Study On The Material Basis And Mechanism Of Action Of Tongfengding Capsule In The Treatment Of Gou

Objective:Tongfengding capsule,included in the Chinese Pharmacopoeia 2020 edition,is composed of 8 herbs,containing Gentiana macrophylla Radix,Phellodendron chinense Cortex,Corydalis Rhizoma,Paeoniae Radix Rubra,Cyathulae Radix,Alismatis Rhizoma,Plantaginis Semen,and Smilacis Glabrae Rhizoma,which has the effect of clearing heat,dispelling dampness,activating blood,clearing collaterals and relieving pain.At present,few qualitative or quantitative studies have been performed on the chemical components in Tongfengding capsule,and only 13 chemical components have been identified.And there is no study on the metabolism of Tongfengding capsule in vivo.On the other hand,Tongfengding capsule is often used in the treatment of gout,hyperuricemia,rheumatoid arthritis and other diseases in clinical practice,but lack of pharmacological mechanism.In conclusion,although Tongfengding capsule has a good therapeutic effect on gout in clinical practice,there are few studies on the chemical components of Tongfengding capsule,and its active ingredients and mechanism of action in treating gout are still unclear.Taking Tongfengding capsule as the research carrier and its treatment of gout as the entry point,this project firstly comprehensively characterized the main chemical components in Tongfengding capsule based on the LC-MS technology.Then the absorption and metabolism of chemical components was systematically studied in Tongfengding capsule in vivo by using sequential metabolism method.The target and action mechanism of Tongfengding capsule in treating gout were predicted with the targeted network pharmacology.Xanthine oxidase is a known key target for the treatment of gout and therefore a variety of experimental techniques and methods were comprehensively applied to screen xanthine oxidase inhibitors from Tongfengding capsule and verified.Meanwhile,in order to reveal the material basis and action mechanism of Tongfengding capsule in treating gout from multiple aspects,a mathematical model of target occupancy in vivo targeting XOD was established to predict the in vivo efficacy of XOD inhibitors.Methods:(1)To establish the chemical constituent database of Tongfengding,the identified chemical constituents in eight Chinese herbal medicines of Tongfengding were obtained through database searches.Ultra-high performance liquid chromatography-high resolution mass spectrum(UPLC-HRMS)technology was used to detect Tongfengding capsule and the test solution of eight Chinese medicinal materials.The retention time and fragment information of each component were compared with compound database,related literature,available reference standards as well as various compounds were identified based on the mass spectrometry fragmentation patterns.(2)Using the intestinal perfusion with venous sampling method,blood was collected from the mesenteric venous plexus and femoral vein,respectively,and plasma samples of intestinal metabolism and liver metabolism were obtained.After intragastric administration,blood was collected from the abdominal aorta,and comprehensive metabolic plasma samples were obtained.UPLC-HRMS technology was used to identify the chemical components in different plasma samples.By comprehensively comparing the chemical components in different plasma samples,the absorption and metabolism of each chemical component at different sites in the digestive tract were clarified.(3)The structural formulas of the prototype blood components identified in Tongfengding capsule were imported into MedChem Studio 3.0 software to predict their potential targets.By searching DrugBank,Online Mendelian Inheritance in Man,and Therapeutic Targets Database database,gout-related disease targets were obtained.Taking the common targets of component targets and disease target as the core targets,the DAVID database was used to conduct Gene Ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analysis for the core targets.Cytoscape 3.6.1 was carried out to construct component-core target-KEGG pathway network,and the potential active components of Tongfengding capsule were screened.Finally,a real-time quantitative polymerase chain reaction was used to verify the effect of apigenin on related targets.(4)First,the inhibitory effect of the water extraction on XOD was investigated in vitro enzyme catalysis.In addition,the XOD inhibitor of Tongfengding capsule was screened out by the method of surface plasmon resonance(SPR)coupled with LC-MS.Then,molecular docking was used to verify and further screen the compounds at the molecular level.Finally,SPR technology was used to experimentally verify some of the compounds after molecular docking verification,so as to screen the inhibitors of XOD in Tongfengding capsule.