Grifola Frondosa Polysaccharide Research Articles

Grifola frondosa polysaccharide's therapeutic potential in oxazolone-induced ulcerative colitis

Grifola frondosa polysaccharide (GFP) is a consumable fungus recognized for its potential health advantages. The present study aimed to investigate the development and potential etiologies of ulcerative colitis (UC) utilizing oxazolone (OXZ) as an inducer in mice, along with assessing the therapeutic effects of GFP at varying doses in UC mice, with sulfasalazine (SASP) serving as the positive control. The obtained results indicated that OXZ intervention in mice induced numerous physical manifestations of UC, including increased disease activity index (DAI), decreased goblet cell division, enhanced fibrosis, reduced expression of Claudin1 and Zona encludens protein1 (ZO-1), decreased proliferative activity of colonic mucosal epithelial cells, disturbed oxidation balance, and alterations in intestinal flora. Nonetheless, GFP intervention significantly ameliorated or even resolved these abnormal indicators to a considerable extent. Consequently, this study suggests that GFP might serve as a prebiotic to regulate intestinal flora, mitigate enterotoxin production, restore oxidative balance, thereby reducing the generation of inflammatory mediators, restoring the intestinal barrier, and ultimately improving OXZ-induced UC in mice. GFP demonstrates promising potential as a candidate drug for colitis treatment and as a dietary supplement for alleviating intestinal inflammatory issues.

The Role of Grifola frondosa Polysaccharide in Preventing Skeletal Muscle Atrophy in Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a burgeoning public health challenge worldwide. Individuals with T2DM are at increased risk for skeletal muscle atrophy, a serious complication that significantly compromises quality of life and for which effective prevention measures are currently inadequate. Emerging evidence indicates that systemic and local inflammation stemming from the compromised intestinal barrier is one of the crucial mechanisms contributing to skeletal muscle atrophy in T2DM patients. Notably, natural plant polysaccharides were found to be capable of enhancing intestinal barrier function and mitigating secondary inflammation in some diseases. Herein, we hypothesized that Grifola frondosa polysaccharide (GFP), one of the major plant polysaccharides, could prevent skeletal muscle atrophy in T2DM via regulating intestinal barrier function and inhibiting systemic and local inflammation. Using a well-established T2DM rat model, we demonstrated that GFP was able to not only prevent hyperglycemia and insulin resistance but also repair intestinal mucosal barrier damage and subsequent inflammation, thereby alleviating the skeletal muscle atrophy in the T2DM rat model. Additionally, the binding free energy analysis and molecular docking of monosaccharides constituting GFP were further expanded for related targets to uncover more potential mechanisms. These results provide a novel preventative and therapeutic strategy for T2DM patients.

Polysaccharide isolated from Grifola frondosa eliminates myeloid-derived suppressor cells and inhibits tumor growth by enhancing T cells responses.

Myeloid derived suppressor cells (MDSCs) are known to accumulate in cancer patients and tumor-bearing mice, playing a significant role in promoting tumor growth. Depleting MDSCs has emerged as a potential therapeutic strategy for cancer. Here, we demonstrated that a fungal polysaccharide, extracted from Grifola frondosa, can effectively suppress breast tumorigenesis in mice by reducing the accumulation of MDSCs. Treatment with Grifola frondosa polysaccharide (GFI) leads to a substantial decrease in MDSCs in the blood and tumor tissue, and a potent inhibition of tumor growth. GFI treatment significantly reduces the number and proportion of MDSCs in the spleen, although this effect is not observed in the bone marrow. Further analysis reveals that GFI treatment primarily targets PMN-MDSCs, sparing M-MDSCs. Our research also highlights that GFI treatment has the dual effect of restoring and activating CD8+T cells, achieved through the downregulation of TIGIT expression and the upregulation of Granzyme B. Taken together, our findings suggest that GFI treatment effectively eliminates PMN-MDSCs in the spleen, leading to a reduction in MDSC numbers in circulation and tumor tissues, ultimately enhancing the antitumor immune response of CD8+T cells and inhibiting tumor growth. This study introduces a promising therapeutic agent for breast cancer.

