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. 2015 Aug 22:172:247-53.
doi: 10.1016/j.jep.2015.06.013. Epub 2015 Jun 18.

Immuno-stimulatory activity of a polysaccharide-enriched fraction of Sutherlandia frutescens occurs by the toll-like receptor-4 signaling pathway

Wei Lei  1 , Jimmy D Browning Jr  1 , Peggy A Eichen  1 , Chi-Hua Lu  1 , Valeri V Mossine  2 , George E Rottinghaus  3 , William R Folk  2 , Grace Y Sun  2 , Dennis B Lubahn  2 , Kevin L Fritsche  4
Affiliations

Immuno-stimulatory activity of a polysaccharide-enriched fraction of Sutherlandia frutescens occurs by the toll-like receptor-4 signaling pathway

Wei Lei et al. J Ethnopharmacol. .

Abstract

Ethnopharmacological relevance: Sutherlandia frutescens (L.) R. Br. is an indigenous plant of southern Africa that has been traditionally used for various cancers, infections, and inflammatory conditions.

Aim of the study: Our aim was to investigate the potential immuno-stimulatory activity of a polysaccharide-enriched fraction (SFPS) from a decoction of S. frutescens.

Materials and methods: RAW 264.7 cells (a murine macrophage cell line) were used to determine the activities of SFPS on macrophage function. The production of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines were evaluated in the cells treated with or without SFPS. CLI-095, a toll-like receptor (TLR) 4-specific inhibitor, was used to identify whether or not SFPS exerts its effects through TLR4. An antagonist of endotoxin, polymyxin B, was used to evaluate whether endotoxin present in SFPS contributed to its immune-stimulatory activity.

Results: SFPS exhibited potent immune-stimulatory activity by macrophages. The production of ROS, NO, and tumor necrosis factor (TNF-α) were increased upon exposure to SFPS in a dose-dependent manner. All of these activities were completely blocked by co-treatment with CLI-095, but only partially diminished by polymyxin B.

Conclusion: We demonstrate for the first time potent immune-stimulatory activity in a decoction prepared from S. frutescens. We believe that this immune stimulatory activity is due, in part, to the action of polysaccharides present in the decoction that acts by way of TLR4 receptors and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. These findings provide a plausible mechanism through which we can understand some of the medicinal properties of S. frutescens.

Keywords: Endotoxin; Macrophage; Polysaccharides; Sutherlandia frutescens; Toll-like receptor 4.

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Figures

Figure 1
Figure 1
Polysaccharides-enriched fraction from S.frutescens (SFPS) was not toxic to murine macrophages, RAW 264.7. Cells were pretreated with 1 μg/mL CLI-095 or 10 μg/mL polymyxin B (PMB) for 1 h prior to incubation with various concentrations of SFPS in DMEM/1%FBS for 20 h. The cell viability was determined by resazurin assay. SFPS with/without co-treatment of CLI-095 or PMB showed no toxicity on murine macrophage (RAW 264.7 cells) after 20 h. The resazurin data was expressed as percentage of untreated control. The data were from four independent experiments conducted in triplicate.
Figure 2
Figure 2
SFPS induced the production of NO and ROS via activation of TLR4 signaling pathway. NO production induced by SFPS was partially inhibited by pretreated cells with polymyxin B (PMB) (A). Meanwhile, SFPS-induced ROS production was completely blocked by treatment of CLI-095, and polymyxin B partially inhibited it (B). Data were from three independent experiments each conducted in triplicate. * p <0.05, ** p <0.01, *** p <0.0001.
Figure 3
Figure 3
Effect of SFPS on NF-κB activity in RAW 264.7 cells. The activation of NF-κB was induced by SFPS. This activity was completely inhibited by CLI-095 and partially diminished by polymyxin B (PMB). * p<0.05, ** p <0.01, *** p <0.0001.
Figure 4
Figure 4
Endotoxin concentration in SFPS. The SFPS was analyzed in triplicate using recombinant factor C endotoxin detection assay.
See this image and copyright information in PMC

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