小鼠、无毛小鼠、豚鼠、Smyth line鸡等。斑马鱼作为国际标准化组织认可的5种鱼类模式生物之一,具有与人类相似的黑色素细胞和黑素体,近年来也越来越多的被用于黑素代谢研究领域。又因周身透明,色素细胞在个体发育过程中易被识别、观察,在黑素细胞遗传学、色素代谢相关模型构建、高通量药物筛选中均已得到大量的应用；此外,斑马鱼属低温低氧鱼,其耐寒性和耐热性都很强,饲养容易,产卵量大,与前几种动物模型相比有特有的优势,在黑素细胞相关研究中将有不可替代的地位。虽然斑马鱼在中医药对血管系统、中药毒理学研究中已有所应用,但作为黑素代谢研究的优势模型,斑马鱼在中医药对黑色素合成中的作用研究尚未有报道。
本研究的目的
明确菟丝子对黑素代谢中的调节作用及机制；探索斑马鱼模型在中医药黑素代谢实验中的应用。
本研究的实验内容及结果
菟丝子临床使用的剂型主要为水煎剂或酊剂,根据这一用药传统,本课题分别用水、95%乙醇对其回流提取制成了菟丝子水提物(WFSC)、菟丝子醇提物(EFSC)。选用蘑菇酪氨酸酶、B16黑素瘤细胞酪氨酸酶提取液、B16黑素瘤细胞、斑马鱼胚胎作为模型,通过体外实验和在体实验两种方式,系统研究菟丝子对黑素细胞色素代谢的作用机制,并借助高效液相色谱法分析WFSC、 EFSC成分的差异。
1.在体外实验,本论文首先以蘑菇酪氨酸酶为研究对象,探讨菟丝子提取物WFSC、EFSC对蘑菇酪氨酸酶活性的作用机理；以粗制的B16黑素瘤细胞酪氨酸酶为研究对象,比较菟丝子不同提取物对这两种酪氨酸酶活性的作用。再将WFSC、EFSC与B16黑素瘤细胞共同孵育,研究其对细胞增殖、酪氨酸酶活性、黑色素合成以及对酪氨酸酶mRNA、蛋白表达的影响,相关研究内容及结果详述如下：
1)分别比较了WFSC、EFSC对蘑菇酪氨酸酶活性的作用。研究结果表明WFSC对蘑菇酪氨酸酶具有抑制作用,EFSC对蘑菇酪氨酸酶则有激活作用。对WFSC, EFSC的作用机理研究显示,二者与蘑菇酪氨酸酶的结合均为可逆性,通过酶动力学实验测试,WFSC对蘑菇酪氨酸酶的活性的抑制作用表现为反竞争型抑制剂,即主要通过与蘑菇酪氨酸酶-底物复合物结合发挥作用；与蘑菇酪氨酸酶相比,WFSC对B16黑素瘤细胞酪氨酸酶提取液的抑制效果相一致。与WFSC表现出抑制作用不同的是,EFSC对蘑菇酪氨酸酶、B16细胞酪氨酸酶提取液体系中均表现为激活作用。
2)研究WFSC、EFSC对B16黑素瘤细胞增殖、细胞内酪氨酸酶活性、黑色素含量的影响。结果表明浓度为0.5～20mg/mL时,WFSC、EFSC对B16细胞增殖均无影响。将WFSC、EFSC配制成不同浓度与B16黑素瘤细胞共同孵育48h后测定细胞内酪氨酸酶的活性。与IBMX作用下的空白对照组比较,WFSC作用下的B16瘤细胞TYR活性有统计学差异(F=31.726, P=0.000), WFSC对酶活性表现为抑制作用,当WFSC>5mg/mL时,差异均有统计学意义(P<0.05),WFSC中剂量组和高剂量组间差异无统计学意义(P=0.784)。与空白对照组比较,EFSC B16黑素瘤细胞酪氨酸酶的活性表现出激活作用(F=8.259,P=0.008),在5～10mg/mL时对,差异均具有统计学意义(P<0.05),但中剂量组与高剂量组间差异没有统计学意义(P=1.000)。与空白对照组比较,WFSC等药物组作用下的B16瘤细胞黑素含量差异有统计学意义(F=9.733,P=0.001), WFSC可下调酪氨酸酶黑素合成量,当WFSC>5mg/mL时,差异均有统计学意义(P<0.05), WFSC中剂量组和高剂量组间差异无统计学意义(P=1.000)。与空白对照组比较,EFSC对B16黑素瘤细胞黑素合成表现出激活作用(F=12.077,P=0.002),在5～10mg/mL时差异均具有统计学意义(P<0.05), EFSC中剂量组与高剂量组间差异没有统计学意义(P=0.18)。
3)研究VFSC、EFSC对TYR的mRNA和蛋白表达的影响,结果表明,WFSC、EFSC对TYR mRNA表达差异无统计学意义(P>0.05)；同样,二者对TYR蛋白合成差异无统计学意义(P>0.05)。
2.在体实验中,以斑马鱼为模型,以熊果苷、对羟基苯甲醚(MQ)为阳性对照,观察WFSC、EFSC对斑马鱼黑素细胞的作用,并通过PTU干预,初步探究菟丝子对斑马鱼黑素细胞发挥作用的机制；检测各受试物对斑马鱼黑素细胞酪氨酸酶活性、黑色素合成的影响,相关研究结果如下：
0.1mg/mL～2mg/mL的EFSC对斑马鱼胚胎生存率没有影响；药物浓度>3mg/mL可延迟斑马鱼胚胎的发育,且随着药物浓度的增高,其生成率降低。经观察发现,WFSC、EFSC均不影响斑马鱼黑素细胞的迁移和分布。较高浓度WFSC作用下可见斑马鱼黑素细胞呈点状改变,与对照药MQ的表现相同,这一现象在加药后6h即可观察到,另一阳性对照药熊果苷也可以使斑马鱼黑素细胞出现同样的改变,但于加药后约20h方出现变化,较WFSC出现形态学改变的时间晚；EFSC对斑马鱼胚胎黑素细胞则没有明显的改变。PTU是酪氨酸酶阻滞剂,在PTU干预下,WFSC、MQ对黑素细胞的作用消失,但熊果苷仍可使黑素细胞形态发生改变,这一作用差异可能因WFSC、熊果苷对酪氨酸酶的抑制作用方式不一样有关。
2)提取斑马鱼胚胎蛋白后测定酪氨酸酶活性,WFSC作用下斑马鱼黑素细胞TYR活性差异有统计学意义(F=13.006,P=0.000)。与空白对照组相比较,WFSC浓度为lmg/mL～3mg/mL时对斑马鱼黑素细胞酪氨酸酶表现出抑制作用,差异均有统计学意义(P<0.05),各浓度组间差异均无统计学意义(P>0.05)。WFSC、熊果苷、MQ对酪氨酸酶的作用差异无统计学意义(P>0.05)。WFSC作用下,斑马鱼黑素细胞黑色合成作用差异有统计学意义(F=16.474,P=0.000)。与空白对照组相比较,WFSC浓度为lmg/mL～3mg/mL时对斑马鱼黑素合成表现出抑制作用,差异均有统计学意义(P<0.05),各浓度组间差异均无统计学意义(P>0.05)。WFSC为3mg/mL时,与熊果苷、MQ相比,对黑素合成的作用差异均无统计学意义(P>0.05)。EFSC对斑马鱼黑素细胞酪氨酸酶活性未表现出影响(F=0.831,P=0.535),不同浓度EFSC对斑马鱼胚胎黑素合成形成的差异无统计学意义(F=0.596,P=0.674)。
3.高效液相色谱法对WFSC、EFSC的成分分析：
在实验过程中我们发现,WFSC在提取初期对酪氨酸酶没有抑制作用,反而表现出轻微的促进作用,至其提取后1个月方表现出明显的抑制作用,抑制效果稳定并维持直至提取后6个月；而EFSC自提取后即对酪氨酸酶活性表现出稳定的激活作用。参考苏薇薇等建立的实验方法,本研究对不同时期WFSC、 EFSC进行了高效色谱法分析,相关结果如下：
1) WFSC、 EFSC的HPLC图谱有明显的差异,WFSC在提取后2周内峰6逐渐低平直至消失,而其提取1个月后与6个月后的HPLC图差异不大,这与WFSC药效变化的时间拐点相符合；与WFSC不同的是,EFSC的HPLC图谱随时间变化差异不大,峰5是EFSC的特有峰,菟丝子主要由多酚类化合物组成,其中含有丰富的黄酮类成分,根据流动相设置条件及出峰时间判断,峰5可能为黄酮类化合物。
2) WFSC对斑马鱼黑素细胞黑色素合成有抑制作用,为研究WFSC在斑马鱼作用下成分的变化,对经或不经斑马鱼胚胎作用的含有WFSC的培养液进行分析,发现经斑马鱼胚胎作用后,23min峰高明显下降,而39min峰高明显升高,推测此两种化合物含量的变化可能与WFSC对斑马鱼胚胎黑素细胞的抑制作用相关。
结论
1.不同的提取方法下,菟丝子的成分变化差异明显,其中水提物表现为抑制作用,醇提物表现为激活作用,菟丝子对黑素代谢的这种双向作用可能是由于各提取部位的成分不同引起的；
2.斑马鱼胚胎在菟丝子对黑素代谢的研究中与其它模型表现出较好的一致性,且更为直观,可应用于中医药对黑素代谢的实验研究。
讨论
通过本课题的研究,体外试验和在体实验结果均表明菟丝子水提物WFSC对黑色素合成具有抑制作用,经蘑菇酪氨酸酶检测,WFSC为反竞争型抑制剂,表明其是通过酪氨酸酶-底物复合物发挥抑制作用的；WFSC对B16黑素瘤细胞及斑马鱼胚胎黑素细胞生物学功能的作用一致,即通过降低酪氨酸酶活性下调黑素合成量,WFSC作用下斑马鱼胚胎黑素细胞的形态发生改变,与熊果苷作用效果相似,但二者的起效时间不同。醇提物EFSC在细胞外酪氨酸酶模型及B16黑素瘤细胞实验中对酪氨酸酶活性及黑色素合成表现出激活作用,然而在斑马鱼黑素细胞却无明显影响,这可能因斑马鱼胚胎发育早期的黑素细胞酪氨酸酶活性本身较高有关,在斑马鱼胚胎发育的早期(12hpf-5dpf),酪氨酸酶活性高于其成熟的黑素细胞,因而EFSC的激活作用无从体现。HPLC图谱表明WFSC的变化与其药效发生改变的时间变化相一致；而峰5为EFSC的特有峰,根据色谱条件及峰5所在的位置,且其紫外吸收图与槲皮素对照品相似,推测其可能为母核与槲皮素相似的黄酮类化合物。WFSC经斑马鱼胚胎作用前后的HPLC图谱显示其组成有明显变化,23min峰明显降低,39min峰明显升高,二者改变是否与WFSC的抑制作用有关?峰5是否是EFSC发挥药效的关键组成?峰6是否为WFSC作用产生变化的关键峰?这些问题尚未解决,有待课题组进一步的深入研究。
