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Mushroom varieties – effects on health

Scientific Investigation from Mushrooms and Health 2008

Background Mushrooms and Health 2008 covered Agaricus bisporus and 17 culinary specialty and nutraceutical specialty mushrooms; however some additional culinary and nutraceutical mushrooms have also been included where significant information was available on consumption and health. The scientific literature is categorised both by health condition and subsequently, the information is also grouped by mushroom variety for readers interested in specific mushroom varieties.

Agaricus bisporus (common white button, brown/crimini, portabella)

It has been demonstrated that dietary supplementation with white button mushrooms (Agaricus bisporus) enhances natural killer (NK) cell activity in C57BL/6 mice, suggesting that increased intake of white button mushrooms may promote innate immunity against tumours and viruses through the enhancement of NK activity (Wu et al. 2007).

Extracts from Agaricus bisporus mushrooms have been suggested as potential breast cancer chemopreventive agents, as they suppress aromatase activity and estrogen biosynthesis. A

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recent study has evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo. Mushroom extract decreased testosterone-induced cell proliferation in MCF-7aro cells but had no effect on MCF-10A, a non-tumourigenic cell line. Most potent mushroom chemicals are soluble in ethyl acetate. The major active compounds found in the ethyl acetate fraction were unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibited aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicated that they are non-competitive inhibitors with respect to androstenedione. As only conjugated linoleic acid was found to inhibit the testosterone-dependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that the mushroom extract decreased both tumour cell proliferation and tumour weight with no effect on the rate of apoptosis (Chen et al. 2006).

The edible mushroom lectin from Agaricus bisporus has been reported to have anti-proliferative effects on a range of cell types. A study has been undertaken to determine whether it might have inhibitory activity on Tenon's capsule fibroblasts in in vitro models of wound healing and therefore have a use in the modification of scar formation after glaucoma surgery. Human ocular fibroblasts in monolayers and in three-dimensional collagen lattices were exposed to Agaricus bisporus (0-100 mg/ml). Agaricus bisporus caused a dose-dependent inhibition of proliferation and lattice contraction without significant toxicity. The data showed that Agaricus bisporus possesses key features required of an agent that might control scarring processes and suggest that Agaricus bisporus may be especially useful where subtle modification of healing may be needed, although further studies are required (Batterbury et al. 2002).

The effect on epithelial cells of a Gal beta-1,3-GalNAc-binding lectin, from the edible mushroom Agaricus bisporus lectin (ABL) has been evaluated. ABL (25mg/ml) inhibited incorporation of [3H]-thymidine into DNA of HT29 colon cancer cells by 87%, Caco-2 colon cancer cells by 16%, MCF-7 breast cancer cells by 50%, and Rama-27 rat mammary fibroblasts by 55% when these cells were grown for 24h in serum-free medium. When

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assessed by cell count, similar inhibition of proliferation of HT29 cells by ABL was found. ABL (0.2mg/ml) caused no cytotoxicity to HT29, MCF-7, and Rama-27 cells as measured by trypan blue exclusion, and inhibition of proliferation in HT29 cells caused by 50mg/ml ABL was reversible after removal of the lectin. A. bisporus lectin appears to be a reversible non-cytotoxic inhibitor of epithelial cell proliferation which deserves further study as a potential anti-cancer agent (Yu et al. 1993).

The reversibility of the anti-proliferative effect of Agaricus bisporus lectin is associated with its release from cancer cells after internalization. The internalization and subsequent slow release, with little degradation of the lectin, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells (Yu et al. 2000).

The three-dimensional structure of the lectin from Agaricus bisporus has been determined by x-ray diffraction. The protein is a tetramer with 222 symmetry, and each monomer presents a novel fold with two beta sheets connected by a helix-loop-helix motif. The T-antigen disaccharide, Gal beta 1-3GalNAc, mediator of the anti-proliferative effects of the protein, binds at a shallow depression on the surface of the molecule. The lectin has two distinct binding sites per monomer that recognize the different configuration of a single epimeric hydroxyl (Carrizo et al. 2005).

