| Morchella esculenta is recognized as one of valuable and rare edible mushrooms worldwide.It is cherished for nutrition and health values owing to the possession of many bioactive substances,including polysaccharides,proteins,trace elements,mineral,dietary fibers and vitamins.M.esculenta has activities against atherosclerosis,fatigue,viral infection,inflammation and tumors.However,no satisfactory yield from this rare wild resource has been accomplished through traditional artificial cultivation.The distribution and consumption of M.esculenta as well as the development of M.esculenta polysaccharides(MEPs)are limited by high price.The current research on MEPs is only preliminary,without mentioning the domain of function mechanism or molecular structure of MEPs.In this study,MEPs were extracted by pulsed electric field(PEF)from the mycelia fermented by submerged culture,during which there was no temperature-elevating to destroy MEPs structure.This is the first time to introduce PEF to extract MEPs to keep intact of molecules.The crude MEPs were seperated and purified into two kinds of neutral polysaccharides and acidic polysaccharides,respectively.Due to the yield and molecular size,the neutral ones with high yield were selected for the next anti-colon cancer tests,finally.By the detections of apoptosis genes and proteins expressions,MEPN-B-? was discovered to induce HT-29 cell apoptosis by two pathways,in which caspase-3 was activated.The basic molecular structure informations of monosaccharides composition and glycosidic bonds were obtained from detailed analysis.For the first time,we got the micromorphology of MEPN-B-? with anti-colon cancer bioactivity.MEPN-B-? was observed to be oval shape of the microspheres,which was closely joined together to form polycrystalline amorphously.Our work can encourage the development of polysaccharides from submerged fermentation of M.esculent,and helps to discover the bioactivities and molecular structure of MEPs.The main findings are listed below.(1)Between ultrasonic-microwave and PEF extractions,we comparatively selected the optimal extraction conditions of either method via single-factor experiments and respond surface methodology.The optimum conditions of the first method were as follows: ultrasonic power = 250 W,microwave power = 220 W,and extraction time = 7 min.Under these conditions,the yield of MEPs was 4.425%.The optimal conditions for PEF were as follows: electric field intensity = 18 k V/cm,pulse number = 7,and material-to-liquid ratio = 1:27 g/m L.The yield of MEP was 5.693%,which was 28.66% larger than the former method.The mycelia cells were broken thoroughly by PEF to release most of the inclusion in cell which could observe by scanning electron microscope(SEM).PEF treatment could improve the yield of MEPs,which reduced the thermal degradation and time at room temperature.Thus,PEF was selected to extract endo-MEP.(2)According to the yield of MEPs,The extracted endo-polysaccharides were precipitated by 80% alcohol,and the yield of MEPs was 13.76%.The crude MEPs were deproteinized by Sevag method,which caused less degradation,and the yield of MEPs was 70.83±1.79%,the deproteinization ratio was 69.29±2.56%.Then MEPs were decolored by an AB-8 macroporous resin column.The endo-polysaccharides were separated and purified by DEAE-cellulose.Then the uronic acid content was determined by m-hydroxybiphenyl assay to be 8.2±0.34%.MEPs were separated by DEAE-cellulose into neutral and acidic polysaccharides,which were graded with Sephadex G-100column chromatography.The neutral polysaccharides were divided to MEPN-A(yield ratio: 56.3%)and MEPN-B(32.5%),and the acidic part was graded into MEPS-A(19.15%)and MEPS-B(8.82%).Given the yields of acidic polysaccharides were too low to be prepared,we only analyzed the neutral polysaccharides.The average molecular weights(MW)of MEPN-A and MEPN-B were calculated via gel permeation chromatography(GPC).The average MW of MEPN-A was over million,indicating MEPN-A could hardly penetrate cell membranes.However,MEPN-B consisted of MEPN-B-?and MEPN-B-?,with average MWs of 222,344 and 81,835 Da,respectively.Thus,MEPN-B might be the main functional component of MEPs.Preparative chromatography showed the yields of MEPN-B-?and MEPN-B-? were 73.56% and 92.12%,respectively.(3)The anti-proliferation activities of MEPN-B-?and MEPN-B-?on HT-29 cells were evaluated by MTT assay,and the results showed the remarkable effect mostly originated from MEPN-B-?,which was then selected in the experiments of anti-cancer mechanism.We find MEPN-B-?could inhibit the proliferation and growth of HT-29 cells in a time- and dose-dependent manner.The apoptosis ratio under the dose of 800 ?g/m L MEPN-B-? reached 22.7%,revealed by Annexin V-fluorescein isothiocyanate/propidiumiodide(FITC/PI)detection.Reverse transcription polymerase chain reaction(RT-PCR)showed that MEPN-B-? could modulate the protein expressionsof apoptosis-related genes(Bax,Bcl-2 and Bid).The MEPN-B-?regulate the relative expressions of Bax,Bcl-2 and Bid in HT-29 cells through the mitochondrial pathway to induce cell apoptosis,in which the relative Bax expression was upregulated 1.07 times,that of Bcl-2 was downregulated 0.36 times and that of Bid was upregulated 0.96 times,but the ratio of Bax and Bcl-2 was elevated.The pathway of MEPN-B- -? induced-apoptosis could be deduced two methods.One was death receptor pathway in HT-29,in which caspase-3 was activated by motivating caspase-8 through MEPN-B- -? intervening.The activated caspase-8 could cut Bid to be activitated one to get into mitochondria signaling pathway,in which Bcl-2and Bax expression could be modulated to decrease the mitochondrial membrane potential and to release Cyto-C.The next step of apoptosis happened after caspase-9 being activated.Both caspase-8 and caspase-9 can activate caspase-3 to induce cells apoptosis.Meanwhile,the MEPN-B--? concentration at anti-proliferative level was non-toxic to normal fibroblasts.It proved that MEPN-B- -? was nontoxic?(4)The acetylization assay and gas chromatography(GC)showed that MEPN-B-? consisted of rhamnose,xylose,mannose,glucose and galactose with the molar ratio of 10.6:13.1:3.2:29.8:43.3.Ultraviolet wave length scanning proved that MEPN-B-? did not contain proteins.The methylation,FT-IR and NMR spectra showed that MEPN-B-?contained ?4)Glc(1?,?4)Gal(1?,Glc(1?,?4,6)Gal(1?,?2)Man(1?,?4)Xyl(1? and ?2,4)Rha(1? residues,which all connected together to form the repeated oval units,as observed by SEM.The main chain was consited of ?-D-Gal and ?-D-Glc,connected by ?,?-(1?4)glycosidic bonds.For ?-D-Glc(1? contributed to 20% of glucose residues,in each oval unit,there were two branced linkages which was connected on O-6 of ?-D-Man,and ?-D-Glc is distributed at the non-reducing terminal.The results of SEM scanning showed that each unit of MEPN-B- -? polymer was close to the oval shape of the microspheres,which was closely joined together.It could be concluded that the MEPN-B--? was composed of a number of repeat units composed of several monosaccharide residues.X-Ray analysis showed that MEPN-B- -? was polycrystalline in amorphous form.In conclusion,the results of this paper will provide the theoretical basis for further research and development of molecular structure and anticancer activity of MEPN-B--?. |
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