Isolation of phosphorylated polysaccharides from algae: the immunostimulatory principle of Chlorella pyrenoidosa.

The major immunostimulatory principle in the hot aqueous extract of Chlorella pyrenoidosa has been isolated by a sequence of ethanol precipitation, precipitation with a cationic surfactant (CTAB), size exclusion chromatography, and anion exchange chromatography. A series of phosphorylated polysaccharides were obtained having different molecular masses but with similar structures. The higher molecular mass fractions showed considerable activity in the stimulation of mouse peritoneal macrophages to synthesize nitric oxide. The structure of the major polysaccharide was established by sugar analysis, configurational analysis, and 1D and 2D NMR experiments at 500 and 800 MHz on the parent polysaccharide, the de-O-acetylated polysaccharide, and on the components obtained after hydrolysis of the phosphate diesters. It had a beta-D-Galp-(1-->3)-beta-D-Galp-(1-->3)-backbone with half of the Galp units substituted at O-6 by terminal beta-D-Glcp units. The remaining Galp units were substituted on O-6 by about equal amounts of alpha-D-Manp-1-phosphate and 3-O-Me-alpha-Manp-1-phosphate diesters. The substituents were not located in a regularly alternating fashion on the backbone. The O-acetyl groups were largely located on O-2 and O-4 of Galp and 35% of the Galp residues were O-acetylated. This is the second observation of a phosphorylated polysaccharide in an alga and the first where it is present to a significant extent.

Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract.

CPE is an aqueous extract of the edible micro alga Chlorella pyrenoidosa, which has been shown to have immunostimulatory effects in vivo. In the present study, CPE was evaluated for an ability to stimulate cytokine production by human peripheral blood mononuclear cells (PBMC). PBMC from healthy individuals were treated ex vivo for 24 hours with 1, 10 and 100 microg/mL CPE. This resulted in a marked increase in the level of IL-10, a regulatory cytokine, and strong stimulation of the T-helper-1 (Th1) cell cytokines, IFN-gamma and TNF-alpha. In contrast, stimulation of representative T-helper-2 (Th2) cell cytokines, IL-4 and IL-13, was minor. CPE (1, 10 or 100 microg/mL) did not cause a proliferation of human PBMC suggesting that enhanced secretion of cytokines was not secondary to an increase in cell number. We conclude that CPE stimulation of human PBMC induces a Th1-patterned cytokine response and a strong anti-inflammatory regulatory cytokine response, observations that await confirmation in vivo.

Immunostimulatory polysaccharides from Chlorella pyrenoidosa. A new galactofuranan. measurement of molecular weight and molecular weight dispersion by DOSY NMR.

Fractionation of the hot water extract of Chlorella pyrenoidosa was performed using a combination of ethanol precipitation, size exclusion chromatography, and anion exchange chromatography. One fraction contained a new polysaccharide, and this compound was shown to be a 1-->2-linked beta-d-galactofuranan from its 1D and 2D (1)H and (13)C NMR spectra, with a molecular weight of 15 kDa from DOSY NMR measurements. A number of other fractions were shown to have the same repeating unit as the previously identified arabinogalactan. However, arabinogalactans from different fractions were shown by DOSY NMR to have different molecular weights, which ranged from 27 to 1020 kDa. Agreement with molecular weights measured for some of these fractions by SEC-MALS was very good, further confirming the relationship established by Viel et al. between molecular weights of neutral polysaccharides and self-diffusion coefficients. The smaller molecular weight polysaccharides, the galactofuranan and the 27 and 50 kDa arabinogalactans, were shown to be close to monodisperse by analysis of the distributions of the self-diffusion coefficients for the polymers. The larger arabinogalactans had considerable variation in their molecular weights (188 +/- 109 kDa and 1020 +/- 370 kDa). Only the two larger arabinogalactans showed immunostimulatory activity.

Isolation, characterization and structural determination of a unique type of arabinogalactan from an immunostimulatory extract of Chlorella pyrenoidosa.

An arabinogalactan was isolated from a hot water extract of freeze-dried cells of the green microalga, Chlorella pyrenoidosa. This hot water extract is a proprietary immunomodulator, with the trademark Respondintrade mark (ONC-107). The arabinogalactan was recovered from the ethanol-soluble fraction of the supernatant resulting from a process that involved controlled ethanol precipitation followed by size exclusion chromatography on Sephadex G-100, then Cetavlon precipitation. Sugar analyses, GC-MS data for (S)-2-octyl glycosides, and 1D and 2D NMR experiments established unambiguously that the repeating unit was -->2)-alpha-L-Araf-(1-->3)-[alpha-L-Araf-(1-->4)]-beta-D-Galp-(1-->. This structure does not fit into any of the known classes of arabinogalactans. SEC/MALS experiments gave a molecular mass for the arabinogalactan isolated as 47 +/- 4 kDa but the original structure was probably larger.