Aqueous extracts of the sporophores of eight mushroom species have been assessed for their ability to prevent H2O2-induced oxidative damage to cellular DNA using the single-cell gel electrophoresis ("Comet") assay. The highest genoprotective effects were obtained with cold (20ºC) and hot (100ºC) water extracts of Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. These edible mushrooms therefore represent a valuable source of biologically active compounds with potential for protecting cellular DNA from oxidative damage (Rocha et al. 2002). A heat-labile protein has also been identified in fruit bodies of Agaricus bisporus which protects Raji cells (a human lymphoma cell line) against H2O2-induced oxidative damage to cellular DNA (Shi et al. 2002).

Lectins from Agaricus bisporus and Agaricus campestris have been shown to stimulate insulin and glucagon release from isolated rat islets in the presence of 2 mM glucose. Maximal stimulation of insulin release was reported at lectin concentrations above 58mg/mL (approximately 1mM). The lectin did not alter islet glucose oxidation to CO2 or incorporation of [3H] leucine into trichloracetic acid-precipitable material, nor did it modify rates of insulin secretion induced by 20 mM glucose. None of nine other lectins tested stimulated insulin release, whereas stimulation of fat cell glucose oxidation was a general property of the lectins. The data also

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suggesting that lectin binding is essential for the expression of insulin-releasing activity. The authors proposed that the specific interaction between mushroom lectin and its receptors may lead to conformational changes in the structure of the membranes of the islet A2- and B-cells that facilitate exocytosis (Ewart et al. 1975).

Agaricus bisporus and Pleurotus sajor caju have been assayed in vitro for their anti-microbial activities using aqueous and organic solvents extracts. It has been shown that Escherichia coli 390, Escherichia coli 739, Enterobacter aerogenes, Pseudomonas aeruginosa and Klebsiella pneumoniae were most sensitive to aqueous, ethanol, methanol and xylene extracts of these mushrooms (Tambekar et al. 2006).

A report on the fractionation of extracts of the edible mushroom, Volvariella volvacea, has shown the isolation of two heterocyclic carboxylic acids, namely pyridine-3-carboxylic acid [nicotinic acid] and pyrazole-3(5)-carboxylic acid and four steroidal metabolites ergosterol, 5-dihydroergosterol, ergosterol peroxide, and cerevisterol. In light of the structural similarity of pyrazole-3(5)-carboxylic acid to pyrazole-3-carboxylic acids, which act as agonists for nicotinic acid receptors, the levels of pyridine-3-carboxylic acid and pyrazole-3(5)-carboxylic acid were estimated in V. volvacea and two other edible mushrooms, namely Agaricus bisporus and Calocybe indica. Significant levels of pyridine-3-carboxylic acid (nicotinic acid) were found in C. indica, and pyrazole-3(5)-carboxylic acid was found in abundance in A. bisporus. Correlations have been suggested between the occurrence of these compounds in mushrooms and consumption as well as beneficial health effects (Mallavadhani et al. 2006).

Plasma cholesterol concentration in rats has been shown to be reduced by feeding of mushroom (Agaricus bisporus) fiber. The results demonstrated that mushroom fiber (and sugar beet fiber) lowered the serum total cholesterol level by enhancement of the hepatic low density lipoprotein (LDL) receptor mRNA (Fukushima et al. 2000). Similar cholesterol-lowering effects in rats of Maitake (Grifola frondosa) fiber, Shiitake (Lentinus edodes) fiber, and Enokitake (Flammulina velutipes) fiber have also been reported (Fukushima et al. 2001).

Long term Agaricus bisporus consumption has been studied in rats. Female Charles River Sprague - Dawley rats were fed a diet containing a 30% dry powder of A. bisporus for 500 days. A control group was given a basal diet without A. bisporus. There was no significant difference in the incidence of tumours between the experimental group and control group. No carcinogenic activity of A. bisporus was observed in this long-term study (Matsumoto et al. 1991).

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A study by Toth and co-workers in which Agaricus bisporus, baked at 220-230ºC for 10 minutes and subsequently fed to mice for 12h each day, five days each week throughout their life and also fed a well-balanced semi-synthetic diet for 12h each day for five days and for the remaining two full days each week, showed no statistically significant difference in tumours in the lungs, blood vessels, cecum, and colon when compared to the untreated controls. The estimated average daily mushroom consumption per animal was 4.8g for female mice and 4.2g for male mice (Toth et al. 1997), which exceeds the average daily consumption of mushrooms by humans (Shephard et al. 1995).

