Novel and Stable Dual-Color IL-6 and IL-10 Reporters Derived from RAW 264.7 for Anti-Inflammation Screening of Natural Products.

Our previous study suggested that the interleukin (IL)-6 and IL-10 could serve as good biomarkers for chronic inflammatory disease. We previously established an IL-6 and IL-10 reporters assay that could examine reporter activity along with the reference gene in LPS-induced RAW 264.7 cells. In this study, we described new and stable RAW 264.7 derived dual-color IL-6/gapdh and IL-10/gapdh reporters. This assay allowed us to easily determine relative IL-6 and IL-10 levels with 96-well plate within one step. We evaluated the relative IL-6 and IL-10 levels in the LPS-induced stable cells testing 52 natural products by real-time bioluminescence monitoring and time-point determination using a microplate luminometer. The relative IL-6 and IL-6/IL-10 values decreased by the crude ethanol extracts from nutmeg and by 1'S-1'-acetoxychavicol from greater galangal using real-time bioluminescence monitoring. At the same time, the relative IL-10 was induced. The relative IL-6 and IL-6/IL-10 decreased by crude ethanol extracts from nutmeg and 1'S-1'-acetoxychavicol acetate at 6 h. Only crude ethanol extract from nutmeg induced IL-10 at 6 h. We suggested that the use of these stable cells by real-time monitoring could serve as a screening assay for anti-inflammatory activity and may be used to discover new drugs against chronic inflammatory disease.

Real-time monitoring of IL-6 and IL-10 reporter expression for anti-inflammation activity in live RAW 264.7 cells.

In previous study, we suggested that the interleukin (IL)-6 and IL-10 could serve as a good biomarker for anti-inflammation that related to chronic inflammatory disease. Recently, we are finding new anti-inflammation compounds from natural products by screening of IL-6 and IL-10 levels. Although, we could measure IL-6 and IL-10 levels by several methods. However, all methods could not measure continuous kinetic of IL-6 and IL-10 levels. Most methods have multiple steps and take a long time. Therefore, there is no a suitable method for screening. To this end, we established IL-6 and IL-10 promoter assay which can monitor with reference gene as Glyceraldehyde 3-phosphate dehydrogenase (gapdh) promoter in living single cell. It could determine IL-6 and IL-10 levels continuously in real-time within two steps. We evaluated IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells with well-known anti-inflammatory compounds such as quercetin, xanthones, ß-D-glucan and dexamethasone. As the results, the expression of IL-6 and IL-10 reporters were strongly induced by LPS. The expression of IL-6 reporter was inhibited by all anti-inflammation compounds in LPS-induced RAW 264.7 cells. The expression of IL-10 reporter was inhibited by quercetin, xanthones and dexamethasone in LPS-induced RAW 264.7 cells. While, expression of IL-10 reporter was induced by ß-D-glucan. These results indicated that this assay could use for determination of IL-6 and IL-10 reporter expression in LPS-induced RAW 264.7 cells for anti-inflammation activity. Moreover, the results showed that natural compounds have an effect on the time course of IL-6 and IL-10 expressions. Therefore, real-time monitoring has a merit for natural compounds screening. We suggested that this assay could serve as a compound screening assay for anti-inflammation activity.

Cordyceps militaris (L.) Link Fruiting Body Reduces the Growth of a Non-Small Cell Lung Cancer Cell Line by Increasing Cellular Levels of p53 and p21.

Cordyceps militaris (L.) Link, an edible entomopathogenic fungus widely used in traditional Chinese medicine, has numerous potential medicinal properties including antitumor activity. The methanolic extract of C. militaris fruiting body was recently shown to have tumor cell growth inhibitory activity in several human tumor cell lines. Nonetheless, the mechanism of action involved is still not known. This work aimed at further studying the effect of the methanolic extract of C. militaris regarding its antitumor mechanism of action, using the non-small cell lung cancer cell line (NCI-H460) as a model. Results showed that treatment with the extract decreased cellular proliferation, induced cell cycle arrest at G0/G1 and increased apoptosis. In addition, the extract increased the levels of p53 and p21. Moreover, an increase in p-H2A.X and 53BP1 levels, together with an increase in the number of 53BP1 foci/cell (all indicative of DNA damage), were also observed after treatment with the extract. This work suggests that this extract affected NCI-H460 cellular viability through a mechanism involving DNA damage and p53 activation. This further supports the potential of this extract as a source of bioactive compounds, which may be used in anticancer strategies.

