基于新型荧光标记技术的羌活主要化学成分研究

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	基于新型荧光标记技术的羌活主要化学成分研究
	张世娟
学位类型	博士
导师	尤进茂
	2014-05
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	植物学
关键词	羌活宽叶羌活化学成分高效液相色谱荧光衍生质谱
摘要	药材羌活为羌活属植物裂叶羌活和宽叶羌活的干燥根茎及根，是我国最早发现和应用的药物之一，药用历史悠久。羌活具有散寒、祛风、除湿、解热、止痛和消炎的功效，主治风寒感冒头痛、风湿麻痹、肩酸背痛和心血管等疾病。中药的药效通常来自多种有效成分的共同作用，药材的化学组成信息对于中药的合理使用具有重要价值。目前羌活化学成分研究主要集中在羌活挥发油和香豆素类物质上，其它非挥发性有效成分研究较少，限制了对羌活的进一步开发利用。本研究针对羌活化学研究中的薄弱环节，利用先进的色谱和质谱技术对羌活的化学成分进行了分析研究，为羌活的综合利用提供科学依据。 1. 以1-(9-咔唑基)-2-丙醇甲磺酸酯(CPMS)为衍生试剂，建立了34 种脂肪酸的液相色谱荧光分析方法。在样品用量为30 mg 的条件下，方法灵敏度为0.032–0.312 μg/g。利用所建立的方法对羌活中的游离脂肪酸含量进行分析，结果表明羌活富含亚油酸等不饱和脂肪酸。 2、以10-甲基-吖啶酮-2-磺酰氯(MASC)为荧光衍生试剂，建立了氨基酸的液相色谱荧光检测方法，在18 min 内实现了对20 种氨基酸的快速分离，方法灵敏度达到nmol/L。利用所建立的方法对羌活中的游离氨基酸进行了定性和定量分析。 3、建立了糖类化合物的柱前衍生–液相色谱荧光分析方法。通过在单糖分子中引入具有优异荧光特性的苯并吖啶酮-5-乙酸酰肼(BAAH)，使得单糖分子的液相色谱灵敏度得到极大提高。同时，强疏水性BAAH 基团的引入增加了单糖类分子的疏水性，使其和液相色谱柱的作用力增大，保留时间得以延长，因此具有相似性质的单糖分子可在普通液相色谱柱上得到有效地分离。利用所建立的方法对羌活粗多糖组成进行了分析，研究结果表明羌活多糖主要由半乳糖、葡萄糖、阿拉伯糖、甘露糖、鼠李糖、岩藻糖、木糖和果糖组成。此外还结合气相色谱–质谱方法对羌活多糖单糖组成进行深入分析。这是首次对羌活多糖的系统研究，将更有利于羌活的质量控制。 4、分别建立了羌活中三萜酸、肉桂酸和香草酸的柱前衍生–液相色谱荧光分析方法。方法灵敏度和准确度较衍生前有较大提高。三萜酸类化合物经手性衍生试剂衍生后，在反相色谱柱上出现保留时间不同，峰面积相近的两个峰，极大地减少了因干扰造成的误判，提高了液相色谱在分析复杂生物样品时的准确度。 5、建立了羌活的液相色谱和液相色谱–质谱指纹图谱，并对羌活药材原植物宽叶羌活根、茎和叶的指纹图谱并行了比较，总结出地上和地下部分的共有峰和特有峰，为羌活的质量控制提供依据。结合相关测定数据(保留时间、紫外光谱和质谱数据等)和文献研究，对部分化合物进行了确认。 6、对羌活甲醇提取物和羌活粗多糖的体外抗氧化能力进行了评价。分别采用FRAP法、DPPH自由基清除法和羟基自由基清除法对羌活多糖和甲醇提取物的总抗氧化能力、清除电子自由基和羟自由基的能力进行了测定。同时对羌活有效成分阿魏酸含量进行了分析并进行了防腐和抑菌实验，结果表明阿魏酸可作为一种有保健价值的防腐剂使用。 7、建立了羌活挥发油的气相色谱–质谱分析方法方法并对羌活中的挥发性成分进行了详细研究。根据气相色谱–质谱数据，结合与国内外文献照，对羌活挥发油的成分进行了详细分析和结构表征，弥补了传统羌活分析中结构性信息缺乏的不足。
其他摘要	Rhizoma et Radix Notopterygii, the dried root and rhizome of Incised Notopterygium ex H. T. Chang and Notopterygium forbesii Boiss, has been widely used as a traditional Chinese herbal medicine for several thousand years. It has been widely used for the treatment of common cold, rheumatism, joint pain and cardiovascular disease. The effect of Chinese herbal medicine often comes from the combined action of many active ingredients. The understanding of the chemical composition of herbal medicine is essential for the good utilization of Chinese herbal medicine. The current study of Rhizoma et Radix Notopterygii mainly focuses on volatile oil and coumarin. Little study was carried out on other nonvolatile chemical ingredients. The further understanding and utilization of Rhizoma et Radix Notopterygii was therefore greatly restricted. The current study was focused on the analysis of active ingredients which were often ignored in Rhizoma et Radix Notopterygii study. High performance liquid chromatography (HPLC) and mass spectrometry were applied in the analysis of chemical ingredients of Rhizoma et Radix Notopterygii. This study provides good material for the comprehensive utilization of Rhizoma et Radix Notopterygii. 1. A sensitive HPLC method using a new synthesized 1-(9H-carbazol-9-yl) propan-2-yl-methanesulfonate (CPMS) as precolumn derivatizing reagent was developed for the analysis of fatty acids. The 34 derivatives of fatty acids were separated on a BDS C8 reversed-phase column. When 30 mg of sample was analyzed, the detection limits at a signal-to-noise ratio of 3 were in the range 0.032–0.312 μg/g. The free fatty acids in Rhizoma et Radix Notopterygii were analyzed by the developed method. The results indicated that Rhizoma et Radix Notopterygii was rich in unsaturated free fatty acids. 2. A sensitive HPLC method was developed for the analysis of amino acids using 10-methyl-acridone-2-sulfonyl chloride (MASC) as pre-column fluorescent derivatizing reagent. Twenty amino acids were separated within 18 min with sensitivity of nmol/L. The proposed method was successfully applied to the analysis of free amino acids in Rhizoma et Radix Notopterygii. 