Lectins from Agaricus bisporus, Phaseolus vulgaris, Momordica charantia, Ricinus communis and its constituent chains have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (Wang and Ng, 2001), while phenolic compounds present in extracts from A. bisporus strongly generated reactive oxygen species (suggesting immunomodulatory effects) in human PBMCs and K 562 cells in vitro(Wei et al. 2008).

Agaricus blazei In general, the anti-tumour activity of Agaricus blazei appears to be mainly due to the activation of the immune system rather than to any direct effects on tumour cells. This is supported by the fact that macrophages derived from rat bone marrow have been shown to be activated and cytokines such as tumour necrosis factor-alpha (TNF-a), interleukin-1 (IL-1) and IL-8, and nitric oxide (NO) were secreted, in

response to water extracts in in vitro experiments. Furthermore, oral administration of Agaricus blazei water extracts to mice has been shown to induce the activation of macrophages and T cells in vivo. Anti-genotoxic, anti-mutagenic and anti-clastogenic effects have also been detected in Agaricus blazei water extracts (Sorimachi and Koge, 2008). Agaricus blazei Murrill extracts, under certain conditions, have also been shown to have anti-mutagenic activities in mice that may contribute to an anti-carcinogenic effect (Delmanto et al. 2001).

Oral administration of dried fruiting bodies of A. blazei has been shown to augment cytotoxicity of natural killer (NK) cells in wild-type (WT) C57BL/6, C3H/HeJ, and BALB/c mice. Augmented cytotoxicity was demonstrated by purified NK cells from treated wild-type (WT) and RAG-2-deficient mice, but not from interferon-gamma (IFN-gamma) deficient

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mice. NK cell activation and IFN-gamma production was also observed in vitro when dendritic cell (DC)-rich splenocytes of WT mice were coincubated with an extract of A. blazei. Both parameters were largely inhibited by neutralizing anti-interleukin-12 (IL-12) monoclonal antibody (mAb) and completely inhibited when anti-IL-12 mAb and anti-IL-18 mAb were used in combination. An aqueous extract of the hemicellulase-digested compound of A. blazei particle (ABPC) induced IFN-gamma production more effectively, and this was completely inhibited by anti-IL-12 mAb alone. NK cell cytotoxicty was augmented with the same extracts, again in an IL-12 and IFN-gamma-dependent manner. These results demonstrate that A. blazei and ABPC augmented NK cell activation through IL-12-mediated IFN-gamma production (Yuminamochi et al. 2007).

Aqueous extracts of Agaricus blazei fruiting body prepared at different temperatures have been fractionated by ethanol precipitation with various ethanol concentrations. The original aqueous extracts of A. blazei failed to stimulate natural killer (NK) cell activity in murine spleen cells in vitro, but the strongest effect was observed in a 30% ethanol-soluble-50% ethanol-insoluble fraction prepared from the extract at 40ºC (fraction A-50). Fraction A-50 also showed the strongest augmenting effect on interferon (IFN)-gamma production. This augmentation of NK activity and IFN-gamma production by fraction A-50 was completely abrogated by heat treatment (Zhong et al. 2005).

Clinical effects and safety evaluation of Agaricus Blazei Condensed Liquid (Agaricus Mushroom Extract; ABCL) on human volunteers with C-type hepatitis has been studied. A total of 20 patients (10 male, 10 female) with chronic C-type hepatitis received the ABCL orally twice per day for 8 weeks. No toxicological effects, nor other side effects were observed (Inuzuka and Yoshida, 2002).

The effects of protein-bound polysaccharides (A-PBP and L-PBP), extracted from the mycelia of Agaricus blazei and Lentinus edodes, on serum cholesterol and body weight have been investigated in 90 female volunteers. The data demonstrated a weight-controlling and hypolipidemic effect of both A-PBP and L-PBP via a mechanism involving absorption of cholesterol (Kweon et al. 2002).

