Preparation, Structural Features and in vitro Immunostimulatory Activity of a Glucomannan From Fresh Dendrobium catenatum Stems

Abstract

Dendrobium catenatum polysaccharides (DCPs) have attracted attention due to their multiple physiological activities and health benefits. In this study, a novel water-soluble DCP was obtained from fresh D. catenatum stems through three-phase partitioning and ethanol precipitation at room temperature. Its structural characteristics, rheological property, and in vitro immunostimulatory activity were evaluated. Results demonstrated that DCP was a homogenous polysaccharide with a carbohydrate content of 92.75% and a weight-average molecular weight of 2.21 × 10⁵ Da. This polysaccharide is an O-acetylated glucomannan comprised by glucose, mannose, and galacturonic acid in a molar ratio of 30.2:69.5:0.3 and mainly comprises (1→4)-β-D-mannopyranosyl (Manp), 2-O-acetyl-(1→4)-β-D-Manp, (1→6)-α-D-glucopyranosyl (Glcp), and (1→4)-α-D-Glcp residues. DCP exhibits an extended rigid chain in an aqueous solution and favorable steady shear fluid and dynamic viscoelastic behaviors. In vitro immunostimulating assays indicated that DCP activates RAW264.7 cells, thus markedly promoting macrophage proliferation and phagocytosis and increasing the levels of nitric oxide, interferon-γ, interleukin-6, and interleukin-1β. Moreover, the presence of O-acetyl group and high M _w in DCP might be responsible for its potent immunostimulatory activity in vitro. Therefore, our data suggested that DCP could be developed as a promising immunostimulant in functional food and pharmaceutical industries.

Keywords: Dendrobium catenatum; immunostimulatory activity; polysaccharide; rheological property; structure; three-phase partitioning.

Figure 1

Extraction and isolation procedures for the DCP from fresh D. catenatum stems by using TPP and ethanol precipitation.

Figure 2

(A) Monosaccharide compositions of DCP and monosaccharide standards, and (B) FT-IR spectrum of DCP.

Figure 3

(A) SEC elution and molar mass profiles, and (B) logarithmic plot of M_w vs. R_z for DCP.

Figure 4

(A) ¹H NMR, (B) ¹³C NMR, (C) HSQC, and (D) HMBC of DCP.

Figure 5

(A) Dependence of apparent viscosity (η_a) on shear rate (γ), and (B) Plots of storage modulus G′ (solid symbols) and loss modulus G^′′ (open symbols) on angular frequency (ω) for DCP solution at different concentrations and 25°C. The DCP concentrations are 20, 30, 40, 50, and 60 mg/mL from bottom to top. The data were shifted along vertical axis by 10^a to avoid overlap.

Figure 6

Effect of DCP at different concentrations on (A) the proliferation of RAW264.7 cells, and (B) NO production in RAW264.7 cells (n = 3, ± SEM). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. the control or LPS group.

Figure 7

A fluorescence microscope image of RAW264.7 cells stained with FITC-labeled E. coli (×400).

Figure 8

Effects of DCP at different concentrations on the levels of (A) IL-6, (B) IL-1β, and (C) IFN-γ in RAW264.7 cells. (n = 3, ±SEM). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control group.