XJIPC OpenIR  > 资源化学研究室
驱虫斑鸠菊治疗白癜风有效部位的研究
李浩榕
学位类型博士
导师阿吉艾克拜尔·艾萨
2015-06-01
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词驱虫斑鸠菊 白癜风 有效部位 黑素合成 抗炎 白癜风动物模型 Nf-κb信号通路 Pka信号通路
摘要

白癜风是一种常见的后天性色素脱失性疾病,主要特征为皮肤和毛发黑素细胞选择性破坏导致的色素脱失。维吾尔医药治疗白癜风有悠久的历史,且疗效显著,驱虫斑鸠菊是常用治疗方剂中的主要药材。为阐明驱虫斑鸠菊治疗白癜风的科学依据,我们开展了如下研究: 1.驱虫斑鸠菊治疗白癜风有效部位的筛选及制备工艺研究 以B16黑素瘤细胞黑素含量及酪氨酸酶的活性为指标,比较水及不同浓度乙醇作为溶剂的驱虫斑鸠菊提取物的促黑素合成活性,发现40 %乙醇提取活性最佳;比较12种大孔吸附树脂纯化后产物的促黑素合成活性,发现HPD300大孔树脂效果最佳。经过40 %乙醇水提取,HPD300大孔吸附树脂纯化,1.6 kg驱虫斑鸠菊种子可得到有效部位68.2 g,提取率为4.3 %。吸光光度法测定显示,该部位中酚酸含量为61 %。UPLC外标法测定表明,3,4-O-二咖啡酰奎宁酸含量为2.9 %,3,5二咖啡酰奎宁酸含量为6.8 %,4,5二咖啡酰奎宁酸含量为7.1 %。其中,3,5二咖啡酰奎宁酸和4,5二咖啡酰奎宁酸属该植物中首次发现。 在此基础上利用正交试验和单因素实验筛选,得到了有效部位的最佳制备工艺:驱虫斑鸠菊种子粉碎后,提取溶剂为60 %乙醇水,料液比1:20,80 ℃回流提取0.5 h,共3次,减压浓缩除醇,调整上样液生药浓度100 g/L,大孔树脂柱径高比为1:8,吸附流速2 BV/h,上样体积1 BV,洗脱流速6 BV/h,2 BV水洗除杂,洗脱溶剂50 %乙醇,洗脱体积4 BV,收集洗脱液,干燥。 2.驱虫斑鸠菊治疗白癜风有效部位的药效学及作用机制研究 动物急性毒性实验结果表明驱虫斑鸠菊有效部位无毒。C57BL/6小鼠氢醌造模20天后,造模成功。从第21天开始,氢醌造模同时,药物处理组灌胃给药。第50天,处死小鼠。肉眼观察及组织切片发现,模型组小鼠造模区域皮肤出现白斑,新生毛发变成白色,皮肤明显变薄,棘细胞层可见水肿,毛囊数量少,且几乎不含黑色素,毛囊中黑素细胞数量显著降低。白癜风胶囊(阳性对照)和驱虫斑鸠菊有效部位能显著恢复模型小鼠的皮肤颜色以及组织形态。在相同临床等效剂量下,驱虫斑鸠菊有效部位疗效更优。生化检测发现,相较模型组,驱虫斑鸠菊有效部位能恢复小鼠造模区域皮肤IL-2、IL-6、IL-10、IL-17、TNF-α、IFN-γ以及酪氨酸酶含量,降低血清中MDA(丙二醛)含量以及酪氨酸酶含量。免疫组化发现,驱虫斑鸠菊有效部位能增加毛囊中黑素干细胞数量,保护毛囊中成熟黑素细胞。有效部位具有减轻氧化损伤和免疫调节作用。 为明确驱虫斑鸠菊有效部位治疗白癜风作用机制,我们研究了其对黑素合成和免疫调节的作用途径。实验发现,有效部位主要通过PKA信号通路,增加B16黑素瘤细胞酪氨酸酶和酪氨酸酶相关蛋白1的表达,促进黑素合成。在RAW264.7巨噬细胞中,有效部位还可以通过抑制IκBα与NF-κB的磷酸化,降低IL-1、IL-6、TNF-α、iNOS和COX-2蛋白表达,抑制炎症反应。 3.酚酸类化合物3-O-咖啡酰奎宁酸对黑素合成作用的研究 3-O-咖啡酰奎宁酸是驱虫斑鸠菊种子的化学成分。3-O-咖啡酰奎宁酸作为酪氨酸酶的底物,并增加细胞黑素含量;另一方面,3-O-咖啡酰奎宁酸能够影响细胞或酶体系中酪氨酸酶的活性。在酪氨酸酶反应体系中,3-O-咖啡酰奎宁酸或有效部位都可作用于酪氨酸酶,促进终产物产生。 综上所述,在白癜风小鼠模型中,驱虫斑鸠菊有效部位能够恢复造模区域皮肤组织形态及肤色,保护成熟黑素细胞,增加黑素干细胞数量,并发挥免疫调节作用,证实了驱虫斑鸠菊酚酸有效部位可用于治疗白癜风。在细胞研究体系中,驱虫斑鸠菊有效部位通过PKA信号通路促进B16小鼠黑素瘤细胞黑素合成;通过NF-κB信号通路抑制RAW264.7小鼠巨噬细胞的炎性反应。在蘑菇酪氨酸酶反应体系中,驱虫斑鸠菊有效部位可促进色素终产物产生,揭示了酚酸可能是驱虫斑鸠菊治疗白癜风中的有效成分。

其他摘要
Vitiligo is a common acquired depigmentation disorder, which is characterized by the selective loss of melanin cells resulting in depigmentation in skin and hair. There is a long history of treatment with vitiligo in the Uygur medicine. In the prescription, the Vernohia anthelmintica Willd is the main ingredient, and its curative effect is distinct. To clarify the mechanism of the V. anthelmintica treated with vitiligo, the following studies were conducted. 1. Effective parts screening and preparation. Taking tyrosinase activity and melanin contents of B16 melanoma cells as indexes, we found that 40 % ethanol was the best extraction solvent among different alcohol concentration, and HPD300 macroporous resin was the best material among 12 kinds of macroporous resin. After extracted with 40 % ethanol and purified by HPD300 macroporous adsorption resin, 68.2 g effective fraction could be gotten from 1.6 kg seeds of V. anthelmintica, the extraction yield was 4.3 %. The total phenolic acids content was determined as 61 % by spectrophotometer method. Using external standard method with ultra performance liquid chromatography, the contents of 3, 4-dicaffeoylquinic acid, 3, 5-dicaffeoylquinic acid and 3, 5-dicaffeoylquinic acid was determined as 2.9 %, 6.8 %, and 7.1 %, respectively. The latter two compounds were