创新及展望
1)本课题因菟丝子对黑素代谢作用描述不清给临床用药带来困惑这一现实问题的目的,以不同模型为对象,研究菟丝子对黑素代谢的作用,实验证实菟丝子对黑色素代谢的作用具有双向性,发现其药效差别的关键因素可能是因提取方法不同导致的有效成分不同,其中水提取产物WFSC对黑素合成具有抑制作用,醇提取物EFSC对细胞内的色素合成有激活作用。
2)首次发现WFSC对黑素合成的抑制作用存在时间拐点,推测与其组成成分发生变化有关。
3)本课题首次将斑马鱼胚胎用于中医药对黑素细胞作用的研究,与其他模型相比更为直观、便利,而且有较好的一致性。
综上所述,本课题具有一定的创新性及科学意义。然而,本课题未能展开菟丝子复方及与不同功效中药配伍对黑素代谢影响的研究,临床应用中药调控色素实践的诸多问题,仍需进一步的探讨。
Pigmented skin disorders (such as vitiligo, melasma) is a common kind of clinic skin disease, which bring shame and enormous psychological pressure to the patient.It has long been the medical community's attention since ancient. And in Asia, the requirements of normal population for skin whitening and other cosmetic are also increasing, from nature to find a safe and effective whitening agent is already a hot topic in the cosmetics industry. With the advent of molecμLar biology techniques and the rapid development of different models, melanin metabolism research has made great progress. The role of Chinese medicine in melanin metabolism has accumulated abudndant research resμLts. However, in the research part of Chinese medicine for melanin metabolism, due to inconsistency of the selected model, or a different extraction methods drugs, traditional Chinese medicine for melanin metabolism sometimes appear inconsistent results, and its mechanism is rarely discussed in depth.
As one of the oldest drug in the treatment of skin pigmentation disorders, Cuscuta seed was recorded in "Shen Nong's Herbal Classic " and described as "juice, get rid of "gan","gan" means dark spots, namely haze shading.However,Cuscuta chinese seeds had not been recorded as the treatment of vitiligo in the ancient books.In the modern times, the function of cuscutae attended national standard records for the first time in1963edition of "Chinese pharmacopoeia", described as: liver or kidney, Yijing, improving eyesight,attending sore waist and knee, spermatorrhea, eye shadow, frequent urination. Topical effect of Cuscutae chinese seeds in vitiligo had not mentioned, since the beginning of1985, after each revision, all revisions, expressing ideas have increased "outside cure vitiligo" standard of attending, but efficacy expressed as "nourishing liver and kidney, solid essence shrinks the urine, fetus, improving eyesight, antidiarrheal", failing to provide theoretical basis for the external treatment of vitiligo. Literature retrieval showing that experimental study abroad believe Cuscutae chinese seeds can inhibit melanin synthesis,and be grace used in the treatment of skin diseases.It hasn't note recorded that Cuscutae seeds can promote the synthesis of melanin. Correspondingly, since the modern times, the domestic study of Cuscutae in chromatodermatosis treatment is less, its application in internal or outside party to cure vitiligo is more common. According to the literature, Cuscutae seeds play a regulatory role in the study of melanin synthesis and inhibit melanin synthesis.However, the mechanism of cuscuta seeds action, there has no further research. Whether the seeds has two-way regulating melanin metabolism role in melanin synthesis? The regulating mechanism is what? As an effective medicinal herbs,Cuscuta seeds were widely used in pigment barrier skin disease in clinical treatment, it was often used in the treatment of performance opposite two kinds of pigment barrier skin disease, but its role has no definite way, which may bring to clinical application a certain confusion. We believe that the research on the mechanism of action of Cuscuta chinese seeds has further important significance.