A detailed comparative study between chemical and bioactive properties of Ganoderma lucidum from different origins.

A detailed comparative study on chemical and bioactive properties of wild and cultivated Ganoderma lucidum from Serbia (GS) and China (GCN) was performed. This species was chosen because of its worldwide use as medicinal mushroom. Higher amounts of sugars were found in GS, while higher amounts of organic acids were recorded in GCN. Unsaturated fatty acids predominated over saturated fatty acids. GCN revealed higher antioxidant activity, while GS exhibited inhibitory potential against human breast and cervical carcinoma cell lines. No cytotoxicity in non-tumour liver primary cell culture was observed for the different samples. Both samples possessed antibacterial and antifungal activities, in some cases even better than the standard antimicrobial drugs. This is the first study reporting a comparison of chemical compounds and bioactivity of G. lucidum samples from different origins.

Effects of polysaccharide from fruiting bodies of Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus on alcoholic liver injury.

In the present study, the curative effects of crude polysaccharides (PSs) from mushrooms on the symptoms of alcoholic liver injury were investigated. PSs from Agaricus bisporus, Agaricus brasiliensis, and Phellinus linteus fruiting bodies were administered by gavage at levels of 100 mg per kg body weight per day for 7 d after the onset of the disease. The caspase-3 activity, mitochondrial membrane potential, mitochondrial outer membrane integrity of the liver tissues of sacrificed rats, and the serum alanine aminotransferase (ALT) levels were determined. In addition, light and transmission electron microscope (TEM) studies were performed for histopathological and cytological evaluations on liver sections. PSs from A. brasiliensis decreased ALT level and mitochondrial membrane potential and increased the outer membrane integrity; microscopic examinations also revealed normal hepatocytes and tissue. On the basis of our data, it can be argued that crude PSs from Agaricus brasiliensis have therapeutic potential for alcoholic liver injury.

Agaricus blazei hot water extract shows anti quorum sensing activity in the nosocomial human pathogen Pseudomonas aeruginosa.

The edible mushroom Agaricus blazei Murill is known to induce protective immunomodulatory action against a variety of infectious diseases. In the present study we report potential anti-quorum sensing properties of A. blazei hot water extract. Quorum sensing (QS) plays an important role in virulence, biofilm formation and survival of many pathogenic bacteria, including the Gram negative Pseudomonas aeruginosa, and is considered as a novel and promising target for anti-infectious agents. In this study, the effect of the sub-MICs of Agaricus blazei water extract on QS regulated virulence factors and biofilm formation was evaluated against P. aeruginosa PAO1. Sub-MIC concentrations of the extract which did not kill P. aeruginosa nor inhibited its growth, demonstrated a statistically significant reduction of virulence factors of P. aeruginosa, such as pyocyanin production, twitching and swimming motility. The biofilm forming capability of P. aeruginosa was also reduced in a concentration-dependent manner at sub-MIC values. Water extract of A. blazei is a promising source of antiquorum sensing and antibacterial compounds.

Consumption of Coprinus comatus polysaccharide extract causes recovery of alcoholic liver damage in rats.

CONTEXT: Excess use of alcohol is known to be associated with liver diseases such as fatty liver, alcoholic hepatitis, and cirrhosis. Various practices may be applied to prevent or treat the damage caused by chronic alcoholism. Coprinus comatus (O.F. Müll.) Pers. (Agaricaceae) is a macrofungus that has been reported to aid the recovery of murine livers damaged by benzopyrene. OBJECTIVE: In this study, the possible therapeutic effects of three different doses (50, 100, and 150 mg/kg) of C. comatus polysaccharide (PS) extract were studied in rats subjected to an alcoholic diet. The histological and biochemical results were compared between the control and experimental groups. MATERIALS AND METHODS: Modified Lieber-Decarli's calorie-adjusted liquid alcohol diet was given orally for 60 d. In addition to histopathology, alanine transaminase (ALT), aspartate transaminase (AST), mitochondrial membrane integrity, total cytochrome-c oxidase activity (TotalStCox), total mitochondrial cytochrome-c oxidase activity (TotalMtStCox), and caspase-3 values were used as liver parameters, and liver sections from all experimental groups were examined by electron microscopy. RESULTS: Using histopathological assessment, it was observed that there was a decline in liver hepatocyte vacuolization in the treatment group fed 50 mg PS/kg. The TotalStCox and TotalMtStCox values of this group differed from the EtOH control group (p < 0.05). DISCUSSION AND CONCLUSION: Daily administration of 50 mg/kg of C. comatus PS extract considerably reduced the negative effects of alcohol on liver structure and function.

