| The research of pharmaceutical material basis of traditional Chinese medicines (TCMs) has been a hotspot for the TCMs’modernization. While we should be clear on chemical components contained in TCM before we start to study that. And the study of chemical compounds contained in TCM was an important prerequisite to elucidate pharmaceutical material basis in essence, also can provide references for formula optimizing, technology developing, and quality of preparation improving. Meanwhile, effective material basis was presented in the prototype components and metabolites biotransformed by organism, thus the study of substance exposed in vivo and metabolites became necessary for the establishment of connection between these compounds and efficacy, and also be of great significance for efficient material basis studies, candidate screening and drug development. But the chemical components of TCM were characterized by complex, various types, constant or trace composition coexistence, and difficulty in detecting ingredients with low concentration. These features have posed a serious challenge for the research of chemical material basis of TCM. Therefore, how to quickly and efficiently clarify chemical components of TCM and solving the problem of material basis research in complex system of TCM depended on the breakthrough of methodology and technology, which has become hot issue scholars continued to explore.Xiao-er-qing-jie (XEQJ) granules, which are composed of eight herbal medicines, viz., Flos Lonicerae Japonicae, Forsythiae Fructus, Cortex Lycii and so on, is a regularly used traditional TCM for the treatment of children with high fever, sore throat. However, the unclarity of chemical composition of this formulation restricted its basic research on effective substance. In order to explore the effective material basis of XEQJ preliminarily, this paper took XEQJ as vehicle and established methods for rapid screening and identification of relevant chemical constituents to study chemical constituents as well as metabolism in vivo of this formulation by the use of U/HPLC-LTQ-Orbitrap high-resolution spectrometer. The main contents and results were described as follows:1. The qualitative study of comprehensive constituents of XEQJIn order to improve the detective efficiency of the compound and achieve high-throughput screening of compound, this present study took Full-scan-Dynamic exclusion (FS-DE) and Data dependent scan (DDS) as well as established LC-MS analytical method based on HPLC-LTQ-Orbitrap for chemical constituents’determination and data acquisition of XEQJ. Different types of compounds’fragment patterns were speculated and summarized according to the relevant references and literatures. For known compounds, it were exactly identified by extract ion chromatograph and reference substances; for unknown compounds, it were rapidly identified based on accurate mass, fragmentation pattern, multi-stage mass fragments, and data reported previously. As a result,91 compounds were unambiguously identified or tentatively characterized, including 33 phenylethanoid glycosides,13 phenolic acids,11 flavonoids,10 alkaloids,9 lignans,9 iridoid glycosides, and 6 saponins. The component herb from which each compound was derived was also analyzed. This part developed a comprehensive understanding of the multiple chemical constituents in XEQJ and could offer chemical basis for the further study.2. Rapid screening and identification of phenylethanoid glycosides of XEQJ based on the diagnostic product ions technology.The diversity of same class chemical composition of TCMs was mainly up to the kinds and number of substituent group. Probable molecular structure and formula were designed first according to the structural feature of phenylethanoid glycosides (PhGs) in this chapter. Then, Full scan-Parent ions list-Dynamic exclusion (FPD) was adopted to pertinently collect the mass data of candidate formula primarily screened by first high-resolution data using UPLC-LTQ-Orbitrap high-resolution mass spectrometer. And again, the common fragmentation pattern of such compounds was summarized by research in mass fragments as well as fragmentation behavior of related reference substances, which could help speculating diagnostic product ions (DPIs) of other compounds belonged to this class. The PhGs were rapidly screened and speculated its’possible structure by means of diagnostic product ions combining high resolution mass data, and rapidly identified by related reference standards, SCI Finder query results, and component herb from which compounds were derived. As a result, a total of 96 compounds which contributed to 40 categories of PhGs were identified in XEQJ through this method. It suggested that compounds belonged to this class in TCMs’complex system could be screened and identified via this method, rapidly and systematically.3. Rapid screening and identification of chlorogenic acids of XEQJ based on the diagnostic product ions bridging technologyThe research method in this part was similar to the previous chapter. The deference was that links between various diagnostic ions and the particular compound were found to build a network of DPIs after the common fragmentation pattern of such compounds was summarized by research in mass fragments as well as fragmentation behavior of related reference substances and subsequently speculate DPIs of other compounds belonged to the same category. The chlorogenic acids (CGAs) were rapidly screened via diagnostic product ions bridging technology combining high resolution mass data, and identified by the bibliography data and reference standards. A total of 73 compounds were identified in XEQJ which contributed to 17 categories of CGAs through this method. The results indicated that this technology improved the efficiency of rapid discovery and simultaneous characterization of groups of compounds including constant and trace compositions. And also provided a new research idea for rapid structural identification of groups of compounds in a complex chemical system.4. The quantitative study of main compounds of XEQJBased on the results analyzed above, this section established an HPLC-DAD method for simultaneous determination of 9 constitutes (neochlorogenic acid, chlorogenic acid, cryptchlorogenic acid, sweroside, secoxyloganin, forsythoside A,3,4-dicaffeoylquinic acid,3, 5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid) in XEQJ. The results revealed that some deference on content of each component was existed in deferent manufacturers and the developed method could provide reference for quality control of multi-index components in XEQJ.5. The metabolic study of main monomers and formulation in ratsThe metabolism of main monomer compounds (chlorogenic acid, isochlorogenic acid B, sweroside, and forsythoside A) and XEQJ water exact in rats were studied respectively after systematically understanding chemical composition of XEQJ. Metabolites of each monomer were identified and metabolic pathway of that were proposed based on characteristic fragmentation pattern, relevant drug biotransformation, and bibliography.50 prototype constituents were discovered in rats’biology samples by comparing blank sample, the retention time and MS fragments of components in vitro and vivo. Besides,19 metabolites of chlorogenic acid,20 of isochlorogenic acid B,8 of sweroside, and 33 of forsythoside A were examined in rats orally administrated with XEQJ water exact through comparative analysis metabolites of XEQJ and monomers. This part provided metabolic basis for mechanism elucidation and further metabolic research of this formulation. |
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