Polysaccharide fractions of Agaricus blazei have been prepared from cultured A. blazei by repeated extraction with hot water (AgHWE), cold NaOH (AgCA), and then hot NaOH (AgHA). By chemical, enzymic, and NMR analyses, the primary structures of AgHWE, AgCA, and AgHA were mainly composed of 1,6-beta-glucan. Among these fractions, the NaOH extracts showed anti-tumour activity against the solid form of Sarcoma

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180 in ICR mice. To demonstrate the active component in these fractions, several chemical and enzymic treatments were applied. These fractions were found to be i) neutral beta-glucan passing DEAE-Sephadex A-25, ii) resistant to periodate oxidation (I/B) and subsequent partial acid hydrolysis (I/B/H), iii) resistant to a 1,3-beta-glucanase, zymolyase, before I/B, but sensitive after I/B/H. In addition, after I/B/H treatment of the neutral fraction of AgCAE, a signal around 86 ppm attributable to 1,3-beta glucosidic linkage was detectable in the 13C-NMR spectrum. These data strongly suggest that a highly branched 1,3-beta-glucan segment forms the active centre of the anti-tumour activity (Ohno et al. 2001).

Agaricus blazei Murill has been reported to possess biological effects that include immunomodulatory activities, although the number of in vivo studies is limited. A recent study has evaluated the immunomodulatory effects of A. blazei in 160 male Balb/cByJ mice. The mice were divided into four groups and treated with various quantities of intragastric A. blazei extract or distilled water for 8 to 10 weeks. Nine parameters, relating to general immune function or adaptive immunity against immunogen chicken ovalbumin, were determined. The mice receiving A. blazei extract exhibited significantly greater serum immunoglobulin G levels, increased T-cell numbers in spleen, and elevated phagocytic capability compared with controls. Consumption of A. blazei was also associated with significant increases in ovalbumin-specific serum immunoglobulin G level, delayed-type hypersensitivity, splenocyte proliferation rate, and tumour necrosis factor-alpha secretion by splenocytes, indicating that A. blazei Murill possesses a wide range of immunomodulatory effects in vivo (Chan et al. 2007). Agaricus blazei has also been reported to have inhibitory effects on mast cell-mediated anaphylaxis-like reactions (Choi et al. 2006b).

Beta-glucans and and their enzymatically hydrolyzed oligosaccharides from Agaricus blazei have anti-hyperglycemic, anti-hypertriglyceridemic, anti-hypercholesterolemic, and anti-arteriosclerotic activity indicating overall anti-diabetic activity in diabetic rats. However, the enzymatically hydrolyzed oligosaccharides have been shown to have around twice the activity of beta-glucans with respect to anti-diabetogenic activity (Kim et al. 2005).

Extracts from Agaricus blazei Murill (AbM) have been evaluated on changes to gene expression on a human monocyte cell line (THP-1). Changes in the levels of mRNA transcripts were measured using 35 k microarrays, and the changes in select cytokine gene products by immunoassays. Lipopolysaccharide (LPS) was included for comparison. Both AbM and LPS had very significant effects on gene expression. Genes

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related to immune function were selectively up-regulated, particularly pro-inflammatory genes such as the interleukins IL1B and IL8. Although most genes induced by AbM were also induced by LPS, AbM produced a unique profile, e.g., as to a particular increase in mRNA for the cytokines IL1A, CXCL1, CXCL2 and CXCL3, as well as PTGS2 (cyclooxygenase2) (Ellertsen et al. 2006)

An extract from Agaricus blazei Murill Kyowa (ABMK), has been reported to possess anti-mutagenic and anti-tumour effects. A study has investigated the effects of ABMK consumption on immunological status and quality of life in cancer patients undergoing chemotherapy. One hundred cervical, ovarian, and endometrial cancer patients were treated either with carboplatin (300mg/m2) plus VP16 (etoposide, 100mg/m2) or with carboplatin (300mg/m2) plus taxol (175mg/m2) every 3 weeks for at least three cycles, with or without oral consumption of ABMK. The authors observed that natural killer cell activity was significantly higher in the ABMK-treated group compared to the non-treated placebo group (n = 61). However, no significant difference in lymphokine-activated killer and monocyte activities was observed in a manner similar to the count of specific immune cell populations between ABMK-treated and non-treated groups. However, chemotherapy-associated side effects such as appetite, alopecia, emotional stability, and general weakness were all reported to be improved by ABMK treatment, with the authors suggesting that ABMK treatment may have some beneficial effects for gynecological cancer patients undergoing chemotherapy (Ahn et al. 2004).

