Study On Pharmacodynamic Substances And Quality Control Method Of Ling-gui-zhu-gan Decoction

Ling-gui-zhu-gan decoction,originated from Shanghan Zabing Lun by Zhang Zhongjing of Han Dynasty,is composed of Poria cocos(Schw.)Wolf,Cinnamomum cassia Presl.,Atractylodes macrocephala Koidz.and Glycyrrhiza uralensis Fisch.It has the effect of warming and Tonifying Qi and diuresis,which is widely used in clinical.However,at present,the research on this prescription is mainly focused on the pharmacodynamics effect and clinical application,lacking the research on relevant quality control and material basis.Therefore,the method of chemical composition analysis,serum pharmacochemistry analysis,drug metabolism analysis,and the technology of UHPLC-ESI-Q-TOF-MS/MS were combined to study the chemical basis and method of quality control of Ling-gui-zhu-gan decoction in this paper.The main research contents are listed as follows:1.Study on HPLC fingerprint of Ling-gui-zhu-gan DecoctionThe HPLC fingerprint of Ling-gui-zhu-gan decoction was established to provide a qualitative method.16 batches of Ling-gui-zhu-gan decoction samples from different origins were tested.After that,the common pattern of fingerprint of Ling-gui-zhu-gan decoction has been established,and 22 common peaks were chosen.The components of 15 peaks in the 22 common peaks were identified by the references,including liquiritin,liquiritigenin,liquirtin apisode,cinnamaldehyde,neoliquiritin,liquiritin,liquiritin apisode,liquiritigenin,licochalcone B,isoliquiritigenin,kaempferol,eugenol,isoliquiritin,glycyrrhizic acid,glycyrrhetinic acid,atractylenolide III,atractylenolide?.The results of the source confirmation experiment of peaks showed that the peaks in HPLC fingerprint of Ling-gui-zhu-gan decoction were mainly from Atractylodes macrocephala Koidz.and Glycyrrhiza uralensis Fisch.At the same time,the methodology was performed for verification.The results of methodology verification shows that the RSD of precision,stability and repeatability data is less than 4.0%,which demonstrated that this method has certain specificity.HPLC fingerprint can provide rich information,which can evaluate the quality of complex decoction comprehensively and completely.It can be used as the qualitative method of the decoction.2.Chemical composition analysis of Ling-gui-zhu-gan DecoctionIn order to conduct a comprehensive analysis of the chemical constituents in Ling-gui-zhu-gan decoction,the technology of UHPLC-ESI-Q-TOF-MS/MS was used to determine the samples in both positive ion mode and negative ion mode.Comparing the data information of fragments of secondary mass spectrometry in this experiment with the database and literature,totally 154 compounds were identified at last,including flavonoids,triterpenoids,organic acids,sesquiterpenoids,coumarins,etc.Therefore,UHPLC-ESI-Q-TOF-MS/MS technology has the characteristic of high sensitivity and high accuracy,which can accurately and efficiently identify the chemical composition of decoction,and providing the basis for its efficacy research and quality control.3.Analysis of the blood components and metabolites of Ling-gui-zhu-gan decoctionIn order to further interpret the pharmacodynamic basis of Ling-gui-zhu-gan decoction,the whole animal model was adopted to simulate the process of oral administration of decoction.The prototype compounds and metabolites in plasma,urine and feces were analyzed.45 prototype compounds were detected from biological samples,including 1 from Poria cocos(Schw.)Wolf,3 from Cinnamomum cassia Presl,3 from Atractylodes macrocephala Koidz.,36 from Glycyrrhiza uralensis Fisch.,which may be the effective components of Ling-gui-zhu-gan decoction.238 metabolites were analyzed by Metabolitepilot and Peakview software from biological samples.Due to the similar metabolism process of the same type of components in vivo,in this chapter,some representative compouds with high content in vivo of each category were also selected as the samples to speculate the metabolic pathway in vivo.The original components and metabolic components detected in this chapter may be the effective components of Lingguizhugan decoction.It also lays a foundation for the further research.4.Study on determination method of Ling-gui-zhu-gan DecoctionQuality control is an important factor to ensure the safety and effectiveness of drug.Ling-gui-zhu-gan decoction,as a traditional Chinese medicine compound,has complex chemical components,and still lacks effective quality control methods.Therefore,HPLC was applied in this chapter to detect four representative components,including liquiritin,glycyrrhizic acid,cinnamic acid and atractylenolide?in Ling-gui-zhu-gan decoction.The results of methodological validation showed that the four components had a good linear relationship within a certain range(R~2?0.9990),the RSD of precision,repeatability and stability data was less than 3.0%,the recovery rate of sample was between 90%~103%,which met the requirements of Chinense Pharmacopoeia.The method is stable,reliable and accurate,and could be used for the quality control of Ling-gui-zhu-gan decoction.