Immunoregulatory Activity And Preparation Of Schizophyllan Under Ultrasonic Treatment

Schizophyllan(SPG) is a water soluble extracellular polysaccharide which can be extracted from the fruit body, mycelium or fermented liquid of Schizophyllum. Immunity and anti-tumor effect can be enhanced by it. But the application of SPG has been limited for high molecular weight(>106 Da) and viscosity. In this study, SPG with solid-state fermentation was degraded by ultrasonic treatment, so we can get different molecular weight of ultrasound-treated Schizophyllan(USPG); then we studied USPG about the primary structure and rheological properties. Through in vivo and in vitro experiment, the immune activity and anti-tumor activity were investigated at last.The main research contents and results were as follows:The molecular weight of SPG was reduced as ultrasonic time and intensity increased, and degradation kinetics conform to second order dynamic model of degradation. Finally ultrasonic degradation parameters were as follows: 10 min, 796 W/cm2. At the same time, with fractional precipitation technique and dialysis, SPG can be separated into three fractions: USPG40, USPG60, USPG80; their molecular weight were concentrated in 106-107 Da, 104-105 Da, 105-106 Da and purifications were 93.15%, 90.56%, 88.77%, respectively.With infrared spectrum and methylation analysis, the result showed that ultrasonic treatment did not change the types and links of glycan chain. The rheological properties of SPG and USPG were studied. It showed that SPG was a pseudoplastic fluid, as the concentration was increased to 10 mg/ml, SPG showed solid characteristics(storage modulus G’ > loss modulus G "). After ultrasonic treatment, USPG40 look like pseudoplastic fluid, but USPG60 and USPG80 were Newtonian fluid. Under the same concentrations(5 mg/ml), USPG40 showed viscosity behavior(storage modulus G’< loss modulus G") when angular frequency was 1.1 s-1, but USPG60 was at 0.05 s-1, and USPG80 completely showed viscosity behavior in any case.The immunity and anti-tumor effects of SPG and USPG fractions were studied. Through in vitro experiment, SPG and USPG fractions(25-600 μg/ml) behaved well in splenic lymphocyte proliferation, promoting macrophages(RAW 264.7) releasing NO and inhibitory activities of breast cancer cells T-47 D. Compared with SPG, USPG fractions showed more activity. USPG60 was best in splenic lymphocyte proliferation, promoting macrophages(RAW 264.7) releasing NO and inhibitory activities of breast cancer cells T-47 D.In vivo experiment, the immunities and anti-tumor functions of SPG and USPG fractions could be further evaluated. Drug serum of SPG and USPG fractions can enhance immune functions of rats. At concentration of 20%, drug serum of USPG60 group was best to promote effects of spleen lymphocyte proliferation, and the immune factors like IL-2(169.85 pg/ml) and TNF-α(85.62 pg/ml) in serum were the highest level. The S180 tumor mice experiments showed that SPG and USPG fractions can enhance delayed type hypersensitivity(DTH), B lymphocyte proliferation induced by LPS and improve the activities of NK cells significantly. The comprehensive performance of USPG60 group behaved well and its inhibitory rate reached 37.75%. Besides, the observation of tumor biopsies showed that USPG60 and USPG80 groups can induce apoptosis of tumor cells and a large number of inflammatory cell infiltration.