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Screening of Active Components of Corydalis Yanhusuo W.T. Wang by Two Dimensional Liquid Chromatography and Proteomic Studies on the Pharmacological and Toxicological Mechanisms of l-THP

Author: WangChen
Tutor: FanGuoRong;WuYuTian;ZouHanFa
School: Second Military Medical University
Course: Pharmaceutical Analysis
Keywords: Traditional Chinese Medicine Corydalis yanhusuo W.T. Wang levo-tetrahydropalmatine comprehensive two-dimensional LC LC-MS/MS microdialysis 2-D nano LC comparative proteomics chemoproteomics formalin test anti-nociception toxicity
CLC: R96
Type: PhD thesis
Year: 2010
Downloads: 334
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Abstract


“The most difficult and most important challenges for Traditional Chinese medicine (TCM) research in order to gain further development is to screen the active ingredients, and illustrate the target proteins and related recognition mechanisms, and ultimately recommend it for clinical application and/or for further structure-activity relationship study”said Chen, Yizhang, the member of the Chinese Academy of Sciences. Despite TCMs’existence and continued use over many centuries and its popularity and extensive use during the last decade, substantial gaps remain both in our understanding of the“real”specific bioactive compounds, and the target proteins and related recognition mechanisms for these compounds. The knowledge on safety and efficacy is lacking because few TCMs have been evaluated by rigorous scientifically-designed trials. Herbal toxicity is increasingly recognized as the use of these medications has increased, and toxicity from TCMs seems to be a serious problem all over world now. We should no longer confine the adverse reactions of TCM to traditional Chinese medicine literature and expansion of basic research into mechanisms of herbal toxicity is warranted. To illustrate the target proteins and related recognition mechanisms of TCM under the guidance of TCM theory is another urgent task.The proteomic approach is widely applied nowadays in the development of novel biomarker candidates for early detection of disease and identification of new targets for therapeutics, mainly by delineation of protein expression changes depending on factors such as the organism’s physiological state and the stage of development of disease. However, traditional proteomics methodology provides information for abundant proteins but only provides limited information for proteins with low abundance. Chemical proteomics which used small molecules as baits to fish for interacting proteins, has emerged as a powerful way to investigate the interacting proteome. By chemical proteomic approach, only a manageable fraction of the proteome interacting with the fixed small molecules will be collected and analyzed and this approach greatly reduces the complexity of a certain proteome and thus enhances the ability to detect and to characterize low-abundance proteins. Furthermore, chemical proteomics becomes a direct readout for the characterization of target proteins and related recognition mechanisms of many drugs whose targets are still unclear, especially for compounds of natural origin.Corydalis yanhusuo W.T. Wang has been widely used to treat spastic pain, abdominal pain and pain due to injury. In this paper, two-dimensional LC was applied to screen the bioactive compounds of Corydalis yanhusuo. L-THP, one of its main active ingredients was selected for further study. Microdialysis combined with LC-MS/MS was applied in the pharmacokinetic studies of l-THP in the straitum, the traget sites, and shotgun-based proteomics was applied to illustrate the detailed mechanisms involved in the l-THP-induced-antinociception and -hepatotoxicology. The detailed results were presented as follows:1. In the present study, an animal mode of nociception based on the formalin injection into the hinder paw of rats was applied to evaluate the anti-nociceptive effect of total alkaloids of Corydalis yanhusuo (TAC), and the results of the formalin test indicated that formalin-evoked spontaneously nociceptive responses (licking behavior) could be inhibited by given (i.g.) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed, and the absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (7 compounds identified) were absorbed into the plasma with appropriate concentrations, and about 20 compounds (4 compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine and corydaline) which were reported to possess profound anti-nociceptive effects, exhibited high concentrations in the striatum, therefore, it appears that they participated synergistically in regulating the formalin-induced nociception. The results indicated that the developed comprehensive two-dimensional biochromatography method is capable of screening the bioactive components in Corydalis yanhusuo, and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.2. Levo-tetrahydropalmatine (l-THP), which is officially listed in the Chinese pharmacopoeia was demonstrated to have excellent analgesic effects and has been in use in clinical practice for years in China. L-THP was recently found to elicit profound effects on the dopaminergic system in the straitum to produce anti-nociception. In the present study, the factors affecting the in vivo recovery from microdialysis probe (drug concentrations and within-day stability) were investigated, and the results indicated that microdialysis was an excellent method for in vivo sampling to measure the concentration of l-THP in the striatum. We developed and validated a sensitive, specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the quantitative determination of l-THP using diphenhydramine as the internal standard. The method combined LC-MS/MS with microdialysis was successfully applied in the pharmacokinetic studies of l-THP in the straitum.3. L-THP, received much attention as an analgesic agent, has been associated with acute or chronic hepatitis in clinical practice. We found that l-THP can induce apoptosis in the hepatocytes of BALB/c mice and human normal liver L-02 (L-02) cells. Several key molecules, including caspase-3, Bcl-2 and Bax, were modulated by l-THP treatment. A novel high-throughput proteomic approach based on shotgun approach was applied to simultaneously evaluate the alterations of global protein expression involved in the response of l-THP treatment in L-02 cells. A total of 156 proteins were differentially expressed, among which 12 proteins play regulatory or constitutive roles in apoptosis pathways. Further analysis of two proteins by Western Blots, mTOR and MEK2, confirmed that these proteins were expressed at lower levels in l-THP treated L-02 cells compared with those of control. The current study provided detailed evidence to support that l-THP is capable of inducing apoptosis in mammalian liver cells and improve the understanding of mechanisms mediating the hepatotoxity of l-THP.4. This study investigated the mechanisms involved in the antinociceptive action induced by l-THP in the formalin test by combined comparative and chemical proteomics. Rats were pre-treated with l-THP by the oral route (40 mg/kg) 1 h before formalin injection. The antinociceptive effect of l-THP was shown in the first and second phases of the formalin test. To address the mechanisms by which l-THP inhibits formalin-induced nociception in rats, the combined comparative and chemical proteomics were applied. A novel high-throughput comparative proteomic approach based on 2D-nano-LC-MS/MS was applied to simultaneously evaluate the deregulated proteins involved in the response of l-THP treatment in formalin-induced pain rats. Thousands of proteins were identified, among which 17 proteins survive the stringent filter criteria were further included for functional discussion. Two proteins (Neurabin-1 and Calcium-dependent secretion activator 1) were randomly selected and their expression levels were further confirmed by Western Blots. The results matched well with those of proteomics. In the present study, we also described the development and application of l-THP immobilized beads to bind the targets. Following incubation with cellular lysates, the proteome interacting with the fixed l-THP was identified. The results of comparative and chemical proteomics are quite complementary. Although the precise roles of these identified moleculars in l-THP-induced antinociception need further study, the combined results indicated that proteins associated with signal transduction, vesicular trafficking and neurotransmitter release, energy metabolism, and ion transport play important roles in l-THP-induced antinociception in formalin test,

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