【作者】 吴宏伟；

【导师】 黄璐琦；

【作者基本信息】 中国中医科学院 ， 中药学， 2011， 博士

【摘要】 药性是中药的基础和核心,一直是中医药研究的热点和难点。中药药性作为中药的重要特征,是对中药化学成分作用于人体后产生的多种医疗作用和效果的高度概括。因此物质基础和药理效应是中药药性的两个本质属性。由于中药来源广泛,物质基础复杂,药理效应涉及多种方面,所以一直以来很难找到合适的研究对象和评价方法来研究中药药性。本课题选取姜黄、郁金为研究对像,是因为二者来源于同一植物的不同药用部位,而二者的寒、热药性却相反,这样使寒、热药性研究更具有针对性和可比性。姜黄为姜科植物姜黄Curcuma longa L.(?)勺根茎,性温,具有破血行气、通经止痛的功能。郁金为姜科植物姜黄Curcuma longa L.的块根,性寒,具有行气化瘀、清心解郁、利胆退黄的功能。中药的药效物质基础主要来源于植物的次生代谢产物,姜黄、郁金作为同一植物的不同器官所产生的次生代谢产物是否存在差异,差异如何是二者形成寒、热药性的根本原因。因此本研究采用HPLC-DAD-MS和GC-MS分析技术对姜科植物姜黄Curcuma longa L.(?)勺根茎(姜黄)和块根(郁金)的次生代谢产物进行分析,采用模式识别技术对分析结果进行比较,整体上评价二者次生代谢产物的异同,确定生物标志物,从物质基础的角度说明姜黄、郁金形成药性差异的原因。“寒者热之,热者寒之”是中医药治疗疾病的过程中对中药药性最朴素的认识。姜黄、郁金的寒、热药性是其对不同症候疾病作用后产生效果的高度概括。因此建立与临床相适应的病症模型是评价姜黄、郁金药理效应的前提,而选择合理的评价方法则是阐释二者寒、热药性差异的关键。临床上姜黄、郁金最主要的用药区别是二者分别用于治疗阴黄证(湿寒黄疸)和阳黄证(湿热黄疸)。前期药理实验证明在某些单一指标上(如：黄疸指标、抗氧化指标)姜黄、郁金对两种寒热模型具有不同的治疗效果,但是同时在某些指标上则无法体现二者的差异。为了从不同层次系统地评价姜黄、郁金的药理效应,阐释二者寒、热药性差异,我们尝试采用代谢组学的研究方法整体上评价姜黄、郁金对两种模型的作用效果,比较其异同。主要研究内容如下：一、基于代谢组学研究方法的姜黄、郁金植物次生代谢产物差异研究1.建立了HPLC-DAD-MS分析方法,对姜黄、郁金样品进行了分析。在检测波长265nm下,建立了姜黄、郁金指纹图谱,并采用主成份分析(PCA)对测定结果进行处理,从二维主成份得分图(Scores Polt)可以看到姜黄、郁金样品分别聚为两类,说明二者存在显著差异,主成份载荷图(Loading Plot)为我们提供了造成这种差异的生物标志物群,经过进一步t检验确定了其中10种主要差异代谢产物,通过质谱和紫外数据对其中7种进行了鉴定,并进行了相对含量的比较,姜黄中姜黄素cuncumin,去甲氧基姜黄素Demethoxycurcumin,双去甲氧基姜黄素bisdemethoxycurcumin,双氢姜黄素Dihydrocurcumin,芳姜黄酮Ar-turmerone,姜黄酮a,B-turmerone(或姜黄醇bisacumol),姜烯Zingiberene(或没药烯B-Bisabolene、姜黄烯B-curcumene)的含量均高于郁金。2.建立GC-MS分析方法,对姜黄、郁金中萜类成分样品进行了分析。样品制备采用石油醚索氏提取,保证了样品制备的重复性。从气质色谱中鉴定了43个化学成分,基于这些成分采用主成份分析(PCA)比较姜黄、郁金二者的差异,从二维主成份得分图(Scores Polt)可以看到姜黄、郁金样品分别聚为两类,说明二者存在显著差异,主成份载荷图(Loading Plot)为我们提供了造成这种差异的生物标志物群,经过进一步t检验确定了其中14种差异代谢产物。3.通过以上两种分析方法系统地对姜科植物姜黄Curcuma longa L的根茎(姜黄)和块根(郁金)的次生代谢产物进行了分析,发现二者所含化合物的种类基本一致,主要为姜黄素类和萜类化合物；通过模式识别对姜黄、郁金进行了区分,并确定了二者间的差异代谢产物。二、基于代谢组学的姜黄、郁金寒热药性评价研究1.针对蛋白质、糖类、脂肪代谢所产生的氨基酸、小分子有机酸、脂肪酸等目标化合物,建立了血浆样品的GC-MS衍生化分析方法。首先对寒湿黄疸和湿热黄疸(以下分别简称为寒证模型和热证模型)进行代谢轮廓分析,结合有监督(PLS-DA)和无监督(PCA)模式识别数据处理方法,能够实现寒、热模型的识别,并确定了寒、热模型的生物标志物,其中寒证模型组与正常对照组比较,升高的标志物有苏氨酸、甘氨酸等,降低的标志物有半乳糖、柠檬酸等。热证模型组与对照组比较,升高的标志物有苏氨酸、苯丙氨酸、半乳糖、柠檬酸、油酸等,降低的有丁二胺。为进一步评价姜黄、郁金的寒热药性奠定了基础。针对寒证模型组,我们分析比较了姜黄、郁金、及主要差异代谢产物(姜黄素)干预前后代谢谱的变化。从PLS-DA得分图上,我们可以从整体上看出各组动物代谢谱变化的轨迹。姜黄素组和姜黄组距离寒证模型组距离最远,郁金组与寒证模型组距离较近并有部分重合,说明姜黄、姜黄素对寒证模型代谢谱的调节作用大于郁金,这一点印证了“姜黄性温”中医理论,也说明了姜黄素具有“温性”的药性。并且通过各给药组与寒证模型组间的两两比较,进一步确认了药物作用前后的差异代谢产物,与寒证模型的标志物大部分一致且变化趋势相反。针对热证模型组,分析比较了姜黄、郁金、姜黄素干预前后代谢谱的的变化。从PLS-DA得分图上,我们可以看到郁金组距热证模型组的距离大于姜黄组。说明郁金对热证模型代谢的影响大于姜黄,反映出郁金具有“寒性”的性质。并且通过各给药组与热证模型组间的两两比较,进一步确认了药物作用前后的差异代谢产物及变化趋势,大部分与热证模型的标志物相一致且变化趋势相反。2.由于文献报道单胺类神经递质与机体的寒、热状态密切相关,因此针对此类物质,采用HPLC-(ESI)MS,建立了大鼠脑中7种单胺类神经递质代谢物色谱分析方法,并对各组动物进行了分析。