3. A sensitive pre-column derivatization method was developed for the analysis of carbohydrates by HPLC with fluorescence detection. The introduction of 2-(12-benzo[b]acridin-5(12H)-yl)-acetohydrazide (BAAH) with excellent fluorescence property into the molecules of monosaccharides greatly enhanced the HPLC sensitivity of the analytes.Meanwhile, derivatization with BAAH also greatly increased the hydrophobicity of the monosaccharides and made them elute at increased retention times. The monosaccharides with similar properties therefore could be completely separated due to the increased interaction between the analytes and the column. Component monosaccharides of the polysaccharides obtained from Rhizoma et Radix Notopterygii were analyzed by the developed method. The results indicated that the polysaccharides of Rhizoma et Radix Notopterygii were mainly composed of d-galactose, d-glucose, l-arabinose, d-Mannose, l-rhamnose, l-fucose, d-xylose and fructose. Gas chromatography and mass spectrometry method (GC–MS) was also applied to the analysis of the components of the polysaccharides of Rhizoma et Radix Notopterygii. Two derivatizing reagents were applied in GC–MS analysis. This is the first systematic study of the sugar composition of the polysaccharides of Rhizoma et Radix Notopterygii. It will be helpful for the quality control of Rhizoma et Radix Notopterygii. 4. A sensitive pre-column derivatization HPLC method with fluorescence detection was applied to the analysis of triterpenic acids, cinnamic acid and vanillic acid in Rhizoma et Radix Notopterygii. The sensitivity and accuracy were greatly enhanced compared to the HPLC ultraviolet detection method. Each triterpenic acid produced two HPLC peaks with similar peak areas after derivatising with chiral CPMS. This phenomenon greatly enhanced the HPLC accuracy due to the double insurance of two retention times and two peak areas. 5. The HPLC–DAD–MS/MS fingerprints were established for the roots, stems and leaves of Notopterygium forbesii Boiss. The similarities and differences among roots, stems and leaves have been studied. Chromatography peaks have been identified by combination of retention time, ultraviolet profile, mass spectrometry data and literature information. The obtained data provide good assurance for the quality control of Notopterygium forbesii Boiss. 6. The vitro antioxidant activity of Rhizoma et Radix Notopterygii including ferricreducing antioxidant power (FRAP), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and hydroxyl (OH·) radical scavenging activities were examined. Meanwhile, the content of ferulaic acid in Rhizoma et Radix Notopterygii was studied. The preservative and antibacterial activity of ferulaic acid was also carried out in detail. The results indicated that ferulaic acid could be used as a healthy preservative. 7. A sensitive GC–MS method was developed for the analysis of volatile oil of Rhizoma et Radix Notopterygii. The chemical composition of Rhizoma et Radix Notopterygii was analyzed in combination of the obtained MS data and published articles. Chemical structures of the ingredients were also included in the conclusion. The absence of structure data was avoided in the current study.
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/4017
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	张世娟. 基于新型荧光标记技术的羌活主要化学成分研究[D]. 北京. 中国科学院研究生院,2014.

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