| 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... |
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