| Objective:The insulin resistance（IR）model of human hepatocellular carcinoma HepG2 cells was established as an in-vitro screening model for the inhibit IR active components of Huangqi san（HQS）,and the active components of HQS that can effectively improve IR were preliminarily screened;A carboxymethyl chitosan-nanoliposome-functionalized hollow fiber liquid phase microextraction（CMCS-NL-HF-LPME）method was established,and combined with high performance liquid chromatography（HPLC）to achieve the concentration,enrichment and content determination of hypoglycemic active components in HQS;The mechanism of HQS on treatment of T2DM was explored based on network pharmacology.This provides a new direction and method for the rational development and utilization of HQS.Methods:（1）Part I:To explore the establishment of a stable IR model of HepG2 cells,the influence of insulin induction concentration and time were investigated,and the activity and glucose consumption（GC）of HepG2 cells were measured by glucose oxidase（GOD-POD）and MTT method,respectively.After successful modeling,12 compounds selected in HQS were used as analysis objects to give drug intervention to IR model HepG2 cells.GOD-POD method was used to determine the effect of drugs on GC of IR model cells,and MTT method was used to evaluate the cell activity after drug intervention.（2）Part II:On the basis of hollow fiber liquid phase microextraction（HF-LPME）,a carboxymethyl chitosan-nanoliposome-functionalized hollow fiber liquid phase microextraction（CMCS-NL-HF-LPME）was established,and combined with HPLC to extract,enrich and determine 7 flavonoids,which had improved IR ability screened out in Part I.The parameters affecting the enrichment efficiency of target analytes were investigated and optimized.The methodological verification was carried out under optimal extraction conditions,and the extraction mechanism of the method was analyzed and discussed.（3）Part III:Through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）,the main chemical components and their targets of Pueraria lobata,Astragalus and Morus alba were obtained,and the active components of Traditional Chinese medicine were selected according to the ADME attributes and the results of the screening of hypoglycemic components in part I;The main target informations of T2DM disease were screened by OMIM,TTD and DRUGBANK databases,and the overlapping HQS-T2DM target was selected to make Venn diagram;Using String platform to construct PPI network;The main biological processes and metabolic pathways were enriched by Metascape platform.The HQS component-T2DM target-pathway network was constructed with Cytoscape3.7.2 software.Results:（1）Part I:IR model of HepG2 cells was established under the conditions of induction concention(10-7mol/L)and induction time（36 h）of insulin.The IR model was used to screen out 7 components that could improve cell IR,which were puerarin,daidzein,genistin,genistein calycosin-7-O-β-D-glucoside,calycoflavone and morusin.When the drug concentration of puerarin,daidzein,calycosin-7-O-β-D-glucoside,calycoflavone and morusin was 2×10-1mg/m L,genistin 2×10-5mg/L,genistein 2×10-3mg/L,they had the most significant effect on improving IR status of HepG2 cells（P<0.01）,and the effect was similar to that of positive control drug metformin.（2）Part II:The EFs of the 7 active components screened by CMCS-NL-HF-LPME ranged from 4.7-112.5,the linear range was 0.4-10000 ng/m L,the limits of quantification（LOQs）were between 0.1-5.0 ng/m L,and the limits of detection（LODs）were not higher than 3 ng/m L,the average recoveries were between 96.7%and 108.8%,and the relative standard deviations（RSDs）of intra-day and inter-day precision were not higher than 11.6%.（3）Part III:The core active components of HQS in the treatment of T2DM are quercetin,kaempferol,genistein,daidzein,puerarin,etc.There were 46 overlapping HQS targets in T2DM,including 7 core targets（PPARG,PTGS1,RXRA,INSR,DPP4,PIK3CG,PPARA）.The biological processes involved include:regulation of hormone levels,hormone response,positive regulation of lipid metabolism,cell response to organic cyclic compound,cell secretion regulation,etc.Related pathways include AMPK signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,insulin resistance,c AMP signaling pathway and AGE-RAGE signaling pathway in diabetic complications.Conclusion:In this paper,the in-vitro IR model of HepG2 cells was used to screen the hypoglycemic active components in HQS that could improve cell IR status.All the screened active components could increase Glucose consumption（GC）of IR HepG2 cells,realizing the in-vitro screening of a variety of hypoglycemic active components in HQS.The CMCS-NL-HF-LPME method combined with HPLC was established,and,it was successfully used for the extraction,enrichment and content determination of hypoglycemic active components.Through network pharmacology,the mechanism of HQS in treating T2DM with multi-component,multi-target and multi-pathway was preliminarily revealed,laying a foundation for the clinical development and utilization of HQS. |
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