【作者】 刘涛；

【导师】 金华； 常艳旭；

【作者基本信息】 天津中医药大学 ， 中药学， 2020， 硕士

【摘要】 随着中医药事业的发展,中药在各种疾病的治疗中显示着很好的应用前景,但是也面临着诸如中药药效物质基础不清楚,作用靶点难以确定,质量评价指标不合理等问题。由于中药基质复杂、化学成分种类多、含量差异大等特点,如何建立适合中医药特点的中药质量评价方法是当前研究的难点和热点。中药中活性物质是其发挥疗效的物质基础。因此,针对中药治疗疾病的关键靶点,开展中药活性物质筛选研究,可为合理地确定中药质量控制指标提供科学依据。传统中药系统化学分离结合药理活性研究模型、活性指导下中药化学成分分离模式具有周期长、成本高、工作量大等缺点。目前,基于高效现代分离与在线活性筛选相结合的技术,能有效克服传统研究方式的不足,引起了研究者关注。样品提取是中药质量评价的关键环节之一。高效快速简单的样品提取方法可实现有效成分从复杂样品向提取溶剂中转移、浓缩或者富集,便于后续检测分析。因此,探索适合中药指标性（活性）成分提取新技术,也是中药质量评价研究重要内容。近年来,随着医疗卫生事业的快速发展,环保节能意识也随之增强,发展中药绿色质量评价方法也将成为中药学科的前沿性任务。本论文拟利用有机溶剂使用少的毛细管电泳现代分离仪器,以槐花、黄连为研究对象,以亚铁离子螯合能力、α-糖苷酶抑制活性为指标,建立在“毛细管”微反应器的基础上的中药中具有亚铁离子螯合能力和降糖活性的活性物质筛选方法及体系,确定与活性相关的中药质量标志物;优化快速简单高效的样品前处理技术,建立中药多成分同时提取及测定新方法,建立一种有效可靠的整合活性在线毛细管电泳分析方法的中药质量控制技术,实现利用活性标志物进行质量控制及质量评价;针对名贵中药,采用固相基质微萃取技术,建立一种适合名贵中药的涡旋辅助基质固相分散样品前处理技术,可极大减少了名贵样品的使用量,为名贵中药质量控制样品处理提供一种可资借鉴的研究策略,以丰富和完善中药绿色质量评价体系,促进中药质量评价技术快速发展。1.建立了一种在毛细管中菲洛嗪-亚铁离子螯合与与毛细管电泳（CE）相结合的在线中药复杂体系中螯合亚铁离子的活性物质筛选新方法(In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD),并以槐米（槐花）为模式药物,验证了方法的可行性。通过优化磷酸氢二钠以及十二烷基硫酸钠（SDS）,β-环糊精（β-CD）的浓度以及p H,分离电压,卡盒温度,实现了槐米（槐花）中的多种成分包括芦丁,槲皮素,山奈酚3-O-芸香糖苷和水仙苷的电泳分离及其活性筛选与样品总活性测定,发现槐花（槐米）样品中具有亚铁离子螯合能力的主要活性成分为芦丁和槲皮素。本研究成功地建立了一种中药复杂体系中具有亚铁离子螯合能力的活性成分分离、在线筛选、定量的一体化研究新技术。2.以槐米（花）提取液的总亚铁离子螯合能力为指标,通过单因素实验以及正交设计实验考察了甲醇浓度、微波功率、萃取时间和固液比对槐米（槐花）的微波辅助提取的影响,确定了槐米（槐花）的最佳微波提取条件为:萃取溶剂为50%（v/v）甲醇,药材质量与溶剂体积的比例为1:10,萃取时间7.5分钟,功率为750 W。与传统的提取方法相比,微波提取提高了螯合亚铁离子的主要活性成分的提取率,同时有机试剂消耗少,提取时间更短。3.利用所优化的微波提取方法及In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD分析方法,测定了来自不同产地的槐米（槐花）的各化合物含量以及总的亚铁离子螯合能力,发现芦丁和槲皮素的总含量和样品总的亚铁离子螯合能力(IC₅₀)存在着良好的线性关系。因此,可以将芦丁和槲皮素作为评价槐米（槐花）的亚铁离子螯合能力的活性标志物,证明了整合活性的在线毛细管电泳分析方法是一种有效可靠的中药质量控制技术,所建立In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD方法具有高效,稳定,灵敏等特点,为中药抗氧化活性成分的筛选以及总活性测定提供了新技术。4.将α-糖苷酶微反应器与毛细管电泳结合,首次将毛细管电泳α-糖苷酶微反应器用于中药中的抑制酶活性成分的筛选。利用多巴胺的多官能团聚合作用将α-葡萄糖苷酶固定于毛细管内壁前端,优化了酶底物反应时间300 s为最佳,α-葡萄糖苷酶的米氏常数为1.85 mm,显示酶与底物的亲和度较高且阳性药阿卡波糖,显示着较强的抑制活性;通过酶微反应器的稳定性和重复性考察,表明该酶微反应器可以用于样品的活性测定。从黄连中筛选出主要抑制α-糖苷酶的活性物质为黄连碱和小檗碱,且利用所建立的方法进行活性测定,黄连碱的α-糖苷酶抑制活性要强于小檗碱而远弱于阳性药阿卡波糖,通过样品测定及分子对接也进一步验证了该筛选结果的准确性。因此,所建立的基于α-葡萄糖苷酶微反应的活性测定和活性物质筛选方法高效稳定,准确可靠,为中药活性物质筛选提供了新策略和新思路。5.提出了一种基于双元洗脱剂联合涡流辅助基质固相分散萃取与气相色谱-质谱法结合的绿色、灵敏、有效的研究策略,并应用于麝香中的多成分（麝香酮,棕榈酸乙酯,油酸乙酯和4-羟基苯甲酸乙酯）定量分析。通过单因素实验和正交设计来优化基质固相萃取的相关实验参数包括吸附剂类型、样品与吸附剂的比例、研磨时间、洗脱剂的类型、混合洗脱液比例、混合洗脱液的体积和涡旋时间。最终确定将C18作为吸附剂,样品与吸附剂的质量比为1:2,研磨2 min,双元洗脱剂（甲醇与乙酸乙酯）体积比为3:7。在GC/MS分析下,12 min即可完成四个目标成分的定量。方法学显示该方法稳定性,精密度,准确度良好,回收率均在92.0%以上,检测限低至4.4 ng/m L,基质效应良好。利用建立的方法测定了八批含有麝香的中成药及三批麝香药材的四个目标化合物含量;与传统方法相比,该方法成本较低,耗时较短,消耗试剂较少,符合分析方法绿色环保的原则。因此本研究所建立的方法在含有挥发性成分的中药特别是珍贵中药的质量控制具有很重要的借鉴意义。综上,本论文建立了基于毛细管电泳色谱分离的中药活性物质筛选方法,实现了基于活性质量标志物的中药质量评价;建立了基于双元洗脱剂联合涡流辅助基质固相分散萃取与气相色谱-质谱法,可为名贵中药质量评价提供一种新样品前处理及质量评价方法。本文所建立的中药质量评价方法可应用于其他中药的质量评价研究以及完善中药的质量评价标准,后续将建立基于功效的中药质量评价和控制体系,以中药有效性和安全性相关的活性（毒性）成分为指标,建立其质量评价方法及标准,更能精准地放映中药临床药理作用、疗效以及药效物质基础,推动中医药事业的发展,加快中医药走向世界的步伐。更多还原

【Abstract】 Rescently,Traditional Chinese medicines（TCMs）showed the great application prospects in the treatment of various diseases.There are some problems such as unclear substance basis of TCM,unknown action targets,and unreasonable quality evaluation markers in the research of TCMs.It is the current research difficulty and hot spot to establish methods for evaluating the quality of TCMs according to the characteristics of TCMs.The active substances in TCMs are the material basis for curative effect of TCMs.Therefore,it should be carried out to screen active substances of TCM according to the key targets of TCMs for disease treatment.It could provide a scientific basis for the reasonable determination of the quality markers of TCM.Some conventional models including systematic chemical separation combined with pharmacological activity research and activity-guided chemical component separation model had the disadvantages of long cycle,high cost,and more work.At present,the technology based on the combination of high-efficiency separation and online activity screening could effectively overcome the shortcomings of conventional research models,which were obbtained researchers’attention.Sample extraction was one of the key links in the quality evaluation of TCM.A fast,simple and efficient