Study On Purification, Modification And Anti-tumor Activity Of Polysaccharides From Auricularia Polytricha

Auricularia polytricha is widely grown throughout China, which belongs to the eumycota, basidiomycetes, auriculariales, auriculariaceae, born in the tropical and subtropical regions and in the warm, wet season plexus. Some physiological functions of polysaccharides from Auricularia polytricha such as antioxidant activity,anticancer, affecting blood glucose and plasma lipid levels have been investigated.Polysaccharides from Auricularia polytricha(APP) have a great potential to be used as clinical therapeutic agents and health products.Auricularia polytricha was used as raw materials, the optimal conditions of utrasonic/microwave assisted extraction(UMAE) of APP were determined. The rheological properties and physiochemical properties of APP were examined. APP was further isolated and purified. Single component named APP3 a was selected with strong antitumor activity. APP3 a was modified with carboxymethyl method and the optimum carboxymethyl modification conditions of APP3 a were studied. At the same time, the effect of before and after the carboxymethyl modification of APP3 a on antitumor activity were also investigated. The main results of the research were listed as follows:1. The optimal conditions of UMAE of APP were obtained as follows: 22(mL/g)of solvent-solid ratio, 15 min of extraction time and 60 W of microwave power,respectively. The yield and purity of polysaccharides were 4.13% and 47.98%,respectively.2. APP was white color with no taste, easy to dissolve in water and could not dissolve in ethanol, methanol and acetone et al. It had no protein and phenols. The study of rheological properties showed that the viscosity of APP increased with the increase of concentration, but the viscosity decreased with the increase of temperature in a certain temperature range. The viscosity of APP decreased with the increase of shear rate. It appeared the phenomenon of shear thinning, characterized by the non-Newtonian fluid and had pseudoplasticity. pH had a great influence on the viscosity. The heating time, the changes of freezing and thawing, the amount of metal ions and sucrose had little effect on the viscosity.3. APP was fractionated by anion-exchange chromatography(DEAE-52cellulose). Four components were obtained. APP1 was neutral acid polysaccharide.APP2, APP3 and APP4 were acid polysaccharides. After fractionation of APP, therecovery rate of polysaccharides was 87.6%. APP3 was choosed because of the higher anti-tumor activity. APP3 was eluted on Sepharose G-200 column. APP3 a was obtained. High performance liquid chromatography(HPLC) indicated that APP3 a was homogeneous. The average molecular weight of APP3 a was 21242 Da.Monosaccharide of APP3 a contained Ara, Man, Glc and Gal, which was determined by gas chromatography. The mole ratio of monosaccharide of APP3 a was1:1.33:1.06:1.23. Thermogravimetic analysis showed that APP3 a decomposed and lost weight rapidly between 160℃ to 500℃.4. The process conditions of APP3 a carboxymethyl modification, the antitumor activity of APP3 a and CM-APP3 a were studied. The optimal conditions of APP3 a carboxymethyl modification were as follows: the reaction medium was isopropanol,APP3 a was 60 mg, the amount of chloroacetic acid was 1.45 g, the dosage of sodium hydroxide was 2.48 g, the reaction temperature was 55.2℃, the reaction time was3.49 h and the predictive value of the degree of substitution degree of polysaccharide was 0.895. Cell proliferation assay showed that compared with the control group,APP3 a and CM-APP3 a after treating HepG2 cells for 48 h could obviously inhibit the growth of HepG2 cells. The inhibitory effects were stronger with the increase of APP3 a and CM-APP3 a concentration. It showed obvious dose effect relationships.APP3 a and CM-APP3 a on HepG2 cell had the maximum inhibitory effect at the concentration of 800 mg/mL. The inhibitory effect on HepG2 cell of CM-APP3 a was higher than APP3 a under the conditions of same concentration and processing time.APP3 a and CM-APP3 a could induce HepG2 cell apoptosis at the concentration of 25 to 800 mg/mL. Cell apoptosis rate was higher with the increase of concentration. At the concentration of 800 mg/mL, the apoptosis rate of APP3 a and CM-APP3 a were19.65% and 35.8%, respectively. There were significant difference compared with the control group(p<0.01). The apoptosis rate of CM-APP3 a was higher than APP3 a under the conditions of the same concentration and processing time.