【作者】 韩乐；

【导师】 刘训红；

【作者基本信息】 南京中医药大学 ， 生药学， 2011， 硕士

【摘要】 本文以地耳草为研究对象,对地耳草的有效成分及有害物质进行了含量分析,对地耳草的指纹图谱进行了研究,并对不同生长期地耳草有效物质的动态积累和变化规律进行了研究,为地耳草药材的质量评价及其质量标准制订提供了可靠依据。主要研究内容与结果如下：1.地耳草商品药材的鉴定研究。用传统鉴定方法及现代鉴定技术,从药材性状、显微特征、薄层色谱及HPLC指纹图谱等方面,建立地耳草及其易混品细叶金丝桃的鉴定方法；以有效成分定量分析结合指纹图谱整体分析的模式,评价商品地耳草的品质。2.UV-VIS法测定地耳草总黄酮的含量。选取槲皮素作对照品,以测定的槲皮素含量来作为地耳草中总黄酮含量的计算指标,测定值与真实值有一定差异,本实验旨在对不同产地地耳草中总黄酮的含量作量化比较,评价药材质量。3.HPLC法测定六种黄酮类单体的含量。供试品制备用80%甲醇50mL,根据本实验供试品制备的优化结果,认为回流提取90min×3所得6种黄酮的总量最高。选取地耳草已知明确的六种黄酮类单体(芦丁、金丝桃苷、异槲皮苷、槲皮苷、槲皮素、山柰酚)作对照品,采用HPLC法同时测定六种黄酮类单体的含量。此测定方法,准确可靠,灵敏度高,重现性良好,为地耳草质量标准评价研究提供了科学依据。4.建立高效毛细管电泳二极管阵列检测法(HPCE-DAD)同时测定地耳草中芦丁、异槲皮苷、金丝桃苷、金丝桃苷、槲皮苷、山奈酚及槲皮素含量的方法。选择毛细管区带电泳分离模式,以40 mmol·L-1硼砂缓冲液(pH8.62)为电泳介质,未涂渍标准熔融石英毛细管(75μmi.d.×64.5 cm,有效长度56 cm)为分离通道,,分离电压为25 kV,检测波长为206nm,毛细管温度为25℃,压力进样为50 mbar,8 s。6种黄酮的峰面积与浓度的线性关系良好(r>0.9953)；加样回收率为98.8～102.9%。该方法简单、准确,重现性较好,可用于地耳草药材质量的评价和控制。5.电感耦合等离子发射光谱法(ICP)和微波消解—原子荧光光谱法测定21种无机元素的含量(16种微量元素和5种重金属元素)。并用SPSS16.0对数据进行分析处理,获得了地耳草无机元素含量的准确信息,可为地耳草微量元素的药效药理研究提供科学依据,同时还将不同产地的微量元素制成元素分布图谱,可以进行地耳草的真伪鉴别。6.建立了地耳草高效液相色谱指纹图谱质控方法,可为地耳草样品的质量和品种鉴定提供全面的信息和检测标准。不同产地及不同采收期地耳草样品的HPLC指纹图谱结果表明,产地不同,地耳草样品之间的相似度有一定的差异,这可能与地耳草生长的不同地理条件,不同气候以及不同的品种有关,同一产地的地耳草样品的高效液相指纹图谱有较好的相似度,实现对地耳草药材质量的整体评价。7.建立了地耳草毛细管电泳指纹图谱质控方法,可为地耳草样品的质量和品种鉴定提供全面的信息和检测标准,实现对地耳草药材质量的整体评价。8. HSGC-MS联用技术对不同产地地耳草进行分析,测定其总离子流指纹图谱,确定共有峰,并对部分色谱峰进行了初步归属,初步建立了以12个共有峰为特征指纹信息的HSGC-MS (?)旨纹图谱,发现地耳草样品之间的相似度有一定的差异。该方法准确可靠,重现性好,可作为地耳草内在质量评价的依据。9. UPLC-MS联用技术对不同产地地耳草进行分析,测定其总离子流图谱,并对部分色谱峰进行了初步归属,通过与对照品及相关质谱数据比较分析,初步鉴定其中8种黄酮类化合物,并用UPLC-DAD法对6种黄酮类化合物的含量同时测定。该方法准确可靠,重现性好,可作为地耳草内在质量评价的依据。10.不同采收期地耳草有效物质的动态积累及变化规律研究。用现代分析技术,以有效成分定量分析结合指纹图谱整体评价的模式,对不同生长时间地耳草中黄酮类成分、挥发性成分、无机元素等有效物质的动态积累及变化规律进行分析研究,为确定药材的适宜采收期提供科学依据。11.气候因子对药材有效物质合成和积累的影响研究。考察气候因子(气温、光照时数、降水量、相对湿度等),以药材中有效物质黄酮类成分(总黄酮和芦丁、异槲皮苷、槲皮苷、槲皮素及山柰酚等)、挥发性成分、无机元素的累积量为考核指标,采用UV和HPLC、HPCE等对不同气候因子作用下药材有效物质的变化及其规律进行研究,用逐步回归法分析了气候因子对有效物质积累的影响,探讨气候因子与有效物质积累的相关性。论文主要创新点：1、探究不同物候期地耳草有效物质的动态积累及变化规律,为确定地耳草的最佳采收期提供依据；分析气候因子与有效物质积累的相关性,揭示影响有效物质含量主导因子,为优质药材的生产提供理论指导。2、以多种有效成分同时定量分析结合指纹图谱整体评价的模式,建立地耳草品质和安全性的科学评价体系。3、首次建立地耳草中芦丁、金丝桃苷、异槲皮苷、槲皮苷、槲皮素及山柰酚6种活性成分同时测定的HPLC-DAD、HPCE-DAD分析方法。4、首次建立不同产地、不同采收期和不同种属地耳草的HPCE-DAD、HSGC-MS指纹图谱分析方法。更多还原

【Abstract】 In this dissertation, isolation of the effective component and harmful substances in Herba Hyperici Japonici, the fingerprinting of Herba Hyperici Japonici based on activity constituent are studied and growth of different effective material accumulation and the dynamic changes were studied.Towards these objectives,the following discussed in the thesis.1.The identification of medicinal products. Identified using traditional methods and modern identification techniques, from the medicinal properties, microscopic characteristics, thin-layer chromatography and HPLC fingerprint, etc., set up Herba Hyperici Japonici and Its Adulterant identification methods; to the active ingredients of quantitative analysis combined with the overall analysis of fingerprint patterns, evaluation of the quality of merchandise Herba Hyperici Japonici.2.Using UV-VIS Determination of total flavonoids content, quercetin select for reference substance to determine the content of