| Polysaccharide is an important active ingredient with a wide range ofpharmacological activities in fungi. The immunomodulatory and anti-tumor activitiesof polysaccharides from fungi have been widely recognized, but the study on thestructure-activity relationship of the fungal polysaccharide is still insufficient now.Pleurotus ostreatus, a tricholomataceae fungus of basidiomycetes, is one of importantmanmade edible fungi with high nutritional value and medicinal value, which is alsocalled oyster mushroom, oyster-cap fugus, green mushroom, jatropha bacteria and etc.It is well known that fungal polysaccharides are usually extracted with waterextraction and there has been no report on the extraction of polysaccharide componentfrom pleurotus ostreatus with alkaline extraction so far. In the present study, in orderto fully explore the medicinal value of pleurotus ostreatus and screen a leadingcompound for anti-tumor agents from pleurotus ostreatu, the pleurotus ostreatuspolysaccharide was separated and purified with alkaline extraction, the structuralcharacterization of the pleurotus ostreatus polysaccharide, WPOP-N1, was analyzedby classical chemical methods and instrumental means, and its immunomodulatoryand anti-tumor effects were investigated with in vitro cytological and in vivo methodsto reveal the structure-activity relationship of anti-tumor and immunomodulatoryeffects of the pleurotus ostreatus polysaccharide.MethodsBy using alkaline extraction, repeated freezing and thawing, deproteinization,ion-exchange chromatography and gel filtration chromatography, WPOP-N1, analkali-extracted and water-soluble polysaccharide, was separated and prepared frompleurotus ostreatus. Phenol-sulfuric acid, Bradford and hydroxyl biphenyls methodswere used to detect total sugar content, protein content, and uronic acid content ofWPOP-N1respectively. High performance liquid chromatography (HPLC) was used to determine the homogeneous degree and the average molecular weight of WPOP-N1.After derivatization by acetylation, the monomer composition of WPOP-N1wasdetermined by gas chromatography (GC). WPOP-N1polysaccharide structure wasanalyzed with combined chemical method such as partial acid hydrolysis, periodateoxidation, Smith degradation, and methylation, as well as analytical measures such asgas chromatography, infrared spectroscopy, and gas chromatography-massspectrometry. In vivo transplanted mouse sarcoma S-180models, as well as the mousespleen lymphocyte transformation experiments, mouse peritoneal macrophagephagocytic capacity, macrophage TNF-a and NO emission analysis by RT-PCR andWestern Blot, and NF-κB activity were applied to evaluate anti-tumor effects ofWPOP-N1and the possible mechanisms.ResultsFirstly, the water-soluble polysaccharide components from the fruiting body ofpleurotus ostreatus were isolated and removed with hot water boiling extraction, andthe residue was extracted by alkaline extraction and ethanol precipitation to obtain thecrude pleurotus ostreatus polysaccharides, from which the total polysaccharides,WPOP, were achieved using the combined deproteinization methods including therepeated freezing and thawing, enzyme methods and Sevag method, then, they wereeluted in a gradient way with DEAE-cellulose ion exchange chromatography indifferent NaCl solutions, and finally, they were graded with Sepharose CL-6B gelfiltration chromatography based on the molecular distribution of the samples to getthe primary polysaccharide fraction, namely WPOP-N1. The HPLC detectionindicated that WPOP-N1showed a simple peak and the average molecular weight was48.2kDa. The phenol sulfuric acid method verified that the total polysaccharidecontent was97.6%. The GC analysis demonstrated that WPOP-N1was composed ofmannose and a small amount of gloucose, and the molar ratio was5.6:3.3. Them-hydroxyl diphenyl method was negative, indicating that WPOP-N1did not containuronic acid, which was in consistent with the result from GC analysis. Moreover,chemical analytical methods such as partial acid hydrolysis, periodate oxidation,Smith degradation and methylation, as well as instrumental analytical methods suchas IR, GC and GC-MS were applied to analyze the chemical structure of WPOP-N1meticulously and finally determine the repetitive unit of WPOP-N1. The resultsshowed that the basic chemical structure of WPOP-N1should include a backbone composed of β-(1â†'3)-mannose and (1â†'3,6)-β-D-mannose, and the molar ratio was1.57:1. In addition, WPOP-N1presented a low branching configuration and there wasa trace of terminated glucose residues on the branch chain. In this study, BALB/cmice were used to establish S-180solid tumor model for the in vivo experiment toexamine the anti-tumor activity. The results showed that WPOP-N1significantlyinhibited the growth of the S-180, an in vivo transplanted tumor, in mice, theinhibition rate reached up to63.1%at the dose of400mg/kg, and the anti-tumoreffect of WPOP-N1was close to that of cyclophosphamide in the positive controlgroup. In the in vitro anti-tumor activity experiments, the results from MTT showedthat WPOP-N1could not inhibit the proliferation of S-180sarcoma cells significantly,suggesting that the anti-tumor activity of WPOP-N1might be due to itsimmunomodulating activity. Our further studies confirmed that WPOP-N1treatmentsignificantly increased the spleen index and thymus index of tumor-bearing mice, andraised the content of tumor necrosis factor-α (TNF-α) in serum. The treatment withWPOP-N1effectively stimulated lymphocyte proliferation, enhanced thephagocytosis of peritoneal macrophages, and increased the secretion of NO andTNF-α by WPOP-N1-treated macrophages. The results from RT-PCR and WesternBlot showed that WPOP-N1could increase the expression of TNF-α and iNOS bothin gene and protein levels, and in the meanwhile, upregulate the phosphorylation levelof p-65of the regulatory subunit of NF-κB, as well as promote the degradation of IκBofthe inhibition subunit of NF-κB, suggesting that WPOP-N1can enhance thetranscriptional activity of NF-κB to promote the secretion of NO and TNFα.Conclusions1.WPOP-N1, the alkali-extracted polysaccharide fraction from pleurotusostreatus, could be isolated and obtained by the systemic grading and purifying withalkaline extraction, freezing and thawing, deproteinization, and ionic and molecularexclusion chromatography.2.Using various chemical methods and instrumental analytical means, WPOP-N1could be identified as a mannose glucan, in which there is a polysaccharide backbonethat is composed of (1â†'3)-β-D-mannose, the branching point sugar residue is (1â†'3,6)-β-D-mannose, and the non-reducing end residue of the main chain is glucose.3.The in vivo experiment demonstrates that WPOP-N1could produce aneffective anti-tumor activity, but the in vitro experiment could not show the cytotoxic effect.4.WPOP-N1can increase the lymphocyte transformation and simultaneouslyinduce the activation of macrophages, improve their phagocytic ability, and promotethe secretion of the corresponding anti-tumor substances through stimulating thetranscriptional activity of NF-κB, suggesting WPOP-N1may exert its anti-tumoractivity by enhancing the immune function. |
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