Methodology Study Of The Screening Of Active Ingredients From Traditional Chinese Medicines By Cell Membrane Chromatography And Identification Of Their Binding Targets

The integration characterization of Traditional Chinese Medicine (TCM) makes itsadvantages of multiple components, multiple targets and the holistic adjustments behaviors.However, due to the complexity of the multiple components and targets, it is still achallenge work to elucidate the active components and the complex mechanism of TCM.Cell membrane chromatography (CMC) is one of the biological chromatography, havingthe characterization of chromatographic separation and biological activity. The activecomponents of TCM could be screened without the separation procedures. This kind ofmethod based on multi-component and multi-target interactions is very suitable for thestudy of the active components from TCM. However, in order to solve the four problems:(1) the column preparation procedure needs to be completely validated and optimized;(2)the analytical techniques need to be further developed;(3) the specificity of CMC needs tobe improved;(4) the target identification of the screened active components is still achallenge work. So this study focuses on the following four aspects：1. Improvements of the reproducibility and efficiency of cell membranechromatographic columnCell membrane chromatography (CMC) is a biological affinity chromatographicmethod using specific cell membrane adsorbed on silica as stationary phase. However, itsshort life span and poor stability have been bothering researchers ever since. In this study,several aspects that influence stability and reproducibility of CMC, including disruptionprocedure, protein quant and time procedure of packing, were investigated and validated.Two novel CMC models (HSC-T6/CMC and SMMC-7721/CMC) were established. Aseries of standard operating procedures and parameters were drawn up to ensure the qualityand reproducibility of CMC columns. Additionally, a novel method by using4%paraformaldehyde (PFA) was employed to improve the column efficiency and prolong thecolumn life span. It was the first time that the methodology of CMC was fully investigatedand optimized for better quality and reproducibility using two different CMC models.2. Comprehensive two-dimensional HepG2/cell membranechromatography/monolithic/time-of-flight mass spectrometry systemfor screening anti-cancer componentsIn this study, a novel comprehensive two-dimensional (2D) chromatography approach was established for screening anti-tumor components from herbal medicines (HMs).HepG2/CMC model was first developed and applied as the first dimensional column.Using an automatic ten-port switching valve equipped with two sample loops, the fractionsof the first-dimension were introduced in the second-dimension consists of a monolithiccolumn and a time-of-flight mass spectrometry (TOFMS) with high resolving ability.Based on the stability, selectivity and suitability assays of the HepG2/CMC/monolithiccolumn/TOFMS system, berberine (BBR) and magnoflorine (MAG) from Cortexphellodendri amurensis, oxymatrine and matrine from Radix sophorae favescentis werescreened and identified as potential active components. The competitive displacementassay suggested that the four components could act on epidermal growth factor receptorregion on the HepG2cell membrane in similar manner of gefitinib. Furthermore, theirinhibiting effects on cell proliferation in vitro were also confirmed and, BBR and MAGshowed concentration dependently inhibitory ability on HepG2cell proliferation (p <0.05).The result demonstrated that the proposed comprehensive2D HepG2/CMC/monolithiccolumn/TOFMS system has the advantages of strong recognition and rapid analysisabilities for the total screening procedure, which will be selectable and practical in drugdiscovery from complex HM samples and can also be applied to other biochromatographymodels.3. Comparative normal/failing rat myocardium cell membranechromatographic analysis system for screening specific componentsthat counteract doxorubicin-induced heart failure from Acontiumcarmichaeli and Sini decoctionCell membrane chromatography (CMC) derived from pathological tissues is ideal forscreening specific components acting on specific diseases from complex medicines owingto the maximum simulation of in vivo drug-receptor interactions. However, there are nopathological tissue-derived CMC models that have ever been developed, as well as novisualized affinity comparison of potential active components between normal andpathological CMC columns. In this study, a novel comparative normal/failing ratmyocardium CMC analysis system based on online column selection and comprehensivetwo-dimensional (2D) chromatography/monolithic column/time-of-flight massspectrometry was developed for parallel comparison of the chromatographic behaviors onboth normal and pathological CMC columns, as well as rapid screening of the specific therapeutic agents that counteract doxorubicin (DOX)-induced heart failure from Acontiumcarmichaeli (Fuzi) and Sini tang (SNT). In total16potential active alkaloid componentswith similar structures in Fuzi were retained on both normal and failing myocardium CMCmodels. Most of them had obvious decreases of affinities on failing myocardium CMCcompared with normal CMC model except for four components, talatizamine (TALA),14-acetyl-TALA, hetisine and14-benzoylneoline. One compound TALA with the highestaffinity was isolated for further in vitro pharmacodynamic validation and targetidentification to validate the screen results. Voltage-dependent K+channel was confirmedas a binding target of TALA and14-acetyl-TALA with high affinities. The onlinehigh-throughout comparative CMC analysis method is suitable for screening specificactive components from herbal medicines by increasing the specificity of screened resultsand can be applied to other biological chromatography models.4. The target identification of active components in SNTIn order to further identify the direct or indirect targets of SNT acting on myocardium,in this study, novel label free proteomics techniques has been adopted to find the proteinbiomarkers of SNT against myocardial infarction (MI). In total590different expressedproteins were identified. Comparing SNT group with MI group, there were30proteinsup-regulated and51proteins down-regulated significantly. The results showed that SNTacting on MI and protecting myocardium cells mainly depending on involving into NADHbiosynthesis, improving myocardium contraction, participating collagen synthesis, energytransport and metabolism, citroyl synthetase etc. activity and adjusting the activity of ionchannels.Through the in-depth study on the cell membrane chromatography, hope tocontributing to the development of the cell membrane chromatography, also forhigh-throughput screening of the active component of TCM and their targets. This canprovide some new ideas and new technologies for the study of CMC and TCM.