quercetin Herba Hyperici Japonici as flavonoid content in the calculation of the total target, determination of value and there is a certain difference between the true value, the experiment aimed at different places on Herba Hyperici Japonici. The content of total flavonoids quantified for comparison, the evaluation of the quality of medicines.3.Determination by HPLC of six monomer flavonoids. under samples prepared with 80% methanol 50mL, According to this study prepared for the trial to optimize the results of goods that reflux extraction 90min×3 to determine. The total content of the amendment to provide a scientific basis. It select six known flavonoid monomers (rutin, hyperoside, isoquercitrin, quercitrin, quercetin and kaempferol) as reference substance, the use of HPLC method for simultaneous determination of six flavonoids monomer content. This method, accurate and reliable, high sensitivity, good reproducibility, quality standards for the evaluation of Herba Hyperici Japonici study provides a scientific basis.4.To establish a high performance capillary electrophoresis method with diode array detection (HPCE-DAD) for simultaneous determination of rutin, isoquercitrin, hyperoside, quercitrin, kaempferol and quercetin in Herba Hyperici Japonici. Based on the mode of capillary zone electrophoresis,40 mmol·L-1 borax was used as buffer solution (pH8.62), uncoated fused silica capillary (56 cm/64.5 cm×75μm id) was used, separation voltage was 25 kV, detection wavelength was at 206nm, column temperature was maintained at 25℃, and sample was injected at 50 mbar,8 s. Six Flavonoids showed good linearity (r >0.9953) in the range of the tested concentration, the average recoveries of the method were between 98.8-102.9%. The method is simple, accurate and reproducible, and can be used for quality control of Herba Hyperici Japonici.5.The content of 21 inorganic elements (11kinds of trace elements and heavy metals five 5) are analyzed with advanced means of ICP and microwave digestion-AFS, use the SPSS 16.0 to accurate information the accurate information of inorganic elements of Herba Hyperici Japonici is obtained.The results provide a scientific basis for the quality evaluating of Herba Hyperici Japonici, but also inorganic elements distribution map is developed, it can identify the authenticity of Herba Hyperici Japonici.6.A simple and reliable high performance liquid chromatographic(HPLC)method has been developed and validated for the fingerprinting of extracts from the Herba Hyperici Japonici. The fingerpints of different samples are different, because of differences in plant species or variations in environmental conditions of the growth locations of the plant.For the Herba Hyperici Japonici from same location, the fingerprints of the samples shoe closely similar fingerprints. So the HPLC fingerprinting technique has high potential in herb autheticaton application.7.A simple and reliable high performance Capillary Electrophoresis method has been developed and validated for the fingerprinting of extracts from the Herba Hyperici Japonici. Herba Hyperici Japonici on the implementation