Study On Extraction And Purification Process,Chemical Constituents Analysis And Network Pharmacology Of Total Flavonoids From Cyclocarya Paliurus Leaves

Objective:To prepare the total flavonoids from Cyclocarya paliurus leaves,to analyze the chemical components in the total flavonoids from Cyclocarya paliurus leaves and to predicts the anti-hepatic fibrosis mechanism of Cyclocarya paliurus leaves,by studying the extraction and purification process and chemical components of the total flavonoids from Cyclocarya paliurus leaves and combining with network pharmacological analysis.It will also provide scientific basis for the development of Cyclocarya paliurus plant resources and the development of anti-liver fibrosis drugs with total flavonoids from Cyclocarya paliurus leaves.Methods:1.By using ultraviolet-visible spectrophotometry and with the content of total flavonoids（calculated as rutin）from Cyclocarya paliurus leaves as the evaluation index,the absorbance value was measured at 410 nm by the aluminum chloride color method.Precision,stability,methodological investigations on repeatability and sample recovery were carried out to establish a method for the determination of total flavonoids from Cyclocarya paliurus leaves.2.By using AHP-CRITIC hybrid weighting method and L₉（3⁴）orthogonal test method,the total flavonoid content,isoquercitrin content and dry paste are used as comprehensive evaluation indicators.The extraction method,ethanol concentration,material-to-liquid ratio,extraction time and the five factors of extraction times were screened to determine the best extraction process for the total flavonoids from Cyclocarya paliurus leaves.3.Using static adsorption and dynamic adsorption-elution test methods,taking total flavonoids content,adsorption rate and elution rate of Cyclocarya paliurus leaves as evaluation indicators,the macroporous adsorbent resin for the purification of total flavonoids from Cyclocarya paliurus leaves was screened,and the influence of diameter-to-height ratio,sample loading speed,sample liquid mass concentration,eluent concentration,eluent dosage and elution speed on adsorption performance was investigated,in order to determine the best purification process for the total flavonoids from Cyclocarya paliurus leaves.4.The UPLC-Q-Exactive quadrupole-electrostatic field orbitrap high-resolution mass spectrometry technology was used to preliminarily analyze the chemical components of the total flavonoids from Cyclocarya paliurus leaves.The 15 components were further confirmed by high performance liquid chromatography,and under the same chromatographic conditions,five chemical components concluding ellagic acid,isoquercitrin,isochlorogenic acid C,quercetin and kaempferol with higher content and related to anti-liver fibrosis were determined.5.Effects of total flavonoids from Cyclocarya paliurus leaves on the proliferation of hepatic stellate cells（HSC-T6）and the expression of fibronectin（FN）--a marker of liver fibrosis in vitro were detected by the tetramethylazolyzol salt trace enzyme reaction colorimetric method（MTT）and immunofluorescence method.A compound-target-pathway network was constructed through network pharmacology platforms（TCMSP,Uniprot,Genecard,TTD,etc.）,and the mechanism of action were predicted by performing functional enrichment analysis of common protein interaction PPI network gene.Results:1.A method for the determination of total flavonoids from Cyclocarya paliurus leaves was established:the aluminum trichloride color method was used to determine the sample at 410 nm by ultraviolet-visible spectrophotometer.The stability of the sample was good.The established method was accurate,reliable,and reproducible.This method can be used for the determination of total flavonoids from Cyclocarya paliurus leaves.2.The extraction process of total flavonoids from Cyclocarya paliurus leaves was confirmed:ethanol concentration 75%,material-to-liquid ratio1:12（g/m L）,extraction time 1.5 h,and 2 extractions.3.The purification process of total flavonoids from Cyclocarya paliurus leaves was determined:AB-8 macroporous adsorption resin,the mass concentration of the sample was 0.6 mg/m L,sample flow rate was 2BV/h,diameter to height ratio 1:15 and using 85%ethanol 2 BV for elution,elution flow rate was 2 BV/h.4.The chemical constituents of total flavonoids from Cyclocarya paliurus leaves were discussed preliminarily:seven compounds were isolated and identified,concluding isoquercitrin,ellagic acid,isochlorogenic acid C,quercetin,kaempferol,hyperoside and chlorogenic acid.The content determination results under the same chromatographic conditions showed that the average contents of isoquercitrin,ellagic acid,isochlorogenic acid C,quercetin,and kaempferol in the total flavonoids of leaves were 366.51 mg/g,99.22 mg/g,38.02 mg/g,30.60 mg/g,and 3.89 mg/g.5.The anti-liver fibrosis mechanism of Cyclocarya paliurus leaves was predicted:total flavonoids from Cyclocarya paliurus leaves can inhibit the proliferation of HSC-T6,significantly reduce the expression of liver fibrosis markers（FN）,indicating that it has anti-liver fibrosis effects;Network pharmacology results showed that Cyclocarya paliurus leaf may regulate tumor necrosis factor（TNF）,interleukin-6（IL-6）,transforming growth factorβ1（TGF-β1）and other targets to regulate cytokine receptor interactions,cancer pathways,hepatitis B and other pathways to achieve effects of anti-liver fibrosis.Conclusion:The method for determination of total flavonoids from Cyclocarya paliurus leaves is stable and reliable.The extraction and purification process is simple and feasible and suitable for large-scale industrial production.The results of chemical composition analysis and the predicted anti-liver fibrosis mechanism of Cyclocarya paliurus leaf can provide scientific basis for the development of anti-hepatic fibrosis drugs with total flavonoids from Cyclocarya paliurus leaves.It is of great significance to the development of Cyclocarya paliurus plant resources.