sample extraction method can realize the transfer,concentration or enrichment of effective components from complex samples to extraction solvents,which is convenient for subsequent detection and analysis.Therefore,it was one of important contents of quality evaluation research of TCMs to explore new technologies for extracting the chemical（active）markers.The awareness of environmental protection and energy saving have been increased with the rapid development of medical and health services.It will also become a cutting-edge task in the discipline of TCMs to develop the green quality evaluation methods of TCMs.This dissertation intends to use modern separation equipment capillary electrophoresis to screen the active components from the complex TCMs.Sophora japonica and Coptis chinensis was selected as the research objects.Ferrous ion chelating ability andα-glycosidase inhibitory activity were selected as indicators to establish some methods and systems based on"capillary"micro-reaction screen active components which have the ability to chelate iron ions and hypoglycemic activity.The quality markers related to activity of TCM need be screened.The fast,simple and efficient sample preparation technology need be optimized.Some effective and reliable quality control technology integrating active online capillary electrophoresis analysis method need be established.The goal of this research was to realize the use of active markers for quality control and quality evaluation.A vortex-assisted matrix solid phase dispersion sample preparation technology was established for quality control of precious TCMs.This technology provides a research strategy that can be used for reference,enriches and improves the green quality evaluation system of TCMs,and promotes the rapid development of quality evaluation technology of TCMs.1.An In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD for screening active components chleating ferrous ion in complex systems of TCM was established by combination of ferrozine-ferrous ion chelation with capillary electrophoresis（CE）.Flos Sophorae Immaturus（Flos Sophorae）as a model drug was used to verify the feasibility of new method.Rutin,quercetin,kaempferol 3-O-rutinoside and narcisin achieved good separation by optimizing the concentration of disodium hydrogen phosphate and sodium lauryl sulfate（SDS）,β-cyclodextrin（β-CD）,p H,separation voltage,cartridge temperature.The screening of active components and the determination of total activity of samples were achieved.It was found that both rutin and quercetin were the main active components chelating ferrous ions in Flos Sophorae Immaturus（Flos Sophorae）.A new integrated research technology was successfully established for the separation,on-line screening and quantification of active components with the ability to chelate ferrous ions in a complex system of TCM.2.The total ferrous ions chelating ability of samples was selected as an indicator.The effects of methanol concentration,microwave power,extraction time and solid-liquid ratio on the extraction of Flos Sophorae Immaturus（Flos Sophorae）were investigated by single factor experiments and orthogonal design experiments.The best microwave extraction conditions for Flos Sophorae Immaturus（Flos Sophorae）were determined as follows.The extraction solvent is 50%（v/v）methanol.The ratio of the mass of medicinal materials to the solvent volume is 1:10.The extraction time is 7.5 minutes,and the power is 750 w.3.Microwave extraction and In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD analysis method were combined to determine the content of active components in Flos Sophorae Immaturus（Flos Sophorae）from different places and the total ferrous ions chelating ability.It was found that there was a good linear relationship between the total contents of rutin and quercetin and the total ferrous ion chelating ability（IC50）of the sample.Rutin and quercetin coud be used as active markers to evaluate the quality of Flos Sophorae Immaturus（Flos Sophorae）.It proved that the on-line capillary electrophoresis analysis method integrated activity was an effective and reliable