Structure Characterization,Modification And Bioactivities Of Exopolysaccharide From Fomes Fomentarius

Fungal polysaccharides feature many bioactivities,such as immune regulation,hypoglycemic,antioxidant and anti-tumor.Fomes fomentarius is a large medicinal fungus that mainly grows in Northeast China and contains glycosides,polysaccharides,organic acids,sterols,terpenoids,and other chemical components.In this paper,to explore the medicinal value of fomes fomentarius exo-polysaccharides（FFEP）,FFEP was obtained utilizing submerged culture in a large tank of liquid with Fomes fomentarius as the experimental material and subjected to purification and structural characterization;the activities of the main component FFEP-1 in hypoxia resistance,lipid-lowering and liver protection and scald wound repair as well as the antioxidant activity and anti-tumor effect of the molecularly modified derivatives were studied;the main results are as follows:（1）Fermentation Conditions for Exopolysaccharide Production by High-yielding Fomes fomentariusThe single factor test,Plackett-Burman design,and the steepest climbing test were employed to determine the central points of response surface optimization for the liquid culture of Fomes fomentarius:sucrose 10.0 g·L^-1,casein peptone 5.0 g·L^-1and inoculum size 6.0%.According to the design principle of response surface methodology（RSM）,the optimal culture conditions that affect the production of Fomes fomentarius exo-polysaccharides were determined as follows:sucrose 15.2 g·L^-1,casein peptone 7.0 g·L^-1,inoculum size 6.2%,KH₂PO₄1.0 g·L^-1,Mg SO₄0.5 g·L^-1,VB₂0.625 mg·L^-1,p H 7.5,culture temperature 30℃,shaker speed 150 rpm,and culture time 10 days.Under these conditions,the content of polysaccharide in the fermentation broth of Fomes fomentarius was 4.02 times higher than that without optimization.（2）Purification and structural characterization of FFEP-1Upon decolorization,deproteinization,and other pretreatment,the crude polysaccharide obtained from the fermentation broth of Fomes fomentarius was subjected to DEAE-52 Column tandem chromatography to obtain three polysaccharides,namely FFEP-1,FFEP-2 and FFEP-3 respectively.In this test,FFEP-1,with the highest content,was selected as the research object,which is a tasteless,light yellow powder with typical characteristic peaks of sugars,easily soluble in water,and has a relative molecular weight of about 3.08×10⁵Da.The results of gas chromatography-mass spectrometry（GC-MS）showed that FFEP-1 was a heteropolysaccharide composed of mannose,glucose,and galactose（with a molar ratio 0.18:0.24:0.58）.Nuclear magnetic resonance（NMR）analysis suggested that the main chain of FFEP-1 may be composed ofβ-1,2-D-galactose（Galp）,β-1,2,3-D-galactose（Galp）,α-1,3-D-glucose（Glcp）,β-1,3,4-D-mannose（Manp）andα-1,3-D-mannose（Manp）,whose O-2 and O-3positions are new branchedα-D-Galp andα-D-Glcp substituted heteropolysaccharides.（3）Bioactivities of FFEP-1After pre-test activity screening,the three bioactivities of anti-hypoxia,reducing lipid and protecting liver,and repairing scalded wound were selected for study.FFEP-1with different concentrations（≥50 mg/kg）can significantly prolong the survival time of mice under different hypoxia conditions,and presents significant anti-hypoxia activity.The number of red blood cells and hemoglobin concentration in the treatment group rose significantly,which was close to that in the positive administration group（phenytoin,PHT）.Different doses of FEEP-1（≥100 mg/kg）produce different lipid-lowering and liver-protecting effects on model mice.When the dosage of FFEP-1was≥100 mg/kg,the contents of total cholesterol（TC）,triglyceride（TG）and fat in serum and liver and the enzyme activity and total bilirubin（T-BIL）of alanine transaminase（ALT）,glutamic oxalacetic transaminase（Glutamic Oxalacetic Transaminase,AST）,lactate dehydrogenase（LDH）,alkaline phosphatase（ALP）,etc.decreased significantly（P<0.01）,and