首先对寒、热证模型和正常对照组进行比较,数据处理采用主成份分析,从主成份得分图可以看出正常对照组、寒证模型组,热证模型组分别聚为3类,说明寒、热模型大鼠脑中单胺类神经递质的表达存在差异,经t-test检验,与正常对照组比较E,3-MT,和5—HT含量在热证模型中均有显著升高,寒证模型显著降低。针对寒证模型,比较了姜黄、郁金、姜黄素对单胺类神经递质表达的影响。经主成份分析姜黄、姜黄素与寒证模型组区分显著,姜黄给药后明显提高了寒证模型中E和5-HT的含量,姜黄素给药后明显提高了寒证模型中E、NE、5-HT,降低了3-MT.而郁金与模型组比较各指标均无显著差异,说明姜黄、姜黄素具有性温的药性。针对热证模型,比较了姜黄、郁金、姜黄素对单胺类神经递质表达的影响。经主成份分析姜黄、郁金、姜黄素对热证模型均有调节作用。姜黄、郁金给药后降低了热证模型中E、3-MT、5-HT、5-HIAA的含量,进一步比较发现,郁金对上上述指标的降低幅度要大于姜黄；姜黄素给药后与热证模型组在载荷图上有部分重叠,与模型组比较,5-HT降低,DA升高。只是在指标的数量和调节的幅度上,郁金大于姜黄和姜黄素。3.通过建立以上两种代谢组学分析方法,从不同的角度对湿热黄疸和寒湿黄疸两种寒热模型进行了研究,确定了二者的生物标志物,并在此基础上评价姜黄、郁金、姜黄素对寒、热模型的不同作用,从整体上说明三者作用的差异,进而从代谢组学的角度阐释姜黄、郁金寒、热药性。本论文的创新点1、研究对象新颖,可比性强。选择来源于同种植物不同部位的郁金和姜黄为研究对象,两者遗传背景、环境因素相同,且炮制方法相同,尽可能地保证了实验结果的可比性。2、采用代谢组学的研究方法系统的比较了姜黄、郁金作为同一植物不同部位所含有的次生代谢产物的表达差异,发现相关生物标志物,说明了二者药性差异的物质基础。3、建立了GC-MS血浆代谢组轮廓分析和HPLC-MS大鼠脑中单胺类神经递质分析的两种代谢组学方法,并将该方法首次应用到姜黄、郁金的药性研究中。通过多元统计分析,确定了湿热黄疸和寒湿黄疸两种寒、热模型的生物标志物,并在此基础上对姜黄、郁金、姜黄素的作用效果进行了评价,从代谢组学的角度阐释了姜黄、郁金的寒、热药性差异,为中药药性的研究提供了新的方法和思路。更多还原

【Abstract】 The properties of tradional Chinese medicinal herbs, which are the essential basis of the analyses and clinical usage of Chinese medicinal herbs, are summarized in medical practice and on the basis of the theories of Yin-Yang, Hot-Cold,Toxicity and other theories of therapeutic principles of traditional Chinese medicine(TCM).The reason that Chinese medicinal herbs can prevent and treat diseases is due to their two internal natures:chemical components and the biological activities. Because the source of tradional Chinese medicinal herbs is widely, the chemical components were various and the biological activities were related with too many active aspects. So it was lacking the proper methods and objects to study and research the property theory of traditional Chinese medicinal herbs by modern scientific language.Jianghuang and Yujin which come from the rhizome and tuberous root of Curcuma longa L. are used as two different Chinese medicinal herbs. In the property theory of traditional Chinese medicinal herbs, it was believed that Yujin has cold nature and Jianghuang has hot nature. Accordingly Jianghuang was used to promote circulation of blood and arrest pain while Yujin was used to circulate the blood and normalize the gallbladder and liver to cure jaundice in clinic. As Jianghuang and Yujin come from the same plant of Curcuma longa L, they have similar chemical components and effective activities. So Jianghuang and Yujin were selected as objects, which makes it easier to study and compare the chemical components and the biological activities so as to explain why they have hot and cold nature.The metabonomics/metabolomics gives a good and new idea to study the plant