Structural characterization and immunomodulatory effect of a starch-like Grifola frondosa polysaccharides on cyclophosphamide-induced immunosuppression in mice

In this study, a pure Grifola frondosa polysaccharide (GFP-1) was extracted and purified from Grifola frondosa. By HPLC, GC-MS, FT-IR, and NMR analysis, GFP-1 was determined to be a starch-like polysaccharide with an average molecular weight of 3370 kDa. It included three monosaccharides, i.e., glucose, galactose, and mannose. The backbone of GFP-1 consisted of →4)-α-Glcp-(1→ and →4,6)-α-Glcp-(1 → . The side branches were composed of →6)-α-Galp-(1→, α-Glcp-(1→, and a small amount of α-Manp-(1 → . By using a cyclophosphamide (CTX)-induced immunosuppressed mice model, we evaluated the immunomodulatory activity of GFP-1. The results showed that GFP-1 increased the thymic and spleen indices, promoted the level of IgG and IgA in serum, and activated the mitogen-activated protein kinase (MAPK) pathway in CTX-induced mice. Also, GFP-1 significantly promoted the mRNA expression of intestinal barrier factors and protected intestinal structural integrity in immunosuppressed mice. In conclusion, the data presented here suggested that GFP-1 might be a potential immune-enhancing supplement.

Gastrointestinal metabolism characteristics and mechanism of a polysaccharide from Grifola frondosa

Grifola frondosa polysaccharide (GFP) is mainly composed of α-1,4 glycosidic bonds and possesses multiple pharmacological activities. However, the absence of pharmacokinetic studies has limited its further development and utilization. Herein, GFP was labeled with 5-DTAF (FGFP) and cyanine 5.5 amine (GFP-Cy5.5) to investigate its gastrointestinal metabolism characteristics and mechanism. Significant distributions of the polysaccharide in the liver and kidneys were observed by near infrared imaging. To investigate the specific distribution form of the polysaccharide, in vitro digestion models were constructed and revealed that FGFP was degraded in saliva and rat small intestine extract. The metabolites were detected in the stomach and small intestine, followed by further degradation in the distal intestine in the in vivo experiment. Subsequent investigations showed that α-amylase was involved in the gastrointestinal degradation of GFP, and its metabolite finally entered the kidneys, where it was excreted directly with urine.

A Se-enriched Grifola frondosa polysaccharide induces macrophage activation by TLR4-mediated MAPK signaling pathway

Se-polysaccharide (Se-GFP-22) from Se-enriched Grifola frondosa has double and cooperative activities of polysaccharide and Se. To delineate the underlying mechanism and signaling cascade involved in immune-stimulatory property of Se-GFP-22, the production of cellular mediators and key proteins in signaling pathway was examined. Results showed that Se-GFP-22 exhibited no cytotoxic and had a high capacity to promote macrophage phagocytosis, up-regulate interleukin-2 (IL-2), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and nitric oxide (NO) productions, as well as the relative messenger RNA (mRNA) expressions. In Se-GFP-22-induced macrophages, intracellular superoxide dismutase (SOD) activity was significantly increased to protect cells from oxidative injury. However, Se-GFP-22 induced macrophage activation was suppressed when the toll-like receptor 4 (TLR4) signaling pathway was blocked by a specific TLR4 inhibitor. According to the western blot analysis and the use of specific inhibitors against the mitogen-activated protein kinases (MAPK) signaling pathway, we speculated that Se-GFP-22 activated RAW264.7 macrophages through the TLR4-mediated MAPK signaling pathway. This study provides a molecular basis for the potential of Se-GFP-22 as a novel immune-stimulatory agent.

