Macrophage-stimulating activities of newly isolated complex polysaccharides from Parachlorella kessleri strain KNK-A001

- Parachlorella kessleri strain KNK-A001 has various water-soluble polysaccharide fractions with different bioactivities.
- The water-soluble polysaccharides can be separated into five fractions by DEAE anion exchange chromatography.
- F5, a fraction isolated from Parachlorella kessleri strain KNK-A001, shows the most potent induction of the secretion of NO and TNF-α from RAW264.7 cells.
- The activity of F5 is mediated through the activation of NF-κB.
- MAP kinase signalling pathways are involved in the macrophage-stimulating activities of F5.

Our previous studies demonstrated that the microalga Parachlorella kessleri (KNK-A001) has immunostimulatory activities, which were observed as an increase in natural killer (NK) cell activity in mice after intraperitoneal injection or as a protective effect on a virus-infected model shrimp after oral administration. In this study, we attempted to gain insight into the constituent substances of KNK-A001 that are responsible for the immunostimulatory activity. First, we obtained five polysaccharide fractions from KNK-A001 by DEAE anion exchange chromatography. Among the fractions, F5 showed the most potent induction of nitric oxide (NO) secretion in RAW264.7 cells, and both mRNA and protein expression levels of inducible NO synthase (iNOS) were increased in F5-treated RAW264.7 cells. A significant increase in the nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) was observed in F5-treated RAW264.7 cells. F5 also induced the secretion of tumor necrosis factor (TNF)-α in RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase were mainly involved in F5-induced NO and TNF-α productions. The compositional analysis of F5 identified the main constituents as galactose, glucose, galacturonic acid, and mannose. Gel-filtration analysis suggested that molecular mass of F5 was approximately 400 kDa.