secondary metabolites and the therapeutic evaluation of TCM. As a part of system biology, metabonomies apply modern chemical analysis technology such as HPLC-MS、GC-MS、NMR to determine active alterations of endogenous metabolites of biological organism and subsequently reveal changes in the whole process, including occurrence, development,and consequences in response to stimuli or drug treatment. Metabonomies, defined as "the quantitative measurement of the multiParametrie time-related metabolic responses of a complex system to a pathophysiological intervention or genetic modification" has been widely used in the researches of pharmacology and biological seiences.In this thesis firstly we established HPLC-DAD-MS and GC-MS methods to analysis and compare the the plant secondary metabolites of the rhizome(Jianghuang) and tuberous root(Yujin) of Curcuma longa L. Principle Component Analysis(PCA) was used to discovery the biomarkers between Jianghuang and Yujin from chromatograms. Secondly we establish a GC-MS method to analyse plasmar metabolites and a HPLC-MS method to determine brain catecholamine metabolic profiling.With the methods we evaluated the Heat-Syndrome model(damp-heat jaundice model) and Cold-Syndrome model(cold-damp jaundice model). Pattern recognition was used to distinguish Heat-Syndrome model and Cold-Syndrome model. Based on this, we evaluated drug effect of Jianghang,Yujin,and curcumin on Heat-Syndrome model and Cold-Syndrome model. According to different effects on Heat-Syndrome model and Cold-Syndrome model, we can explain and illustrate hot and cold nature of Jianghuang and Yujin.Main contents are summarized below:1. The plant secondary metabolites analysis of Jianghang and Yujin by HPLC-DAD-MS and GC-MS.In order to analyse and compare the the plant secondary metabolites including volatile terpenes and nonvolatile curcuminoids of Jianghuang and Yujin, the metabolite profilings was established by HPLC-DAD-MS and GC-MS.18 samples of Curcuma longa L inluding the rhizome and tuberous roots were collected from the regions in Sichuan province of China. The samples analyzed by HPLC-DAD-MS were processed by ultrasonic extraction in methanol.While the samples analysed by GC-MS were processed by Soxhlet extraction with light petroleum. After peaks of every chromatograms synchronized, multivariate analysis was performed using the SIMCA-P 12 version (Umetrics AB, Ume□, Sweden) based on the all peak areas(peak area/sample weight). Principal component analysis (PCA) was used and reveal the differences between the the rhizome and tuberous roots.Form Scores Polts