of the overall evaluation of the quality of medicin.8.HSGC-MS in conjunction with technology from different areas of Herba Hyperici Japonici analysis, determination of the total ion current fingerprint to determine the total peak, and part of chromatographic peaks for the initial vesting, the initial set up a peak for a total of 12 fingerprint HSGC-MS information of the fingerprint, found a small number of samples HSGC-MS fingerprint of a significant difference. This method is accurate and reliable, reproducible and can be used as the internal quality evaluation of leaf venetum basis.9.UPLC-MS in conjunction with technology from different areas of Herba Hyperici Japonici analysis, determination of the total ion-current figure to determine the total peak, and 8 chromatographic peaks for the initial vesting, at the same time, determination by UPLC-DAD of six monomer flavonoid. This method is accurate and reliable, reproducible and can be used as the internal quality evaluation of leaf venetum basis.10O.Harvesting period of different dynamic analysis of changes in modern technology, combined with quantitative analysis of active ingredient overall evaluation of fingerprint pattern of growth for different time Herba Hyperici Japonici, volatile components, and other inorganic elements in the dynamic accumulation of effective substances and Analysis of changes in laws, in order to determine the best ingredients to provide a scientific basis for harvest time.11.Climatic factors on the synthesis of medicinal substances and the accumulation of the effective impact of research. Study climatic factors (temperature, light hours, precipitation, relative humidity, etc.) to medicinal substances in the effective flavonoids (total flavonoids and rutin, isoquercitrin, quercitrin, quercetin and kaempferol, etc.), volatile component of the cumulative target of evaluation, using UV and HPLC, HPCE, such as the role of climatic factors on the different ingredients of change and its effective material to study the law, using stepwise regression analysis of climatic factors on the effective impact of the accumulation of material to explore the climate factor and effective accumulation of the relevance of the material.The main feature and innovation in this thesis are presented:1 To explore the different phenology of Herba Hyperici Japonici effective substances and changes in the dynamic accumulation, Climatic factors and the effective accumulation of the relevance of the material, for the production of high-quality ingredients to provide theoretical guidance.2 To establish the quality and safety of Herba Hyperici Japonici scientific evaluation system by Quantitative analysis of the active ingredient in combination fingerprint pattern of the overall evaluation.3 To establish the method of rutin, hyperoside, isoquercitrin, quercitrin, quercetin and kaempferol 6 Determination of the active ingredient at the same time by the HPLC-DAD, HPCE-DAD analysis first time. 4 To esstablish the HPCE-DAD、HSGC-MS finger print analytical method of Herba Hyperici Japonici in differet habit, collection period and kind.更多还原