Model pigment metabolism can be divided into three categories:cell-model test systems, test cell model system, animal model test systems. Mushroom Tyrosinase is an enzyme inhibition studies on the mechanism of the test drug most commonly used non-cell test model, there is no need to test the advantages of enzymatic cell culture, is simple and fast acting type of the test substance can make a preliminary determination, the disadvantage is system is too simple, the degree of inhibition of the test substance alone tyrosinase activity to determine its effects seem one-sided; cell test system can react in different receptor gene expression changes, to some extent, make up for the deficiencies of mushroom tyrosinase. The most commonly used mouse B16melanoma cells are cells whose genetic makeup of human epidermal melanocytes have high similarity, compared to more accessible than the latter, training, test substance is used to test the melanocytes one of the biological function of the preferred model. Animal testing system includes C57mice, hairless mice, guinea pigs, Smyth line chicken, zebrafish recognized as one of the five International Organization for Standardization species of fish model organisms, with human melanoma cells and similar melanosomes. In recent years, more and more are used to melanin metabolism studies in the field, because the whole body transparent and easily identify the individual pigment cells during development made observation easily. And with the cell labeling technique, the target gene-directed mutagenesis techniques, genetically modified technologies, such as gene overexpression of mature development, development of melanocytes, the pigment metabolism-related modeling, high-throughput drug screening have been a large number of applications.Addition, zebrafish are hypothermia and hypoxia fish, its cold resistance and resistance thermal resistance are strong, easy feeding, spawning large, compared with the previous several animal models have unique advantages, there are an irreplaceable position in the relevant research in melanocytes. Although zebrafish vascular system in Chinese medicine, Traditional Chinese medicine toxicology studies have been applied, but the advantages of the model as melanin metabolism studies in zebrafish on the role of research in medicine melanin synthesis has not yet been reported.
The porpuse of this study was to clear Cuscuta chinesis seeds of melanin metabolism biphasic regulation mechanism; To explore the zebrafish model in the role of melanin metabolism experiment of traditional Chinese medicine.
The experiment contents and results of this study recorded as follows:Cuscuta seeds clinical use of dosage forms are mainly water decoction or tincture. According to the traditional medication, this topic will use the water and95%ethanol reflux extraction and respectively made the water fraction from Semen cuscutae (WFSC), the ethanol fraction from Semen cuscutae (EFSC). Choose the mushroom tyrosinase, tyrosinase of B16melanoma cells extract, B16melanoma cells, zebrafish embryos as the model. Through the experiment in vitro and in vivo experiment, system research the mechanism of action of cuscutae on melanin cell metabolism of pigment. With the analysis of High-performance liquid chromatography fingerprinting of WFSC and EFSC, to compare the composition differences.
1.In vitro experiment, this paper first using the mushroom tyrosinase as the research object, explored the Cuscutae seeds extract WFSC, EFSC mechanism of mushroom tyrosinase activity.With rough B16melanoma cells tyrosinase as the research object, comparison of different extracts of the Cuscutae seeds effect on tyrosinase activity. Then the B16melanoma cells incubated in WFSC, EFSC, detected the research on cell proliferation, tyrosinase activity and melanin synthesis and tyrosinase mRNA and protein expression, the influence of the related research contents and results are described below:
1) Compare the WFSC, EFSC role of mushroom tyrosinase activity respectively. Results indicate that WFSC has inhibitory effect on mushroom tyrosinase, through the enzyme kinetics experiment test WFSC, EFSC mechanism of enzyme. According to the results that both the combination of WFSC, EFSC and mushroom tyrosinase were reversible. Inhibition of WFSC showed the type of competitive inhibitor, which means that it work with mushroom tyrosinase substrate complex combination. Compared with mushroom tyrosinase, WFSC consistently inhibited the tyrosinase extract of B16melanoma cells. With WFSC showed an obvious inhibition, EFSC showed an activation function on mushroom tyrosinase and B16cells in the extraction of tyrosine enzyme system.The activation of EFSC, effect on the enzyme kinetics showed the mechanism of mixed activation.
2) With study the role of WFSC, EFSC on B16melanoma cell proliferation rate, cell tyrosinase activity and melanin content. Results showed that WFSC, EFSC had no significant effect on the proliferation of B16cells. At the concentration>5mg/mL, WFSC showed an inhibition function on the intracellular enzyme of tyrosinase. At the concentration10mg/mL,the inhibition effect of which similar to that of50ug/mL arbutin.While EFSC showed an obvious activation function on enzyme activity,both of which worked dose-dependent effect on B16melanoma cell. Consistenting with the role of the activity of tyrosinase, WFSC inhibits melanin synthesis, and EFSC can promote the synthesis of melanin. According to the results of Western blot, QRT PCR,neither WFSC nor EFSC effect on tyrosinase mRNA and protein expression.
2. In vivo experiments, taking zebrafish as the model, with arbutin, Methoxyphenol (MQ) as the positive control, observated the effect of WFSC, EFSC on Zebrafish melanocyte. The study detected various subjects:including melanocyte tyrosinase activity and melanin synthesis. Under the PTU intervention, to explore the mechanism of Cuscutae seeds on zebrafish melanin cell function, related research results are as follows:
1)0.1mg/mL～2mg/mL WFSC, EFSC had no effect on zebrafish embryo survival rate. When drug concentration>3mg/mL, extracts would delay the development of zebrafish embryos. As the drug concentration increased, Livability of Zebrafish embryo is reduced.According to the observation we found that24hpf-5dpf was the best working time for the research of zebrafish melanocyte tyrosinase activity of optimal detection period. Larval melanocytes with punctate melanocyte morphology under the action of WFSC,which were same as the control medicine MQ performance. This phenomenon at6h after dosing can be observed that the positive control drug arbutin can also make the same change on the zebrafish melanocyte, but afer the it working20h change, later than onset time of the WFSC. After incubated with EFSC,there is no obvious change on zebrafish embryos melanocyte. Determinated the tyrosine enzyme activity with the extraction of zebrafish embryo protein, the results revealed that WFSC could inhibit tyrosinase activity of zebrafish embryo, reduced the melanin synthesis. While zebrafish embryo incubated in EFSC, zebrafish tyrosinase activity and pigment synthesis of melanocyte are similar with the blank controller. PTU is tyrosinase blockers, under the PTU intervention, the effect of WFSC, MQ on melanin synthesis disappeared, but arbutin could still make the melanocyte with punctate melanocyte morphology, the role differences might because of WFSC and arbutin on tyrosinase inhibitory effect way were different, WFSC as competitive inhibitors, While arbutin as competitive inhibitors.
