Study On Isolation, Purification, Structural Identification And Antitumor Activity Of Polysaccharides From Agaricus Blazei Murill

Agaricus blazei Murill(ABM) is originated from countries in southern North America, such as Brazil and Peru. It has become a precious fungus, and processes edible and medical values, since it was cultured by Japanese in 1972. ABM has a strong flavor of almond and tastes delicious. Its soft cap and crispy stipe contain high amount of proteins and other nutrients, which are of high nutrition value. Moreover, ABM has quite high therapeutic potential. It is reported that the polysaccharides from ABM is effective in inhibiting tumor (especially ascites liver cancer), improving stamina, decreasing cholesterol, blood pressure and blood sugar content, curing diabetes and preventing atherosclerotic and cardiovascular illness. The fruiting body and mycelia of ABM and fermented medium of ABM contain active polysaccharide. So far the studies on polysaccharides from ABM have been focused on crude polysaccharides. There are few reports regarding chemical structures on the homogenous components from polysaccharides. The present project studied the procedure how to purify the multiple polysaccharide components from the fruiting body and mycelia of ABM and fermented medium of ABM, their chemical structural characterization and pharmacological activity.This dissertation consists of six chapters, which are extraction of polysaccharides from the fruiting body, extraction and purification of polysaccharides from the mycelia fermentation, comparison of the polysaccharide components from different ABMs, purification of polysaccharides from the fruiting body, physical characterization and chemical structural analysis of polysaccharides from ABM, and anti-tumor activity of different polysaccharides of ABM.The results are shown as following.1. Development of extract technique for polysacchadde from ABM fruiting bodyThrough single-factor experiments and orthogonal experiments, it was found that best yield could be obtained by using raw material: water ratio 1: 30 at 120℃for incubation for 3 h. Pretreatment of the raw material by microwave for 8-12 min could increase the polysaccharide yield from 8.12% to 9.15%. Multiple extractions of 2 times resulted an optimal yield of 93.10%. Extracted solution was concentrated, then precipitated after incubation for 6 h with 3 volumes 95% ethanol.2. Development of technique for extraction and purification of polysaccharide from deep fermentation of ABMThe medium for ABM submerged fermentation and the fermentation conditions were studied, in order to increase the biomass of ABM mycelia and the yield of extracellular polysaccharides. The results from these experiments paved the way for bioactivity assays of polysaccharides from ABM mycelia and extracellular polysaccharides, as well as replacing culture of fruiting body with submerged fermentation.2.1 The components in the medium for submerged fermentation have been optimized. Raw corn powder and bean cake were chosen as the suitable carbon and nitrogen source. The optimal medium consists of 1% sugar, 2% corn powder, 0.5% bean cake, 0.3% KH₂PO₄ and 0.2% MgSO₄·7H₂O.2.2 Conditions for ABM submerged fermentation were studied. The suitable condition is using 100 mL liquid culture in 500 mL flask, 10% inoculation amount, rotation speed 120 r/min and the fermentation undergoes for 140 h. Under this condition, ABM biomass of 1.21g/100mL and extracellular polysaccharides of 0.205g/100mL was obtained.2.3 According to the extraction and purification methods used for fruiting body, two homogeneous components of polysaccharides were purified from the ABM mycelia and named ABMM1 and ABMM2, having the yield of 34.5% and 25.1% respectively.3. Component comparison of ABM from different places3.1 Nutrient components from ABM fruiting body and mycelia were compared. ABM mycelia contained slightly higher amount of sugars and proteins than those in fruiting body, while the latter had more ash.3.2 The amounts of micro-nutrients in ABM fruiting body and mycelia were measured by emission spectroscopy. Both parts contained multiple essential micro-nutrients, especially with Zn and Se in high amount.3.3 Fruiting body had higher amount of crude polysaccharides than those in mycelia; while it had lower amount of proteins than the amount of crude polysaccharides from mycelia. However, after extraction, the yield of crude polysaccharides from mycelia was higher than that from fruiting body.3.4 By comparison HPLC spectra of crude polysaccharides from different sources, polysaccharides from mycelia and extracellular polysaccharides contained relatively higher amount of lower molecular weight components than that of higher molecular weight ones, while this situation was opposite for crude polysaccharides from fruiting body.4. Extraction and purification of polysaccharides from ABM fruiting bodyTechnical procedure for extraction and purification of polysaccharides from ABM fruiting body was developed, including ultra-filtration and purification, protein removal by ion-exchange resin, multiple precipitation by ethanol and gel purification.4.1 It was concluded that such technical procedures can be used to remove proteins from polysaccharides of ABM, as ABM polysaccharide solution→filtration through 10 KDa membrane→filtrate→ion-exchange resin→freeze-drying→fine polysaccharide powder without proteins.4.2 Removing proteins from crude polysaccharides by ion-exchange resin produced polysaccharide powder with high purity. This method has not been reported before. The crude polysaccharides were treated by weak basic resin negatively charged followed by weak acid one charged with cation, then washed by deionized water. The condition of the treatment was mild, and the rate of protein removal and the yield of polysaccharides could be as high as 89.7% and 82.1% respectively, in addition having some effect of color removal. This method has the advantages of large exchange capacity, easy scale-up for industrial production, reusable resin, simple operation and low cost.4.3 By ethanol precipitation, polysaccharides from ABM were separated in two groups, ABMF1 and ABMP-2, of yield 48% and 46%, respectively. Two fractions of homogeneous polysaccharide, ABMPâ… and ABMPâ…¡, 65.1%, were obtained from ABMF1 through Sepharose 6 F. F. gel filtration.4.4 Another homogeneous fraction ABMPâ…¢was separated from ABMF2 through Sephacryl S-300 gel filtration, yielded 28.6% of ABMF2.5. Structural analysis of homogeneous component from polysaccharides of ABM fruiting body5.1 ABMPâ… is a white solid, and ABMPâ…¡is a subtle yellow solid. Both are soluble in water, having optical activities of＋62.5°and＋44.3°respectively, and molecular weight about 20×10⁵ and14×10⁵ Dalton respectively.5.2 Determined by HPLC measurement after complete acid hydrolysis, ABMPâ… and ABMPâ…¡both consist of only glucoses.5.3 The main chain of ABMPâ… is made of 1,4-glucopyranose, together with small amount (1.5%) of non-starchα-glucan, which can react with proteins.5.4 ABMPâ…¡is a glucosan withβ-glucopyranosyl group.6. Study of anti-tumor activity from ABM polysaccharides6.1 The growth of S180 tumor in mice was inhibited when mice was fed by ABMPâ… and ABMPâ…¡. The former one had better inhibition than the other. The inhibition rate was positively correlated to the amount of the both components fed to mice. When the amount was 200 mg/kg·d, the inhibition rate was 46.3% and 38.7% for ABMPâ… and ABMPâ…¡, respectively.6.2 ABMPâ… and ABMPâ…¡also inhibited the growth of ascites liver cancer in mice by 53.4% and 40.1%, respectively, when the amount was 200 mg/kg·d.6.3 The growth of S 180 tumor in mice was inhibited when mice was fed by crude polysaccharides of mycelia and extracellular polysaccharides from ABM, by 52.8% and 54.6% respectively, when the dosages were 500 mg/kg·d and 300 mg/kg·d respectively. The inhibition rate was negatively correlated with the dosage, when the polysaccharides were fed in higher amount. This indicates that extracellular polysaccharides of ABM inhibit the immune system in mice at such high dosage.