Purification Of Polysaccharides And Oligosaccharides From Fermentation Concentrate Of Hericium Caput-medusae (Bull.:Fr) Pers. And Its Protective Effects On Oxidative Injured RAW264.7 Cells

Hericium caput-medusae?Bull.:Fr.?pers.is a valuable edible and medicinal fungus.The common method for its production is liquid deep fermentation technology,and fermentation extract is used for the treatment of atrophic gastritis,peptic ulcer and other gastrointestinal diseases.Studies have shown that polysaccharide and oligosaccharide are the major active components,and the anti-inflammatory,antitumor activities and modulatory effect on intestinal flora have been confirmed in plenty researches,however reports on the antioxidant activity,especially on cell level,are yet to be revealed.In addition,the occurrence and development of such diseases as chronic inflammation,ulceration,as well as tumor are related with the disorder of redox equilibrium.Therefore,polysaccharide and oligosaccharide from fermentation concentrate of Hericium caput-medusae?Bull.:Fr.?Pers.were isolated and purified in the present study.The chemical properties,the chemical and cell antioxidant activities were investigated in order to clear the exact antioxidant component and the mechanisms.We hope this study could provide reference for the development and utilization of related products.Alcohol precipitation,dialysis and column chromatography were used for the separation of polysaccharide and oligosaccharide with different molecular weight.Next we analyzed the chemical composition by measuring the contents of reducing sugar,the soluble protein,the uronic acid,sulfate group and the amino sugar of each component.Then we used PMP pre-column derivatization HPLC and FI-TR to analyze the composition and structure of oligosaccharide.Chemical reaction system was further used to detect the antioxidant ability of polysaccharide and oligosaccharide.RAW264.7 macrophage cell model for H2O2 oxidative damage was established and the cell viability,antioxidant enzyme activities,apoptosis rate,MDA and ROS levels were also investigated to further confirm the protective effect of each component on cell oxidative damage.The effect of polysaccharide and oligosaccharide on genetic transcription of antioxidant enzymes was analyzed using q-PCR,and the activation effects of MAPK signaling and Nrf2 were investigated by western blot.The main achievements are as follows:?1?The present study achieved 4 crude sugar components:d-HFCO1?<500 Da?,d-HFCO2?500-5,000 Da??d-HFCP1?5000-50,000 Da??d-HFCP2?>50,000 Da?,13 purified components,among them are 2 neutral oligosaccharides d-HFCO1-1,d-HFCO2-1with high purity?total sugar content 88.90%and 97.43%?and 1 neutral oligosaccharide with lower purity?total sugar content 55.43%?.?2?HFCO has more reducing sugar,proteins are mostly present in HFCP,and small amounts of uronic acids,amino acids and sulfate group are in crude sugar components.Glc is the major component in monosaccharide,the variety and Gal ratio increase with the molecular weight.Both?and?glucosidic bonds exist in d-HFCO1-1 and d-HFCO2-1,and the the major one in d-HFCP1-1 is?glucosidic bond.?3?The capacity of HFC on ABTS and DPPH radical scavenging are:d-HFCP1<d-HFCO2<d-HFCP1-1<d-HFCO2-1<d-HFCO1<d-HFCO1-1;the IC50 values of HFC on hydroxyl radical scavenging are:d-HFCO1<d-HFCO2-1<d-HFCO2<d-HFCP1-1<d-HFCP1;the ability of HFC on superoxide anion scavenging is:d-HFCP1-1<d-HFCP1.Other IC₅₀ values of saccharide components are beyond the measuring scope.?4?Both d-HFCO2 and d-HFCP1 could significantly improve cell viability,decrease MDA levels and increase T-SOD,Gpx and CAT activities;2 neutral saccharide components could significantly improve cell viability and decrease MDA level?p<0.05?,in which d-HFCO2-1significantly increased CAT acitivity,d-HFCP1-1 significantly increase T-SOD and CAT activities?p<0.05?.Both d-HFCO2-1 and d-HFCP1-1 inhibited ROS generation and reduced cell death and apoptosis.?5?Both d-HFCO2-1 and d-HFCP1-1 significantly improved the relative content of Mn-SOD,GPx1 and CAT mTNA?p<0.05?,and enhanced gene transcription of three antioxidant enzymes.The activity of d-HFCP1-1 was better than d-HFCO2-1,which is consistent with its tendency in improving antioxidant enzyme activity.?6?d-HFCP1-1 treatment promoted the dissociation of Nrf2 protein and Keap1,and the nuclear translocation of Nrf2.The effect reached maximum at 1.5 h?p<0.05?.Conclusions:?1?The major component of HFC is neutral oligosaccharides,with small amount of acidic sugar and high content of monosaccharides.?2?The protective activity of HFCO and HFCP on H₂O₂ oxidative damaged RAW264.7cells is due to scavenging free radicals,improvement of antioxidant enzyme activity,inhibition of MDA and ROS generation and reduction of cell apoptotic rate,with better effects of polysaccharides than oligosaccharides,crude saccharides than neutral saccharides.?3?Reduction of MAPK phosphorylation and improvement of Nrf2 nuclear translocation might account for the antioxidant effects of HFCO and HFCP.In summary,our study uncovered the major antioxidant components in polysaccharides and oligosaccharides from polysaccharides and oligosaccharides Hericium caput-medusae?Bull.:Fr.?pers.fermentation concentrate,revealed the underlying antioxidant mechanism,and provides theoretical evidence for the development and utilization of Hericium caput-medusae?Bull.:Fr.?pers.