【作者】 王晓华；

【导师】 朱华；

【作者基本信息】 成都中医药大学 ， 中药学， 2013， 博士

【摘要】 桂郁金为广西莪术(Curcuma kwangsiensis S.G.Lee et C.F.Liang)的干燥块根。是《中国药典》收录的四种郁金药材来源之一。广西为广西莪术的道地产区,全国60%的桂郁金来自该产区。本论文主要以广西桂郁金为研究对象,探讨了桂郁金的高效液相色谱(High performance liquid chromatography, HPLC)指纹图谱、药理作用及其药效物质基础。1主要研究方法(1)利用HPLC分析技术对广西道地产区的10批次桂郁金样品进行比较分析,采用国家药典委员会推荐的“中药指纹图谱计算机辅助相似度评价软件”来进行模式分析及相似度计算,建立了桂郁金HPLC特征指纹图谱；(2)对桂郁金及对照药材人参、黄芪、刺五加进行抗脊髓损伤的筛选。从SD大鼠胚胎(妊娠17天)的大脑皮质分得神经细胞,采用原代培养的方式,进行体外细胞培养实验：应用磷酸化神经丝H (Phosphorylated neurofilament, pNF-1抗体(轴突的标记)和微管相关蛋白2a,2b (Microtubule associated protein2, MAP2a&2b)抗体(树突的标记)进行免疫染色；采用显微摄影及图像分析技术,定量测得神经细胞轴突和树突的长度。然后采用硫酸软骨素蛋白(chondroitin sulfate proteoglycan, CSPG)抑制剂对初筛结果进行复核实验,并进一步将初筛具有活性的药材采用系统溶剂提取法进行提取,对体外筛选出的活性部位采用柱色谱及制备色谱法进一步进行系统的化学成分分离和纯化,运用质谱(Mass spectrometry, MS)、核磁共振氢谱(Nuclear magnetic resonance spectroscopy of1H,1H-NMR)、核磁共振碳谱(Nuclear magnetic resonance of13C,13C-NMR)、异核多量子相关二维谱(Heteronuclear multiple quantum coherence,HMQC)、异核多键相关二维谱(Heteronuclear Multiple Bond Correlation,HMBC)等方法进行结构鉴定。(3)采用噻唑蓝法检测桂郁金不同部位对宫颈癌Hela细胞的生长增殖抑制作用,找出了对癌细胞具有抑制作用的活性部位：通过免疫荧光染色观察细胞自噬出现：通过Western blotting检测微管相关蛋白(Light chain3,LC3)蛋白的转化情况,探讨桂郁金活性部位提取物对细胞自噬的影响;检测桂郁金诱导宫颈癌Hela细胞自噬的可能的机制。2研究的结果(1)建立了桂郁金HPLC指纹图谱,从指纹图谱中检测出12个共有特征峰,10批郁金HPLC指纹图谱中,各批次样品间共有指纹峰的相对保留时间吻合,10批样品具有较高的相似度(>0.9)；10批次桂郁金的指纹图谱中各特征峰得到了较好的分离,具有特征性和唯一性。(2)在原代培养的初筛中,与其它三种对比药材人参、黄芪、刺五加相比,桂郁金的水提取物显示较强的活性；在CSPG实验中,桂郁金的水提取液及萃取后的水部位都显示较强的活性。从桂郁金水提取物中分离得到16个化合物,鉴定了其中4个化合物,分别为葡萄糖、柠檬酸、顺丁烯二酸及以葡萄糖为单元的二糖。(3)体外实验表明,桂郁金的4种提取物中乙酸乙酯和石油醚部位对宫颈癌Hela细胞有增殖抑制作用,且具有一定的量效关系。乙醇部位和水溶性部位对这种癌细胞没有作用。免疫荧光染色观察发现桂郁金的石油醚部位能增加Hela细胞中自噬小体的数量,在石油醚作用24h后,LC3-Ⅱ蛋白表达增强,通过流式细胞仪检测,桂郁金石油醚部位能导致Hela细胞内的ROS水平升高。本研究初步证实桂郁金石油醚部位通过提高活性氧簇(Reactive oxygen species.ROS)水平以诱导宫颈癌Hela细胞自噬。3结论本学位论文建立了桂郁金HPLC指纹图谱,首次采用CSPG斑点筛选法在脊髓损伤体外培养中进行运用,首次确定了桂郁金的水部位是其促进脊髓损伤修复的活性部位,并从水部位中分离鉴定了4种化合物。同时对桂郁金抗肿瘤活性部位及初步机制进行研究,为桂郁金的质量控制和药效评价标准的制定提供了一定的依据。更多还原

