Three-phase partitioning technique for the green separation of crude polysaccharides from Schizophyllum commune and its effect on macrophage activation

Abstract

This study aimed to investigate a non-chromatographic method for separation polysaccharides from Schizophyllum commune (ScP1) using three-phase partitioning (TPP) for facile large-scale implementation. A crude slurry was extracted with hot water extraction, then separated using TPP at the following conditions: 40% (w/v) (NH4)2SO4, 1:2 (v/v) slurry:t-butanol ratio, pH 6, temperature of 40 °C, and incubation duration of 75 min to form the three phases. The results showed that extraction yield of the ScP1 under the optimized conditions was 20.08%. The ScP1 contained the most abundant mannose, with minor amounts of glucose and galactose. Two different equivalent molecular weights were found in ScP1 (709.54 ± 20.04 and 8.10 ± 0.31 kDa). An analysis of the Fourier transform-infrared spectrometer spectra, X-ray diffraction, and Congo red reaction indicated that the ScP1 were polysaccharides with semi-crystalline and random coil conformations. The solubility and microstructure of the ScP1 revealed a rough surface with small cavities features. Nuclear magnetic resonance analyses suggested that the backbone of ScP1 was primarily (1→3)-α-d-mannose and (1→3,6)-linkage residues. ScP1 enhanced the production of nitric oxide (NO) and mRNA expression of iNOS and TNFα of RAW264.7 cell in a dose-dependent manner. Moreover, ScP1 likely plays an immune stimulation role through MR, GR and DC-1 receptors on RAW264.7 cell. Thus, this technology may encourage economical and environmentally friendly separation of immune-enhancing agent polysaccharides from S. commune, which can be applied in the downstream industrial processing of functional foods and pharmaceuticals.