Preparation, chemical structure and α-glucosidase inhibitory activity of sulfated polysaccharide from Grifola frondosa

• Sulfated Grifola frondosa polysaccharide was prepared and characterized. • Sulfation and degradation could exist concurrently during the sulfation reaction. • S-GFP is a noncompetitive inhibitor of α-glucosidase. • S-GFP could significantly quench the endogenous fluorescence of α-glucosidase. Sufalted modification of Grifola frondosa polysaccharide (GFP) was performed using the chlorosulfonic acid-pyridine (CSA-Pyr) method. The chemical structure and α-glucosidase inhibitory activity of sulfated Grifola frondosa polysaccharide (S-GFP) were investigated. FT-IR and NMR spectra both revealed C-6 as the main substituted position owing to steric hindrance. Molecular weight ( Mw ) analysis indicated that sulfation and degradation could exist concurrently during the sulfation reaction given that the Mw of S-GFP was different from that of natural GFP. Inhibition test of α-glucosidase activity implied that S-GFP is a noncompetitive inhibitor of α-glucosidase. S-GFP effectively inhibited the activity of α-glucosidase with an IC 50 value of 3.353 µg mL −1 . The results of inhibitory mechanism indicated that S-GFP quenched the endogenous fluorescence of α-glucosidase and changed the backbone structure of the protein. Circular dichroism (CD) analysis showed that treatment with S-GFP changed the conformation of α-glucosidase, increased the content of the α-helix structure, and destroyed the hydrogen bond system of the enzyme. This experiment aims to elucidate the potential α-glucosidase inhibitory activity of sulfated polysaccharides and to provide new insights into the targets of their bioactivities.

Grifola frondosa Polysaccharide Ameliorates Early Diabetic Nephropathy by Suppressing the TLR4/NF-κB Pathway.

Grifola frondosa is a medicinal macro-fungus with a wide range of biological activities. Polysaccharides from Grifola frondosa (PGF) play a positive role in regulating blood glucose and alleviating kidney injury. Here, we investigated the exact mechanism of action by which PGF ameliorates diabetic nephropathy. Our results showed that PGF effectively improved glucose tolerance and insulin sensitivity in streptozocin (STZ)-induced DN mice. Additionally, administration of PGF also ameliorated renal function and inflammatory response in STZ-induced DN mice. Consistent with the in vitro results, the high glucose-induced inflammatory response and apoptosis of renal tubular epithelial cells were decreased by PGF treatment. Furthermore, PGF not only suppressed the expression of TLR4, but also more effectively protected the kidney and reduced the inflammatory response when TLR4 was inhibited. All these data revealed that PGF alleviates diabetic nephropathy by blocking the TLR4/NF-κB pathway.

Aqueous two-phase simultaneous extraction and purification of a polysaccharide from Grifola frondosa: Process optimization, structural characteristics and antioxidant activity

Traditional method for Grifola frondosa polysaccharide (CGFP) separation is water extraction or other method assistance, ethanol precipitation and further purification via column chromatography. Due to the strict control conditions, inefficient and not environmental-friendly, the method is hard to scale up and implement, and even limited the biological activity study and application of Grifola frondosa polysaccharide. Therefore, an ammonium sulfate/ethanol aqueous two phases (ATPS) was developed to extract and preliminary purify the polysaccharide from Grifola frondosa (GFPA). The optimized conditions were concluded as the ATPS composition of ammonium sulfate 17% and ethanol 27.9%, extraction pH 3.22 based on single-factor and response surface methodology experiments. Under this condition, the recovery yield and content of GFPA were 90.21% and 66.7%, respectively. The structural characteristics investigation revealed that GFPA exhibited a slight change in weight average molecular weight (Mw), molar ratio of monosaccharide and microstructure as compared to CGFP. Results indicated that the Mw of GFPA was 2.954 × 106 Da, which was lower than that of CGFP. Both of them are heteropolysaccharides with pyranose ring, and mainly composed of glucose. GFPA exhibited a rod shape and reticular structure with smooth surface, while CGFP showed a rough surface. Also, Congo red test indicating the presence of triple-helix structure in both of them, while AFM observation suggesting a spherical structure tended to aggregate occurred. The chemical antioxidant activities of GFPA were increased in a dose-dependent manner. Moreover, GFPA can effectively prevent RAW264.7 cells from H2O2-induced damage through reducing the release of MDA, LDH and ROS, and enhancing the levels of SOD, CAT and GSH-Px. Furthermore, GFPA showed more significant and effective than CGFP at all the tested dosages. Thus, it can be seen that the ATPS method established in this paper possess the merits of efficient, environmental-friendly and simple operation, which can successfully be applied for Grifola frondosa polysaccharide large scale separation, and make the application of GFPA possible. It has reference value for other large-scale polysaccharide separation. The resulting also revealed that GFPA has the potential value of developing as a natural antioxidant agent.

