Studies On The Antitumor And Immunocompetent Polysaccharides From Mycelium Of Agaricus Blazei Murill

Agaricus blazei Murill (AbM) is a kind of fungus used for food and medicine. Itexhibited a diverse bioactivities. Now, it has been used as an important nature source ofanticarcinogens, immunomodulators, antimutigens and bactericidal substances. Beausefungus submerged fermentation posess characteristics of low-cost, short-period and yearround producing, so , it has becoming to be a promising way to develop antitumorpolysaccharides from AbM. Few papers were reported about extraction, isolation,purification and structure determination of polysaccharides from submerged fermentationmycelium of AbM.In this paper, three valueble fractions were extracted and screened from submergedfermentation mycelium of AbM by hot water and cold NaOH.The water-soluble fraction(HWSP) extracted by hot water received a yield of 7.73g/100g dried mycelium powder under the technology conditions optimizated by RSA.HWSP exhibited a significant immunocompetence and inhibitive rate of 59.84% againstS180 at a dose of 20 mg/Kg.d in mice. Also, HWSP had significant promoting proliferationof splenic lymphocytes and selective tumoricidal effect on liver cancer cells of Bel-7402 invitro.A homogeneous fraction(DP-HWSP30-21) was obtained by alcohol fractionalprecipitation and column chromatography from HWSP. It has a yield of 1.55g/100g driedmucelium power. It was an acidic proteoglycan with 67.39% polysaccharide, 29.69%protein and 21.41% uronic acid content respectively. The protein and polysaccharide moietywere linked by O-glycopeptide bond of glycan chain to Serine. The relative molecularweight of DP-HWSP30-21 was 218KDa. The sugar composition of it was arabinose,mannose, xylose, glucose with a molar ratio of 0.05:0.16:0.32:1.It was vertified that linkage of protein moiety had a positive effect on the bioactivity ofpromoting splenic lymphocytes proliferation of polysaccharides moiety fromDP-HWSP30-21, but protein moiety had no any effect itself alone. However,DP-HWSP30-21 would lose the inhibitive effect on Bel-7402 when the protein andpolysaccharide moiety were dissociated with each other. DP-HWSP30-21 had no significantinhibitive effect on human normal liver cells and was of synergism with chemotherapeuticsof DHI(Doxorubicin Hydrochloride for injection).The water-soluble fraction of CAWSP extracted by cold alkali received a yield of 5.73g/100g dried mycelium powder under the technology conditions of 5% NaOH(0.1%NaBH4), 6h, 4℃, 2 times. CAWSP exhibited an significant immunocompetence andinhibitive rate of 64.66% against S180 at a dose of 20 mg/Kg.d in mice. CAWSP hadsignificant promotive proliferation of splenic lymphocyte and had no tumoricidal effect onexperimental cancer cells. A homogeneous fraction , CAWSP211, was isolated fromCAWSP by column chromatography. It contained no nucleic and protein with a relativemolecular weight of 587KDa, the sugar composition and molar ratio were Glc: Gal: Xyl:Ara=1.1: 1.8: 5.0:5.7. The structure of CAWSP211 was elucidated as follows:The water-insoluble fraction of CAASP extracted by cold alkali received a yield of8.58 g/100g dried mycelium powder under the same technology conditions as CAWSP. Thecombinative method of twice alkalinization and microwave radiation was applied incarboxymethylation of CAASP. Carboxymethyl-CAASPs (CM-CAASP) with differentdegree substitution(DS) could been obtained by adjusting preparation technologyparameters. The mutual blank determing methods of FTIR(fourier transform infraredspectroscopy) and ABT(acid-base titration) for DS were indicated brief, precise andpracticable.CM-CAASPs presented significant promoting proliferation of splenic lymphocytes anda dose-effect relationship when DS lower than 0.70 in vitro. The CM-CAASP with DS of0.70 exhibited a significant antitumor activity against S180 and immunocompetence in mice.It had no acute toxic reaction in mice. When undertake the technological conditions as:add15 mL 75% alcholo to 1g CAASP powder, stirring for 30min, then add 3.5g NaOH tothe blend slowly during 40min, after alkalified for 50min, react for 15min undermicrowave output power of 200W, then add 1g NaOH and 5 mL 75% alcholo again, reactfor 10min under the same conditions as above, then end the reaction, neutralization withacetic acid, dialysis, concentrate, precipitate and freeze drying, the CM-CAASP with adesirable DS of 0.70 could be got.