Extraction Of Total Flavonoids Of Astragali Radix By Ultrasonic-assisted Deep Eutectic Solvents Method And Therapeutic Effect On Chronic Fatigue Syndrome

Objective:The material basis for treating chronic fatigue syndrome(CFS)by total flavonoids of Astragali Radix(TFAR)was explored through network pharmacology.To study the optimal extraction process of TFAR by ultrasonic-assisted deep eutectic solvents(UDES)method,and to compare the effective components and contents extracted by traditional reflux(TR)method.The therapeutic effect of UDES-TFAR on CFS was studied through cell experiments and animal experiments.Methods:(1)The flavonoids of Astragali Radix and their targets were collected by TCM-ID,TCMSP and HIT databases,and CFS-related genes were collected by Genecards database,so as to obtain potential targets for drug treatment of diseases.The protein-protein interaction(PPI)network of potential targets was constructed using String database,and key targets were found through network topology analysis.Biofunctional enrichment analysis of potential targets were performed to find potential biological processes and signaling pathways.Construction of compound regulatory target network.Docking with SYBYL-X2.0 software to combine flavonoids in Astragalus with core targets at the lowest free energy.(2)The system of deep eutectic solvents(DES)was screened,and the effects of solid-liquid ratio,molar ratio of H-bond donor and H-bond acceptor in DES,water content,ultrasonic-time,ultrasonic-power and ultrasonic-temperature on the extraction amount of TFAR were investigated by single factor experiments.A box-behnken design(BBD)-response surface methodology(RSM)was used to optimize the extraction process.(3)UDES-TFAR was analyzed by UPLC-MS and selective reaction monitoring/multi-reaction monitoring to techniques,and the different components from TR-TFAR were screened.(4)In vitro and in vivo experiments: MTT assay was used to detect the toxicity of UDES-TFAR,TR-TFAR and their different components to RAW 264.7 macrophages.Lipopolysaccharide(LPS)stimulated RAW 264.7 cells for 24 h,and UDES-TFAR,TR-TFAR and differential components intervened.Cell activity was detected by MTT,nitric oxide(NO)was detected by Griess,tumor necrosis factor-α(TNF-α),nterleukin-6(IL-6),transforming growth factor beta(TGF-β),superoxide dismutase(SOD),malonaldehyde(MDA)and glutathione(GSH)levels in the cell supernatant were detected by Elisa.After adaptive feeding,specific pathogen free mice were randomly divided into 6groups,including blank group,model group,positive group and medicated group.Body weight changes and organ coefficients after dissection were recorded,immobility time(IT)and exhaustive swimming time(EST)were measured.Elisa was used to detect the serum levels of TNF-α,IL-6,TGF-β,MDA,SOD and GSH.Results:(1)A total of 63 flavonoids Astragali Radix were collected in the network pharmacology study,corresponding to 348 action targets.Among the 2859 related targets for CFS,232 potential targets were obtained from intersection,and 54 flavonoids were mapped.PPI analysis identified 16 core targets,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses by R language collected 2,403 GO entries and 191 KEGG pathways.Fifty-four flavonoids were molecular docked with the core targets,and the results showed that 97.7% of the compounds had binding activity with the core targets.(2)The optimum conditions for extracting TFAR by UDES method are as follows:solid-liquid ratio 1:25 g/m L,molar ratio of choline chloride to glycerol 1:3 in DES,ultrasonic power 60%,water content 50%,ultrasonic temperature 60℃,and ultrasonic time 40 min.The extraction amount of TFAR was 2.50 ± 0.12 mg/g,which was significantly higher than that by TR method of 2.12 ± 0.10(p < 0.05).(3)UDES-TFAR detection based on UPLC-MS technology,compared with TR method,six flavonoids prepared by UDES method had significantly increased contents(p < 0.05),namely quercetin,rutin,glycitin,luteolin,daidzein and genistein.(4)In vitro experiments showed that the six differential components all significantly improved cell viability and reduced intracellular NO level(p < 0.05).UDES-TRAF and TR-TFAR significantly reduced the increases in TNF-α,IL-6,and MDA contents induced by RAW264.7 cell injury,and significantly increased TGF-β,GSH content,and SOD activity(p < 0.05).The effect of UDES-TFAR was better than TR-TFAR.In vivo experiments showed that UEDS-TFAR could significantly increase the body weight of CFS mice,reduce the liver coefficient of mice,shorten IT and prolong EST,significantly reduce the serum TNF-α,IL-6 and MDA contents of mice,and significantly increase the TGF-β,GSH content and SOD enzyme activity(p < 0.05).The above results indicated that the flavonoids from Astragali Radix can alleviate CFS symptoms by anti-inflammatory immunity and regulating oxidative imbalance in vitro and in vivo.Conclusion:Network pharmacology and molecular docking have revealed the flavonoids of Astragali Radix in the treatment of CFS,and this therapeutic effect might be achieved by regulating the oxidative stress,inflammation,viral infection and other related signaling pathways.DES combined with ultrasound-assisted technology could quickly and effectively extract the flavonoids from Astragali Radix.UDES-TFAR may have a more potent biological activity than TR-TFAR,it has the potential to treat CFS.This study has provided a theoretical basis for the clinical application of UDES-TFAR in the treatment of CFS.