2.High performance liquid chromatography (HPLC) method for WFSC,EFSC composition analysis:
We found that in the process of experiment,WFSC of the early extraction period didn't show an inhibition effect on tyrosine enzyme, but showed slight promoting effect. However,1month after WFSC extracted,it showed an obvious inhibitory effect, which maintained the inhibition effect stably even6months after extraction. It's intersting that EFSC kept a stable activation function on the tyrosine enzyme since the extraction of EFSC was prepared. According to the chromatography analysis experimental methods of Su Weiwei established, the study determined the difference between WFSC and EFSC during different periods, the results are as follows:
1) High performance liquid chromatography finger print of WFSC, EFSC had obvious difference. Peak6of High performance liquid chromatography finger print of WFSC within2weeks after extracting became lower day by day until it disappeared, but then WFSC extraction kept the similar high performance liquid chromatography finger print from1month to the6month. Its change of time corresponds with the change of WFSC efficacy.Unlike WFSC, EFSC kept the similar High performance liquid chromatography finger print during the whole experiment process. Peak5are the characteristic peak of EFSC.Considering that Cuscuta chinensis seeds are mainly composed of polyphenols and flavonoids, according to the mobile phase condition, peak5was judgmented as one kind of flavonoids.
2) WFSC showed an obvious inhibitory effect on zebrafish melanocyte melanin synthesis. To compare the change of composition of WFSC consumed with or without Zebrafish, we analyzed the WFSC culture medium with the role of zebrafish embryos.It's found that after zebrafish embryos consumption,23min peak of WFSC culture medium decreased obviously, and39min peak rised obviously.It's speculated that the change of the two compounds may be concerned with WFSC melanocyte inhibitory effect on zebrafish embryos.
Through this topic research, in vitro and in vivo experimental results show that the Cuscuta chinensis seeds water extract WFSC has inhibitory effect on melanin synthesis, by the mushroom tyrosinase, WFSC play a role in the form of competitive inhibitors, suggesting that it worked through the tyrosinase-substrate compounds and play an inhibitory effect on melanin synthesis. The role of WFSC on B16melanoma cells and biological functions of zebrafish embryos melanocyte is consistent, namely cut melanin synthesis by lowering the activity of tyrosinase.Zebrafish embryos under WFSC effects showed the action of melanocyte morphological changes, the effects of which are similar with arbutin, but the onset time is different. Zebrafish embryo incubated with WFSC showed the change within6h, but showed the same changes after20h while incubated with arbutin, the difference may be associated with inhibition mechanism of WFSC and arbutin,which is different from each other. Because arbutin also play an inhibiton role on tyrosinase single phenol enzyme inhibition, which can prolong the lag time of response. EFSC showed obvious activation tyrosine on tyrosinase activity and melanin synthesis either on the enzyme model outside the cell or the experiment of B16melanoma cells. However it had no obvious effect on zebrafish melanin cell morphological changes. This may be due to the hightyrosinase activity itself in the early development of zebrafish embryos of melanocyte, EFSC activation cannot reflect on it. It was reported that in the early developed time of zebrafish embryo (12hpf～5dpf) tyrosine enzyme activity is much higher than the mature melanocyte. High performance liquid chromatography finger print showed that the time change of WFSC consistent with its efficacy change. And peak5is the characteristic peak for EFSC. According to the chromatographic conditions and5location, and the ultraviolet absorption figure is similar to quercetin reference substance, We preliminary judgment for the structure similar to that of quercetin flavonoids. Through the zebrafish embryo comsumed, HPLC of WFSC showed that the composition has obvious change before and after, wether the changes of39min peak and23min peak associated with the inhibition of WFSC? Weather Peak5play efficacy of key in the role of EFSC? If the peak6is the key peak of WFSC changes? All of the problems needs to be researched further in-depth study.
Discussion
This topic worked on the problem that the effect of Cuscuta chinensis seeds on melanin metabolism had not been described clearly in the clinical medicine.The purpose of this study was to explain the role of Cuscuta chinensis seeds on metabolism of melanin. Using different models as the object, we found that the key factors of Cuscuta chinensis seeds with bidirectional regulation of melanin metabolism was the different extract method. The water fraction from Semen cuscutae has inhibitory effect on melanin synthesis, and95%ethanol fraction from Semen cuscutae has the effect of the activation on melanin synthesis. Both of them worked through tyrosinase.Neither of them have effect on the expression of tyrosinase. It was the first time that we discovered WFSC in inhibition of melanin synthesis with time turning point, as well as its composition was changed; This topic first determinated that zebrafish embryos can be used to study the role of melanin synthesis of traditional Chinese medicine, which compared with other models has good consistency. To sum up, this topic has certain innovative and practical application value.
Conclusion
1)Under different extraction methods, the different effect may caused by the difference composition. It showed that WFSC inhibited while EFSC show an activation function. The two-way role of Cuscuta on melanin metabolism may be due to the different component parts of each extract.
2) Zebrafish embryos in our research were consist with other models, which were more intuitive, the experimental study showed that Zebrafish could be used in correlational study of TCM.
Innovation and Prospects
1) WFSC inhibit melanogenesis, the EFSC activated the intracellular pigment synthesis. While the precise factor of Cuscutae in the metabolism of melanin is unclear.
2) It was first discovered the inhibition function of WFSC on the melanin synthesis depend on the time of inflection, presumably related to its composition changes.
3) It's the first time in zebrafish embryos for research on the role of TCM on melanin cells, compared with other models which was more intuitive, convenient.
