Speciation Analysis Of Selenium In Cardamine Violifolia And Its Selenium Enriched Peptides Activity In Hepatoma Cells

Selenium is a kind of trace element which makes a great contribution to human body and has a variety of health care functions.Cardamine violifolia,as one of the good sources of selenium,has a high selenium content,and most of them are organic selenium.At present,the studies on C.violifolia were mainly focused on the preparation of its selenoproteins,selenium polysaccharides and the exploration of its antioxidant function,while the mechanism of its selenium-enriched peptides on hepatoma cells was rarely reported.In this paper,a method for the determination of selenium speciation in C.violifolia was established;the Se-enriched peptides were prepared by protease enzymolysis and ultrafiltration,separated and purified by preparative high performance liquid chromatography(prep-HPLC),and the amino acid sequences of the components with the best inhibitory effect on the survival of HepG2 cells were identified;the effect and mechanism of Se-enriched peptides on hepatoma cells were studied in vitro.The main conclusions are as follows:A high performance liquid chromatography inductively coupled plasma mass spectrometry(HPLC-ICP-MS)method was established for the determination of selenium speciation in Se-enriched C.violifolia,including selenate[Se(?)],selenite[Se(?)],selenocysteine(SeCys₂),methylselenocysteine(MeSeCys),and selenomethionine(SeMet).Different speciations of selenium compounds were extracted by adding flavor protease and protease E successively,and diluted to appropriate concentration after twice enzymatic hydrolysis.The samples were separated with a Thermo Hypersil GOLD C8(250 mm×4.6mm,5?m)column by isocratic elution using 20 mmol/L potassium dihydrogen phosphate containing 0.05%(v/v)heptafluorobutyric acid and 3%(v/v)methanol as mobile phase.The five speciations of selenium could be completely separated within 6 min.There were linear relationships for five selenium speciations with correlation coefficients(r²)more than 0.999.Recoveries for five speciations of selenium were in the range of 85.8%?106.3%with relative standard deviations(RSD)less than 5%.The limits of detection(LOD)and quantitation(LOQ)were 0.08?0.25?g Se/L and 0.25?0.54?g Se/L,respectively.Using this method,it was found that Se Cys₂was the main selenium speciations in C.violifolia.Se-enriched peptides of C.violifolia were prepared by protease hydrolysis,and three components(S1-1?S1-3)were obtained by ultrafiltration.Comparing the effects of each component on the survival rate of HepG2 cells,S1-3 with the best effect was selected and further purified by prep-HPLC,and 12 components(SP1?SP12)were obtained.The selenium content and peptide content of 12 components and their effects on the survival rate of HepG2 cells were compared.It was found that there was a negative correlation between selenium content and the survival rate of HepG2 cells,among which the component SP9 had the highest effect.The main amino acid sequences of SP9 identified by LC-MS/MS were IVSCT,LPLVKCP,KDACYAYGLL,CFPIKIRQNPSP,LKECASP,KFMEIPIP,MHPNISDVEPI,QLLLCPAPLP,and IDESLICQGFVDP.HepG2 and Huh-7 cells were used as models to explore the mechanism of the effect of S1-3 on hepatoma cells.The results showed that S1-3 had a certain effect on the survival rate of HepG2 and Huh-7 cells in a dose-dependent manner.S1-3 inhibited the proliferation of hepatoma cells by arresting the S phase of HepG2 cells and the G2 phase of Huh-7 cells,and promoted the late apoptosis of the two kinds of cells.After S1-3 treatment,the levels of ROS in hepatoma cells increased,the permeability of mitochondria increased,the influx of calcium ions,and the activity of Caspase-3 and 9 increased,which significantly affected the intracellular mitochondrial pathway.The transcriptome analysis have shown that there were305 up-regulated genes and 629 down-regulated genes.Many pathways were affected,of which the PI3K-Akt pathway was most significantly affected.Finally,PCR was used to further verify that the relative mRNA expression of genes CCND3,BAD and BAX increased,while the expression of genes PIK3R3 and BCL2 decreased,which was consistent with the results of transcriptome analysis.