Characterisation of exopolysaccharides isolated from Dunaliella salina exhibiting immunomodulation in peripheral blood mononuclear cells and RAW 264.7 macrophages

Abstract

Dunaliella salina (D. salina) is the green halotolerant algae known for its growth in hypersaline environment. Recently, the algae have been explored for the extracellular polysaccharides (EPS) and their various industrial applications. The scope of this study is characterization and immunological assessment on peripheral blood mononuclear cells and RAW 264.7 of EPS isolated from D. Salina and enhancement of the production of EPS by cultivating in stress conditions. Column chromatography of crude EPS resulted in five fractions where DsF1 and DsF4 were observed to contain high sugar content. Cell proliferation assay showed that DsF1 and DsF4 increased the cell growth of PBMC in a dose dependent manner. Three cytokines (IFN-γ, TNF-α, TGF-β) were also estimated in PBMC culture by ELISA and observed to be increased with increase in concentration for DsF1 and DsF4 indicating the immunostimulatory effect. In case of RAW cells, both DsF1 and DsF4 showed a decreasing trend of cell growth and nitric oxide production (griess reagent) with increasing concentration. Mass spectroscopy results revealed the presence of hetero polysaccharides composed of various hexoses (glucose, galactose, and fructose) and pentoses (xylose) both in DsF1 and DsF4. The presence of halide associated polysaccharides was also suggested which was confirmed by FTIR analysis. Oligosaccharides (eight monosaccharide units) were also found to be present in fraction DsF4 as per the mass spectroscopy results. FTIR analysis have shown the presence of proteoglycans due to the detection of N=O and N-H group. Enhanced production of EPS was obtained when grown in stress conditions i.e. nitrate (KNO3:14.7 mM and 24.5 mM), phosphate (KH2PO4: 4.2 mM and 7 mM) and sodium salt (NaCl: 3 M and 3.5 M) stress as compared with normal conditions (KNO3: 4.9 mM, KH2PO4: 1.4 mM and NaCl: 1.7 M).Hence, it is concluded that two fractionated EPS isolated from D. salina possess immunomodulatory activities and has the potential to be used for therapeutic purpose.