quality control technology for TCM.The established In-Capillary[Fe（ferrozine）₃]²⁺-CE-DAD method is characterized by its high efficiency,stability,and sensitivity.It could provide a new technology for the screening of antioxidant active component and the determination of total activity of TCM.4.Theα-glucosidase microreactor was combined with capillary electrophoresis.The capillary electrophoresis withα-glucosidase microreactor was firstly used to screen the active ingredients from Chinese herb.The polyfunctional polymerization of dopamine was used to fixα-glucosidase to the inner wall of the capillary.The optimal reaction time of substrate and enzyme was 300s.The value of Mie constant（Km）ofα-glucosidase was 1.85 mm,which showed that the enzyme had a higher affinity with the substrate.The positive drug acarbose showed a strong inhibitory activity.It can be seen that the enzyme microreactor could be used for the activity determination of samples by examining the stability and repeatability of the enzyme microreactor.Coptisine and berberine were screened to be the active components mainly inhibitingα-glucosidase in Coptis chinensis by the established method.The inhibitory activity of Coptisine was stronger than berberine while it was much weaker than positive drug.The accuracy of the screening results was further verified by molecular docking.Therefore,the capillary electrophoresis withα-glucosidase microreactor is efficient stable accurate and reliable and provides new strategies and new ideas for the screening of active components from TCMs.5.A green flexible and effective strategy based on vortex-assisted matrix solid-phase dispersion（MSPD）extraction coupled with GC/MS was proposed for the quantification of four target compounds（muscone,ethyl palmitate,ethyl oleate and ethyl 4-hydroxybenzoat）from musk.Single factor tests and orthogonal design were applied to determine the reasonable experimental parameters of MSPD in the study.C18 as a dispersant and sample powder（2:1 of mass ratio）was ground for 2 min and the mixture was eluted by employing double eluents methanol and ethyl acetate（V:V,3:7）under vortexing forcing for 3 min.The analysis of the samples was eventually completed within 10 min by GC/MS.The total four target components own good linear equations that were expressed with all correlation coefficients（r²）over 0.9991.Four recoveries ranged from 92%-101%with RSD%to less than 7%after repeated tests under the optimal pretreatment conditions.Matrix effects ranging from 83-90%do not exhibit significant matrix interference during the analysis.Eight batches of Chinese patent musk medicine and three batches of musk samples were accurately analyzed with RSD%within 5%.Compared with the traditional method,it can be seen that this method is lower in cost,shorter in analysis time and less in consumption of reagents.Therefore,it conformed to the principle of green environmental protection and provided the important reference significance for the quality control of TCM containing volatile components especially precious Chinese medicinal materials.In summary,a screening method of active substances of TCMs based on capillary electrophoresis chromatography was established in this thesis.The quality evaluation of TCMs based on active quality markers was realized.A new sample pretreatment and quality evaluation method based on dual eluent assisted matrix solid phase dispersion extraction and gas chromatography-mass spectrometry was established.The quality evaluation method of TCMs established in this paper can be applied to the quality evaluation research of other TCMs and improve the quality evaluation standard of traditional Chinese medicine.In the future,we will establish the quality evaluation and control system of TCMs based on the efficacy and safety related activity（toxicity）components as indicators,and establish its quality evaluation methods and standards,which can more accurately show the clinical pharmacology of TCMs,function,curative effect and the material basis of efficacy,will promote the development of TCMs and accelerate the pace of Chinese medicine going to the world更多还原