色素障碍性皮肤病(如白癜风、黄褐斑)是临床中常见的皮肤疾病,其对形象的损毁给患者带来巨大的心理压力,自古以来一直受到医界的重视,而在亚洲,普通人群对于皮肤美白等美容方面的要求也越来越高,从自然界寻找安全有效的美白剂早已是化妆品界的研究热点。随着分子生物学技术的迅速发展及不同模型的出现,黑素代谢的相关研究取得了极大的进步,中医药在黑素代谢中的作用也积累了丰富的研究成果。然而,由于所选模型不一致,或者药物提取方法不同,部分中医药对黑素代谢的作用时而出现不一致的结果,究其机制却鲜有深入的探讨。
本课题在结合古代和现代中医药对黑素代谢作用的基础上,选用菟丝子为实验药物。早在《神农本草经》中菟丝子就被记载有“汁去面皯”的作用,“皯”即黑斑、黧黑斑的意思。菟丝子的功能主治国家标准首次收载于1963年版《中国药典》,记载为：补肝肾,益精,明目。主治腰膝痠痛,遗精,目暗,尿频余沥。未提及菟丝子的外用功效,自1985年修订版开始,历经各次修订,各版均增加了“外治白癜风”的主治标准,2010年版则修订为“外用消风祛斑…外治白癜风”,但功效表述为“滋补肝肾,固精缩尿,安胎,明目,止泻”,未能为外治功效提供理论依据。文献检索提示,国外的实验研究认为菟丝子能抑制黑色素合成,可用于增色性皮肤病的治疗,未见菟丝子促进黑色素合成的报道；与之相对应的,近现代以来,国内对菟丝子治疗增色性皮肤病的研究较少,其应用于内服方或外治白癜风则更为常见。根据文献,在促进黑色素合成和抑制黑色素合成的研究中菟丝子均被认为发挥调节作用,但菟丝子的作用机理尚无进一步的揭示。菟丝子是否具有双向调节黑素代谢的作用?其调节机制如何?作为色素障碍性皮肤病临床治疗中应用广泛而且有效的一味中药,菟丝子常用于治疗表现完全相反的两类色素障碍性皮肤病,但其发挥作用的机制并不明确,这给临床用药带来了一定的混乱,我们认为对菟丝子的作用机制进一步深入研究具有非常重要的现实意义。
色素代谢的研究模型可以分为三类：非细胞模型测试系统,细胞模型测试系统,动物模型测试系统。蘑菇酪氨酸酶是研究受试药物对酶抑制机理最常用的非细胞测试模型,酶法测试的优点是无需细胞培养、简单快速,可对受试物的作用类型进行初步的判断,缺点是体系过于简单,单凭受试物对酪氨酸酶活性的抑制程度判定其作用效果显得片面；细胞测试系统可反应不同受体基因表达的变化,一定程度上弥补了蘑菇酪氨酸酶的缺陷。最常用的细胞是小鼠B16黑色素瘤细胞,其基因组成与人表皮黑素细胞有较高的相似性,较后者相比更容易获取、培养,是用于测试受试物对黑素细胞生物学功能影响的首选模型之一。动物测试系统主要包括C57小鼠、无毛小鼠、豚鼠、Smyth line鸡等。斑马鱼作为国际标准化组织认可的5种鱼类模式生物之一,具有与人类相似的黑色素细胞和黑素体,近年来也越来越多的被用于黑素代谢研究领域。又因周身透明,色素细胞在个体发育过程中易被识别、观察,在黑素细胞遗传学、色素代谢相关模型构建、高通量药物筛选中均已得到大量的应用；此外,斑马鱼属低温低氧鱼,其耐寒性和耐热性都很强,饲养容易,产卵量大,与前几种动物模型相比有特有的优势,在黑素细胞相关研究中将有不可替代的地位。虽然斑马鱼在中医药对血管系统、中药毒理学研究中已有所应用,但作为黑素代谢研究的优势模型,斑马鱼在中医药对黑色素合成中的作用研究尚未有报道。
本研究的目的
明确菟丝子对黑素代谢中的调节作用及机制；探索斑马鱼模型在中医药黑素代谢实验中的应用。
本研究的实验内容及结果
菟丝子临床使用的剂型主要为水煎剂或酊剂,根据这一用药传统,本课题分别用水、95%乙醇对其回流提取制成了菟丝子水提物(WFSC)、菟丝子醇提物(EFSC)。选用蘑菇酪氨酸酶、B16黑素瘤细胞酪氨酸酶提取液、B16黑素瘤细胞、斑马鱼胚胎作为模型,通过体外实验和在体实验两种方式,系统研究菟丝子对黑素细胞色素代谢的作用机制,并借助高效液相色谱法分析WFSC、 EFSC成分的差异。
1.在体外实验,本论文首先以蘑菇酪氨酸酶为研究对象,探讨菟丝子提取物WFSC、EFSC对蘑菇酪氨酸酶活性的作用机理；以粗制的B16黑素瘤细胞酪氨酸酶为研究对象,比较菟丝子不同提取物对这两种酪氨酸酶活性的作用。再将WFSC、EFSC与B16黑素瘤细胞共同孵育,研究其对细胞增殖、酪氨酸酶活性、黑色素合成以及对酪氨酸酶mRNA、蛋白表达的影响,相关研究内容及结果详述如下：
1)分别比较了WFSC、EFSC对蘑菇酪氨酸酶活性的作用。研究结果表明WFSC对蘑菇酪氨酸酶具有抑制作用,EFSC对蘑菇酪氨酸酶则有激活作用。对WFSC, EFSC的作用机理研究显示,二者与蘑菇酪氨酸酶的结合均为可逆性,通过酶动力学实验测试,WFSC对蘑菇酪氨酸酶的活性的抑制作用表现为反竞争型抑制剂,即主要通过与蘑菇酪氨酸酶-底物复合物结合发挥作用；与蘑菇酪氨酸酶相比,WFSC对B16黑素瘤细胞酪氨酸酶提取液的抑制效果相一致。与WFSC表现出抑制作用不同的是,EFSC对蘑菇酪氨酸酶、B16细胞酪氨酸酶提取液体系中均表现为激活作用。
2)研究WFSC、EFSC对B16黑素瘤细胞增殖、细胞内酪氨酸酶活性、黑色素含量的影响。结果表明浓度为0.5～20mg/mL时,WFSC、EFSC对B16细胞增殖均无影响。将WFSC、EFSC配制成不同浓度与B16黑素瘤细胞共同孵育48h后测定细胞内酪氨酸酶的活性。与IBMX作用下的空白对照组比较,WFSC作用下的B16瘤细胞TYR活性有统计学差异(F=31.726, P=0.000), WFSC对酶活性表现为抑制作用,当WFSC>5mg/mL时,差异均有统计学意义(P<0.05),WFSC中剂量组和高剂量组间差异无统计学意义(P=0.784)。与空白对照组比较,EFSC B16黑素瘤细胞酪氨酸酶的活性表现出激活作用(F=8.259,P=0.008),在5～10mg/mL时对,差异均具有统计学意义(P<0.05),但中剂量组与高剂量组间差异没有统计学意义(P=1.000)。与空白对照组比较,WFSC等药物组作用下的B16瘤细胞黑素含量差异有统计学意义(F=9.733,P=0.001), WFSC可下调酪氨酸酶黑素合成量,当WFSC>5mg/mL时,差异均有统计学意义(P<0.05), WFSC中剂量组和高剂量组间差异无统计学意义(P=1.000)。与空白对照组比较,EFSC对B16黑素瘤细胞黑素合成表现出激活作用(F=12.077,P=0.002),在5～10mg/mL时差异均具有统计学意义(P<0.05), EFSC中剂量组与高剂量组间差异没有统计学意义(P=0.18)。
3)研究VFSC、EFSC对TYR的mRNA和蛋白表达的影响,结果表明,WFSC、EFSC对TYR mRNA表达差异无统计学意义(P>0.05)；同样,二者对TYR蛋白合成差异无统计学意义(P>0.05)。
2.在体实验中,以斑马鱼为模型,以熊果苷、对羟基苯甲醚(MQ)为阳性对照,观察WFSC、EFSC对斑马鱼黑素细胞的作用,并通过PTU干预,初步探究菟丝子对斑马鱼黑素细胞发挥作用的机制；检测各受试物对斑马鱼黑素细胞酪氨酸酶活性、黑色素合成的影响,相关研究结果如下：
0.1mg/mL～2mg/mL的EFSC对斑马鱼胚胎生存率没有影响；药物浓度>3mg/mL可延迟斑马鱼胚胎的发育,且随着药物浓度的增高,其生成率降低。经观察发现,WFSC、EFSC均不影响斑马鱼黑素细胞的迁移和分布。较高浓度WFSC作用下可见斑马鱼黑素细胞呈点状改变,与对照药MQ的表现相同,这一现象在加药后6h即可观察到,另一阳性对照药熊果苷也可以使斑马鱼黑素细胞出现同样的改变,但于加药后约20h方出现变化,较WFSC出现形态学改变的时间晚；EFSC对斑马鱼胚胎黑素细胞则没有明显的改变。PTU是酪氨酸酶阻滞剂,在PTU干预下,WFSC、MQ对黑素细胞的作用消失,但熊果苷仍可使黑素细胞形态发生改变,这一作用差异可能因WFSC、熊果苷对酪氨酸酶的抑制作用方式不一样有关。