The edible mushroom Laetiporus sulphureus as potential source of natural antioxidants.

Hot water extract (LN), partially purified polysaccharides (LP) and hot alkali extracted polysaccharides (LNa) obtained from fruiting bodies of the wild basidiomycete Laetiporus sulphureus were examined for their antioxidant activities. LNa was the most active antioxidant, as shown by the median effective concentrations (EC50 values) of 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity (0.5 ± 0.2 mg/ml), reducing power (4.0 ± 0.3 mg/ml) and ferrous ion-chelating ability (1.5 ± 0.1 mg/ml). LNa contained the highest level of α-glucan (17.3 ± 1.2 g/100 g dw), whereas LP contained mostly ß-glucans (66.8 ± 1.3 g/100 g dw). The prevalent monosaccharide in all extracts was glucose. The EC50 values of all three antioxidant activity assays were well-correlated with the α-glucan content. Strong and significant correlation was found between total phenolic compounds and DPPH scavenging ability and also reducing power. The three investigated extracts (at concentrations of 0.1-10 mg/ml) were not toxic to HTR-8/SVneo trophoblast cell line.

Flow cytometric evaluation of the effects of 3-bromopyruvate (3BP) and dichloracetate (DCA) on THP-1 cells: a multiparameter analysis.

Two human leukemia cells K562 and THP-1, the breast cancer lines MCF-7 and ZR-75-1, and the melanoma line MDA-MB-435S were compared by flowcytometry for their behaviour at increasing levels of 3BP. K562 and THP-1 responded to 3BP by membrane depolarization and increased ROS; MCF-7 and ZR-75-1 showed decreased polarization and low ROS increase; MDA-MB-435S had limited depolarization and no ROS increase. THP-1 cells exposed to a range of 3BP concentrations in combination with DCA showed increase of polarization, slight ROS increase, and weakened nuclear integrity. 3BP and DCA show no synergism, but have complementary destructive effects on THP-1 cells. The data led to the conclusion that the THP-1 cells do not carry a functional membrane monocarboxylate transporter (MCT) or that 3BP circumvents MCT binding and can enter these cells independently.

Polysaccharides from Agaricus bisporus and Agaricus brasiliensis show similarities in their structures and their immunomodulatory effects on human monocytic THP-1 cells.

BACKGROUND: Mushroom polysaccharides have traditionally been used for the prevention and treatment of a multitude of disorders like infectious illnesses, cancers and various autoimmune diseases. Crude mushroom extracts have been tested without detailed chemical analyses of its polysaccharide content. For the present study we decided to chemically determine the carbohydrate composition of semi-purified extracts from 2 closely related and well known basidiomycete species, i.e. Agaricus bisporus and A. brasiliensis and to study their effects on the innate immune system, in particular on the in vitro induction of pro-inflammatory cytokines, using THP-1 cells. METHODS: Mushroom polysaccharide extracts were prepared by hot water extraction and precipitation with ethanol. Their composition was analyzed by GC-MS and NMR spectroscopy. PMA activated THP-1 cells were treated with the extracts under different conditions and the production of pro-inflammatory cytokines was evaluated by qPCR. RESULTS: Semi-purified polysaccharide extracts of A. bisporus and A. brasiliensis (= blazei) were found to contain (1→6),(1→4)-linked α-glucan, (1→6)-linked ß-glucan, and mannogalactan. Their proportions were determined by integration of 1H-NMR signs, and were considerably different for the two species. A. brasiliensis showed a higher content of ß-glucan, while A. bisporus presented mannogalactan as its main polysaccharide. The extracts induced a comparable increase of transcription of the pro-inflammatory cytokine genes IL-1ß and TNF-α as well as of COX-2 in PMA differentiated THP-1 cells. Pro-inflammatory effects of bacterial LPS in this assay could be reduced significantly by the simultaneous addition of A. brasiliensis extract. CONCLUSIONS: The polysaccharide preparations from the closely related species A. bisporus and A. brasiliensis show major differences in composition: A. bisporus shows high mannogalactan content whereas A. brasiliensis has mostly ß-glucan. Semi-purified polysaccharide extracts from both Agaricus species stimulated the production of pro-inflammatory cytokines and enzymes, while the polysaccharide extract of A. brasiliensis reduced synthesis of these cytokines induced by LPS, suggesting programmable immunomodulation.

