Extraction Optimization and Bioactivity of Polysaccharides from Ganoderma leucocontextum Spores

Abstract

Background: Oxidative stress is associated with the occurrence and progress of aging. Natural polysaccharides have attracted considerable attention in the field of antioxidants and anti-aging products due to their superior biological activity and low toxicity. Ganoderma leucocontextum is primarily found in the Tibetan plateau region and is classified as a subspecies of Ganoderma. Known as the famous white Ganoderma, it is a precious food and medicine that has potent biological activity, including antitumor, hypoglycemic, and immune regulation. Since available resources are limited, there are few studies on the spore of Ganoderma leucocontextum. Methods: In this work, a polysaccharide (named GLSP) was extracted from the spore of Ganoderma leucocontextum using a fast, simple, efficient, and environmentally friendly extraction process: the three-phase partitioning (TPP) method. Results: The extraction condition was optimized under the Box-Behnken design (BBD): ratio of the solute to the solvent, 1:21.126 (w/v); (NH₄)₂SO₄ concentration, 30% (w/v); ratio of the slurry to tert-butanol, 1:1.945 (v/v); and shaking temperature, 54.136 °C. Furthermore, a polysaccharide termed GLSP-A1 was purified from GLSP by column chromatography. The basic physicochemical properties were analyzed by molecular weight, Fourier transform infrared spectroscopy, monosaccharide composition, and scanning electron microscopy. Conclusions: GLSP-A1 down-regulated the expression of the pro-inflammation cytokines interleukin-6 and interleukin-1β, indicating favorable in vitro anti-inflammatory properties. In vivo, the effect of GLSP-A1 on aging was examined using the Caenorhabditis model. The results showed that GLSP-A1 reduced reactive oxygen species levels and lipofuscin accumulation. In general, these findings improve our understanding of the chemical content and bioactivity of a polysaccharide from Ganoderma leucocontextum spore and highlight the possibility of GLSP-A1 being utilized in dietary supplements for its anti-aging properties.

Keywords: Ganoderma leucocontextum spore; anti-aging; anti-inflammation; polysaccharide; three-phase partitioning.

Figure 1

Effect of GLSP-HW and GLSP-TPP on LPS-induced IL-6 relative expression in RAW264.7 macrophages compared to the blank group (^&&&& p < 0.001), the LPS group (^#### p < 0.001), and the GLSP-HW group (* p < 0.05). The above values are expressed as mean ± SD (n = 3).

Figure 2

Infrared spectroscopy of GLSP-TPP and GLSP-HW.

Figure 3

Single-factor experiment on the extraction yield of GLSP. (A) Solute-to-solvent ratio. (B) (NH₄)₂SO₄ concentration. (C) Slurry-to-tert-butanol ratio. (D) Shaking temperature. Different letters (a–d) indicate that the difference was statistically significant (p < 0.05).

Figure 4

Response surface (A–C) and contour plots (D–F) showing the effect of the solute-to-solvent ratio (A, g/mL), slurry-to-tert-butanol ratio (B, mL/mL), and shaking temperature (C, °C) on the extraction yield of GLSP (Y, %).

Figure 5

(A) DEAE-52 anion exchange column chromatography elution curve. (B) Experdex 75 gel osmotic column elution curve. (C) HPGPC spectrum and peak Mw of GLSP-A1. (D) HPLC chromatograms of standard monosaccharides and GLSP-A1: 1, Fuc; 2, GalN; 3, Rha; 4, Ara; 5, GlcN; 6, Gal; 7, Glc; 8, Xyl; 9, Man; 10, Fru; 11, Rib; 12, GalA; 13, GulA; 14, GlcA; and 15, ManA. (E) UV spectra of GLSP-A1. (F) FT-IR of GLSP-A1. (G) Congo Red experimental analysis of GLSP-A1. (H) Thermal analysis of the GLSP-A1.3.3.8. FT-IR analysis.

Figure 6

Scanning electron micrographs of GLSP-A1 at magnification ((A): 10,000×, (B): 5000×, (C): 2000×, (D): 1000×, and (E): 500×). The lyophilized samples were coated with a thin layer of gold and photographed by a scanning electron microscope.

Figure 7

(A) Effect of GLSP-A1 on the viability of RAW264.7 macrophages. (B) Effect of GLSP-A1 on LPS-induced IL-6 relative expression in RAW264.7 macrophages. (C) Effect of GLSP-A1 on LPS-induced IL-β relative expression in RAW264.7 macrophages compared to the blank group (### p < 0.001) and the LPS group (* p < 0.05, ** p < 0.01, and *** p < 0.001). The above values are expressed as mean ± SD (n = 3).

Figure 8

(A–C) ROS fluorescence levels in the nematodes of each group. (D) Relative fluorescence intensity of ROS in nematodes compared to the blank group (** p < 0.05). The above values are expressed as mean ± SD (n = 3).

Figure 9

(A–C) Blue fluorescence produced by lipofuscin in the nematodes of each group under fluorescent irradiation. (D) Effects of 1 mg/mL GLSP-A1 and 1 mg/mL VC on lipofuscin in nematodes compared to the blank group (* p < 0.1, ** p < 0.05). The above values are expressed as mean ± SD (n = 3).