Several species of microalgae have been known to produce exopolysaccharides (EPS) with potential immune activity. In the present investigation, ethyl acetate fraction of crude EPS secreted by Dunaliella salina was explored for immunomodulatory activity against peripheral blood mononuclear cells (PBMC) and RAW 264.7 macrophages. Effect of EPS on cell growth and cytokines production were measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ELISA respectively. Griess reagent was used for measuring the nitric oxide production in RAW 264.7 macrophages. FTIR analysis and mass spectroscopy were carried out for the characterization. Ethyl acetate fraction exhibited dose dependent increase in proliferative index and cytokines production (IFN-γ, TNF-α, TGF-β). At low concentration (250 and 500 µg/mL), it showed growth inhibition and at higher concentration (1000 and 1500 µg/mL), it enhanced the cell growth. Interestingly, the pronounced increased TNF-α production was observed in ethyl acetate fraction treated PBMC cells at higher concentration (750 and 1000 µg/mL) indicating the immunostimulatory effect. In RAW cells, concentration dependent diminished cell growth (IC₅₀ = 691 µg/mL) and nitric oxide production (IC₅₀ = 630 µg/mL) was observed. FTIR analysis showed the presence of polysaccharides due to the detection of hydroxyl (–OH), Carbonyl (C–O) and alkyl (C–H) groups. Mass spectroscopy results revealed ethyl acetate fraction as penta-saccharide (m/z = 887.56 and 886.54) which are confirmed to be hetero-polysaccharides consisting of hexoses and pentoses along with association of ions. These results suggest that penta-saccharide (ethyl acetate fraction) isolated from D. salina may have the potential to be used for therapeutic purpose as immunomodulatory agent.

中文翻译：

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杜氏盐藻分泌的杂多糖表现出针对外周血单个核细胞和RAW 264.7巨噬细胞的免疫调节活性。

已知几种微藻可产生具有潜在免疫活性的胞外多糖（EPS）。在本研究中，杜氏盐藻分泌的粗EPS的乙酸乙酯级分研究了针对外周血单核细胞（PBMC）和RAW 264.7巨噬细胞的免疫调节活性。分别使用MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）测定法和ELISA测量EPS对细胞生长和细胞因子产生的影响。Griess试剂用于测量RAW 264.7巨噬细胞中一氧化氮的产生。进行了FTIR分析和质谱分析。乙酸乙酯级分显示出增殖指数和细胞因子产生（IFN-γ，TNF-α，TGF-β）的剂量依赖性增加。在低浓度（250和500 µg / mL）下，它显示出生长抑制作用；在高浓度（1000和1500 µg / mL）下，它增强了细胞生长。有趣的是 在较高浓度（750和1000 µg / mL）的乙酸乙酯级分处理的PBMC细胞中观察到明显增加的TNF-α产生，表明具有免疫刺激作用。在RAW细胞中，浓度依赖性减弱了细胞生长（IC₅₀  = 691 µg / mL）和一氧化氮的产生（IC ₅₀  = 630 µg / mL）。FTIR分析表明，由于检测到羟基（–OH），羰基（C–O）和烷基（CH–H），因此存在多糖。质谱分析结果表明，乙酸乙酯级分是五糖（m / z = 887.56和886.54），已确认是由己糖和戊糖以及离子缔合组成的杂多糖。这些结果表明，从盐藻（D. salina）分离的五糖（乙酸乙酯级分）可能具有作为免疫调节剂用于治疗目的的潜力。