The effect of royal sun agaricus, Agaricus brasiliensis S. Wasser et al., extract on methyl methanesulfonate caused genotoxicity in Drosophila melanogaster.

The effect of culinary-medicinal Royal Sun Agaricus (Agaricus brasiliensis) hot water extract on methyl methane sulfonate (MMS) induced mutagenicity/genotoxity in Drosophila melanogaster was studied using a quick and broadly applicable in vivo assay, i.e., the wing somatic mutation and recombination test. We used 2nd instar larvae, trans-heterozygous for the third chromosome recessive markers, i.e., multiple wing hairs (mvh) and flare-3 [flr (3)], and fed them for 24 h with the aqueous extract of A. brasiliensis. For antigenotoxicity studies a 24-h pretreatment with the extract was done, followed by a 48-h treatment of the then 3rd instar larvae with MMS. The frequency of mutations of the wing blade changes (i.e., of the number of wing spots of different sizes) induced in somatic cells was determined as a parameter of genetic changes of the wing imaginal discs. The results showed that A. brasiliensis extract did not cause any genotoxic or mutagenic effects. No antigenotoxic and/or protective effect against the induction of mutations by MMS was observed. Instead, a possible enhanced mitotic recombination frequency by MMS was seen after pretreatment of the larvae with A. brasiliensis extract. Possible mechanisms of action are discussed.

High molecular weight glucan of the culinary medicinal mushroom Agaricus bisporus is an alpha-glucan that forms complexes with low molecular weight galactan.

An alpha-glucan was isolated from the culinary medicinal mushroom A. bisporus by hot water extraction, ethanol precipitation and DEAE-cellulose chromatography. The resulting material showed a single HMW peak excluded from a Sephadex G50 column that could completely be degraded by alpha-amylase treatment. After heating in 1% SDS a small additional peak of low MW eluted from the G50 column. The monosaccharide composition of the main peak was evaluated by HPLC, and was found to consist of a majority of glucose (97.6%), and a minor proportion of galactose (2.4%). Methylation analysis and degradation by alpha-amylase indicated the presence of an alpha-glucan with a main chain consisting of (1(R)4)-linked units, substituted at O-6 by alpha-D-glucopyranose single-units in the relation 1:8. Mono- (13C-, 1H-NMR) and bidimensional [1H (obs.),13C-HSQC] spectroscopy analysis confirmed the alpha-configuration of the Glcp residues by low frequency resonances of C-1 at delta 100.6, 100.2, and 98.8 ppm and H-1 high field ones at delta 5.06, 5.11, and 4.74 ppm. The DEPT-13C-NMR allowed assigning the non-substituted and O-substituted -CH(2) signals at delta 60.3/60.8 and 66.2 ppm, respectively. Other assignments were attributed to C-2, C-3, C-4, C-5 and C-6 of the non-reducing ends at delta 71.8; 72.8; 70.0; 71.3 and 60.3/60.8 ppm, respectively. The minor proportion of galactose that was demonstrated was probably derived from a complex between the alpha-glucan and a low molecular weight galactan.

Antibacterial effects of the essential oils of commonly consumed medicinal herbs using an in vitro model.

The chemical composition and antibacterial activity of essential oils from 10 commonly consumed herbs: Citrus aurantium, C. limon, Lavandula angustifolia, Matricaria chamomilla, Mentha piperita, M. spicata, Ocimum basilicum, Origanum vulgare, Thymus vulgaris and Salvia officinalis have been determined. The antibacterial activity of these oils and their main components; i.e. camphor, carvacrol, 1,8-cineole, linalool, linalyl acetate, limonene, menthol, a-pinene, b-pinene, and thymol were assayed against the human pathogenic bacteria Bacillus subtilis, Enterobacter cloacae, Escherichia coli O157:H7, Micrococcus flavus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella enteritidis, S. epidermidis, S. typhimurium, and Staphylococcus aureus. The highest and broadest activity was shown by O. vulgare oil. Carvacrol had the highest antibacterial activity among the tested components.

Chemical composition of essential oils of Thymus and Mentha species and their antifungal activities.

