Research On Quality Evaluation And High Throughput Sequencing Based Screening Of Bioactive Compounds Of Moutan Cortex

Moutan Cortex(MC),the dried root bark of Paeonia suffruticosa Andr.(family Ranunculaceae),shows the effects of clearing excessive heat,cooling the blood,promoting the blood circulation,and removing blood stasis according to the traditional Chinese medicine(TCM)theory.MC is one of the commonly used crude drugs in TCM that has a variety of chemical components and a wide spectrum of pharmacological activities.MC is extensively used in TCM prescriptions and has a long history of use in China and other Eastern countries such as Japan and Korea.Furthermore,MC was first recorded in Shennong's Classic of Materia Medica and classified as one of the middleleveled herbs.Description of the first processing method of MC can be found in Essential Prescriptions from the Golden Cabinet(Jingui Yaolue),composed by Zhong-jing Zhang at the end of the Eastern Han Dynasty.Modern pharmacological studies have shown that MC has anti-inflammatory,anti-bacterial,regulating the cardiovascular system,regulating immune function,and liver protection effects.However,there are still some deficiencies in the current research on MC.For example,the method of quality evaluation is too dependent on one or two compounds,and the mechanism of action of the pharmacological molecule is unclear.Objective: The quality of MC was supposed to be systematically evaluated based on morphological,microscopic,multi-component contents and fingerprints methods.In ombination with the newly proposed concept of "quality marker",the effective components of MC with traceability and transmissibility were selected to establish a fast and reliable multi-component quantitative method by UHPLC.Untargeted plant metabolmics was employed to profile the root bark metablomics of different species in Genus Paeonia,Sect.Moutan,and to provide a new perspective and scientific basis for the classification of the species from the metablomics level.The next generation highthroughput sequencing,refered as HTS2,of the Hep G2 cell model administered with main components and extracts of MC was carried out to investigate the pharmacological mechanism of the traditional curative effect of MC and the exploration of the potential pharmacological effects.Methods: In this study,the content of several components of MC was quantified by HPLC;the total ion flux of the extract and the content of six main constituents was quantified by HPLC-MS/MS;the metabolomics profile of different samples was acquired by HPLC-Qtof-MS;virtual screening and high-throughput sequencing were employed to the pharmacological screening of the active ingredients of MC and its extraction.Besides,a variety of software,such as SIMCA-P,Chem Pattern,Graphpad prism 5,Metaboanalyst 3.0,Cytoscape,R language pack GO plot,were used for data analysis and data visualization.Results:1.The root system of Paeonia suffruticosa was measured to compare the morphological variation and the chemical composition of different grades of MC was discussed according to previous studies.The difference between the main microscopic features of MC powder and the xylem powder is dramatic,the MC powder contains great amount of starch granules and clusters of calcium oxalate,while the xylem powder displays considerable vessels.Interestingly,the growth rings of Paeonia suffruticosa as first reported in the xylem of the root transection,this can help to determine the growth years of the plant.Moreover,through the assay of 17 component,MC produced in Tongling and Bozhou in Anhui province were compared,content of PGG in MC produced in Bozhou was significantly higher than MC produced in Tongling(P < 0.01).MC with different growth years,MC with xylem and unprocessed MC and MC decoction pieces were compared respectively by combining the results of 17 compounds assay and fingerprinting.2.The holistic metablomics profile of different parts of Paeonia suffruticosa and different species in Genus Paeonia,Sect.Moutan was completed,and a chemical database of Paeonia was established.This provided the metabolomic evidence for the taxonomic relationship among different species in Sect.Moutan.3.Eight major components are selected as quality markers of MC,such as oxypaeoniflorin,apiopaeonoside,albiflorin,paeonolide,paeoniflorin,PGG,mudanpioside C and paeonol.These eight quality markers are successfully applied to the quality evaluation of MC,and could be useful in improving the current quality standards of MC.4.Different groups of Hep G2 cells were treated with paeonol,paeoniflorin,PGG,apiopaeonoside,galloylpaeoniflorin and MC extract respectively.Then HTS2 analysis was performed on these Hep G2 cells,and genes with significant fold change within each group were quantified at the transcriptional level in Hep G2 cell model after the administraton of different compounds and MC extract.Through the functional enrichment analysis,GO and KEGG,we found that differentially expressed genes and their related pathways and diseases phenotype.In the light of the reported results of the activity and mechanism studies in the previously published literature,new perspective for the study of the TCM theory and future pharmacology of MC have been offered.Besides,our Western blotting results suggest that paeonol could significantly down-regulate the expression of necroptosis-related proteins in xtracellular acidification-induced rat articular chondrocytes.Conclusion: In summary,this study includes a combination of methods to evaluate the overall quality of MC more systematically and comprehensively.It is suggested that paeoniflorin should be added to MC quality evaluation standards and the minimum level should be recommended.In addition,we also proposed 8 quality markers of MC after systematically summarize the biosynthetic pathways,pharmacological activities,and in vivo metabolites of 8 quality markers.A high-throughput sequencing study of the active constituents of MC was conducted,and its possible pharmacological effects and potential mechanisms for the treatment of various diseases were elucidated.