栀子黄色素对小鼠B16黑素瘤细胞增殖、酪氨酸酶活性及黑素生成影响的实验研究

发布时间：2018-03-19 09:23

本文选题：中药　切入点：中药色素　出处：《湖南中医药大学》2016年硕士论文　论文类型：学位论文

【摘要】：1.研究背景与意义色素异常性皮肤病可分为色素增加性皮肤病(如黄褐斑)和色素减退性皮肤病(如白癜风)两大类,其发病率呈不断上升趋势。由于色素异常性皮肤病主要影响人的外观,因此给患者造成较大的社会压力,亦明显降低患者的生活质量。色素异常性疾病的具体发生原因尚未明确,但与多种因素相关,如遗传、免疫、紫外线照射和内分泌失调等。西医药治疗色素异常性皮肤病药物种类较多,费用高且疗程长,给患者带来较大的精神压力和经济负担。中医药在治疗色素异常性皮肤病的过程中积累了丰富的经验,欧阳恒教授创立“以色治色”疗法,并研发创制紫铜消白方(紫铜、丹参、紫草、红花等多味紫红色药材)治疗白癜风获得了较好的临床疗效,并荣获1994年度国家中医药管理局科技进步奖。有些学者考虑中药本身的颜色与色素异常性皮肤病的治疗存在一定相关性,认为深色类中药对治疗白癜风等色素障碍性皮肤病有良好疗效,浅色类中药对治疗黄褐斑等色素增加性皮肤病疗效颇佳。杨柳教授[6]把这种表明药材颜色与疾病疗效相关的现象称之为“中药色象”。那么,单纯的中药的色素成分与色素异常性皮肤病之间是否存在直接相关性,目前尚未十分明确,相关研究报道亦偏少。因此,中药色素成分与色素异常性皮肤病之间的相关性是一个值得研究的问题。2.研究目的:2.1通过实验研究证明中药色素成分与人体皮肤色素代谢之间的相关性,直接证明中医“以色治色”理论的正确性,并提供有力的理论依据和实验依据。2.2为中医药治疗疾病提供新的治疗方向和研究领域,促进新药研发。2.3发掘出天然、绿色、无毒害的色素,拓展中药色素的应用范围,产生理想的社会效益和经济效应。3.研究方法3.1查阅关于治疗白癜风、黄褐斑等色素异常性皮肤病的中医药研究类文献,筛选出常用于治疗色素异常性皮肤病的中药材,根据中药外观的颜色将中药分为深色组(黑色、褐色、紫色、红色)和浅色组(白色、黄色)。在深色类中药组和浅色类中药组的每种色系中分别选取2种目前成分研究较为明确的中药色素进行实验研究。3.2以中药色素干预细胞,采用现代研究技术较为成熟的MTT法检测中药色素对细胞增殖的影响,多巴氧化法检测中药色素对酪氨酸酶活性的影响和NaOH裂解法测定其对黑素含量生成的影响。3.3统计学处理:运用配伍组设计方法,色素为处理因素,浓度为配伍因素。实验结果中计量资料以“均数标±准差“(x±s)表示,采用SPSS19.0软件包进行统计分析。进行单应变量方差分析的最小显著差异法(LSD)检验。4.研究结果4.1栀子黄色素作用于小鼠B16黑素瘤细胞的安全浓度为4mg/ml。4.2栀子黄色素对细胞增殖的影响实验结果12h:与阴性对照组相比,栀子黄色素在安全浓度范围内(0.25mg/ml~4mg/ml)对细胞增殖无明显抑制作用;24h:栀子黄色素溶液浓度在0.25mg/ml~1mg/ml范围内对细胞增殖无明显抑制作用;浓度在2mg/ml~4mg/ml范围内对细胞增殖有较明显的抑制作用,其抑制作用随栀子黄色素溶液浓度的增大而增加;48h:栀子黄色素溶液浓度为0.25mg/ml时对细胞增殖无明显作用;浓度在0.5mg/ml~4mg/ml范围内对细胞增殖有明显抑制作用,其抑制作用随栀子黄色素溶液浓度的增大而增加。4.3栀子黄色素对小鼠B16黑素瘤细胞酪氨酸酶活性影响栀子黄色素溶液在浓度为0.25mg/ml时对酪氨酸酶活性无明显作用,在0.5mg/ml~4mg/ml浓度范围内时对酪氨酸酶活性有明显的抑制作用。4.4栀子黄色素对小鼠B16黑素瘤细胞黑素生成影响栀子黄色素溶液在浓度为0.25mg/ml时,对黑素生成无明显抑制作用。栀子黄色素溶液在浓度0.5mg/ml~4mg/ml范围内时,对黑素生成具有明显抑制作用。4.5栀子黄色素对细胞外酪氨酸酶活性影响栀子黄色素溶液各浓度组OD值与阴性对照组OD值相比,其差异有明显统计学意义(P0.01)表明栀子黄色素对蘑菇酪氨酸酶的活性有显著的抑制作用,且随着栀子黄色素浓度的增加,酪氨酸酶活性抑制率增大,测定的IC50分别为50mg/ml,100mg/ml。结论1.细胞实验1.1栀子黄色素作用于小鼠B16黑素瘤细胞的安全浓度为4mg/ml。1.2栀子黄色素在12h时间点对细胞增殖无明显抑制作用,在24h、48h时间点,能够抑制小鼠B16黑素瘤细胞的增殖,其抑制作用呈浓度依赖性,即随栀子黄色素溶液浓度的增大其抑制作用越明显。1.3栀子黄色素能够抑制小鼠B16黑素瘤细胞酪氨酸酶活性,其抑制作用呈浓度依赖性,即随栀子黄色素溶液浓度的增大其抑制作用越明显。1.4栀子黄色素能够抑制黑素生成,其抑制作用呈浓度依赖性,即随栀子黄色素溶液浓度的增大其抑制作用越明显。2.细胞外实验栀子黄色素对蘑菇酪氨酸酶的活性有显著的抑制作用,且随着栀子黄色素浓度的增加,酪氨酸酶活性抑制率增大,测定的IC50分别为50mg/ml,100mg/ml。
[Abstract]:1. research background and significance of abnormal pigment skin disease can be divided into hyperpigmentation disease (such as chloasma) and hypopigmented skin (vitiligo) two categories, the incidence rate showed a rising trend. Due to abnormal pigmentation skin disease mainly affects the appearance of people, thus causing great social pressure to patients were also significantly lower the quality of life of patients. The specific causes of pigment abnormalities is not yet clear, but with many factors, such as genetic, immune, ultraviolet radiation and endocrine disorders. Western medicine treatment of pigmented skin disease abnormal many drugs, high cost and long course of treatment, great mental pressure and the economic burden for patients. Traditional Chinese medicine has accumulated rich experience in the treatment of abnormal pigmentation of skin diseases, Professor Ouyang Heng founded "by the color for color therapy, and created research white copper, copper consumption (Salvia miltiorrhiza Shikonin, safflower, multi flavor