Sodium pyroglutamate isolated from Agaricus blazei has been shown to have potent anti-tumour and anti-metastatic actions, as well as immune-modulatory activity, in tumour-bearing mice (Kimura et al. 2004).

Oral administration of ergosterol, isolated from the lipid fraction of Agaricus blazei Murill has also been shown to have anti-tumour activity in Sarcoma 180-bearing mice. Ergosterol reduced tumour growth at doses of 400 and 800mg/kg. Administration of ergosterol for 20 days was reported to be without side effects, such as decreases in body, epididymal adipose tissue, thymus, and spleen weights and leukocyte numbers induced by cancer chemotherapy drugs. Ergosterol had no cytotoxicity against tumour cells and it appears as though the antitumour activity of ergosterol might be due to direct inhibition of angiogenesis induced by solid tumours (Takaku et al. 2001).

The effects of low molecular weight products extracted from Agaricus blazei Murill on MethA tumour cell growth have been studied. Inoculation of a low molecule fraction (LM) into the primary tumour of a two-tumour mouse model resulted in a marked inhibition of the tumour, not only in

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the right flank, but also in the non-injected left flank. Chromatographic purification and physicochemical characterization showed the main tumouricidal activity to be located in a low molecule fraction-3 (LM-3), containing alpha-1,4-glucan-beta-1,6-glucan complex with an average molecular weight of 20kDa. Serum levels of immunosuppressive acidic protein (IAP) in mice receiving LM fractions, particularly LM-3, significantly increased, indicating the possible activation of granulocytes (Fujimiya et al. 1999).

Induction of apoptosis in human gastric epithelial AGS cancer cells by an aqueous extract of Agaricus blazei has been demonstrated. It was found that an Agaricus blazei extract could inhibit cell growth in a dose-dependent manner, which was associated with the arrest of G2/M phase and the induction of apoptotic cell death via caspase-3 activation (Jin et al. 2006). A subsequent study by the same group has shown a similar effect on apoptosis by Agaricus blazei on human leukemic U937 cells via similar mechanisms (regulation of Bcl-2 and caspase-3) (Jin et al. 2007).

The effect of an RNA-protein complex isolated from A. blazei Murill, on human leukemia HL-60 cells has been studied. Typical apoptotic characteristics were determined by morphological methods using DNA agarose gel electrophoresis and flow cytometry. The data showed that the fraction from Agaricus blazei induced HL-60 cell apoptosis and that the combined effect of down-regulation of telomerase activity and up-regulation of mRNA expression of the caspase-3 gene could be the primary mechanism of induction of apoptosis. These findings provide good evidence that the isolated fraction may be of value for the clinical treatment of acute leukemia (Gao et al. 2007).

An extract from fruit bodies of Agaricus blazei has been evaluated in a "double grafted tumour system" mouse model and reported to cure the primary tumour and inhibit the growth of metastatic tumours in this model. A separate extract from Agaricus blazei (Himematsutake) inhibited the growth of the primary tumour. An immuno-suppresive acidic protein (IAP) was induced by both the Agaricus and Himematsutake preparations but not by Lentinus edodes. Lentinus edodes had no effect on the growth of either the primary or metastatic tumours (Ebina, 2005).

An aqueous extract of Agaricus blazei has been shown to exert a hepato-protective effect on both liver toxicity and hepato-carcinogenesis on rat liver toxicity induced by different doses of diethylnitrosamine (Barbisan et al. 2002).