the antioxidant indexes superoxide dismutase（SOD）,glutathione peroxidase（GPX）and catalase（CAT）increased significantly,the antioxidant indexes superoxide dismutase（SOD）,glutathione peroxidase（GPx）and catalase（CAT）increased significantly,while the content of malondialdehyde（MDA）in tissues was significantly reduced,and the levels of the anti-inflammatory related factor（Nuclear Factor Kappa-B,NF-κB）,interleukin 1β（IL-1β）,cytochrome P4502E1（CYP2E1）,and interleukin-6（IL-6）declined significantly and were close to those of the positive group.It is suggested that FFEP-1 can improve the antioxidant defense system of mice,effectively reduce the peroxidation of liver tissue caused by high fat,weaken the injury of inflammatory factors of liver injury to mice,and promote liver tissue remodeling.Further,the study on the activity of FFEP-1 in repairing deep second-degree scalded wound indicated that 5.0%FFEP-1 ointment could greatly promote the scab healing of scalded wound and shorten the repair time of scalded surface;the secretion of Matrix metalloproteinase（MMP-1）and transforming growth factorβ（TGF-β）in scalded skin treated with 5.0%FFEP-1 grew significantly,the release of IL-6 accelerated the metabolism of wound cells.Then it promoted the wound healing of the scalded wound,demonstrating that FFEP-1 can inhibit the initial infection of the wound and effectively promote the later repair and regeneration of scalded wound.（4）Molecular modification of FFEP-1 and its antioxidant activity in vitroFFEP-1 was modified by phosphorylation,sulfation and selenization.Fourier transform infrared（FT-IR）analysis verified that the product was successfully modified.The results of in vitro antioxidant activity stated that the antioxidant activity of FFEP-1was significantly enhanced after phosphorylation,sulfation and selenization.Phosphated polysaccharide（PFFEP-1）presented the most significant scavenging effect on hydroxyl radical and O₂^-,and the scavenging rates were 91.55%and 57.85%higher than those of FFEP-1,respectively;sulfated polysaccharide（SFFEP-1）significantly inhibited lipid peroxidation and DPPH,with the inhibition rates being 99.78%and41.09%higher than those of FFEP-1,respectively.The polysaccharide reducing the power of FFEP-1 and its derivatives was SFFEP-1>PFFEP-1>Se FFEP-1>FFEP-1,which gradually increased with the rise of concentration.（5）Antitumor effects of FFEP-1 and its derivativesThe anti-tumor effects of FFEP-1 and its SFFEP-1 and PFFEP-1 on human esophageal cancer Eca109 cells were studied.The results revealed ed that Eca109 cell apoptosis was evident with the increase of the concentration of the test substance;when the concentration was 400μg/m L,SFFEP-1,PFFEP-1 and FFEP-1 after treatment for48 hours produced the inhibition rates of Eca109 cells 52.45%,69.47%and 41.73%respectively,The apoptosis rate presented a specific dose-dependent relationship.Different concentrations of FFEP-1,SFFEP-1 and PFFEP-1 can induce ROS production in Eca109 cells and regulate antioxidant enzymes such as NADPH Quinine-Oxidoreductase-1（NQO-1）,heme oxygenase 1（HO-1）,glutathione peroxidase（GPx）,manganese superoxide dismutase 2（SOD-2）in Eca109 cells,while significantly inhibiting Nrf2（NF-E2-related factor 2）-mediated antioxidant defense system.Moreover,FFEP-1,SFFEP-1 and PFFEP-1 can induce the change of mitochondrial membrane potential in Eca109 cells and then activate the mitochondrial apoptosis pathway,leading to a significant decrease in the expression of anti-apoptotic proteins Bcl-2 and Bcl XL in the B-cell lymphoma/leukemia-2 gene（Bcl-2）protein family,and a significant rise in the expression of pro-apoptotic proteins Bax and Bak and inhibiting the proliferation of Eca109 cells.Additionally,SFFEP-1 can cause the Eca109 cells to stagnate in G2/M phase,and PFFEP-1 can make Eca109 cells stagnate in S phase,thereby prolonging the cycle of tumor cells and suppressing DNA repair of damaged cells.