Immunostimulatory and antioxidant activities of the selenized polysaccharide from edible Grifola frondosa.

Grifola frondosa polysaccharide (GFP2) was extracted and purified by anion‐exchange chromatography. A selenized G. frondosa polysaccharide, SeGFP2, was modified in selenylation by nitric acid–sodium selenite (HNO3‐Na2SeO3) method. Structural features were investigated, and the lymphocyte proliferation and antioxidant activities were compared taking GFP2 as control. SeGFP2 with a molecular weight of 2.12 × 104 Da was composed of mannose, glucose, and galactose with a ratio of 3.5:11.8:1.0. A typical absorption of selenium ester was observed in SeGFP2 molecule. SeGFP2 was proposed as a branched polysaccharide, which consisted of 1,3‐D‐Glcp, 1,6‐D‐Glcp, 1,4,6‐D‐Galp, and 1,3,6‐D‐Manp. SeGFP2 showed a linear filamentous structure with some branches. SeGFP2 could significantly promote T‐ or B‐lymphocyte proliferation and the enhancement was higher than GFP2. The in vitro antioxidant activities of SeGFP2 were more potent than GFP2. These present data suggested that selenylation could significantly improve the lymphocyte proliferation and in vitro antioxidant activities of GFP2.

Anti-tumor and immunomodulatory effects of Grifola frondosa polysaccharide combined with vitamin C on Heps-bearing mice: Based on inducing apoptosis and autophagy

• Grifola frondosa polysaccharide combined with vitamin C has the same obvious anti-tumor effect in mouse liver cancer models as in in vitro experiments, and the tumor inhibition rate exceeds 50%. The mechanism of action is to induce tumor apoptosis and autophagy at the same time. • The combined application of GFP/VC can also enhance the cellular immune function of mice by increasing the percentage of CD4 + CD8 + T cells,B cells and Treg cells. • The combined application of GFP/VC can also enhance the humoral immune function of mice by increasing the secretion of various cytokines (IL-2,IL-12p70,TNF-α and IFN-γ). The aim of this study was to detect the anti-tumor and immunomodulatory effects of Grifola frondosa polysaccharide (GFP) combined with Vitamin C (VC) on Heps-bearing Mice. The tumor inhibition rate of GFP/VC intragastric administration or intraperitoneal injection were 57.92% and 69.58% respectively. Immunohistochemical and Western Blot results showed the expression of apoptosis-related and autophagy-related proteins (PARP, Caspase3, Beclin-1 and LC-3) all increased. FCM results indicated that the percentages of CD4 + T, CD8 + T, B lymphocytes, and Treg cells in the GFP/VC treatment group all increased. And the secretion of GM-CSF, IL-4,IL-10 and MIP-3a/CCL20 in peripheral blood significantly reduced. On the contrary, the secretion of IFN-γ, TNF-α, IL-2 and IL-12p70 increased after GFP/VC treatment. And in the above indicators, the differences between the experimental group and the negative control group were statistically significant ( P all < 0.05). So GFP combined with VC have obvious anti-tumor and immunomodulatory effect in mouse liver cancer models.