【Abstract】 Guiyujin is the dry root derived from Curcuma kwangsiensis S.G.Lee et C.F.Liang,which is one of the origins of Radix curcumae. Guangxi is the authentic region of Guiyujin, and where about60%Guiyujin Supplied around the whole nation. This paper based on Guiyujin in Guangxi province. High Performance Liquid Chromatography (HPLC) fingerprint and the pharmacological activity and fundamental substance were studied.1Main content is as follows:(1) In this paper, HPLC analysis was performed for10batches of authentic region in Guangxi province from curcuma kwangsiensis. The pattern analysis and similarity calculation were evaluated by using the national pharmacopoeia committee recommended " The computer aided similarity evaluation software of Traditional Chinese medicine (TCM) fingerprint ", the HPLC fingerprint of Guiyujin was established.(2) In this paper, a screening on drugs against spinal cord injuries was studied.Neurite outgrowth activity was evaluated for extracts of natural medicines by using primary cultured cortical neurons (SD rat, E17); Extention of axons and dendrites were quantified by immunocytochemistry using pNF-H antibody (axon marker) and MAP2a&2b (dendrite marker); microscopic analysis and image analysis were used for measure the length of axons and dendrites.Then the experiment of CSPG inhibitors was used to check on the preliminary screening results, and some nature medicine that have some active from the first screening were further separate by system solvent extraction methods, then test the active site. Effective parts were further isolated and purified by using the column chromatography and preparation chromatography, compounds were identified by using MS,1H-NMR,13C-NMR. HMQC, HMBC for the structure identification.(3)In this paper the effect of inhibiting proliferation of Guiyujin to Hela cells and MCF-7cells were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, the active sites were found out; the autophagy body were observed and analyzed by immunofluorescence staining, the expression of LC3protein was detected with western blotting; explored the influence of active extract of Guiyujin against autophagy; explored the possible mechanism to Hela cells.2The results of the study are as follows:(1) set up HPLC fingerprint of Guiyujin, and12feature peaks in fingerprints were identified, the relative retention time between samples was consistent, the resemblance was higher among10samples(>90%).The characteristic peak area from10sample of authentic region had good separation, characteristics and uniqueness.(2) The preliminary experiments indicated that:compare to the other three extracts, The water extracts of Guiyujin have effect to extend axons in cortical neurons. in CSPG experiment, Guiyujin water extract and the remain water extraction indicate strong activity.From Guiyujin water extract16compounds have been isolated, and4have been identified:Glucose, citric acid, fumaric acid, and the disaccharide composed of glucose.(3) In vitro experiments showed that:only petroleum ether and ethyl acetate parts had inhibiting proliferation to Hela cells among four kinds of extracts of Guiyujin, and the effect of inhibiting proliferation was dose-effect relationship, ethanol and water soluble parts had no inhibiting proliferation against this kinds of cancer cells. The petroleum ether part of Guiyujin increased the the number of autophagic vacuole in the Hela cells from immunofluorescence staining. And increased the expression of LC3-Ⅱ protein after24h.The petroleum ether part of Guiyujin elevated ROS levels in Hela cells through flow cytometry instrument, so this study preliminary confirmed Guiyujin petroleum ether part induced autophagy by improve the level of ROS in cervical cancer.3Conclusions:To sum up, this dissertation establishes the HPLC fingerprint of Guiyujin, at the first time the experiment of CSPG inhibitor spot was used on screening as vitro model for spinal cord injury. And at the first time the water part of Curcumae Radix is determined to be the active part which can promote spinal cord injury repair, and4compounds were identified from the active site. At the same time. the active site of Guiyujin and Preliminary mechanism of antitumor were studied, which provide a reference for the quality control and the establishment of evaluation standards of Guiyujin.更多还原