Purification, characterization and in vitro and in vivo immune enhancement of polysaccharides from mulberry leaves

Abstract

Mulberry leaf polysaccharide (MLP) was extracted and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography to afford two major purified polysaccharides (MLP-1 and MLP-2). The purified polysaccharides were characterized, and their immune-enhancing properties were investigated. MLP-1 had a molecular weight of 9.31×104 Da and was composed of mannose, rhamnose, glucose, galactose, xylose, and arabinose in a molar ratio of 0.71:1.00:2.76:1.13:3.70:2.81. The molecular weight of MLP-2 was 2.22×106 Da, and its monosaccharide constituents were mannose, rhamnose, glucose, galactose, and arabinose in a molar ratio of 1.31:8.45:6.94:1.00:11.96. Infrared spectroscopy showed that each MLP had a typical absorption peak characteristic of sugars, and ultraviolet (UV) spectroscopy showed that neither MLP contained nucleic acid or protein components. Then, the abilities of these polysaccharides to stimulate spleen lymphocyte proliferation in mice in vitro were compared by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. MLP-2 was more effective than MLP-1; therefore, MLP-2 was chosen for the study of its immune-enhancing effects in vivo. For the in vivo experiments, 14-day-old chickens immunized with Newcastle disease (ND) vaccine were orally administered MLP-2, and Astragalus polysaccharide (APS) was used as the control. Each chicken was orally administered 4 mg or 8 mg of MLP-2 for seven consecutive days starting three days before ND vaccine immunization. MLP-2 significantly improved the ND serum antibody titer and interleukin-2 (IL-2), interferon-γ (IFN-γ) and immunoglobulin A (sIgA) concentrations in tracheal and jejunal wash fluids, and increasing numbers of immune globulin A-positive (IgA+) cells in cecal tonsils and increased body weight. These results indicated that MLP-2 could significantly enhance immune activity and could therefore be utilized as an immunopotentiator drug candidate.

Fig 1. Elution chromatograms of the polysaccharides from mulberry leaves.

(a) Elution curve of crude MLP from a DEAE-52 cellulose column; (b) elution curve of the MLP-1 fraction from a Sephadex G-100 column; and (c) elution curve of the MLP-2 fraction from a Sephadex G-100 column.

Fig 2

HPGPC chromatograms of MLP-1 (a) and MLP-2 (b).

Fig 3

HPLC chromatograms of the PMP derivatives of the monosaccharide standards (a) and the hydrolysates of MLP-1 (b) and MLP-2 (c).

Fig 4

Infrared spectra of MLP-1 (a) and MLP-2 (b).

Fig 5. UV spectra of MLPs.

Fig 6. The sIgA levels in the jejunal wash fluids from each group (μg/mL) (n = 6).

Bars without the same superscript (a-c) differ significantly (p< 0.05).

Fig 7. The sIgA levels in the trachea washing liquids from each group (μg/mL) (n = 6).

Bars without the same superscript (a-c) differ significantly (p< 0.05).

Fig 8. IgA⁺ cell numbers in the cecal tonsils of each group (n = 6).

Bars without the same superscript (a-d) differ significantly (p< 0.05).