2)提取斑马鱼胚胎蛋白后测定酪氨酸酶活性,WFSC作用下斑马鱼黑素细胞TYR活性差异有统计学意义(F=13.006,P=0.000)。与空白对照组相比较,WFSC浓度为lmg/mL～3mg/mL时对斑马鱼黑素细胞酪氨酸酶表现出抑制作用,差异均有统计学意义(P<0.05),各浓度组间差异均无统计学意义(P>0.05)。WFSC、熊果苷、MQ对酪氨酸酶的作用差异无统计学意义(P>0.05)。WFSC作用下,斑马鱼黑素细胞黑色合成作用差异有统计学意义(F=16.474,P=0.000)。与空白对照组相比较,WFSC浓度为lmg/mL～3mg/mL时对斑马鱼黑素合成表现出抑制作用,差异均有统计学意义(P<0.05),各浓度组间差异均无统计学意义(P>0.05)。WFSC为3mg/mL时,与熊果苷、MQ相比,对黑素合成的作用差异均无统计学意义(P>0.05)。EFSC对斑马鱼黑素细胞酪氨酸酶活性未表现出影响(F=0.831,P=0.535),不同浓度EFSC对斑马鱼胚胎黑素合成形成的差异无统计学意义(F=0.596,P=0.674)。
3.高效液相色谱法对WFSC、EFSC的成分分析：
在实验过程中我们发现,WFSC在提取初期对酪氨酸酶没有抑制作用,反而表现出轻微的促进作用,至其提取后1个月方表现出明显的抑制作用,抑制效果稳定并维持直至提取后6个月；而EFSC自提取后即对酪氨酸酶活性表现出稳定的激活作用。参考苏薇薇等建立的实验方法,本研究对不同时期WFSC、 EFSC进行了高效色谱法分析,相关结果如下：
1) WFSC、 EFSC的HPLC图谱有明显的差异,WFSC在提取后2周内峰6逐渐低平直至消失,而其提取1个月后与6个月后的HPLC图差异不大,这与WFSC药效变化的时间拐点相符合；与WFSC不同的是,EFSC的HPLC图谱随时间变化差异不大,峰5是EFSC的特有峰,菟丝子主要由多酚类化合物组成,其中含有丰富的黄酮类成分,根据流动相设置条件及出峰时间判断,峰5可能为黄酮类化合物。
2) WFSC对斑马鱼黑素细胞黑色素合成有抑制作用,为研究WFSC在斑马鱼作用下成分的变化,对经或不经斑马鱼胚胎作用的含有WFSC的培养液进行分析,发现经斑马鱼胚胎作用后,23min峰高明显下降,而39min峰高明显升高,推测此两种化合物含量的变化可能与WFSC对斑马鱼胚胎黑素细胞的抑制作用相关。
结论
1.不同的提取方法下,菟丝子的成分变化差异明显,其中水提物表现为抑制作用,醇提物表现为激活作用,菟丝子对黑素代谢的这种双向作用可能是由于各提取部位的成分不同引起的；
2.斑马鱼胚胎在菟丝子对黑素代谢的研究中与其它模型表现出较好的一致性,且更为直观,可应用于中医药对黑素代谢的实验研究。
讨论
通过本课题的研究,体外试验和在体实验结果均表明菟丝子水提物WFSC对黑色素合成具有抑制作用,经蘑菇酪氨酸酶检测,WFSC为反竞争型抑制剂,表明其是通过酪氨酸酶-底物复合物发挥抑制作用的；WFSC对B16黑素瘤细胞及斑马鱼胚胎黑素细胞生物学功能的作用一致,即通过降低酪氨酸酶活性下调黑素合成量,WFSC作用下斑马鱼胚胎黑素细胞的形态发生改变,与熊果苷作用效果相似,但二者的起效时间不同。醇提物EFSC在细胞外酪氨酸酶模型及B16黑素瘤细胞实验中对酪氨酸酶活性及黑色素合成表现出激活作用,然而在斑马鱼黑素细胞却无明显影响,这可能因斑马鱼胚胎发育早期的黑素细胞酪氨酸酶活性本身较高有关,在斑马鱼胚胎发育的早期(12hpf-5dpf),酪氨酸酶活性高于其成熟的黑素细胞,因而EFSC的激活作用无从体现。HPLC图谱表明WFSC的变化与其药效发生改变的时间变化相一致；而峰5为EFSC的特有峰,根据色谱条件及峰5所在的位置,且其紫外吸收图与槲皮素对照品相似,推测其可能为母核与槲皮素相似的黄酮类化合物。WFSC经斑马鱼胚胎作用前后的HPLC图谱显示其组成有明显变化,23min峰明显降低,39min峰明显升高,二者改变是否与WFSC的抑制作用有关?峰5是否是EFSC发挥药效的关键组成?峰6是否为WFSC作用产生变化的关键峰?这些问题尚未解决,有待课题组进一步的深入研究。
创新及展望
1)本课题因菟丝子对黑素代谢作用描述不清给临床用药带来困惑这一现实问题的目的,以不同模型为对象,研究菟丝子对黑素代谢的作用,实验证实菟丝子对黑色素代谢的作用具有双向性,发现其药效差别的关键因素可能是因提取方法不同导致的有效成分不同,其中水提取产物WFSC对黑素合成具有抑制作用,醇提取物EFSC对细胞内的色素合成有激活作用。
2)首次发现WFSC对黑素合成的抑制作用存在时间拐点,推测与其组成成分发生变化有关。
3)本课题首次将斑马鱼胚胎用于中医药对黑素细胞作用的研究,与其他模型相比更为直观、便利,而且有较好的一致性。
综上所述,本课题具有一定的创新性及科学意义。然而,本课题未能展开菟丝子复方及与不同功效中药配伍对黑素代谢影响的研究,临床应用中药调控色素实践的诸多问题,仍需进一步的探讨。
Pigmented skin disorders (such as vitiligo, melasma) is a common kind of clinic skin disease, which bring shame and enormous psychological pressure to the patient.It has long been the medical community's attention since ancient. And in Asia, the requirements of normal population for skin whitening and other cosmetic are also increasing, from nature to find a safe and effective whitening agent is already a hot topic in the cosmetics industry. With the advent of molecμLar biology techniques and the rapid development of different models, melanin metabolism research has made great progress. The role of Chinese medicine in melanin metabolism has accumulated abudndant research resμLts. However, in the research part of Chinese medicine for melanin metabolism, due to inconsistency of the selected model, or a different extraction methods drugs, traditional Chinese medicine for melanin metabolism sometimes appear inconsistent results, and its mechanism is rarely discussed in depth.
