A Poly-D-Mannose Synthesized by a One-Pot Method Exhibits Anti-Biofilm, Antioxidant, and Anti-Inflammatory Properties In Vitro

Abstract

In this study, D-mannose was used to synthesize poly-D-mannose using a one-pot method. The molecular weight, degree of branching, monosaccharide composition, total sugar content, and infrared spectrum were determined. In addition, we evaluated the safety and bioactivity of poly-D-mannose including anti-pathogen biofilm, antioxidant, and anti-inflammatory activity. The results showed that poly-D-mannose was a mixture of four components with different molecular weights. The molecular weight of the first three components was larger than 410,000 Da, and that of the fourth was 3884 Da. The branching degree of poly-D-mannose was 0.53. The total sugar content was 97.70%, and the monosaccharide was composed only of mannose. The infrared spectra showed that poly-D-mannose possessed characteristic groups of polysaccharides. Poly-D-mannose showed no cytotoxicity or hemolytic activity at the concentration range from 0.125 mg/mL to 8 mg/mL. In addition, poly-D-mannose had the best inhibition effect on Salmonella typhimurium at the concentration of 2 mg/mL (68.0% ± 3.9%). The inhibition effect on Escherichia coli O157:H7 was not obvious, and the biofilm was reduced by 37.6% ± 2.9% at 2 mg/mL. For Staphylococcus aureus and Bacillus cereus, poly-D-mannose had no effect on biofilms at low concentration; however, 2 mg/mL of poly-D-mannose showed inhibition rates of 33.7% ± 6.4% and 47.5% ± 4%, respectively. Poly-D-mannose showed different scavenging ability on free radicals. It showed the best scavenging effect on DPPH, with the highest scavenging rate of 74.0% ± 2.8%, followed by hydroxyl radicals, with the scavenging rate of 36.5% ± 1.6%; the scavenging rates of superoxide anion radicals and ABTS radicals were the lowest, at only 10.1% ± 2.1% and 16.3% ± 0.9%, respectively. In lipopolysaccharide (LPS)-stimulated macrophages, poly-D-mannose decreased the secretion of nitric oxide (NO) and reactive oxygen species (ROS), and down-regulated the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). Therefore, it can be concluded that poly-D-mannose prepared in this research is safe and has certain biological activity. Meanwhile, it provides a new idea for the development of novel prebiotics for food and feed industries or active ingredients used for pharmaceutical production in the future.

Keywords: anti-biofilm; anti-inflammatory; antioxidant; poly-D-mannose.

Figure 1

Macrostructure and microstructure of poly-D-mannose: (A): poly-D-mannose powder; (B): microstructure of poly-D-mannose at 30× magnification; (C): microstructure of poly-D-mannose at 150× magnification; (D): microstructure of poly-D-mannose at 400× magnification.

Figure 2

Results of FT-IR, molecular weight, and monosaccharide composition of poly-D-mannose synthesized by the one-pot method: (a): FT-IR comparison spectra of D-mannose, citric acid, and poly-D-mannose; (b): HPLC chromatogram of molecular weight distribution; (c): HPLC chromatogram of monosaccharide composition and monosaccharide standards.

Figure 3

NMR spectra of poly-D-mannose (500 MHz, 298 K): (a): ¹H NMR spectrum; (b): ¹³C NMR spectrum.

Figure 4

Cell viability of Vero cells after treatment with different concentrations of poly-D-mannose. Standard deviation of the samples (n= 3) is shown by vertical bars. ^a Significant differences (p < 0.05) are indicated with different lowercase letters.

Figure 5

Anti-biofilm effects of poly-D-mannose on Bacillus cereus, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli O157: H7. Standard deviation of the samples (n = 3) is shown by vertical bars. ^a,b,c,d Significant differences (p < 0.05) are indicated with different lowercase letters.

Figure 6

Scavenging activities of poly-D-mannose on ABTS, DPPH, superoxide anion radicals, and hydroxyl radicals. Standard deviation of the samples (n = 3) is shown by vertical bars.

Figure 7

Effects of poly-D-mannose on RAW 264.7 macrophages: (a): proliferation of macrophages; (b): NO production; (c): ROS production; (d): ROS production observed by fluorescence microscopy (20×). (e): TNF-α production; (f): IL-6 production. Standard deviation of the samples (n = 3) is shown by vertical bars. ^a,b,c,d,e,f Significant differences (p < 0.05) are indicated with different lowercase letters.