| BackgroundWith the improvement of human life and the changes of diet structure, especially high purine diet intake such as seafood and beer, the incidence of hyperuricemia rises every year and it has become one of metabolic diseases that serious threat to human health, and presents the trend of younger age in recent years. A number of epidemiological reports have revealed that hyperuricemia is not only has a close relation with obesity, diabetes, cardiovascular disease, but also an independent risk factor for hypertension, coronary heart disease, kidney disease and type 2 diabetes. Therefore, its become an important clinical significance to carry out the prevention and treatment of hyperuricemia.In previous studies, we found chicory could significantly reduce the hyperuricemia model of serum uric acid (UA) levels in quails, which were induced by high purine diet. However, to the best of our knowledge, the major effective components in the UA-lowering remain unclear so far, it also become a bottleneck when making chicory quality evaluation. Thus, chicory treatment of hyperuricemia was used as the breakthrough point, we adopt the "spectrum-effect relationship" to carry on chicory to research the material basis in the treatment of hyperuricemia, aims to clarify the material basis in the treatment of hyperuricemia from overall level; And on this basis, combining with quantitative analysis of multi-components by single marker (QAMS) technology, to establish the QAMS method for chicory, further provide a basis and reference for the quality evaluation of chicory, so as to make it better applied to clinical.Objectives1. By constructing the "spectrum-effect relationship" chicory in the treatment of hyperuricemia, we are aim to clarify the material basis of chicory in UA-lowering.2. On the base of pharmacodynamics material basis of chicory and combined the technology of QAMS, establish the QAMS method for chicory, so as to provide a basis and reference for the quality evaluation of chicory.Method1. The material basis research of chicory in the treatment of hyperuricemia:Choose different sources of chicory as the research object, adopt the HPLC fingerprint technology to obtain chemical information from chemical level; adopt high purine diet induced quail hyperuricemia to obtain the UA-lowering value from animal level. Through correlation analysis (CA) and partial least squares regression (PLSR) method to build the "spectrum-effect relationship" of chicory in the treatment of hyperuricemia, in order to screen out the characteristic peak which is close to UA-lowering.2. Verify the rationality of "spectrum-effect relationship" results:Adopt the LC-MS/MS technology, qualitative analysis the chemical structure of each characteristic peak in chicory, using chemical monomer to carry on the UA-lowering pharmacodynamics experiment, verifying the rationality of the results by the "spectrum-effect relationship".3. The establishment of chicory by QAMS:Three different HPLC instruments and three different C18 chromatographic columns were used to investigate the durability of relative correction factor (RCF) for each compound. Using RCF to calculate the content of each effective compound from 10 batches of chicory, and compared with external standard method (ESM) measured content, further evaluate the accuracy of the QAMS.Research contents:This study was composed of two main parts:literature review and experimental research.Literature reviewIn the literature review part, we mainly discusses the research progress of chemical constituents and pharmacological effects of chicory, and made a systematically reviewed and summary for the "spectrum-effect relationship", including research contents, data processing technologies, and structure determination.Experimental research:Chapter One HPLC fingerprint and UA-lowering efficacy research on different regions of chicoryThe HPLC fingerprint technology was applied to establish analysis method of chicory HPLC fingerprint and then conduct methodologically inspected, build the different regions of chicory HPLC fingerprint. Mining chemical information that can reflects component of chicory as much as possible; in addition, high purine diet was used to induce quail hyperuricemia model, then different origins of chicory were administrate to quails to obtain UA-lowering value, so that provide "spectrum" and "effective" information for the "spectrum-effect relationship".Chapter Two Study on the spectrum-effect relationship of chicory in UA-loweringAdopted CA and PLSR method to build the "spectrum-effect relationship" between the fingerprint information and UA-lowering value of chicory, screened characteristic peak by combining two methods of data processing results which is associated with UA-lowering. By LC-MS/MS acquire the information of each characteristic peak fragment ions and retention time, compared with literature reports and standard reference information, then determine the chemical of each characteristic peak, and then carries on the preliminary UA-lowering validation use part of the chemical composition which was screened by "spectrum-effect relationship" to further verify the rationality of the "spectrum-effect relationship" screening results.Chapter Three The quality evaluation model for chicory by QAMSOn the basis of predecessors research combined with the above experimental results, choose a proper amount and stable compound as internal content, establish the RCF between the internal content and the rest of the compound, calculate the content of the rest of the compounds according to their RCF, and ESM method is used to measure the content of each compounds, and compare their difference, so as to verify the accuracy and feasibility of QAMS method.Research results1. Similarity analysis results show that the 10 batches of chicory with chicory contrast diagram (R) spectral similarity were greater than 0.759, and cluster analysis show that the samples could be divided into three clusters when L=11.10 batches of chicory are show different levels of UA-lowering over time, especially the origin from Beijing haidian, changji, xinjiang chicory sample, which show good effectiveness.2. The active components of chicory was screened by the "spectrum-effect relationship" study, included esculine, chlorogenic acid, chicory acid, chlorogenic acid B, chlorogenic acid A and characteristic peak of 11; Esculine, chlorogenic acid, chicory acid was used for preliminary to verify the efficacy UA-lowering, and the results showed that the three above have good UA-lowering effect, verify the rationality of the results by the "spectrum-effect relationship".3. The values of RCF of chlorogenic acid, chlorogenic acid B and chlorogenic acid A to esculine is 1.3339、0.8721、0.7658 respectively, no significant differences were observed between the quantitative results of QAMS and ESM, further verify the rationality of the QAMS method.Research conclusions1. Different regions of chicory fingerprint show differ in chemical composition, and its may be the basis of difference in UA-lowering.2. "Spectrum-effect relationship" study shows esculine, chlorogenic acid, chicory acid, chlorogenic acid B, chlorogenic acid A and 11 are the pharmacodynamics material basis.3. Established the QAMS method for the quality control of chicory, provide basis and reference for the quality control and evaluation of chicory, so as to make it better applied to clinical.Innovation1. "Spectrum-effect relationship" was the first time for study chicory in UA-lowering, revealed the efficacy material base of chicory in UA-lowering.2. Combined with QAMS technology for the first time, established the QAMS method for the quality control of chicory, and provided basis and reference for the quality evaluation of chicory. |
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