Based On The Technology Of Lc - Ms ~ N Clear Open Spirit Injection The Research Of Chlorogenic Acid

Qingkailing injection is composed of Fructus Gardeniae, baicalin, cholic acid, hyodeoxycholic acid, Radix Isatidis, Flos Lonicerae Japonicae, Cornu Bubali and Concha Margaritifera, with the effects of clearing away the heat-evil and expelling superficial evils, eliminating phlegm, dredging collaterals and inducing resuscitation. The principle chemical components in the injection contain iridoid glycosides, flavonoids glycosides and amino acids etc. Chlorogenic acids, as the main representative organic acids in the injection, mainly come from Flos Lonicerae Japonicae and Fructus Gardeniae herbs. These compounds are greatly contributed to clear heat-evil and detoxification effect.Chlorogenic acids, in Qingkailing injection and related herbal medicines in the prescription, were studied systematically in this project. And the quality control of these compounds in Qingkailing was evaluated. What’s more, a preliminary study for the regularity of changes in these compounds was taken out in our laboratory. The main research contents and results are as follows:1. The analysis and identification of chlorogenic acids in Qingkailing injectionAn HPLC-DAD-ESI-MSn method for analyzing chlorogenic acids in Qingkailing injection was developed. Retention time, molecular weight and MS/MS fragmentation of all kinds of chlorogenic acids were obtained with the developed method and13chlorogenic acids, including1caffeic acid, were identified in the injection eventually. This work was greatly contributed to clarify the chlorogenic acids material basis of Qingkailing injection.2. The analysis and identification of chlorogenic acids in related herbal medicines in the prescriptionTo demonstrate the source and belonging of the chlorogenic acids in Qingkailing injection, the developed method in first chapter was applied in analyzing these compounds in related herbal medicines in the prescription. Finally,22and18chlorogenic acids, including1caffeic acid, were identified in Flos Lonicerae Japonicae and Fructus Gardeniae herbs, separately. Compared with the results got from product and herbs, all chlorogenic acids in Qingkailing injection were detected in Flos Lonicerae Japonicae and Fructus Gardeniae herbs expect1,3-dicaffeoylquinic acid. It was speculated that it may come from production process through isomerization.3. Simultaneous determination of chlorogenic acids in Qingkailing injection by HPLC-DADIn this section, an HPLC-DAD method for simultaneous determination of chlorogenic acids was developed. And this method was successfully applied for determination of3-caffeoylquinic acid,5-caffeoylquinic acid,4-caffeoylquinic acid, caffeic acid,3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid and4,5-dicaffeoylquinic acid in Qingkailing injection of5different batches which had great variation on the contents.4. The application of quantitative analysis of multi-component with a single-marker (QAMS) in simultaneous determination of chlorogenic acids in Qingkailing injection by HPLC-DADHPLC-DAD method was applied to the determination of seven ingredients by QAMS. The factors effecting reproducibility of the relative correction factors were investigated systematically, including experimental instruments, chromatographic columns, flow rate and column temperature. The results indicated that the reproducibility of the RCFs was satisfactory and the RSD was less than5%. The contents of3-caffeoylquinic acid,4-caffeoylquinic acid,3,5-dicaffeoylquinic acid,3,4-dicaffeoylquinic acid and4,5-dicaffeoylquinic acid were determined by both external standard method and QAMS. The method was evaluated by Pearson correlation analysis and/-test of the quantitative results between external standard method and QAMS. The results of QAMS method were not significantly different from that of external standards method. As the complimentary method of multi-component analysis, it was feasible to control the quality of Qingkailing injection by QAMS. QAMS method can overcome the challenge of unstability and high cost of reference substance.5. Regularity of changes in chlorogenic acid in Flos Lonicerae Japonicae by HPLC-DADWith Flos Lonicerae Japonicae as the research object,3-caffeoylquinic acid,5-caffeoylquinic acid,4-caffeoylquinic acid,3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid and4,5-dicaffeoylquinic acid as evaluation index, we investigated the regularity of changes in these compounds preliminarily. During water extraction process, the contents of5-caffeoylquinic acid and3,5-dicaffeoylquinic acid tended to stabilize or even decrease gradually after reaching a high level during the extraction. There existed a rising trend for the other components, but their variation rates were decreasing. In this section, we studied the effect of alcohol concentration on the content of chlorogenic acids. The results showed the optimal alcohol concentration was70%for each compound. By comparing with other compounds, dicaffeoylquinic acids were more sensitively effected by alcohol concentration. Affected by temperature and illumination, chlorogenic acids could break up into caffeic acid through hydrolysis reaction. What’s more, dicaffeoylquinic acids could transform into caffeoylquinic acids affected by temperature through ester bond rupture. In our study,1,3-dicaffeoylquinic acid failed to detected in the samples above-mentioned by the established method. Speculationsareas are as follows:firstly, isomerization of chlorogenic acids were happened in the other production process of Qingkailing injection; secondly, as the storage time passed, isomerization of chlorogenic acids in Qingkailing injection could take place affected by other environmental factors or internal environment changes.