Immunomodulatory Activity of Polysaccharides Isolated from Saussurea salicifolia L. and Saussurea frolovii Ledeb

Abstract

The genus Saussurea has been used in the preparation of therapies for a number of medical problems, yet not much is known about the therapeutic high-molecular-weight compounds present in extracts from these plants. Since polysaccharides are important in immune modulation, we investigated the chemical composition and immunomodulatory activity of Saussurea salicifolia L. and Saussurea frolovii Ledeb polysaccharides. Water-soluble polysaccharides from the aerial parts of these plants were extracted using water at pHs of 2 and 6 and subsequently precipitated in ethanol to obtain fractions SSP2 and SSP6 from S. salicifolia and fractions SSF2 and SSF6 from S. frolovii. The molecular weights of fractions SSP2, SSP6, SFP2, and SFP6 were estimated to be 143.7, 113.2, 75.3, and 64.3 kDa, respectively. The polysaccharides from S. frolovii contained xylose (67.1-71.7%) and glucose (28.3-32.9%), whereas the polysaccharides from S. frolovii contained xylose (63.1-76.7%), glucose (11.8-19.2%), galactose (4.7-8.3%), and rhamnose (6.8-9.4%). Fractions SSP2, SSP6, and SFP2 stimulated nitric oxide (NO) production by murine macrophages, and NO production induced by SSP2, SSP6, and SFP2 was not inhibited by polymyxin B treatment of the fractions, whereaspolymyxin B treatment diminished the effects of SFP6, suggesting that SFP6 could contain lipopolysaccharide (LPS). The LPS-free fractions SSP2, SSP6, and SFP2 had potent immunomodulatory activity, induced NO production, and activated transcription factors NF-κB/AP-1 in human monocytic THP-1 cells and cytokine production by human MonoMac-6 monocytic cells, including interleukin (IL)-1α, IL-1β, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF). These data suggest that at least part of the beneficial therapeutic effects reported for water extracts of the Saussurea species are due to the modulation of leukocyte functions by polysaccharides.

Keywords: Saussurea; cytokine; macrophage; nitric oxide; plant polysaccharide; polymyxin B; xyloglucan.

Figure 1

Effect of the Saussurea polysaccharides on macrophage NO production. Mouse macrophages were treated for 48 h with the indicated polysaccharide fractions, media alone (0; negative control), or 100 ng/mL LPS (positive control). NO production was quantified by measuring nitrite in the cell-free supernatants. The data are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of two independent experiments. Statistically significant differences (* p < 0.01) between cells treated with media alone (0) and cells treated with SSP2, SSP6, SFP2, SFP6, or LPS are indicated.

Figure 2

Effects of polymyxin B on NO production by polysaccharide-treated macrophages. Mouse macrophages pretreated with media (w/o PMB) or with 50 μg/mL polymyxin B (PMB) for 48 h were incubated with 20 μg/mL of the indicated polysaccharide fractions, media alone (negative control), or 100 ng/mL LPS (positive control). NO production was quantified by measuring nitrite in the cell-free supernatants. The data in each panel are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of two independent experiments. Statistically significant differences (* p < 0.05) between samples treated with media and samples treated with polymyxin B are indicated.

Figure 3

Effect of the Saussurea polysaccharides on AP-1/NF-κB activation. Human THP-1Blue monocytes (10⁵ cells/well) were incubated for 24 h with the indicated concentrations of polysaccharide, media alone (0; negative control), or 100 ng/mL LPS (positive control). Alkaline phosphatase activity was analyzed spectrophotometrically (absorbance at 655 nm) in the cell supernatants, as described. Values are the mean ± S.D. of triplicate samples from one experiment, which is representative of three independent experiments. Statistically significant differences (* p < 0.01) between cells treated with media alone (0) and cells treated with SSP2, SSP6, SFP2, or LPS are indicated.

Figure 4

Effect of the Saussurea polysaccharides and LPS on IL-6 production. MonoMac-6 cells were pretreated with the indicated concentrations of polysaccharide, 100 ng/mL LPS, or media alone (0) for 24 h. Production of IL-6 in the supernatants was evaluated by ELISA. The data in each panel are presented as the mean ± S.D. of triplicate samples from one experiment that is representative of two independent experiments. Statistically significant differences (* p < 0.01) between cells treated with media alone (0) and cells treated with SSP2, SSP6, SFP2, or LPS are indicated.

Figure 5

Effect of Saussurea polysaccharides on cytokine production by human MonoMac-6 cells. MonoMac-6 cells were incubated for 24 h with 20 μg/mL of the indicated polysaccharide fractions or 100 ng/mL of LPS (positive control), and production of cytokines in the supernatants was evaluated using a Multiplex Human Cytokine ELISA kit. The data are presented as mean ± SD of duplicate samples from one experiment that is representative of two independent experiments. Statistically significant differences (* p < 0.01) between cells treated with media alone and cells treated with the polysaccharide fractions or LPS are indicated.