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A subsequent study from another group has also demonstrated the chemo-preventative potential of an Agaricus blazei (Ab) Murrill mushroom meal in a medium-term rat liver carcinogenesis assay. Male Wistar rats initiated for hepatocarcinogenesis with diethylnitrosamine (DEN, 200mg/kg i.p.) were fed during a 6-week period with dry powdered mushroom strains Ab 29 or 26, each one with opened (OB) or closed basidiocarp (CB), mixed at a level of 10% in a basal diet. Chemo-preventative activity of the mushroom meal was observed for the Ab 29 (OB and CB) and Ab 26 (CB) strains in terms of the number of putative pre-neoplastic altered foci of hepatocytes which express either the enzyme glutathione S-transferase, placental form (GST-P+) or the transforming growth factor-alpha, and for the Ab 29 (OB) and Ab 26 (CB) strains on the size of GST-P+ foci. This was associated with inhibition of foci cell proliferation in the animals fed the Ab 29 (OB) and Ab 26 (CB) strains. The results suggest that the protective influence of the Ab meal against the DEN potential for rat liver carcinogenicity depends on both the strain and period of mushroom harvest (Pinheiro et al. 2003).

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) have also been investigated in adult male Wistar rats. The data indicated that previous treatment with the highest concentration of Agaricus blazei (11.5mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity (Barbisan et al. 2003a) while in a subsequent study, the same group reported that treatment with aqueous extracts of Agaricus blazei does not exert a protective effect against the development of GST-P-positive foci induced by DEN (Barbisan et al. 2003b).

Anti-bacterial effects of Agaricus blazei Murill (AbM) have been investigated. The AbM extract protected against systemic Streptococcus pneumoniae 6B infection in mice and was most effective when given 24h before inoculation but it also had protective effects when given together with challenge compared with control. The lack of an antibiotic effect on pneumococci in vitro and increased levels of cytokines MIP-2 and TNF in the serum of mice receiving AbM extract, indicated that the protective effect of AbM was due to the involvement of the native immune system. The anti-infection properties of AbM have therefore been shown in vivo and the results suggest that AbM extract may be useful as an additional prophylactic and possibly therapeutic treatment against bacterial infections in humans (Bernardshaw et al. 2005).

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A subsequent study by the same group has shown that an extract of Agaricus blazei Murill can protect against lethal septicemia in a mouse model of faecal peritonitis. Bacterial septicemia can occur during gastroenterological surgery. The putatively anti-infective immunomodulatory action of Agaricus blazei Murill (AbM) has been studied in an experimental peritonitis model in BALB/c mice. The mice were orally given an extract of AbM or phosphate-buffered saline 1 day before the induction of peritonitis with various concentrations of faeces from the mice. The state of septicemia, as measured by the number of colony-forming units of bacteria in blood, and the survival rate of the animals were compared between the groups. Mice that were orally treated with Agaricus blazei Murill extract before bacterial challenge showed significantly lower levels of septicemia and improved survival rates (Bernardshaw et al. 2006).

A randomized, double-blinded, and placebo-controlled clinical trial has evaluated the effects of Agaricus blazei Murill in combination with metformin and gliclazide on insulin resistance in type 2 diabetes. Supplementation of Agaricus blazei Murill extract improved insulin resistance among subjects with type 2 diabetes. The increase in adiponectin concentration after taking Agaricus blazei Murill extract for 12 weeks may be the mechanism that results in the observed effect (Hsu et al. 2007).

A recent study has demonstrated that ultrafiltration, in combination with spray-drying, is applicable for the preparation of protein-bound polysaccharide powders with higher anti-tumour activities from Agaricus Blazei Murill (Hong et al. 2007). Thermostable antioxidant activity has also been reported from Agaricus blazei Murill (Izawa and Inoue, 2004).

A study to evaluate the chronic toxicity and oncogenicity of Agaricus blazei Murill in F344 rats has been reported (Lee et al. 2008). Long-term (2 years) feeding of rats of a powdered diet containing Agaricus blazei at levels up to 25,000 ppm (parts per million) revealed no remarkable change in mean body weight, body weight gain, hematologic or serum chemistry parameters, or absolute or relative organ weights in control or treatment groups. Mortality in male treatment (mushroom) groups was significantly lower than in controls. Histopathological studies showed no increased incidence of tumours.

A recent study has also reported good bioavailablity of both copper and zinc from mycelium of Agaricus blazei Murrill equating to very good levels of recommended daily intakes of these minerals from small amounts of (1g) of this mushroom (Rabinovich et al. 2007).

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Anti-viral activities of Agaricus blazei Murill have been demonstrated against cytopathic effects induced by western equine encephalitis (WEE) virus by the mycelial fractions but not those of fruiting bodies (Sorimachi et al. 2001).

