Isolation And Purification Of Corn Peptide And Preliminary On Anti-HepG2 Cells Activity

Corn peptides come from corn protein,a by-product which is purified by the food industry product to produce starch or brewing industry.Corn peptides are relatively non-toxic and have many biological activities such as protecting liver,reducing blood sugar,promoting alcohol metabolism,anti-hypertensive,anti-fatigue,anti-oxidant and so on.The protective effect on liver of corn peptides is more prominent among these activities.Studies have shown that corn peptides can inhibit the proliferation of human liver cancer cells HepG2,but the objects of those studies are mixtures of corn peptides.There is currently no report on a pure peptide and its inhibitory effect on human hepatoma cells HepG2.This study aims to isolate,purify,and identify a hexapeptide that have the ability to inhibit the proliferation of HepG2 cells from corn peptides,and preliminary studied on its mechanism of inhibition on HepG2 cells.In this study,active anti-tumor corn peptides were evaluated by MTT assay.The corn peptides were isolated and purified by DEAE-FF and Sephdax-G15 column chromatography.Using LC-ESI-MS/MS to identify the structure of its active fragments.Short peptides were synthesized by solid phase peptide synthesis,and the anti-tumor activity on HepG2 cell of the synthesized peptide CPs-6 was evaluated by MTT assay.Cell morphological changes were observed,cell apoptosis was observed by AO/EB double staining assay,DCFH-DA staining assay was used to detect intracellular reactive oxygen species(ROS),PI staining assay was used to detect cell cycle,Annexin V-FITC/PI double staining assay was used to detect cell apoptosis,JC-1 staining assay was used to detect intracellular mitochondrial membrane potential(MMP),RT-PCR analysis the relative mRNA expression levels of cytokines in HepG2 cells.As results,an active peptide inhibiting the proliferation of HepG2 cells was isolated and purified from corn peptides with the sequence of LPPYLP(CPs-6);and the toxicity of CPs-6 to L-02 cells is low.The mechanism of CPs-6 inhibiting the antitumor activity of human hepatomar cells HepG2 shows that CPs-6 inhibited HepG2 cells proliferation in a dose-dependent manner.The IC50 value of CPs-6 after 24 hours treatment is 1015.16 μg/mL.CPs-6 can make HepG2 no longer adherent,shrink,round,reduce cell membrane integrity,and induce the accumulation of intracellular ROS.Flow cytometry results showed that CPs-6 can block the cell cycle in G1 phase,increase the apoptosis rate of the cells,and decrease the mitochondrial membrane potential.RT-qPCR results showed that CPs-6 increased the relative transcription levels of the pro-apoptotic factor Fas,Bid,and Caspase-9 in HepG2 cells.In conclusion,this study found a corn peptide CPs-6 that can inhibit the proliferation of HepG2 cells for the first time,CPs-6 can induce intracellular ROS aggregation,block HepG2 cells cycle,and induce HepG2 cells apoptosis through the mitochondrial pathway.