Pre-clinical Pharmacokinetic And Network Pharmacological Studies Of Total Flavonoids Of Desmodii Styracifolii

Objective:High-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）method was established to study the pharmacokinetics of the absorption,distribution,metabolism and excretion（ADME）process after oral administration of total flavonoids of Desmodii styracifolii（TFDS）in rats,and to jointly explore the material basis and mechanism of action of TFDS through network pharmacological study.Methods:According to the previous studies,the HPLC-MS/MS method was established for the detection of Schaftoside,Vicenin-1,Vicenin-2,Vicenin-3 and Isovitexin,which are the main components of TFDS.Agilent 1260-QTRAP 5500LC-MS with Waters ACQUity UPLC HSS T3 column（50 mm×2.1 mm,1.8μm）was used to separate the ingredients.The gradient elution of 0.1%formic acid in aqueous solution（A）-methanol（B）was performed.The mass spectrometry was performed using an electrospray ion（ESI）source,negative ion mode,and multiple reaction monitoring（MRM）ion scanning mode.The content of five ingredients in TFDS were determined after methodology validation;SD rats were given low（50 mg/kg）,medium（100 mg/kg）and high（200 mg/kg）doses of TFDS by gavage,respectively.Plasma was collected at different time points,and pharmacokinetic analysis was performed after the detection method was validated by the methodology.By searching Pub Chem,compound 3D structures were obtained.The relevant targets of urinary system lithiasis（USL）were retrieved and screened through Mala Cards Database,Uniprot Database,String Database and Pub Med.PDB proteins were obtained through Protein Data Bank（PDB）,and the pocket parameters were obtained by docking with reference ligands through TCM Network Pharmacological Analysis System.Then molecular docking technology was used to dock the compound with the protein to screen the target with the standard of the affinity was less than-5 kcal/mol and less than the reference ligand binding energy.“Component-target”network was constructed,and enrichment analysis of gene ontology,signal pathway and disease ontology was conducted to explain the possible mechanism of TFDS in the treatment of USL under the P value was adjusted less than 0.05.Results:Methodological investigation was carried out to determine the content of TFDS.The method showed good specificity.The five flavonoids had good linear relationship in the concentration range of 1～200 ng/mL,and the correlation coefficient r were all above 0.999.The limits were 70,50,30,70,30 pg/mL of detection;200,250,100,200,100 pg/mL of quantification.In precision tests,RSDs were 1.46%,1.03%,1.34%,2.21%and 0.93%,respectively,and the RSDs were all less than 2.3%.RSDs of the reproducibility tests were 2.60%,1.49%,2.18%,2.39%and 2.70%,respectively,and RSDs were all less than 3%.The RSDs of the peak area of the components measured in the stability experiment were 0.99%,1.09%,1.19%,3.68%and 1.23%,respectively,and the RSDs were all less than 3.7%.The recoveries were96.57%,97.26%,98.31%,95.33%and 95.31%,respectively,when 100%of the reference solution was added.Methodology verification results showed that the method was suitable for the determination of TFDS content.The results showed that the contents of Schaftoside,Vicenin-1,Vicenin-2,Vicenin-3 and Isovitexin in 2batches of TFDS were 60.63,23.60,31.03 68.63,9.42 mg/g,and 68.70,27.85,32.30,76.05,13.40 mg/g,respectively.Plasma determination method showed good specificity.The linear relationship was good in the plasma sample of 0.5-200 ng/mL,and r were all above 0.999.The precision were all less than 15%,and accuracy were between87.27%and 108.27%.The recoveries ranged from 100.15%to 104.33%,and the matrix effect ranged from 95.30%to 102.18%.The RSDs of stability were all less than 8.3%.The results showed that this method was well to be used in the pharmacokinetic study of TFDS in rats.Pharmacokinetic detection showed that the absorption of each component was faster after the rats were given different doses of TFDS intragastrically,the active component could be detected 5 min after administration,and the detection limit was close to 12 h after administration.Among them,the plasma concentration of Schaftoside was the highest,followed by Vicenin-3,Vicenin-2 and Vicenin-1,and the concentration of Isovitexin was the lowest.The linear kinetic characteristics of C_max were observed in the low,medium and high dose groups.A total of 112 genes related to USL were retrieved from Mala Cards Database.PDB proteins were obtained from PDB Database,Uniprot Database,String Database and Pub Med literature search.The proteins were molecular docked with 5 compounds,and20 proteins were successfully docked.The“component-target”network shows that the targets were mainly concentrated on the Schaftoside and Vicenin-3,and the compounds mainly focused on the genes PRPS1,UMOD,KLK3,Ca MKMT,AGT,LCN2.A total of 82 gene ontology entries,1 KEGG signaling pathway,5 Reactome signaling pathways and and 23 co-associated diseases were obtained from biological function enrichment.Conclusion:The pharmacokinetic parameters of TFDS were obtained by pre-clinical study through pharmacokinetics,and the ADME of TFDS in rats was observed.Combined with network pharmacology,we get the way to explore the potential mechanism of USL treatment,and provide a basis for further research.