Ethanol extracts and hot water extracts of Agaricus blazei, Agrocybe cylindracea, and Boletus edulis have been shown to have significant antioxidant properties. The ethanolic extracts were more effective than hot water extracts in antioxidant activity using the conjugated diene method and scavenging ability on 1,1-diphenyl-2-picrylhydrazyl radicals whereas hot water extracts were more effective in reducing power, scavenging ability on hydroxyl radials and chelating ability on ferrous ions as demonstrated by their lower EC50 values. Naturally occurring antioxidant components including total tocopherols (3.18-6.18mg/g) and total phenols (5.67-5.81mg/g) were found in the extracts and their contents were associated with the EC50 value of the antioxidant properties (Tsai et al. 2007).

Differences of the pharmacological effects of Agaricus blazei cultured on various materials have been examined. Agaricus blazei mushrooms were prepared on culture media composed of 1) tops of sugar cane shoots (stems and leaves), 2) rice straw 3) wheat straw, 4) broad leaf tree bark, and 5) used substrate after Pleurotus ostreatus cultivation. The pharmacological effects of this mushroom were examined by the following methods; 1) anti platelet aggregation stimulated by PAF or arachidonic acid Na, 2) inhibition of IL-8 gene expression stimulated by TNF-alpha, 3) improvements of rough surfaces by using replica method. In both the anti-platelet aggregation test and chemokine gene revelation control test, A. blazei cultured on the top shoot of sugar cane medium showed the most effective results compared with that cultured on other media. The results suggested that the A. blazei cultured on the top shoot of sugar cane medium has increased pharmacological activity compared to mushrooms cultured on rice or wheat straw or broad leaf bark (Yoshimoto et al. 2005).

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Agaricus brasiliensis

Agaricus brasiliensis (previously named Agaricus blazei ss. Heinem), also known as the sun mushroom, is native to Southeastern Brazil, and is widely consumed, mainly in the form of tea. Aqueous (AqE) and ethanol (EtOHE) extracts and an isolated polysaccharide (PLS) from the fruiting body of A. brasiliensis have been evaluated for anti-viral activity against poliovirus type 1 in HEp-2 cells. The evaluation of the time of addition by plaque assay showed that when AqE, PLS and EtOHE were added, just after the virus inoculation (time 0 h), there was a concentration-dependent reduction in the number of plaques by up to 50%, 67% and 88%,

respectively. The test substances showed anti-viral activity and were more effective when added during the poliovirus infection. As the extracts had little effect on reducing viral adsorption and did not show any virucidal effect, the authors suggested that they may act at the initial stage of the replication of poliovirus (Faccin et al. 2007).

The mycelium polysaccharide and exo-polysaccharide (EPS) of Agaricus brasiliensis LPB 03 produced by submerged fermentation has shown strong inhibition against Sarcoma 180 in mice, reaching 72.19% inhibition compared to a control group. Furthermore, 50% of mice in the test group demonstrated total tumour regression (Fan et al. 2007).

The structure and anti-tumour activity of polysaccharide fractions of the fruit body of Agaricus brasiliensis have been studied in cold and hot water extracts (CWE and HWE) on a mouse diabetic model (C57BL Ksj-db/db). Compared to the water administered control group, the body weight, urinary glucose exclusion, urinary pH, blood glucose level, and organs weight were comparable. The splenocytes of CWE administered mice produced a higher concentration of interleukin-6. By megascopic and microscopic examinations of renal sections, the number of the mice having abnormal kidney was 3/5 (control), 2/5 (HWE), and 0/5 (CWE) suggested the activity of the renal protection in the cold water extract. The results strongly suggest that the pharmacological action of the cold water extract of A. brasiliensis is significantly stronger than that of the hot water extract (Furukawa et al. 2006).

The administration of ethanol-soluble, boiling water-soluble, and ammonium oxalate-insoluble fractions isolated from the fruiting bodies of

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Agaricus brasiliensis into C3H/HeN mice has been shown to play an important role in the maintenance of hematopoietic cells for compromised patients at the risk of infection associated with malignancy (Fujimiya et al. 2006).

