Comprehensive comparison of polysaccharides from Ganoderma lucidum and G. sinense: chemical, antitumor, immunomodulating and gut-microbiota modulatory properties

Abstract

Both Ganoderma lucidum (GL) and G. sinense (GS) are used as Lingzhi in China. Their functions are assumed to mainly derive from triterpenes and polysaccharides; however, the two species have very different triterpenes profiles, if this was the case, then the bioactivity of these two species should differ. Instead, could the polysaccharides be similar, contributing to the shared therapeutic basis? In this study, two main polysaccharide fractions from different batches of GL and GS were systematically compared by a series of chemical and biological experiments. The results showed that the polysaccharides from two species shared the same structural features in terms of mono-/oligo-saccharide profiles, molecular size, sugar linkages, and IR/NMR spectra. In addition, these polysaccharides showed similar tumor-suppressive activity in mice. Further study on RAW264.7 cells indicated that these polysaccharides exhibited similar inducing effects to macrophages, as evaluated in the phagocytosis function, NO/cytokines production, inhibition against the viability and migration of cancer cells. Mechanistic investigation revealed the identical activation via TLR-4 related MAPK/NF-κB signaling pathway and gut-microbiota modulatory effects. In summary, GL and GS polysaccharides presented similar chemical features, antitumor/immunomodulating activities and mechanism; this establishes polysaccharides as the active principles and supports the official use of both species as Lingzhi.

Figure 1

Research scheme and methodology.

Figure 2

General chemical characterization of GLW, GSW, GLA and GSA. (a) Typical HPGPC chromatograms of molecular weight distribution; (b) Typical monosaccharide compositional profiles. Monosaccharides were detected by UPLC-UV (λ = 245 nm) after 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization Mixed standards (Mix Std) contained the following saccharides: Mannose (Man); Ribose (Rib); Rhamnose (Rha); Glucuronic acid (GlcA); Galacturonic acid (GalA); Glucose (Glc); Galactose (Gal); Arabinose (Ara); Fucose (Fuc); (c) Typical TFA induced partial hydrolysates profiles containing oligosaccharides by HPTLC analysis; Samples were applied on 0.2 mm silica gel 60 HPTLC plates (Merck, Germany) with an automatic TLC sampler (CAMAG, Switzerland). Then the plate was developed with n-butanol–ethanol–water 5: 3: 2: (v/v) and colorized with 5% H₂SO₄ in ethanol solution and heated to make bands colored clearly. Then the plate was photographed and scanned by a TLC visualizer and scanner (CAMAG, Switzerland).

Figure 3

Structural related characterization of GLW, GSW, GLA and GSA. IR spectra (a) were got by KBr pellet method with the scan range from 4000 to 400 cm⁻¹ and typical spectra were shown. ¹H-NMR and ¹³C-NMR (b) spectra were recorded at 400 MHz and 100 MHz, respectively; Linkage types distribution chromatograms (c) were got by GC-MS analysis after methylation.

Figure 4

Polysaccharides inhibits carcinogenesis in 4T1-bearing BABL/C mice. 4T1 cells (2 × 10⁴) were implanted into mice mammary fat pad tissue (n = 8 in each group). After 6 days intragastrical administration in advance with the polysaccharides 200 mg/kg, mice were treated for another 18 days. Cis-Di-chiorodiamineplatinum (II) (CDDP) as positive control was intraperitoneally injected with 4.0 mg/kg CDDP every four days after 4T1 cells implantation. Control: 4T1 tumor bearing mice with water gavage; GLW, GSW, GLA and GSA: polysaccharide of GLW-2, GSW-2, GLA-2 or GSA-2 gavage treatment group, respectively. (a) Experiment process; (b) body weight; (c) tumor images captured by the end of experiment. The black frame label was denoted as the dead subject before the end of experiment; (d) tumor weight; (e) tumor weight/body weight. Data are shown as mean ± SD. Significant difference *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group or GSA vs GLA, GSW vs GLW.

Figure 5

Effect of the polysaccharides from two species on cell viability by MTT assay and suppressing migration by scratch wound healing assay in 4T1 cell. (a) 4T1 cells were treated with 400 μg/mL GLW-2, GSW-2, GLA-2 and GSA-2 or 500 ng/mL LPS for 24 h; (b) 4T1 cells were exposed to supernatants of RAW264.7 cells after treatment with above sample with 10 μg/ml polymyxin B (PolyB) or 500 ng/mL LPS treatment for 24 h; (c) scratched 4T1 cells were treated with above supernatants of RAW264.7 cells incubated in DEME medium with 2% FBS. Images were photographed, and scratch distance was measured at 0, 6, 12, 24 h. All results are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 compared with the control group or GSA vs GLA, GSW vs GLW.

Figure 6

Effect of the polysaccharides from two species on activation on murine macrophage RAW264.7 cells. (a) Phagocytic activity. Seeded RAW264.7 cells were exposed to 400 μg/mL GLA-2, GSA-2, GLW-2 and GSW-2 with 10 μg/mL polymyxin B (PolyB) for 24 h, respectively. 0.1% neutral red was added as phagocytic substance. The phagocytosis of RAW 264.7 cells was observed under a microscope and the neutral red uptake measurement was at 570 nm; The production of NO (b), IL-6 (c) and TNF-α (d). Seeded RAW264.7 cells were treated with 400 μg/mL GLA-2, GSA-2, GLW-2 and GSW-2 with 10 μg/mL polymyxin B (PolyB), respectively and positive control LPS (500 ng/mL) with the presence or absence of PolyB for 24 h; (e) phosphorylation of ERK, JNK, and p38 MAPK and p65 in RAW264.7 macrophages cells after incubation with above sample for 30 min. All Western blots presented in were cropped to improve clarity. Full-length blots are displayed in Supplementary Figure 5; Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 compared with the control group or GSA vs GLA, GSW vs GLW.

Figure 7

Taxonomic classification of gut microbiota in the six group mice by the relative abundances of phylum (a) and (b), genus (c) and (d) (n = 5); Normal: mice without tumor or any polysaccharide treatment; Control: 4T1 tumor bearing mice; GLW, GSW, GLA and GSA: polysaccharide of GLW-2, GSW-2, GLA-2 or GSA-2 treatment group, respectively. Data shown are the mean ± SD. Significant difference *p < 0.05, **p < 0.01, ***p < 0.001 compared with control group or GSA vs GLA, GSW vs GLW.