Health benefits of Grifola frondosa polysaccharide on intestinal microbiota in type 2 diabetic mice

Grifola frondosa polysaccharide (GFP) as the natural compounds have been reported to exert diverse bioactivities. The aim of this study was to investigate the regulatory effects of GFP on intestinal microbiota in type 2 diabetic mice. The changes of microbiota in faeces of the diabetic mice upon high fat diet were determined and the V3 region of the 16S rRNA was sequenced by Illumina MiSeq high-throughput sequencing platform. Eighty operational taxonomic unit were identified to be shared by all samples, and the quality and richness of sequencing were assessed via rarefaction and rank abundance curves. The diversity of gut flora was slightly improved in diabetic mice after GFP treatment. The composition and relative abundance of gut microbiota at phylum and genus levels were altered by GFP. The abundance of Robinsoniella, Flavonifractor, Anaerotruncus, and Desulfvibrio were found to concentrate on diabetic mice through LEfSe analysis. Furthermore, Alloprevotella and Burkholderia had strong relevancy with other gut flora. Based on the findings, GFP might be a desired candidate on ameliorating the intestinal unbalance in diabetes.

Optimization of Extraction Technology, Structure, and Antioxidant Activity of Polysaccharide from Grifola frondosa

AbstractUsing DMSO as solvent, ultrasonic‐assisted extraction of polysaccharides from Grifola frondosa is studied. The effects of different extraction conditions on the yield of polysaccharides are investigated. The optimal extraction conditions are found by a Box‐Behnken design. The results showed that the yield of Grifola frondosa polysaccharide can reach 6.94% under the following conditions: liquid–solid ratio of 33.8 mL g‐1, ultrasonic power of 104.6 W, extraction temperature of 35.6°C, and extraction time of 4.7 h. The structure of the polysaccharides is investigated by infrared spectroscopy, nuclear magnetic resonance, molecular weight, monosaccharide composition, and methylation. The purified polysaccharides are found to have good antioxidant activity in vitro.

Meta-analysis on effect of Grifola frondosa polysaccharide in regulating in vivo immunoregulatory function on animal disease models

The study aimed to explore the in vivo immunoregulatory function of Grifola frondosa polysaccharide( GFP) on animal disease models. Databases of PubMed,Embase,Web of Scinece,CNKI,CBM and Wan Fang Data were searched from the date of their establishment to February 2018. Two reviewers independently screened included studies and evaluated their quality by using SYRCLE&apos;s risk of bias tool. R software was used to analyze the data. Finally,20 animal experiment studies were included. According to Metaanalysis. For cellular immunity,GFP could effectively enhance the proliferation of effect or T cells,natural killer cells and macrophages in mice. The percentage of CD4+T cells( MD = 1. 89,95% CI [0. 94,2. 83],P &lt; 0. 000 1),CD8+T cells( MD = 8. 46,95% CI[5. 93,11. 00],P&lt;0. 000 1),NK cells( MD= 2. 67,95% CI [0. 23,5. 11],P= 0. 03),and macrophages( MD= 14. 09,95% CI[0. 84,27. 34],P= 0. 04) were all higher than those in control group. For humoral immunity,GFP could increase the secretion of TNF-α and INF-γ． The secretion of TNF-α( SMD = 15. 92,95% CI [9. 07,22. 76],P&lt;0. 000 1) and INF-γ( SMD = 5. 34,95% CI[3. 42,7. 26],P&lt;0. 000 1) were all higher than those in control group. In conclusion,GFP could regulate immunologic function by enhancing the proliferation activity of immune cells( CD4+T cells,CD8+T cells,NK cells and macrophages) and the secretion of immune factors( TNF-α and INF-γ) ． However,it is necessary to further standardize the selection of specific surface markers of immune cells and the administration of GFP,in order to reduce the heterogeneity among the studies. At the same time,more attention shall be paid to experimental design,implementation and full report,especially to the establishment and implementation of animal experimental registration system,so as to improve the transparency and quality of the whole process of animal experimental research,enhance the value of basic research ultimately,and provide a reliable theoretical basis for the transformation of basic research into clinical research.