The potential antifungal effects of Thymus vulgaris L., Thymus tosevii L., Mentha spicata L., and Mentha piperita L. (Labiatae) essential oils and their components against 17 micromycetal food poisoning, plant, animal and human pathogens are presented. The essential oils were obtained by hydrodestillation of dried plant material. Their composition was determined by GC-MS. Identification of individual constituents was made by comparison with analytical standards, and by computer matching mass spectral data with those of the Wiley/NBS Library of Mass Spectra. MIC's and MFC's of the oils and their components were determined by dilution assays. Thymol (48.9%) and p-cymene (19.0%) were the main components of T. vulgaris, while carvacrol (12.8%), a-terpinyl acetate (12.3%), cis-myrtanol (11.2%) and thymol (10.4%) were dominant in T. tosevii. Both Thymus species showed very strong antifungal activities. In M. piperita oil menthol (37.4%), menthyl acetate (17.4%) and menthone (12.7%) were the main components, whereas those of M. spicata oil were carvone (69.5%) and menthone (21.9%). Mentha sp. showed strong antifungal activities, however lower than Thymus sp. The commercial fungicide, bifonazole, used as a control, had much lower antifungal activity than the oils and components investigated. It is concluded that essential oils of Thymus and Mentha species possess great antifungal potential and could be used as natural preservatives and fungicides.

Simple and effective purification approach to dissociate mixed water-insoluble α- and ß-D-glucans and its application on the medicinal mushroom Fomitopsis betulina.

Differences in anomericity and in the branching degree of glucans lead to characteristic intermolecular association that influences their solubility in water or other solvents. A simple purification approach, based on the glucan solubility in aq. 0.1 M NaOH solution, was applied for the separation of mixed water-insoluble α-D-glucans from ß-D-glucans extracted from fruiting bodies of Fomitopsis betulina, which is an underexploited medicinal mushroom. The results indicated that the ß-D-glucan is constituted by (1→3)-linked ß-D-Glcp units substituted at O-6 by non-reducing ß-D-Glcp and (1→6)-linked ß-D-Glcp units, while the α-D-glucan has a linear (1→3)-linked glucan structure. Thus, the 0.1 M NaOH treatment proved to be a simple, efficient and low-cost purification method for separation of water-insoluble glucans with different anomeric configurations and degree of branching that were interacting by intermolecular forces.

Chemical characterization and wound healing property of a ß-D-glucan from edible mushroom Piptoporus betulinus.

A water-soluble ß-D-glucan was obtained from fruiting bodies of Piptoporus betulinus, by hot aqueous extraction followed by freeze-thawing procedure and dialysis. Its molar mass distribution and conformational behavior in solution was assessed by size-exclusion chromatography coupled with multiangle laser light scattering, showing a polysaccharide with an average molecular weight of 2.5 × 105 Da with a random coil conformation for molecular weights below 1 × 106 Da. Typical signals of ß-(1 → 3)-linkages were observed in NMR spectrum (δ 102.7/4.76; 102.8/4.74; 102.9/4.52; and δ 85.1/3.78; 85.0/3.77) and also signals of O-6 substitution at δ 69.2/4.22 and 69.2/3.87. The analysis of partially O-methylated alditol acetates corroborates the NMR results, indicating the presence of a ß-D-glucan with a main chain (1 → 3)-linked, substituted at O-6 by single-units of glucose. The ß-D-glucan showed no toxicity on human colon carcinoma cell line (Caco-2) up to 1000 µg mL-1 and promoted cell migration on in vitro scratch assay, demonstrating a potential wound healing capacity.

The anti-inflammatory effect of Agaricus brasiliensis is partly due to its linoleic acid content.

For hundreds of years mushrooms have been used as functional food for health. The basidiomycete Agaricus brasiliensis (A. brasiliensis) is famous for the medicinal properties of its beta glucans and of its antioxidants. Most researchers have studied polysaccharides from A. brasiliensis for their anti-inflammatory activity. However, active compounds from this mushroom have not yet been studied for the inactivation of NO inhibitory activity. The present study aimed to find the active compounds from A. brasiliensis for their NO inhibitory activity related inflammatory activity. This study found that linoleic acid isolated from A. brasiliensis inhibited NO production and suppressed the expression of pro-inflammatory cytokines including TNF-α, IL-6, IL-1ß, and NOS2 in RAW 264.7 cells. Linoleic acid also suppressed the expression of NF-κB subunit p50 and restored PPARα. This leads to the conclusion that linoleic acid from A. brasiliensis could reduce NO production and inflammatory activity in RAW 264.7 cells by the inhibition of p50 and via the activation of PPARα. This study suggests that linoleic acid present in A. brasiliensis could play a role in the prevention of inflammatory diseases for which this edible mushroom is already known.

