Biological Activities Of Chemically Modificated Polysaccharides Extracted From Morchella Angusticeps Peck

Morchella esculenta(L.)Pers.is a rare edible fungus,there are more than 20 varieties such as Morchella angusticeps Peck,Morchella costata Pers.,Morchella conica Pers.,Morchella crassipes(Vent.)Pers.and so on.At present,mainly due to its rich in protein,phenolic acids,polysaccharides and other bioactive substances,Morchella angusticeps Peck has been shown that exhibited a wide range of biological activities such as antioxidant,anti-fatigue,anti-tumor,immunoregulatory activity,etc.Many studies have confirmed that natural polysaccharides after chemical modification can significantly enhance the biological activities of polysaccharides and even produce a new activity,which has become a research hotspot in the field of glycobiology and nutrition.Therefore,the Modified PMEP of Sulfation,carboxymethylation,acetylation was prepared by using Morchella angusticeps Peck(PMEP)as raw material,then to study the activity of anti-oxidation,anti-proliferation and immunoregulation of unmodified PMEP and chemically modified PMEP,which contribute to provide theoretical basis for the development and utilization of Morchella angusticeps Peck.The main studies and results are as follows:(1)The sulfated,carboxymethylatiedand acetylated PMEP were prepared by Chlorosulfonic acid-pyridine method,monochloroacetic acid method and acetic anhydride method,respectively.The results of the determination of polysaccharide content,yield and degree of substitution showed that the higher the proportion of the modified reagent(dose)was,the higher the degree of substitution was.But the higher degree of substitution may lead to degradation of the polysaccharide,resulting in a lower polysaccharide content or yield.The results of UV scanning showed that no new absorption peak appeared;the results of infrared spectroscopy showed that PMEP and chemically modified PMEP all had the characteristic absorption peaks of polysaccharides,and the chemically modified polysaccharides had the specific absorption peaks of the modified groups.The above results indicate that the PMEP has been successfully modified and the polysaccharide structure has not been changed to a great extent.(2)The anti-proliferation of unmodified PMEP and chemically modified PMEP were studied by using HepG2 cells and Caco-2 cells as models.The results showed that PMEP had significant inhibitory effect on HepG2 cells and Caco-2 cells,and there was a good dose-effect relationship.Sulfated and carboxymethylated modified PMEP can not inhibit the proliferation of HepG2 cells and Caco-2 cells.Acetylated modified PMEP(Ac-PMEP1、Ac-PMEP2 and Ac-PMEP3)has obvious inhibitory effect on HepG2 cells and Caco-2 cells,and there is a good dose-effect relationship(P<0.05).In particular,the cell proliferation activity of Ac-PMEP3 was significantly higher than PMEP(P<0.05),and the anti-proliferative capacity of HepG2 cells and Caco-2 cells was 0.71,1.229 mg/mL,respectively.These results indicated that the chemical modification method and the degree of substitution of PMEP is different,its anti-proliferative activity is also different.(3)The chemical antioxidant activities of PMEP and chemically modified PMEP were evaluated by ABTS free radical scavenging Capacity and oxygen radical absorbance capacity(ORAC).Then,the cellular antioxidant activity(CAA)was determined by using HepG2 cells as a model.The ABTS+.Radical scavenging capacity of PMEP was 0.697 mg/mL,the ORAC value of PMEP was 372.04 μmol TE/g,Which indicated that PMEP has good chemically antioxidant activity.The CAA value was 3.08 μmol of QE/g(no PBS wash),1.96 μmol QE/g(PBS wash).The results showed that PMEP outside the cell membrane has more antioxidant activity than inside the cell membrane.Compared with PMEP,the antioxidant activity of the sulfated and carboxymethylated PMEP had decreased significantly;the ABTS+.Radical scavenging capacity,ORAC values and CAA values of the acetylated PMEP(Ac-PMEP1、Ac-PMEP2 and Ac-PMEP3)was enhanced significantly.It indicated that the acetylated modification of PEMP can significantly improve its chemical antioxidant activity and cell antioxidant activity.And it positively correlated with the degree of polysaccharide acetyl substitution.(4)The immunoregulatory activity of PMEP and chemically modified PMEP were studied by using mouse macrophage RAW264.7 as a model.When RAW264.7 cells were in resting state,PMEP and chemically modified PMEP could promote cell proliferation,enhance cell phagocytosis and increase the release of NO and TNF-α to a certain extent(Immune up-regulation).Compared with PMEP,in a certain concentration range,sulfated PMEP(S-PMEP1:12),carboxymethylated PMEP(CM-PMEP1)and acetylated PMEP(Ac-PMEP1,Ac-PMEP2,Ac-PMEP3)could significantly promote the proliferation of RAW264.7 cells and improve the phagocytic activity(P<0.05).Especially,Ac-PMEP3 could significantly increase the release of NO and TNF-α(P<0.05).When RAW264.7 cells were in the over-induced state by LPS,PMEP and sulfated PMEP(S-PMEP1:3,S-PMEP1:12)and acetylated PMEP(Ac-PMEP1,Ac-PMEP3)can down-regulate cell phagocytosis and decrease the release of NO and TNF-α(Immune down-regulation).Compared with PMEP,sulfated PMEP(S-PMEP1:3,S-PMEP1:12)and acetylated PMEP(Ac-PMEP1,Ac-PMEP3)can obviously downregulate the excessive activation of RAW264.7.Especially,Ac-PMEP3 had the best effect.(5)The mechanism of immunomodulatory activity of PMEP and Ac-PMEP was studied by using mouse macrophage RAW264.7 as a model.When the RAW264.7 cells were in resting state,the high dose group of PMEP(PMEP-H,50 μg/m L)and the high dose of acetylated PMEP(Ac-PMEP-H,50 μg/mL)could be significantly increased the levels of p65(nucleus)in the nuclear transcription factor-kappa B(NF-κB)dimer,inducible nitric oxide synthase(iNOS)and phosphorylated p38 protein kinase(p-p38),and significantly reduced the content of inhibitor of NF-κB(IκB-α)(P<0.05).When RAW264.7 cells were in over-induced state by LPS,both PMEP-H and Ac-PMEP-H significantly inhibited the increase the levels of p65(nucleus),iNOS and p-p38,and significantly inhibited the decrease of IκB-α.This suggests that the mechanism of immune regulation of macrophage RAW264.7 may be through NF-κB and p38 MAPK pathway to regulate the expression of related cytokines.Compared with PMEP,the immune down-regulation of acetylated PMEP(Ac-PMEP3)on RAW264.7 cells is more prominent.