purple herbs) in the treatment of vitiligo has better clinical efficacy, and won the 1994 annual National Administration of traditional Chinese medicine science and technology progress award. Some scholars consider the treatment of traditional Chinese medicine itself abnormal color and pigment skin diseases there is a certain correlation, that the dark kind of traditional Chinese medicine has a good effect on the treatment of vitiligo. Pigmented skin disorder. The light of Chinese traditional medicine on the treatment of chloasma and hyperpigmentation disease has good curative effect. It shows that the medicine Professor Yang Liu [6] color associated with disease phenomenon known as the "Chinese medicine color like. So, whether there is a direct correlation between pigment pigment composition and abnormal skin disease pure Chinese medicine at present, is not clear, relevant research reports are few. Therefore, the correlation between the traditional Chinese medicine pigment pigment composition and abnormal skin disease is a problem worthy of study.2 Objective: 2.1. Through the experimental study proved the correlation between traditional Chinese medicine and pigment composition of human skin pigment metabolism, direct proof of correctness of traditional Chinese medicine to cure color color "theory, and provides a strong theoretical basis and experimental basis for.2.2 provides a new treatment direction and research in the field of Chinese medicine for the treatment of diseases, promote new drug research and development.2.3 discover the natural, green, non-toxic pigment, expand the scope of application of traditional Chinese medicine pigment, social benefit and economic effect of the.3. generation of ideal 3.1 consulting on the treatment of vitiligo, traditional Chinese medicine research literature of pigment chloasma and other abnormal skin disease, were often used in the treatment of pigmented skin disease abnormal herbs according to the traditional Chinese medicine, Chinese medicine will be divided into the appearance of the color of dark (black, brown, purple group, red light) and group (white, yellow). In the dark class Chinese medicine group and Chinese medicine group each kind of light color In traditional Chinese medicine were selected 2 kinds of components of pigment is clear for experimental study of.3.2 with traditional Chinese medicine intervention by pigment cells, modern research technology influencing pigment MTT assay on cell proliferation of mature Chinese medicine, traditional Chinese medicine pigment DOPA oxidation detection effect on tyrosinase activity and cleavage of NaOH method for the determination of its impact on the formation of melanin content.3.3 statistics processing: using the design method of compatibility group, pigment treatment, concentration of compatibility factors. The experimental results of the measurement data to mean + standard deviation standard "(x + s), analyzed by SPSS19.0 software. The single variable ANOVA least significant difference method (LSD) the results of.4. test in the safe concentration of B16 murine melanoma cells 4.1 Gardenia Yellow Gardenia Yellow effect of 4mg/ml.4.2 on cell