Hypoglycemic activity and gut microbiota regulation of a novel polysaccharide from Grifola frondosa in type 2 diabetic mice

GFP-N, a novel heteropolysaccharide with a molecular weight of 1.26 × 107 Da, was isolated from maitake mushroom and purified by anion-exchange chromatography on a DEAE cellulose-52 column and gel-filtration chromatography on a Sephadex G-100 column. Its structure was characterized by Fourier transform infrared spectroscopy and one-dimensional (1H- and 13C-) NMR spectra, 1H1H correlation spectroscopy, and 1H13C heteronuclear single-quantum coherence spectroscopy. The structure of GFP-N consisted of L-arabinose, D-mannose and D-glucose and mainly contained three kinds of linkage type units as →2,6)-α-D-Manp-(1 → 4, α-L-Araf-C1→, and →3,6)-β-D-Glcp-(1 → . GFP-N could activate insulin receptor substrate 1, phosphatidylinositol-3-kinase, and glucose transporter 4 and inhibit c-Jun N-terminal kinase 1/2 for hypoglycemic effects in diabetic mouse livers. This is also the first report of the regulatory efficacy of Grifola frondosa polysaccharide on intestinal microflora in vivo using single-molecule real-time sequencing. These results indicated that polysaccharide from maitake mushroom could be as an enhancer to improve type 2 diabetes and a healthy food option to help regulate gut microbiota in diabetic individuals.

The biological activities of the antitumor drug Grifola frondosa polysaccharide.

Grifola frondosa, a polypore fungus that grows at the base of trees, is an edible and medicinal mushroom with a large fruiting body characterized by overlapping caps. Japanese scholars found that Grifola frondosa polysaccharide or D-fraction is the major biologically active ingredient, which is a protein-bound glucan or proteoglucan, consisting of β-glucan (either β-1,6-linked glucan with β-1,3 branches or β-1,3-linked glucan branched with β-1,6 glucosides, but it also contains d-xylose, d-fucose, d-mannose, l-arabinose, uronic acid, and galactose with largely unknown linkages. The Grifola frondosa polysaccharides with different molecular weight and monosaccharide compositions can be obtained by using different extraction methods, such as hot water extraction, acid or alkaline extraction, or microwave extraction methods from the fruiting bodies, mycelia or the fermentation broth. Grifola frondosa polysaccharide-based drug was developed in China and approved as an adjunctive therapeutic drug for cancer treatment by the State Food and Drug Administration (SFDA) in 2010. There are eight fungal glycan-based drugs, some of them have to be injected in order to be effective in vivo but the Grifola frondosa polysaccharide-based drug can be taken either orally or by injection. In this article, based on the search results of Chinese VIP, CNKI, Wanfang database and PubMed database, 108 independent studies were summarized. The chemical structure, the antitumor, immunomodulatory, anti-diabetic, anti-hyperlipidemia, and antiviral activity and molecular mechanisms of GFP are reviewed and discussed. Our goal is to provide a molecular picture that would allow in-depth evaluation of Grifola frondosa polysaccharide as an adjunctive drug for cancer therapy.

Effect of Grifola frondosa polysaccharide on anti-tumor activity in combination with 5-Fu in Heps-bearing mice

GP11 had been reported to have effectively anti-tumor activity by improving the immune function in our previous study. To avoid drawbacks of the 5-Fu, GP11 in combination with 5-Fu was investigated in this study. The results demonstrated that such synergism displayed enhance the anti-tumor activity of 5-Fu. Additionally, a strength effect was also observed in regulating immune function of GP11 and 5-Fu simultaneous administration, such as enhancing serum interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF-α) secretion, and increasing immune organs weights. Moreover, GP11 could improve the haematological and biochemical parameters deterioration, superoxide dismutase (SOD) activities reduction and malondialdehyde (MDA) levels enhancement in non-immune organs induced by 5-Fu. All these results illustrated that GP11 exhibited attenuated and synergized effect on 5-Fu by improving the immune function. It could be developed as an auxiliary preparation for chemotherapeutic drugs.