(5)Three kinetics(PK,BK and TK)processes affecting drugs in vivo were systematically integrated.Taking XOD as the target and 4 small molecules(febuxostat(positive drug),engeletin,astilbin,berberine)as the research objects,based on the PK-BK-TK model,the in vivo target occupancy mathematical mode of XOD was established to predict the time course of target occupancy in vivo after oral administration.Results:(1)A total of 141 chemical components were identified from Tongfengding capsule by UPLC-MS technology,including 52 alkaloids,22 flavonoids,38 terpenoids,23 organic acids and 6 other components.131 compounds were identified from Tongfengding capsule for the first time,and 33 compounds were accurately identified by comparison with reference standards.(2)A total of 100 chemical components were identified from different plasma samples,containing 64 prototype blood components and 36 metabolites.76 chemical components were identified in intestinal metabolism samples,including 58 prototype absorption components and 18 metabolites.A total of 53 chemical components were identified in liver metabolism samples,including 30 prototypes absorbed into the blood and 23 metabolites.A total of 74 chemical components were identified in comprehensive metabolic samples,including 45 prototype blood components and 29 metabolites.In addition,through comparative analysis of chemical components in different plasma samples,the absorption and metabolism of 64 prototype components at different sites in the digestive tract were elucidated.(3)753 component targets and 222 gout-related disease targets were obtained respectively,and 50 core targets and 35 chemical components acting on the key core targets were further screened.The results of enrichment analysis demonstrated that 35 chemical constituents mainly significantly modulated 113 GO items,involving multiple metabolism-related biological processes,such as steroid metabolism,exogenous metabolism and progesterone metabolism,and regulated 20 KEGG pathways,including multiple metabolism-related metabolic pathways and so on.In addition,the arachidonic acid metabolic pathway in the metabolic pathway was highly enriched,involving 6 core targets of PTGS1,PTGS2,AKR1C3,CYP2B6,ALOX5 and PLA2G1B.Further mechanism analysis showed that it was the key pathway of Tongfengding capsule in the treatment of gout by reducing inflammatory response.By analyzing the component-core target-KEGG pathway network,it was found that 12 components,including amaranthine,astilbin,astilbin,apigenin and so on,could map multiple targets,including the above six targets.It can be considered a potential active ingredient in the treatment of gout in Tongfengding capsule.RT-qPCR in vitro results confirmed that apigenin could significantly reduce the mRNA expression levels of PTGS2,TNF-α and IL-6 in the arachidonic acid metabolic pathway.(4)The water extraction of Tongfengding capsule performed good XOD inhibitory activity.Additionally,32 compounds combined with XOD were preliminarily screened from Tongfengding capsule by SPR coupled with LC-MS.According to the active site molecular docking result,15 XOD inhibitors bound tightly to the active site of XOD from the above 32 compounds screened out.Finally,6(engeletin,astilbin,berberine,tetrahydro berberine,dehydrocorydaline and 4-O-feruloylquinic acid)of the above 15 inhibitors were experimentally verified by SPR technology,and the affinities of the above 6 inhibitors to XOD ranged from 1.12 to 8.53×107,indicating all of them can be closely combined with XOD.(5)The marketed drug febuxostat could occupy 80%of the XOD target at the clinical dose for 18 h,which was in good agreement with its clinical efficacy duration,proving that the established PK-BK-TK mathematical model can accurately predict the occupancy process of XOD in vivo after oral administration.When rats were given berberine by a single gavage of 100 mg/kg,the duration of target occupancy>70%in vivo was approximately 2.2 h.When rats were given engeletin or astilbin by a single gavage of 100 mg/kg,the duration of target occupancy>80%in vivo was approximately 5.4 h and 1.7 h,respectively.Conclusion:In this study,UPLC-MS technology was used to comprehensively characterize the main chemical components in Tongfengding capsule.At the same time,combined with the sequential metabolism method,the absorption and metabolism of chemical components in Tongfengding capsule at different sites in the digestive tract were systematically studied,which laid a foundation for comprehensive quality control and in-depth scientific research of Tongfengding capsule.In addition,based on targeted network pharmacology,it is proven that some of the potential active ingredients in Tongfengding capsule could reduce inflammation by acting on the metabolic pathway of arachidonic acid,thereby exerting a therapeutic effect on gout.At the same time,a variety of technical methods were used to screen out some inhibitors that could act on XOD,and a mathematical calculation model was established to predict the target occupancy closely related to in vivo activity,to clarify that Tongfengding capsule may inhibit the activity of XOD and reduce the production of uric acid in the treatment of gout.In conclusion,this study performed in-depth research on the pathway of anti-inflammatory and reducing uric acid production,and initially revealed the medicinal components and mechanism of action of Tongfengding capsule,providing the theoretical basis and data support for the clinical application and drug development of Tongfengding capsule.