first discovered in V. anthelmintica. The orthogonal test and dynamic condition screening were used to optimize preparation technology of the effective fraction. The seeds of V. anthelmintica were smashed, extracted by 1:20 60 % ethanol. The reflux extraction was conducted at 80 ℃ for 3 times, 0.5 h every time. The optimal purification was dryness of the extraction dissolved and diluted to 100 g crude drug/L, subjected to the HPD300 macroporous resin column. The diameter-height ratio of column was 1:8. The flow rate was 2 BV/h, and solution treatment capacity was 1 BV. After washing with 2 BV water, 50 % ethanol was used as eluent solvent, and the eluent volume was 4 BV at the flow rate 6 BV/h. 2. Pharmacodynamics and mechanism of effective fraction treated with vitiligo The results of acute animal toxicity test showed that the effective fraction. After 20 days of treating with hydroquinone, C57BL / 6 mouse skin and hair showed the spots of depigmentation. The mice of drug-treated groups were smeared with hydroquinone and gave effective fraction between days 21 and 50. The results of naked eye and tissue sections indicated that modeling areas of skin and new hair turned white and thinner. Many edemas were visible in Prickle cell layer. The number of hair follicles and melanocytes were significantly reduced. Few melanin was observed in hair. The color and morphology of mice skin restored normal by Vitiligo capsules (positive control) and the effective fraction significantly. In the same clinical equivalent dose, the therapeutic effect of effective fraction was better. Results of biochemical tests showed that the effective fraction can increase contents of IL-2, IL-6, IL-10, IL-17, TNF-α, IFN-γ, and tyrosinase of modeling area in skin, while decreased contents of MDA and tyrosinase in serum. Immunohistochemistry results demonstrated that effective fraction could increase the number of melanocyte stem cells and protect hair follicles mature melanocytes in hair follicle. The effective fraction could relieve oxidative damage and inflammatory response. To clarify the mechanism of effective fraction in therapy of vitiligo, melanin synthesis and immune regulation were studied. In B16 melanoma cells, it was mainly through PKA signaling pathway that the effective fraction increased protein expression of tyrosinase and tyrosinase-related protein-1, and promoted melanin synthesis. In RAW264.7 macrophages, effective fraction inhibited the phosphorylation levels of IκBα and NF-κB, thus reduced IL-1, IL-6 TNF-α, iNOS and COX-2 protein expression, and inhibited inflammation. 3. Effect on melanin synthesis of 3-O-caffeoylquinic acid 3-O-caff
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4260
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
推荐引用方式
GB/T 7714
李浩榕. 驱虫斑鸠菊治疗白癜风有效部位的研究[D]. 北京. 中国科学院大学,2015.
条目包含的文件
文件名称/大小 文献类型 版本类型 开放类型 使用许可
驱虫斑鸠菊治疗白癜风有效部位的研究.pd(4843KB)学位论文 开放获取CC BY-NC-SA浏览 请求全文
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[李浩榕]的文章
百度学术
百度学术中相似的文章
[李浩榕]的文章
必应学术
必应学术中相似的文章
[李浩榕]的文章
相关权益政策
暂无数据
收藏/分享
文件名: 驱虫斑鸠菊治疗白癜风有效部位的研究.pdf
格式: Adobe PDF
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。