As one of the oldest drug in the treatment of skin pigmentation disorders, Cuscuta seed was recorded in "Shen Nong's Herbal Classic " and described as "juice, get rid of "gan","gan" means dark spots, namely haze shading.However,Cuscuta chinese seeds had not been recorded as the treatment of vitiligo in the ancient books.In the modern times, the function of cuscutae attended national standard records for the first time in1963edition of "Chinese pharmacopoeia", described as: liver or kidney, Yijing, improving eyesight,attending sore waist and knee, spermatorrhea, eye shadow, frequent urination. Topical effect of Cuscutae chinese seeds in vitiligo had not mentioned, since the beginning of1985, after each revision, all revisions, expressing ideas have increased "outside cure vitiligo" standard of attending, but efficacy expressed as "nourishing liver and kidney, solid essence shrinks the urine, fetus, improving eyesight, antidiarrheal", failing to provide theoretical basis for the external treatment of vitiligo. Literature retrieval showing that experimental study abroad believe Cuscutae chinese seeds can inhibit melanin synthesis,and be grace used in the treatment of skin diseases.It hasn't note recorded that Cuscutae seeds can promote the synthesis of melanin. Correspondingly, since the modern times, the domestic study of Cuscutae in chromatodermatosis treatment is less, its application in internal or outside party to cure vitiligo is more common. According to the literature, Cuscutae seeds play a regulatory role in the study of melanin synthesis and inhibit melanin synthesis.However, the mechanism of cuscuta seeds action, there has no further research. Whether the seeds has two-way regulating melanin metabolism role in melanin synthesis? The regulating mechanism is what? As an effective medicinal herbs,Cuscuta seeds were widely used in pigment barrier skin disease in clinical treatment, it was often used in the treatment of performance opposite two kinds of pigment barrier skin disease, but its role has no definite way, which may bring to clinical application a certain confusion. We believe that the research on the mechanism of action of Cuscuta chinese seeds has further important significance.
Model pigment metabolism can be divided into three categories:cell-model test systems, test cell model system, animal model test systems. Mushroom Tyrosinase is an enzyme inhibition studies on the mechanism of the test drug most commonly used non-cell test model, there is no need to test the advantages of enzymatic cell culture, is simple and fast acting type of the test substance can make a preliminary determination, the disadvantage is system is too simple, the degree of inhibition of the test substance alone tyrosinase activity to determine its effects seem one-sided; cell test system can react in different receptor gene expression changes, to some extent, make up for the deficiencies of mushroom tyrosinase. The most commonly used mouse B16melanoma cells are cells whose genetic makeup of human epidermal melanocytes have high similarity, compared to more accessible than the latter, training, test substance is used to test the melanocytes one of the biological function of the preferred model. Animal testing system includes C57mice, hairless mice, guinea pigs, Smyth line chicken, zebrafish recognized as one of the five International Organization for Standardization species of fish model organisms, with human melanoma cells and similar melanosomes. In recent years, more and more are used to melanin metabolism studies in the field, because the whole body transparent and easily identify the individual pigment cells during development made observation easily. And with the cell labeling technique, the target gene-directed mutagenesis techniques, genetically modified technologies, such as gene overexpression of mature development, development of melanocytes, the pigment metabolism-related modeling, high-throughput drug screening have been a large number of applications.Addition, zebrafish are hypothermia and hypoxia fish, its cold resistance and resistance thermal resistance are strong, easy feeding, spawning large, compared with the previous several animal models have unique advantages, there are an irreplaceable position in the relevant research in melanocytes. Although zebrafish vascular system in Chinese medicine, Traditional Chinese medicine toxicology studies have been applied, but the advantages of the model as melanin metabolism studies in zebrafish on the role of research in medicine melanin synthesis has not yet been reported.
The porpuse of this study was to clear Cuscuta chinesis seeds of melanin metabolism biphasic regulation mechanism; To explore the zebrafish model in the role of melanin metabolism experiment of traditional Chinese medicine.
The experiment contents and results of this study recorded as follows:Cuscuta seeds clinical use of dosage forms are mainly water decoction or tincture. According to the traditional medication, this topic will use the water and95%ethanol reflux extraction and respectively made the water fraction from Semen cuscutae (WFSC), the ethanol fraction from Semen cuscutae (EFSC). Choose the mushroom tyrosinase, tyrosinase of B16melanoma cells extract, B16melanoma cells, zebrafish embryos as the model. Through the experiment in vitro and in vivo experiment, system research the mechanism of action of cuscutae on melanin cell metabolism of pigment. With the analysis of High-performance liquid chromatography fingerprinting of WFSC and EFSC, to compare the composition differences.
1.In vitro experiment, this paper first using the mushroom tyrosinase as the research object, explored the Cuscutae seeds extract WFSC, EFSC mechanism of mushroom tyrosinase activity.With rough B16melanoma cells tyrosinase as the research object, comparison of different extracts of the Cuscutae seeds effect on tyrosinase activity. Then the B16melanoma cells incubated in WFSC, EFSC, detected the research on cell proliferation, tyrosinase activity and melanin synthesis and tyrosinase mRNA and protein expression, the influence of the related research contents and results are described below:
1) Compare the WFSC, EFSC role of mushroom tyrosinase activity respectively. Results indicate that WFSC has inhibitory effect on mushroom tyrosinase, through the enzyme kinetics experiment test WFSC, EFSC mechanism of enzyme. According to the results that both the combination of WFSC, EFSC and mushroom tyrosinase were reversible. Inhibition of WFSC showed the type of competitive inhibitor, which means that it work with mushroom tyrosinase substrate complex combination. Compared with mushroom tyrosinase, WFSC consistently inhibited the tyrosinase extract of B16melanoma cells. With WFSC showed an obvious inhibition, EFSC showed an activation function on mushroom tyrosinase and B16cells in the extraction of tyrosine enzyme system.The activation of EFSC, effect on the enzyme kinetics showed the mechanism of mixed activation.
2) With study the role of WFSC, EFSC on B16melanoma cell proliferation rate, cell tyrosinase activity and melanin content. Results showed that WFSC, EFSC had no significant effect on the proliferation of B16cells. At the concentration>5mg/mL, WFSC showed an inhibition function on the intracellular enzyme of tyrosinase. At the concentration10mg/mL,the inhibition effect of which similar to that of50ug/mL arbutin.While EFSC showed an obvious activation function on enzyme activity,both of which worked dose-dependent effect on B16melanoma cell. Consistenting with the role of the activity of tyrosinase, WFSC inhibits melanin synthesis, and EFSC can promote the synthesis of melanin. According to the results of Western blot, QRT PCR,neither WFSC nor EFSC effect on tyrosinase mRNA and protein expression.
