| Malignant tumors seriously endanger human health and are one of the most important fatal diseases.Chemotherapy is an important method for the treatment of malignant tumors,but the selectivity of chemotherapy drugs is poor,which can cause toxic and side effects in multiple organs and tissues of the whole body,and seriously affect the quality of life of patients.Therefore,it is necessary to find novel antitumor drugs with high efficiency and low toxicity.In our laboratory,crude extracellular polysaccharides were isolated from the fermentation broth of Rhizopus nigricans.After purification,two polysaccharides were obtained,which were named EPS1-1 and EPS-2,respectively.The antitumor activity of EPS 1-1 has been extensively studied.However,EPS-2 is a mixed component,and its structure and activity have not been studied.In this study,a single polysaccharide(EPS2-1)was isolated and purified from EPS-2 by a variety of column chromatography.Its physicochemical properties.structural characterization,and inhibitory effects on human lung adenocarcinoma A549 cells were studied.1.Isolation,purification,structural analysis and antioxidant activity of EPS2-1The crude polysaccharide was separated and purified by DEAE-52,Sephadex G-100 and G-75 chromatographic columns to obtain EPS2-1.The molecular weight and polydispersity index were detected by high performance gel permeation chromatography,the monosaccharide composition was determined by ion chromatography,the appearance of polysaccharide was observed by transmission electron mircoscope and atomic force microscope,and the structure of EPS2-1 was analyzed by infrared spectroscopy,methylation assay and nuclear magnetic resonance.Antioxidant detection assay were used to detect the scavenging activity of EPS2-1 on three kinds of free radicals,and iron ion reducing ability.The results showed that the weight-average molecular weight of EPS2-1 was 32803 Da and the polydispersity index was 1.345.EPS2-1 did not contain starch,protein or nucleic acid.EPS2-1 is mainly composed of mannose,galactose,glucose,arabinose and fucose in a molar ratio of 5.19:2.04:0.65:0.31:0.29.EPS2-1 is a linear polysaccharide with branched chains,the main chain is composed of→2)-α-D-Manp-(1→ and→3)-β-D-Galp-(1→.In addition.EPS2-1 can scavenge DPPH,ABTS and hydroxyl radicals,and keeps a certain ability to reduce iron ions.2.Inhibitory effect of EPS2-1 on human lung adenocarcinoma A549 cellsA549 cells were cultured in vitro,the cell viability was detected by cell counting kit 8,and the inhibitory effects of EPS2-1 on cell migration and proliferation were detected by cell scratch and clone formation assays.The effects of EPS2-1 on intracellular reactive oxygen species,mitochondrial membrane potential and cell cycle were detected by fluorescence microscopy or flow cytometry.Hoechst 33258 staining and Annexin-FITC/Propidium iodide double staining were used to detect cell apoptosis,and the effect of EPS2-1 on the activity of Caspase 3,Caspase 8 and Caspase 9 was detected.The results showed that EPS2-1 could significantly inhibit the proliferation of A549 cells,and the inhibition rate was 60.29%at 0.8 mg/mL after 48 h treatment.EPS2-1 inhibited cell scratch healing ability,indicating that which could inhibit migration of A549 cells.Meanwhile,EPS2-1 could inhibit the ability of cell clone formation.EPS2-1 can increase intracellular reactive oxygen species levels and reduce mitochondrial membrane potential.EPS2-1 condenses chromatin in the nucleus,produces apoptotic bodies,and increases the proportion of early and late apoptotic cells.Meanwhile,EPS2-1 could increase the activity of Caspase 3 and Caspase 8 in A549 cells,suggesting that EPS2-1 may induce apoptosis through mitochondrial pathway.In summary,EPS2-1 with a molecular weight of 32803 Da was isolated from the fermentation broth of R.nigricans.The main chain is composed of→2)-α-D-Manp-(1→and→3)-β-D-Galp-(1→EPS2-1 can inhibit the proliferation and migration of A549 cells and induce cell apoptosis in cells.It deserves further research to develop drugs or functional food for the treatment or adjuvant treatment of lung cancer. |
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