proliferation effect of experimental results of 12h: and negative control Compared the gardenia yellow pigment in the safe concentration (0.25mg/ml~4mg/ml) had no significant inhibitory effect on the proliferation of 24h: cells; gardenia yellow pigment concentration in the range of 0.25mg/ml~1mg/ml on cell proliferation is not inhibited; the concentration in the range of 2mg/ml~4mg/ml on cell proliferation was restrained significantly, with the inhibition of gardenia yellow pigment solution the concentration increases with the increase of the concentration of 48h: solution; gardenia yellow is no obvious effect on the proliferation of 0.25mg/ml cells; the concentration has obvious inhibitory effects on cell proliferation in the range of 0.5mg/ml~4mg/ml, the inhibitory effect of gardenia yellow pigment with the concentration increasing and the increase of.4.3 of gardenia yellow on mice B16 melanoma cells tyrosinase activity effect of gardenia yellow pigment solution at the concentration of 0.25mg/ml had no obvious effect on tyrosinase activity, 0.5mg/ml~4mg/ml concentration in the range of tyrosine The enzyme activity significantly inhibited.4.4 of gardenia yellow on melanogenesis in B16 melanoma cells of gardenia yellow pigment solution at the concentration of 0.25mg/ml, generation of black pigment is not inhibited. Gardenia yellow pigment solution concentration in the range of 0.5mg/ml~4mg/ml, the melanogenic has obvious inhibitory effect on.4.5 of gardenia yellow the extracellular activity of tyrosinase effect of Gardenia yellow pigment solution with different concentration group was compared with the negative control group OD value, there was significant difference (P0.01) showed that the inhibition activity of gardenia yellow on mushroom tyrosinase is significant, and with the increasing concentration of gardenia yellow, tyrosinase activity inhibition rate increase, determination of IC50 respectively. For 50mg/ml, the safe concentration in B16 murine melanoma cells 100mg/ml. cells in experiment 1.1 conclusion 1. Gardenia Yellow Gardenia Yellow role for 4mg/ml.1.2 in 12h Time no obvious inhibition on cell proliferation in 24h, 48h time point, can inhibit the proliferation of B16 murine melanoma cells, the inhibitory effect was concentration dependent, with the increase of solution concentration of gardenia yellow pigment in the more obvious inhibition of gardenia yellow.1.3 can inhibit B16 melanoma cells tyrosinase activity in mice and its inhibition in a concentration dependent manner, with the gardenia yellow pigment concentration increased more obvious inhibition of gardenia yellow.1.4 can inhibit melanin production, its inhibitory effect in a concentration dependent manner, the inhibition increased significantly inhibited the more obvious.2. cell experiment of gardenia yellow on mushroom tyrosinase activity with gardenia yellow in the solution concentration, and with the increasing concentration of gardenia yellow, tyrosinase activity inhibition rate increase, determination of IC50 were 50mg/ml, 100mg/ml.

【学位授予单位】：湖南中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R285.5

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