2. In vivo experiments, taking zebrafish as the model, with arbutin, Methoxyphenol (MQ) as the positive control, observated the effect of WFSC, EFSC on Zebrafish melanocyte. The study detected various subjects:including melanocyte tyrosinase activity and melanin synthesis. Under the PTU intervention, to explore the mechanism of Cuscutae seeds on zebrafish melanin cell function, related research results are as follows:
1)0.1mg/mL～2mg/mL WFSC, EFSC had no effect on zebrafish embryo survival rate. When drug concentration>3mg/mL, extracts would delay the development of zebrafish embryos. As the drug concentration increased, Livability of Zebrafish embryo is reduced.According to the observation we found that24hpf-5dpf was the best working time for the research of zebrafish melanocyte tyrosinase activity of optimal detection period. Larval melanocytes with punctate melanocyte morphology under the action of WFSC,which were same as the control medicine MQ performance. This phenomenon at6h after dosing can be observed that the positive control drug arbutin can also make the same change on the zebrafish melanocyte, but afer the it working20h change, later than onset time of the WFSC. After incubated with EFSC,there is no obvious change on zebrafish embryos melanocyte. Determinated the tyrosine enzyme activity with the extraction of zebrafish embryo protein, the results revealed that WFSC could inhibit tyrosinase activity of zebrafish embryo, reduced the melanin synthesis. While zebrafish embryo incubated in EFSC, zebrafish tyrosinase activity and pigment synthesis of melanocyte are similar with the blank controller. PTU is tyrosinase blockers, under the PTU intervention, the effect of WFSC, MQ on melanin synthesis disappeared, but arbutin could still make the melanocyte with punctate melanocyte morphology, the role differences might because of WFSC and arbutin on tyrosinase inhibitory effect way were different, WFSC as competitive inhibitors, While arbutin as competitive inhibitors.
2.High performance liquid chromatography (HPLC) method for WFSC,EFSC composition analysis:
We found that in the process of experiment,WFSC of the early extraction period didn't show an inhibition effect on tyrosine enzyme, but showed slight promoting effect. However,1month after WFSC extracted,it showed an obvious inhibitory effect, which maintained the inhibition effect stably even6months after extraction. It's intersting that EFSC kept a stable activation function on the tyrosine enzyme since the extraction of EFSC was prepared. According to the chromatography analysis experimental methods of Su Weiwei established, the study determined the difference between WFSC and EFSC during different periods, the results are as follows:
1) High performance liquid chromatography finger print of WFSC, EFSC had obvious difference. Peak6of High performance liquid chromatography finger print of WFSC within2weeks after extracting became lower day by day until it disappeared, but then WFSC extraction kept the similar high performance liquid chromatography finger print from1month to the6month. Its change of time corresponds with the change of WFSC efficacy.Unlike WFSC, EFSC kept the similar High performance liquid chromatography finger print during the whole experiment process. Peak5are the characteristic peak of EFSC.Considering that Cuscuta chinensis seeds are mainly composed of polyphenols and flavonoids, according to the mobile phase condition, peak5was judgmented as one kind of flavonoids.
2) WFSC showed an obvious inhibitory effect on zebrafish melanocyte melanin synthesis. To compare the change of composition of WFSC consumed with or without Zebrafish, we analyzed the WFSC culture medium with the role of zebrafish embryos.It's found that after zebrafish embryos consumption,23min peak of WFSC culture medium decreased obviously, and39min peak rised obviously.It's speculated that the change of the two compounds may be concerned with WFSC melanocyte inhibitory effect on zebrafish embryos.
Through this topic research, in vitro and in vivo experimental results show that the Cuscuta chinensis seeds water extract WFSC has inhibitory effect on melanin synthesis, by the mushroom tyrosinase, WFSC play a role in the form of competitive inhibitors, suggesting that it worked through the tyrosinase-substrate compounds and play an inhibitory effect on melanin synthesis. The role of WFSC on B16melanoma cells and biological functions of zebrafish embryos melanocyte is consistent, namely cut melanin synthesis by lowering the activity of tyrosinase.Zebrafish embryos under WFSC effects showed the action of melanocyte morphological changes, the effects of which are similar with arbutin, but the onset time is different. Zebrafish embryo incubated with WFSC showed the change within6h, but showed the same changes after20h while incubated with arbutin, the difference may be associated with inhibition mechanism of WFSC and arbutin,which is different from each other. Because arbutin also play an inhibiton role on tyrosinase single phenol enzyme inhibition, which can prolong the lag time of response. EFSC showed obvious activation tyrosine on tyrosinase activity and melanin synthesis either on the enzyme model outside the cell or the experiment of B16melanoma cells. However it had no obvious effect on zebrafish melanin cell morphological changes. This may be due to the hightyrosinase activity itself in the early development of zebrafish embryos of melanocyte, EFSC activation cannot reflect on it. It was reported that in the early developed time of zebrafish embryo (12hpf～5dpf) tyrosine enzyme activity is much higher than the mature melanocyte. High performance liquid chromatography finger print showed that the time change of WFSC consistent with its efficacy change. And peak5is the characteristic peak for EFSC. According to the chromatographic conditions and5location, and the ultraviolet absorption figure is similar to quercetin reference substance, We preliminary judgment for the structure similar to that of quercetin flavonoids. Through the zebrafish embryo comsumed, HPLC of WFSC showed that the composition has obvious change before and after, wether the changes of39min peak and23min peak associated with the inhibition of WFSC? Weather Peak5play efficacy of key in the role of EFSC? If the peak6is the key peak of WFSC changes? All of the problems needs to be researched further in-depth study.
Discussion
This topic worked on the problem that the effect of Cuscuta chinensis seeds on melanin metabolism had not been described clearly in the clinical medicine.The purpose of this study was to explain the role of Cuscuta chinensis seeds on metabolism of melanin. Using different models as the object, we found that the key factors of Cuscuta chinensis seeds with bidirectional regulation of melanin metabolism was the different extract method. The water fraction from Semen cuscutae has inhibitory effect on melanin synthesis, and95%ethanol fraction from Semen cuscutae has the effect of the activation on melanin synthesis. Both of them worked through tyrosinase.Neither of them have effect on the expression of tyrosinase. It was the first time that we discovered WFSC in inhibition of melanin synthesis with time turning point, as well as its composition was changed; This topic first determinated that zebrafish embryos can be used to study the role of melanin synthesis of traditional Chinese medicine, which compared with other models has good consistency. To sum up, this topic has certain innovative and practical application value.
Conclusion
1)Under different extraction methods, the different effect may caused by the difference composition. It showed that WFSC inhibited while EFSC show an activation function. The two-way role of Cuscuta on melanin metabolism may be due to the different component parts of each extract.
2) Zebrafish embryos in our research were consist with other models, which were more intuitive, the experimental study showed that Zebrafish could be used in correlational study of TCM.
Innovation and Prospects
1) WFSC inhibit melanogenesis, the EFSC activated the intracellular pigment synthesis. While the precise factor of Cuscutae in the metabolism of melanin is unclear.
2) It was first discovered the inhibition function of WFSC on the melanin synthesis depend on the time of inflection, presumably related to its composition changes.
3) It's the first time in zebrafish embryos for research on the role of TCM on melanin cells, compared with other models which was more intuitive, convenient.
引文
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