The ornithine cycle enzyme arginase from Agaricus bisporus and its role in urea accumulation in fruit bodies.

An extensive survey of higher fungi revealed that members of the family Agaricaceae, including Agaricus bisporus, accumulate substantial amounts of urea in their fruit bodies. An important role of the ornithine cycle enzymes in urea accumulation has been proposed. In this work, we present the cloning and sequencing of the arginase gene and its promoter region from A. bisporus. A PCR-probe based on fungal arginase was used to identify the A. bisporus arginase gene from a cDNA library. The arginase cDNA encodes a 311-aa protein which is most likely expressed in the cytosol. Expression of the cDNA in Escherichia coli was established as a His-tagged fusion protein. The arginase gene was used as a molecular marker to study expression and regulation during sporophore formation and postharvest development. The expression of the arginase gene was significantly up-regulated from developmental stage 3 onwards for all the tissues studied. A maximum of expression was reached at stage 6 for both stipe and cap tissue. In postharvest stages 5, 6 and 7 the level of expression observed was similar to normal growth stages 5, 6 and 7. A good correlation was found between arginase expression and urea content of stipe, velum, gills, cap and peel tissue. For all tissues the urea content decreased over the first four stages of development. From stage 4 onwards urea accumulated again except for stipe tissue where no significant changes were observed. The same trend was also observed for postharvest development, but the observed increase of urea in postharvest tissues was much higher.

Effects of Black Hoof Medicinal Mushroom, Phellinus linteus (Agaricomycetes), Polysaccharide Extract in Streptozotocin-Induced Diabetic Rats.

In this article we report the healing effects of a Phellinus linteus fruiting body hot water extract (PLE) in streptozotocin (STZ)-induced diabetic rats. PLE was given before and after STZ. The preprotective, protective, and postprotective effects of PLE on STZ-induced oxidative stress were studied using biochemical (caspase 3 activity, cytosolic-to-lysosomal ratio of cathepsin B and L, DNA fragmentation levels), ordinary histological and immuno-histochemical investigation parameters. Following oral administration of PLE after STZ application, the serum glucose concentration significantly decreased up to 41.13% compared with the control group (P < 0.05). The hypoglycemic potential of the PLE was further supported by an increase of insulin secretion in the islets of Langerhans. In addition, the number of cells in Langerhans islets increased by 45.89% when PLE was given after STZ application. On the other hand, the use of PLE before oxidative stress could not prevent the onset of diabetes. This is, to our knowledge, the first study of the effect of application time of orally administered Ph. Linteus hot water extract on STZ-induced diabetes.

Structural characterization and immunomodulatory effects of polysaccharides from Phellinus linteus and Phellinus igniarius on the IL-6/IL-10 cytokine balance of the mouse macrophage cell lines (RAW 264.7).

Phellinus linteus and igniarius (L.) Quel. have been used in traditional Asian medicine for over two centuries against a variety of diseases. Polysaccharides from their fruiting bodies show strong immunomodulatory activity. In this study we characterized the structure and composition of polysaccharides from Phellinus linteus and Phellinus igniarius by HPLC, GC-MS and NMR (1-H, 13-C, COSY, NOESY and TOCSY). The polysaccharides from P. linteus and P. igniarius mainly contained glucose with minor proportions of mannose, galactose, xylose, arabinose and rhamnose. Methylation analyses showed that the glycosidic linkages were mostly 1 → 3, 1 → 6 or 1 → 3,6. The two-dimensional COSY, NOESY and TOCSY confirmed that these polysaccharides have a main chain of →3)-ß-D-Glcp-(1→ with →6)-ß-D-Glcp-(1→ side chain. In vitro assays by RT-PCR and ELISA showed that (1 → 3; 1 → 6)-ß-D-polysaccharides from P. linteus and P. igniarius decreased TNF-α in RAW 264.7 cells, suggesting an immuno-suppressive activity. Furthermore, these polysaccharides stimulated a high IL-10 response and induced strong suppression of transcription of IL-6. The results suggest that polysaccharides from P. linteus and P. igniarius could possibly find applications in restoring the IL-6/IL-10 balance, the disturbance of which is thought to be related to chronic inflammatory disease, obesity, diabetes type 2, and to mania and depression.