of PCA based on HPLC or GC, there are both obvious difference between Jianghuang and Yujin. Based on chromatograms of HPLC-DAD-MS, there are 10 biomarkers found between Jianghuang and Yujin. Seven of them were identified as Bisdemethoxycurcumin、Demethoxycurcumin、Curcumin Dihydrocurcumin Ar-turmerone、a,β-turmerone (bisacumol)、Zingiberene (B-bisabolene, B-curcumene),which are all higher in Jianghuang than Yujin.Based on chromatograms of GC-MS,14 biomarkers were found and identified whose contents are obvious different between Jianghuang and Yujin.2. Evaluation of Hot and cold nature of Jianghuang and Yujin by metabonomics method of GC-MS technologyThe GC/MS method was established to analyse the blood plasma samples and the Heat-Syndrome model(damp-heat jaundice model) and Cold-Syndrome model(cold-damp jaundice model) rats blood plasma was introdued to GC/MS. Then applying the GC/MS method, the blood plasma samples from the Heat-Syndrome model and Cold-Syndrome model rats treated by Jianghuang、Yujin and curcumin were detected by the machine.With the PCA and PLS-DA, the status of drug effect was evaluated.With the multianalysis to the metabonomic datas of GC/MS, the Heat-Syndrome group、Cold-Syndrome group and the normal group can be divided into three groups accordingly. And compared with normal group, the biomarkers of the Heat-Syndrome and Cold-Syndrome group were found and identified such as aminoacids, organic acids. Then we compared the efficiency of jianghuang,Yujin and curcumin on the Cold-Syndrome model and the Heat-Syndrome model. The result suggest that Jianghuang and curcumin have more regulation effect on Cold-Syndrome model than Yujin based on the metabonomic datas. While Yujin have more regulation effect on Heat-Syndrome model than Jianghuang and curcumin.3. Evaluation of Hot and cold nature of Jianghuang and Yujin Based on brain catecholamine Metabolic ProfilingHPLC-MS method for the quantitative determination of 7 catecholamines in brain samples was developed. And Principal Component Analysis(PCA) was processed to analyze the catecholamine metabolic profiling. Heat-Syndrome model and Cold-Syndrome model rats was firstly introdued to HPLC-MS. Then applying the the method, the samples of the Heat-Syndrome model and Cold-Syndrome model rats treated by Jianghuang、Yujin and curcumin were detected.The results suggest Heat-Syndrome model and Cold-Syndrome model can be distingushed by catecholamine metabolic profiling. Jianghuang、Yujin and curcumin has different effect for the Heat and Cold Syndrome model based on Brain catecholamines Metabolic Profiling.更多还原