Qualitative And Quantitative Study On Effective Compounds Of Fangji Huangqi Tang Against Adriamycin-induced Nephropathy Based On The Process In Vivo

Rationales : Fangji Huangqi Tang was from ? Synopsis Golded Chamber?,which has the effects of inducing diuresis to alleviate edema,and replenishing wei and consolidating the exterior.In recent years,the clinical application of Fangji Huangqi Tang mainly focuses on cardiogenic edema,nephrotic syndrome and other diseases.The exact pharmacological effects of promoting water and reducing swelling have made Fangji Huangqi Tang more and more popular in recent years.So far,the research group has identified the effective components of Astragali Radix in Fangji Huangqi Tang against the adriamycin-induced rat nephropathy model by spectrum-effect correlation in our previous study.At the same time,the research group established the analysis method of chemical components of Fangji Huangqi Tang in vivo and in vitro in the early stage,and preliminarily studied the metabolism of Fangji Huangqi Tang in physiological state.However,the identified components were less,the analysis was not comprehensive,and the absorption of components in the pathological state was not covered.Therefore,it is very important to further identify the effective components of Fangji Huangqi Tang in adriamycin nephropathy and quantitative determine the content of the effective components based on the process in vivo.In this study,firstly,based on UHPLC-Q-Exactive MS,the components of Fangji Huangqi Tang in blood,urine and feces were analyzed to clarify the metabolism process of Fangji Huangqi Tang in vivo;secondly,animal experiments were carried out to construct adriamycin nephrotic rat model,and metabolomics combined with multivariate statistical analysis was used to compare the absorption differences between the two body states.Finally,simultaneous determination of 19 components in Fangji Huangqi Tang based on UPLC-Qtrap-MS.The purpose of this study is to clarify the material basis of Fangji Huangqi Tang in the treatment of nephrotic syndrome,whichwill lay a foundation for the further development of Fangji Huangqi Tang into a compound with clear composition and controllable quality,and provide a basis for the classification and evaluation of Astragali Radix.Objective: In this study,the in vivo process analysis method was used to identify the effective components of Fangji Huangqi Tang in treatment of adriamycin nephropathy rats,which will provide ideas for the material basis research of traditional Chinese medicine,and at the same time provide a research basis for the classification and application of astragali Radix.Methods:(1)UPLC-Q-Exactive MS was used to Analysis the metabolic components of serum samples containing Fangji Huangqi Tang given by gavage to normal rats.After several times administration of high-dose Fangji Huangqi Tang to SD rats,the serum samples was pretreated by methanol protein precipitation method,and urine and feces were pretreated by solid-phase extraction.The prototype compounds and their metabolites of Fangji Huangqi Tang in the physiological state were assigned in combination with secondary ion fragments in vitro and Compound Discover 2.0 software prediction;(2)The serum samples of C,M,C-FHT and M-FHT were analyzed by metabolomics and multivariate statistical analysis technology to determine the efficacy markers.At the same time,multivariate statistical analysis was used to analyze the metabolic spectra of Fangji Huangqi Tang under physiological and pathological conditions,and to determine the absorption difference of components from the compound under the two conditions,which was defined as potential effective components.Finally,the correlation between the potential effective components and the efficacy markers was analyzed to determine the effective components and their related metabolites of the compound;(3)A method of the simultaneous determination of 19 components in Fangji Huangqi Tang was established based on UPLC-MS/MS byoptimizing the parameters of chromatography and mass spectrometry.Results:(1)A total of 100 compounds were identified by UPLC-Q-Exactive MS,including 22 prototypes and 78 metabolites.The results showed that the main chemical components in the blood,urine and feces of rats were flavonoids and their metabolites.Among them,the main metabolites are the phase ? metabolites of glucuronidation from Calycosin and Liquiritin.These results suggest that these components may be the potential active components of Fangji Huangqi Tang in the treatment of adriamycin nephropathy.(2)Metabolomics was performed on the 4 groups of serum samples including C,M,C-FHT and M-FHT,and the multivariate statistical analysis was applied to screen differential metabolites.According to the content change of endogenous differential metabolites,FHT can significantly reduce the contents of 10-HDA,and significantly increase the content of LPE(18:2),but has no significant effect on other metabolites in physiological state.At the same time,6 biomarkers,including 10-DHA,aspartyl-L-proline,2-methyl-butyryl carnitine,safingol,uric acid and Bis(4-ethylbenzylidene)sorbitol,showed a tendency of regression only in the pathological state,which were efficacy markers of NS.Further analysis of metabolic pathways showed that phenylalanine,tyrosine and tryptophan biosynthesis,linoleic acid metabolism,phenylalanine metabolism,sphingolipid metabolism was the key pathways.The prototype components with different absorption under physiological and pathological conditions are Fan Ghinoline ?(+)-Tetrandrine?Atractylenolide III?Atractylenolide I?Liquiritin and Liquiritin.The differential metabolites were M1,M2,M3,M4,M5,M6 and M7.Through correlation analysis,we can conclude that 14 components,such as fanghinoline,(+)-tetrandrine,atractylenolide III,atractylenolide I,Liquidin,isoliquidin and M1,are highly positively and negatively correlated with efficacy biomarkers.However,the specificrelationship of its regulation in vivo needs further study.A method for simultaneous determination of 19 components in Fangji Huangqi Tang based on UPLC-Qtrap-MS technology was successfully constructed.These ingredients include Ononin ? Formononetin ?Calycosin ? Calycosin-7-O-?-D glucoside ?(6a R,11 a R)-3-hydroxy-9,10-dimethoxypterocarpan ?(6a R,11 a R)-3-hydroxy-9,10-dimethoxypterocarpan-7-O-?-D-glucoside ?7,2'-Dihydroxy-3',4'-dimethoxyisoflavan ? Liquiritigenin ? Liquiritin ?Glycyrrhetinic acid?(+)-Tetrandrine?Fan Ghinoline?Atractylenolide I?Atractylenolide ???Astraisoflavan-7-O-?-D-glucoside?Astragaloside I?Astragaloside II ? Astragaloside ? and Astragaloside IV.The results showed that the linear relationship was good(R2 ? 0.9990),and the recovery was between 96.10% and 101.70%.The method is accurate and rapid,and the content of the sample based on the method is stable,which can be used for the quality control of Fangji Huangqi Tang.The compound compatibility of AR from different producing areas was quantitative determination and the proportion of 19 components in Fangji Huangqi Tang was determined as Liquiritin(34.15%),(+)-Tetrandrine(15.97%),Fan Ghinoline(16.23%),Calycosin-7-O-?-D glucoside(10.19%),and Ononin(7.16%).This method quantitatively characterizes the chemical composition of the sample,provides a basis for the evaluation of Astragalus resources,and also provides a method reference for subsequent pharmacokinetic studies.Conclusion:In this study,firstly,based on UHPLC-Q-Exactive MS,the components of Fangji Huangqi Tang in blood,urine and feces were analyzed to clarify the metabolic process of Fangji Huangqi Tang in vivo.Secondly,animal experiments were carried out to construct adriamycin nephrotic rat model,and metabolomics and multivariate statistical analysis were used to compare the absorption differences between the two body states.Finally,a method of simultaneous determination of 19 components in Fangji Huangqi Tang based on UPLC-Qtrap-MS was successfully established.