Purification, Structural Analysis And Antitumor Activity Of Polysaccharides From Tricholoma Matsutake

Tricholoma matsutake is a kind of exotrophic mycorrhiza edible mushroom, havingmutualistic symbiosis with Loose genus or oak plants. The main active ingredient ofTricholoma matsutake is polysaccharide. At present, studies on polysaccharide of Tricholomamatsutake in China focus on antitumor ability and immunoregulation effect. There is littlereport on purification and structure of polysaccharide of Tricholoma matsutake. In this paper,based on extraction and purification of polysaccharide of Tricholoma matsutake, physical andchemical characteristics and antitumor activity of TM-P were investigated.The chemical composition of Tricholoma matsutake were analyzed, the results were:9.14%of moisture,19.21%of protein,7.40%of fiber,4.00%of fat,8.28%of ash,48.37%of total sugar,10.61%of crude polysaccharide. On basis of the results of singlefactors test, response surface analysis was used to optimize process condition according to theresponse of polysaccharide yield and ORAC value. The optimal condition of ultrasonic power,process time and solvent-material ratio was243W,6min and16, respectively. Under theoptimal conditions, the yield and ORAC value of polysaccharides were5.76±0.05%and423.80±9.43μmol Trolox/g.TM-P was first purified through DEAE Sepharosse Fast Flow column, as a result,TM-P1, TM-P2and TM-P3were obtained. TM-P2was the fraction with highest ORAC value2100.44±25.32μmol Trolox/g. TM-P1was consisted of fucose, mannose, glucose andgalactose with mol ratio of1.0:1.9:7.7:4.0. TM-P2was consisted of mannose, glucose andgalactose with mol ratio of1.0:5.9:1.1. TM-P3was consisted of fucose, mannose, glucoseand galactose with mol ratio of1.0:2.2:5.1:4.9.With methylation analysis, it was shown that: TM-P1were mainly linked withâ†'3,6)-Glcp-(2â†',â†'6)-Glcp-(1â†'andâ†'6)-Galp-(1â†'; TM-P2were mainly linked withâ†'6)-Glcp-(1â†'and Glcp-(1â†'; TM-P3were mainly linked withâ†'3,6)-Glcp-(2â†',â†'6)-Galp-(1â†'and Galp-(1â†'. At the same time, antitumor activity in vitro of TM-P1,TM-P2and TM-P3were evaluated by determining their inhibitory rates against humanhepatoma cell HepG2and human lung cancer cell A549. The result implied that TM-P2showed strongest inhibitory rates. At a concentration of4mg/ml, the inhibitory rates ofTM-P2against human hepatoma cell HepG2and human lung cancer cell A549were67.98±1.08%and59.04±3.78%respectively.After purified by Sephadex G-75column, TM-P1A was obtained from TM-P1. Withmolecular weight361309Da, TM-P1A had certain antioxidant ability, the ORAC value of which was211.80±4.68μmol Trolox/g. TM-P2was separated into TM-P2A and TM-P2B bySephadex G-75column. Molecular weights of TM-P2A and TM-P2B were372443Da and4796Da, and the ORAC value were268.66±5.02μmol Trolox/g and2475.33±15.32μmolTrolox/g respectively.TM-P1A was consisted of mannose, glucose and galactose with mol ratio of1.0:8.7:1.8.TM-P2A was consisted of fucose, mannose, glucose and galactose with mol ratio of1.0:3.9:17.7:7.9. TM-P2B was consisted of mannose, glucose and galactose with mol ratio1.0:8.9:1.3.The structure characterizations of TM-P1A, TM-P2A and TM-P2B were studied withNMR. The results manifested that TM-P1A was α-1,6-pyranoid glucans; TM-P2A wasβ-1,6-pyranoid glucans with a few α-1,6-pyranoid glucose and β-1,6-pyranoidgalacose;TM-P2B was β-1,6-pyranoid glucans.