Screening The Active Components From TFDS Against Calcium Oxalate Induced HK-2 Cells Injured

ObjectiveIn this study,the ethanol extract of Desmodium styracifolium was separated and purified to obtain the purified total flavonoids of Desmodium styracifolium?TFDS?,and then TFDS were separated by ODS column chromatography to obtain the different polar fractions.Two fractions with strong antioxidant activity were indentified by antioxidant experiments in vitro.The cellular experiments were performed to examine the efficacy of TFDS and two different polar fractions,and to explore of the mechanism.Cell membrane chromatography was used to isolate and identify the attached components on cells to identify the effective components.Metabolism of TFDS in vivo was studied to look for its metabolism ways and changes in efficacy components.Methods1.Purification of TFDS and preliminary screening of active fractionsIn this study,highly purtied TFDS was isolated and purified by the macroporous resin combined with polyamide,and purified by ODS column chromatography using methanol and water as eluting solvents to obtain different polarities.The antioxidant capacity of different polarities were determined with DPPH,ABTS,and reducing power(Cu²⁺),two fractions were screened out for follow-up studies.2.Protective effect of TFDS and different fractions on the treatment of calcium oxalate monohydrate?COM?injured HK-2 cellsTFDS,25%and 85%elution fraction were selected as study subjects.MTT,MDA,GSH,SOD and LDH assays were used to examine and compare TFDS and different elution fractions for the treatment of COM injured cells.The effects on the reactive oxygen species and mitochondrial membrane potential in damaged cells were examined.The apoptotic rate of the cells was also examined to the effects of the drugs and to compare the efficacy.3.Effects of TFDS and different eluted fractions on occludin and ZO-1 Proteins in HK-2 cells membraneThe mechanism of TFDS and different eluted fractions protecting HK-2 cells were investigated by Western Blot.The ZO-1 and occludin proteins on cell membranes were tested to explore whether TFDS and different elution fractions could protect damaged-cells by regulating the expressions of ZO-1 and occludin proteins in TJ proteins.4.Isolation and identify attached components of TFDS in HK-2 by cell membrane chromatographyAfter co-cultivation of HK-2 with TFDS,the cell lysates were detected by UPLC-Q-TOF-MS to identify the attached components.5.Metabolism study of TFDS on calcium oxalate kidney stone rat modelsThe experiment established calcium oxalate kidney stone rat models and treated with TFDS.Plasma,urine and feces were collected to analyze by UPLC-Q-TOF-MS.Metabolynx software was used to extract metabolites and further analyze the identified metabolites'ways.Results1.The purified TFDS is more than 75%by separating and purifying with macroporous resin and polyamide.5%,25%,85%,and 100%methanol eluted fractions are separated by ODS column chromatography.The content of flavonoids in each fraction were 41.56%,77.93%,79.30%,and 63.72%,respectively.Through the experiment of in vitro antioxidant capacity:DPPH,ABTS and reducing power(Cu²⁺),two stronger elution fractions of antioxidant capacity were screened out,which were 25%and 85%elution fractions.2.The cell were cultured with 100?g/mL COM when exposed for 24h,which the cells viability are about 70%.According to the determination of MDA,GSH,SOD,and LDH,it was found that 85%elution fraction is better;In the TFDS and different elution fractions for the detection of ROS in COM-injured cells,it was found that 85%elution fraction regulated the activation of ROS more strongly than the 25%;The MMP measurement showed that high-dose 85%elution fraction had the strongest effect,but the low and medium dose was not as good as the 25%elution fraction;85%elution fraction were found to have a strongest effect on inhibiting apoptosis.It found that TFDS and two different elution fractions protect the cells through the regulation of ZO-1 and occludin protein expressions.Among them,85%elution fraction had significantly stronger protein expression of ZO-1 and occludin in TJ than the other two fractions.3.In the study of cell membrane chromatography,vicenin-2,vicenin-1,carlinoside,isoschaftoside,schaftoside,iisovitexin,astragalin,genistein and diosmetin,schaftoside I and schaftoside II were found..4.By studying the metabolism of TFDS in the rat models,it was respectively found that there were 5,11 and 10 flavonoids in the plasma,urine and feces,and the same prototype products in the three metabolites were:vicenin-2,isovitexin,schaftoside,isoschaftoside,genistein.The urine also contains:luteolin,geraniol,carlinoside,naringenin,daidzin,hesperidin;The prototype products contained in the feces are:luteolin,isoorientin,diosmetin,naringenin and daidzin.Most of the components in TFDS exist in the form of metabolites.The experiment identified 71 metabolites,most of which are metabolized by reduction,hydrolysis,oxidation and methylation.ConclusionIn this study,85%elution fraction of TFDS is the active fraction.11 compounds which were attached on cells membrane chromatography were identified by UPLC-Q-TOF-MS.In the metabolism study of Desmodium styracifolium,5 prototypes and16 metabolites were detected in the plasma samples.11 prototypes and 21 metabolites were detected in the urine samples.10 prototypes and 43 metabolites were detected in the fece samples.