| OBJECTIVE: The macroporous adsorption resin was used to enrich and separate the different parts of the FTZ extract and combined with the lipid accumulation cell model to selected the best lipid-lowering activity site;optimized the refining process of the macroporous resin for the FTZ extract;Material basis and network pharmacology research;which aims to provide a certain experimental basis for the secondary development of FTZ.METHODS:(1)Screening of the optimal elution site for FTZ lipid-lowering activity.After the FTZ extract was adsorbed on the column by macroporous resin,it was desorbed by simple elution and gradient elution respectively,and different elution sites were collected.The fat accumulation in HepG2 cells model was combined to screen out the elution site with the best lipid-lowering activity.(2)Study on the refining process of FTZ extract.The optimum parameters of refined FTZ by macroporous adsorption resin were optimized by single factor investigation and verified by the fat accumulation in HepG2 cells model.(3)Study on material basis of FTZ refined parts.UPLC-Q-TOF-MS method was used to qualitatively identify the chemical components of the most powerful lipid-lowering parts of FTZ,and UPLC method was used to simultaneously determine the content of 8 chemical components in the strongest lipid-lowering parts of FTZ.(4)Study on the content determination of FTZ refined parts.The total phenolic acid,total saponin,and total alkaloid of the FTZ purified fraction were measured by the UV method.(5)Through network pharmacology,constructed a network of components and targets,a network of disease and target interactions,a medicinal component-target-pathway network,and predicted lipid-lowering active components,target sites,and action signaling pathways in lipid-lowering sites.RESULTS:(1)oil red O staining showed that FTZ elution site could reduce cell lipid droplets,but with the increase of administration concentration,gradient elution of T6 ~ T10 site inhibited cell growth.In the T5 site of gradient elution,the decrease of lipid droplets and the increase of nucleus were observed,and the TG clearance rate reached more than 60%,higher than that of other sites.(2)It showed a good purifying profile on A6 macroporous resin with the conditions as follows: the concentration of the sample solution is 0.30 g /mL of FTZ,the amount of saturated adsorption at 6 body volumn(BV),the flow rate was 2 BV/h,then eluting successively with 15 BV/h 44 BV water and 3 BV/h 8 BV 30% ethanol solution to remove impurities,elution with 50 % ethanol at length.50% ethanol eluate was collected and dried.Eventually,the total mass fraction of 8 index components in the solid sample was 23%.(3)Using UPLC-Q-TOF-MS technology to qualitatively analyze the elution site of FTZ optimal lipid-lowering activity,a total of 43 components were identified,and the retention time and mass spectrometry fragment ion information of 8 components were consistent with the reference material.They were the 4-Hydroxycinnamic acid,Jatrorrhizine,Oleuropein,Lithospermic acid,Berberine,Ginsenoside Rg1,Linarin and Ginsenoside Rb1,and the contents of the above eight components were successfully tested.(4)The content of total phenolic acid in the refined fraction of FTZ was determined by UV-VIS method to be 10.13%,the content of total alkaloid was 21.86%,and the content of total saponin was 40.09%.(5)26 of the 43 ingredients were the main medicinal ingredients,namely Berberine,Buddleoside,Palmatine,Oleuropein,Lithospermic acid,etc.;network pharmacology research predicted FTZ refining site for treatment of hyperlipemia 16 target genes of the disease;KEGG pathway enrichment analysis,a total of 7 related pathways were obtained.CONCLUSIONS:(1)The elution site with the lowest lipid-lowering activity at T5 site has a good inhibitory effect on the lipid accumulation model of HepG2 cells,and significantly reduces the intracellular TG content.(2)A6 macroporous resin can be used for the purification of FTZ extract,and the refined process obtained is stable and feasible.(3)The established fingerprint method is stable and feasible,and can identify and quantitatively analyze the main chemical components of FTZ,and provide a solid foundation for the quality assurance of FTZ.(4)The total content of phenolic acid,alkaloids and saponins is more than 50%,and the established detection method is stable and feasible.(5)The treatment of hyperlipidemia in the refined part of FTZ may be through the combination of multi-component,multi-target and multi-pathway. |
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