Study On Structure And Anti-tumor Activities Of Exopolysaccharides From Alternaria Mali Roberts

Objective: This research conducted a systematic study on exopolysaccharides from Alternaria mali Roberts(a plant pathogenic fungus),which contained extraction,isolation,purification,determination.In addition,this research studied on the structure and anti-tumor activities of exopolysaccharides from Alternaria mali Roberts.Methods:1.The AMEP was separated and purified by the DEAE-Sepharose Fast Flow anion-exchange chromatography,Sephadex G-100 and Sephacryl S-400 gel-filtration chromatography.2.The total sugar content,thermal stability,light stability,acid and alkal stability and metals ion stability of AMEP were determined by anthrone-sulfuric acid method.The protein and starch content were determined by Coomassie brilliant blue G-250 staining reaction and iodine-potassium iodide reaction.3.The purity and average molecular weight of AMEP were measured by high performance size exclusion chromatography(HPSEC).The monosaccharide components of AMEP was analysised by high performance liquid chromatography(HPLC).The IR spectrum of AMEP was determined with a Fourier transform infrared spectrophotometer(FTIR).The NMR spectroscopy of AMEP was determined with a nuclear magnetic resonance spectrometer.4.The BGC-823,K562 cells were treated with AMEP-2 at various concentrations for different determined duration such as,24,48 and 72 hours.The proliferations in vitro of the BGC-823,K562 cells were measured by a way called MTT assays.5.The morphological changes after treatment were evaluated by Hoechst 33258 staining.6.The apoptosis-induction effect was evaluated by confocal laser microscopy and flow cytometry.The changes of cell cycle was evaluated by RNAase/PI dyeing.The changes of intracellular reactive oxygen species(ROS)and mitochondrial membrane potential were respectively assessed by fluorescence probe DCFH-DA and JC-1,then assessed it with flow cytometry.Results:1.The AMEP has better thermal stability at 20-80℃ and remain stable in weak acid(p H=6)and Na+ ion solution.And strong acid and alkali solution or Cu2+、Fe3+ ions had an impact on the stability of exopolysaccharide,the light could also influence the stability of it.2.AMEP-1 、 AMEP-2 were eluted as a symmetrical peak on HPSEC,and their average molecular weight were estimated to be 38.61 k Da、46.89 k Da.Infrared spectrum showed that AMEP-1、AMEP-2 all had characteristic absorption peaks of polysaccharides.Monosaccharide analysis showed that AMEP-1 was composed of mannose,glucose and galactose with a molar ratio of 12.9:1.6:1.0,and AMEP-2 was composed of mannose,glucose and galactose with a molar ratio of 9.1:2.0:1.0.NMR spectroscopy analysis showed that AMEP-1,AMEP-2 were pyranose.AMEP-1 was determined to contain 1-6,1-2glycosidic bonds,AMEP-2 was determined to contain1-6 glycosidic bonds.3.AMEP-2 displayed a dose-and time-dependent inhibition of tumor cells proliferations in vitro.It was obviously that the growth of BGC-823 cells was significantly inhibited.After treated with increasing concentration of AMEP-2 for 72 h,the growth of inhibition ratio has increased from 8.5 to 46.84 %.The treatment with 2.0 mg/m L AMEP-2for 24 h、48 h、72 h,the corresponding inhibition rates were 25.05 %、35.86 %、46.84 %respectively.4.AMEP-2-treated cells showed significant morphological changes including cell shrinkages,chromatin compaction and nuclear fragmented.The nuclei appeared to be slightly smaller and had a brighter fluorescence than the control.5.The apoptotic rate of BGC-823 cells treated with AMEP-2 at the concentration of0.4,0.8 and 1.6 mg/m L for 48 h was significantly increased to 6.29 %、9.65 %、15.6 %respectively.AMEP-2 could induce apoptosis and cause accumulation in the S-phase of the cell cycle.Furthermore.AMEP-2 induced accumulation of intracellular ROS.In addition,AMEP-2 induced alterations in mitochondrial membrane potential,indicating that mitochondrial apoptotic pathways areinvolved.Conclusion:1.AMEP has better thermal stability,but strong acid,alkali and metal ions would affect the stability of it.2.Monosaccharide analysis showed that AMEP-1 was composed of mannose,glucose and galactose with a molar ratio of 12.9:1.6:1.0,and AMEP-2 was composed of mannose,glucose and galactose with a molar ratio of 9.1:2.0:1.0.They contained both alpha-and betaglycosidic bonds,and they were pyranose.AMEP-1 was determined to contain 1-6,1-2glycosidic bonds,AMEP-2 was determined to contain1-6 glycosidic bonds.3.AMEP-2 displayed a dose-and time-dependent inhibition of tumor cells proliferations in vitro.4.AMEP-2 could induce apoptosis and cause accumulation in the S-phase of the cell cycle.Furthermore,AMEP-2 induced accumulation of intracellular ROS.In addition,AMEP-2 induced alterations in mitochondrial membrane potential,indicating that mitochondrial apoptotic pathways areinvolved.