Study Of Extraction,Structure Analysis And The Effects Against Oxidative Stress Of Morchella Conica Polysaccharide

Morchella is an edible fungus and medicinal fungus,which is nutritional,rare and with high medical value.With unique flavor and form,it has attracted attention in the fields of functional foods,traditional Chinese medicines and health care products.Morchella contains many biologically active substances,including polysaccharides,proteins,fats,mineral elements,dietary fibers and so on.Among them,polysaccharide is the most abundant and bioactive nutrient.The bioactivity of it includes antioxidant properties,immunomodulatory activity,hepatoprotective activity and antitumor activity.Morel polysaccharides are mostly extracted by conventional extraction methods,such as hot water extraction.However,the hot water extraction method has the disadvantages of high temperature,long extraction time and more energy consumption.A new extraction method should be applied to replace it.In this paper,Ultrasonic microwave synergistic extraction?UMSE?is used to extract the polysaccharide of Morchella.In addition,although studies have shown that the antioxidant activity of the crude polysaccharide of Morchella,the antioxidant activity and the inhibition of oxidative stress for the purified Morchella polysaccharide are rarely studied in depth.In this paper,the molecular structure and antioxidant activity of pure polysaccharides from Morchella conica fruit body were systematically studied.The main research contents and results are as follows?1?Optimization of process conditions of Morchella conica polysaccharides by UMSE and comparison of other extraction methods.After single factor experiment,the response surface Box-Behnken design was used to optimize the experiment.The optimal process conditions for Morchella conica polysaccharides extraction process conditions were as follows:microwave power was211 w,liquid-to-material ratio was 41:1,and extraction time was 127 s.The average yields of Morchella conica polysaccharides extracted by UMSE,UAE and MAE were7.53±0.17%,6.62±0.08%and 5.86±0.03%,respectively.UMSE has the highest polysaccharide yield of Morchella conica compared to ultrasonic extraction?UAE?and microwave extraction?MAE?.In the analysis of the Morchella conica extract,the UMSE extract has a higher turbidity value,and the solution is darker than the other two extraction methods.This indicates that UMSE has more dissolution than the extracts of UAE and MAE.In the analysis of morel residue,the microscopic morphology of the hyphae of Morchella conica was observed by SEM.It was found that the UAE extract showed the crack characteristics of the hyphae.While the MAE extract showed the pore characteristics and the general morphology of the hyphae was distinguished.After the UMSE treatment,the morel hyphae were completely broken.The outline of the hyphae can not be completely distinguished,which exhibited both a porous structure and cracks with the characteristics of MAE and UAE.It indicates that UMSE has more damage efffect to the hyphae of morel.?2?In vitro antioxidant evaluation of polysaccharide fraction of Morchella conica and preliminary study on protective effect of Morchella conica crude polysaccharide on oxidative stress injury.Two polysaccharides components of Morchella conica?acidic polysaccharide AMCP and the neutral polysaccharide NMCP?were separated from the by the cellulose DEAE-52 ion exchange column.The NMCP was purified by Sephadex G100 gel column to separate NMCP-1 and NMCP-2.The in vitro antioxidant activity of Morchella conica crude polysaccharides?MCP?,NMCP-1,and NMCP-2 was evaluated by DPPH free radical scavenging activity,ferrous ion chelation,reducing power and inhibition the linoleic acid auto-oxidation ability test.Among the four groups of experiments,NMCP-2 was the strongest antioxidant activity,followed by NMCP-1,and finally MCP.The protection effects of oxidative stress on the MCP was studied.When MCP was added to H₂O₂ treated cells,it could significantly inhibit the lipid oxidation induced by H₂O₂ in cells.When the concentration of polysaccharide increased to 50?g/mL,the Malondialdehyde?MDA?content decreased significantly to 5.10±0.45 nmol/mg.MCP is able to increase the activity of GSH-px in cells.When the MCP concentration reached 50?g/mL,the enzyme activity was 40.4±3.8 U/mg.?3?Mechanism of NMCP-2 on protecting oxidative stress from HEK-293T cells.NMCP-2 can significantly reduce the production of ROS in H₂O₂-induced oxidative stress cells,significantly reduce intracellular mitochondrial membrane potential?MMP?levels and significantly reduce H₂O₂-induced apoptosis.Transmission electron micrographs showed NMCP-2 significantly improved mitochondrial rupture in HEK-293T cells induced by H₂O₂.NMCP-2 can significantly reduce the expression of proapoptotic factors Caspase-9,Bax,Caspase-3and increase the expression of anti-apoptotic factor Bcl-2 mRNA in H₂O₂-treated cells.NMCP-2 can significantly reduce the expression of Bax and Cleaved Caspase-3protein in H₂O₂-treated cells,and the expression of Bcl-2 is increased.It can be confirmed that after treatment of H₂O₂-induced HEK-293T cells by NMCP-2,the expression of proteins that promote apoptosis in the cells is decreased,the expression of proteins that inhibit apoptosis is further increased and the oxidative stress of cells is inhibited.?4?Identification of the primary structure of NMCP-2.The structure of NMCP-2 was analyzed by liquid chromatography,gas chromatography,infrared spectroscopy and nuclear magnetic resonance detection.NMCP-2 has a relative average molecular weight of 48.3 kDa and is composed of mannose,glucose,galactose and xylose in a molar ratio of 3.35:19.57:1.00:3.14.NMCP-2 consists of 7 monosaccharide residues,respectively?4)-?-D-Glup-?1?,?6?-?-D-Glup-?1?,?4,6?-?-D-Glup-?1?,?-T-Manp,?3?-?-Manp-?1?,?4?-?-D-Xylp-?1?and?4?-?-D-Galp-?1?.NMCP-2 is based on?-glucopyranose residue as the main chain,and a small amount of?-galactopyranosylose,xylose residue is present in the main chain;?-mannose is located at the end of the sugar chain;?4,6?-?-D-Glup-(1?is the branch point of the sugar chain.The results obtained in this study provide a theoretical basis for the development of polysaccharides from Morchella conica,the study of molecular structure and the inhibition of oxidative stress.