A novel iron supplements preparation from Grifola frondosa polysaccharide and assessment of antioxidant, lymphocyte proliferation and complement fixing activities

Grifola frondosa polysaccharide-iron (III) complex (GFP-iron) was synthesized and characterized. Based on single factor and orthogonal optimization experiments of GFP-iron (III) complex synthesis, the optimum conditions were the reaction temperature 80°C, pH 8, reaction time 90min and ratio of catalyst to GFP 1:1.0gg−1. The iron content of GFP-iron (III) complex reached 24.15% under the optimums and characterized by fourier transform infrared (FTIR), scanning electron microscopy (SEM), X-Ray Diffraction (XRD), thermogravimetry (TG) and iron release in vitro assay.By 5-axe cobweb charts, the antioxidant activity of GFP-iron (III) complex was comprehensively evaluated. The results showed GFP-iron (III) complex retained a certain antioxidant activity. The lymphocytes proliferation of GFP-iron (III) complex was increased by 61.68% comparing with that of GFP at the sample concentration of 62.5μg/mL. At giving 50% haemolysis, the concentration of GFP-iron (III) complex was 0.261mg/mL. Therefore, GFP-iron (III) complex would be expected to exploit into a new-type comprehensive iron supplements.

Structure and immunomodulatory activity of extracellular polysaccharide from Grifola frondosa

We aimed at analyzing the structure of extracellular polysaccharide A from Grifola frondosa (EXGFP-A) and testing its immunomodulatory activity. Structural analysis shows that EXGFP-A was a contained α-D-glucoside bond and pyranose ring. GC analysis reveals that EXGFP-A was mainly composed of rhamnose, arabinose, xylose, mannose, glucose, galactose, by the molar ratio of 0.28:0.31:0.30:0.06:7.98:0.61. The results of MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay indicates when EXGFP-A was at a concentration of 80 μg/mL and treatment time of 48 h, RAW264.7 cells proliferation index reached a maximum of 137.5%. Meanwhile, the AO staining showed that EXGFP-A activated RAW264.7 cells and improved the level of intracellular nucleic acid metabolism. In addition, in a certain range of concentration, EXGFP-A was able to increase the release of NO in RAW264.7 cells, and upregulate the mRNA expression of immunological factor TNF-α, IL-1β, IL-6, IL-12, IFN-γ and iNOS of RAW264.7 cells. Our results confirm that EXGFP-A had immunomodulatory activity. Our findings provided scientific basis for the structural analysis and application of Grifola frondosa polysaccharide.

Structural characterization and immunomodulatory activity of Grifola frondosa polysaccharide via toll-like receptor 4-mitogen-activated protein kinases-nuclear factor κB pathways.

We isolated a neutral polysaccharide from the fruiting body of a mushroom Grifola frondosa (GFP-A). The aim of this study was to characterize a neutral α-d-polysaccharide derived from G. frondosa and evaluate its immunomodulatory effect on toll-like receptor 4, mitogen-activated protein kinases and nuclear factor κB pathways of protein expression in macrophages. The structural features of GFP-A were characterized by physicochemical and instrumental analyses. Its molecular weight was found to be 8.48 × 10(2) kDa. The main chain of GFP-A consisted of (1 → 4)-linked and (1 → 6)-linked α-d-glucopyranosyl, and (1 → 3,6)-linked α-d-mannopyranosyl residues, which branched at C-3. The branches consisted of (1 → 6)-linked α-d-galactopyranosyl and t-l-rhamnopyranosyl residues. An in vitro immunomodulatory assay for pro-inflammatory cytokines (interleukin-1β, interleukin-2, tumor necrosis factor alpha, etc.) using the macrophage cell line, RAW 264.7, revealed that GFP-A exhibited significant immunomodulatory activity by stimulating the toll-like receptor 4, mitogen-activated protein kinases to nuclear factor κB/pathway.