Assessment Of Cuscuta Chinensis Seeds’ Effect On Melanogenesis In Vitro And In Vivo

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.DiscussionThis 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.Conclusion1)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 Prospects1) 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.