Study On The Chemical Material Basis Of Chinese Classical Formula Qingxin Lianzi Yin Decoction And Evaluation Of Its Efficacy In Diabetic Nephropathy Model

BackgroundQingxin Lianzi Yin Decoction(QXLZY),a Chinese classical formula for clearing the heart and nourishing the Yin,is made up of nine herbs(Scutellariae Radix,Ophiopogonis Radix,Lycii Cortex,Plantaginis Semen,Glycyrrhizae Radix et Rhizoma Praeparata Cum Melle,Nelumbinis Semen,Poria,Astragall Radix Praeparata Cum Melle and Ginseng Radix et Rhizoma).QXLZY is coming from the Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy.It can clear heart fire,enrich yin,boost qi,and stop strangury-turbidity.It was used to treat white turbidity of urination,seminal emission,irritability,restlessness,wasting-thirst and so on.However,the current studies on QXLZY were mostly focused on its clinical efficacy,lacking systematic material basis research on constituents.There were few reports on its overall chemical composition,pharmacodynamic substance basis and pharmacological activity.Aim of the studyIn order to break through the bottleneck of quality evaluation of the classical formula QXLZY,this study clarified the historical evolution,prescription dosage and the processing of single herb,etc.Fifteen batches of medicinal materials from different producing areas were collected to prepare reference materials.This work aims to elucidate and quantify the chemical constituents,clarify the blood-absorbed components and excretion pathways,predict major bioactive constituents and discover potential therapeutic targets.The pharmacodynamic evaluation on mice with diabetic nephropathy(DN)model was conducted to provide scientific basis for the promotion and application of QXLZY.Methods1)Referring to ancient books and literatures and fully considering the current production and application practice,this study clarified the processing of single herb,prescription dosage and the historical evolution of formula,etc.2)UHPLC-LTQ-Orbitrap HRMS was employed to clarify the chemical constituents and metabolites of QXLZY.It was suitable for the analysis of trace component in complicated samples for providing accurate full MS and MSn fragment information with high sensitivity and precision.Data-dependent acquisition method was employed to acquire multi-stage mass spectra.And dynamic ion exclusion method was aimed to obtain the MSn spectra of co-eluted minor compounds.The extraction of diagnostic ion and neutral loss fragment was aimed for searching specific type of constituents.The uses of MCI gel column and liquid-liquid extraction technology achieved the enrichment of minor constitutes.3)UHPLC-QQ-MS/MS was employed to simultaneously detect different types of constitutes.The method was carried out systematic method validation.The contents of 15 batches of reference substances were determined.4)By comparing with the blank group,based on the identification results of QXLZY formula as well as the literatures of the single drug metabolism,the plasma,urine,bile and feces samples of rats after oral administration of QXLZY were systematically studied.5)During the experiment,db/db mice,a model of DN,were used to observe the biochemical indexes and histopathological changes of the kidneys,to evaluate the pharmacological effects of QXLZY on DN.6)Based on the analysis of ingredients in vivo,the bioactive constituents and potential therapeutic targets in the treatment of DN was investigated by using network pharmacological analysis.Results1)We have systematically combed and clarified the QXLZY in terms of the origin of single herb,the processing of medicinal slices,the historical evolution,composition,preparation,interpretation,function,and modern clinical application of the formula.2)Under the optimized experimental conditions,a total of 220 compounds were identified or tentatively characterized,including 119 flavonoids,34 triterpenes and steroids,18 alkaloids,26 organic acids,12 amino acids,two phenylethanoid glycosides,two coumarins,one iridoid and six other constituents.Among them,59 compounds were identified unambiguously by comparing with the retention time and mass spectrometric data of reference standards.The other 161 compounds were tentatively deduced by their CID pathways referring to previous literatures.All the compounds could be detected in the individual constituent herbs.3)The 21 components covered nine herbs.The analysis time was shortened to 15 minutes.The validated UHPLC-QQQ-MS/MS method was used for the quantification analysis of 21 constituents in 15 batches of QXLZY samples under the MRM mode.4)A total of 122 components(73 prototypes as well as 49 metabolites)were identified or tentatively characterized.A total of 68 components were identified in plasma samples,including 40 metabolites.98 components were excreted by urine,68 components were excreted by bile,63 components were excreted by urine and bile,and 67 components could be detected in feces.5)QXLZY could improve the general condition of db/db mice and reduce the histopathological conditions of kidney such as collagen deposition in glomeruli and tubular interstitium,glomerular hypertrophy,basement membrane and thylakoid stromal hyperplasia,and the degree of improvement of renal histopathological damage was dose dependent.6)By using network pharmacological analysis,baicalein,liquiritigenin,succinic acid,formononetin,wogonin might be the major effective constituents in QXLZY during the treatment of DN.MAPK3,PIK3CA,EGFR,RAF1,IKBKB,TNF,MAPK14,IGF1R,VEGFA,SRC were predicted the main targets.ConclusionsQXLZY has widely clinical application and is suitable for the treatment of chronic urinary system diseases and diabetic nephropathy.Flavonoids,triterpenes and organic acids were the major small molecule chemical ingredients of QXLZY.The content of flavonoids displayed the highest of QXLZY,followed by triterpenes.For the first time,trace elements such as poricoic acid B,methylophiopogonanone A,lotusine and coclaurine were tested simultaneously.Flavonoids were the main components absorbed into blood,followed by organic acids.Phase Ⅱ conjugation reaction was the major metabolic type.The constituents of QXLZY were excreted through urine and bile,in which urine excretion accounted for a large proportion,but no class of compounds was found to have a specific excretion pathway.Low dose of QXLZY had a certain improvement effect on DN in db/db mice.The pathways which QXLZY treated DN were related to glucose and lipid metabolism,oxidative stress and inflammation by network pharmacological analysis.Overall,this study provides valuable information and support for the use of QXLZY in clinic.In addition,this work could provide informative groundwork for further studies on QXLZY and build a strategy for systematic study on the chemical substance and the mechanism of action of other Chinese classical formula.