Phenolic compounds present in mushroom extracts from A. brasiliensis strongly generated reactive oxygen species (suggesting immunomodulatory effects) in human PBMCs and K 562 cells in vitro (Wei et al. 2008).

Agrocybe aegerita (Black Poplar, Piopinno, Chiodini)

Agrocybe aegerita is an edible mushroom with reported anti-tumour properties. An investigation of bioactivity gave positive results for ceramide, methyl-beta-D-glucopyranoside and alpha-D-glucopyranoside, along with linoleic acid and its methyl ester. Ceramide inhibited the cyclooxygenase (COX) enzymes, COX-1 and -2 and its anti-

cancer potential was investigated against five human cancer cell lines in vitro and it was found to inhibit the proliferation of stomach, breast and central nervous system cancer cell lines suggesting that the compound may be useful in alleviating inflammatory conditions, as well as possibly reducing the development of the above cancers (Diyabalanage et al. 2008).

A lectin from Agrocybe aegerita (AAL) has been found to possess potent tumour-suppressing function and tumour cell apoptosis-inducing activity. Its full sequence has been published. It has been reported that AAL is a member of the galectin family and the dimeric form is the active unit for functional performance. The recombinant AAL showed comparable tumour cell apoptosis-inducing activity with the wild AAL but no DNase activity (Yang et al. 2005).

Antioxidant activity of a methanol crude extract and its fractions, from the fruiting bodies of Agrocybe aegerita, has been evaluated by scavenging activity of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid) radical cation (ABTS(+)) and inhibition of lipid peroxidation of rat brain homogenate. The ethyl acetate (EA) fraction, which showed the most potent antioxidant activity in the above two assays, was further

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fractionated by a Sephadex LH-20 column into four subfractions (EA1-EA4). Significant correlation was found between the total phenolic content and the antioxidant activity in the EA fraction and its sub-fractions (Lo and Cheung, 2005).

Proliferation of human leukemic U937 cells has been shown to be significantly inhibited by conditioned medium of human peripheral blood mononuclear cells stimulated with cold-water extracts (10-800 mg/mL of medium) of dietary mushrooms, Hypsizigus mamoreus, Agrocybe aegerita and Flammulina velutipes (Ou et al. 2005).

Agrocybe chaxingu

Osteoclast forming suppressive compounds (important in osteoporosis) have been isolated from the mushroom Agrocybe chaxingu (Abel et al. 2007).

Auricularia auricular (Wood Ear)

In vitro evaluation of antioxidant activities of Auricularia auricular has shown significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with the drug catechin. The IC50 value of crude, boiled and ethanolic extracts of A. auricula represented 403, 510, and 373mg/ml respectively of hydroxyl radical scavenging activity and 310, 572 and 398mg/ml respectively of lipid peroxidation, while crude, boiled and ethanolic extracts were shown to significantly increase nitric oxide production (664,

191 and 850pmole/mg dry wt/h respectively) over the control (Acharya et al. 2004).

Auricularia auricular has been shown to have hypocholesterolemic properties (Cheung, 1996b).

Constipation is one of the most prevalent gastrointestinal complaints and high fiber intake is recommended as an initial therapy for constipation. Ear mushrooms (Auricularia) are known to have higher fiber contents (by

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~50%) than other mushroom varieties. In patients with functional constipation, fiber supplements using ear mushrooms has been shown to significantly improve constipation related symptoms without serious side effects (Kim et al. 2004).

An acidic polysaccharide with anti-coagulant activity has been isolated from Auricularia auricula using water, alkali or acid extracts. The alkali extract showed the highest anti-coagulant activity and was further purified using gel filtration chromatography. The specific anti-coagulant activity of the purified polysaccharide was 2 international units (IU)/mg and its average mass was approximately 160kDa. The polysaccharide from this species of mushroom contains mainly mannose, glucose, glucuronic acid and xylose but no sulfate esters. Its anti-coagulant activity was due to catalysis of thrombin inhibition by anti-thrombin, but not by heparin cofactor II. Inhibition of Factor Xa by anti-thrombin was not catalyzed by the polysaccharide. The glucuronic acid residues were essential for the anti-coagulant action of the mushroom polysaccharide since the activity disappeared after reduction of its carboxyl groups. In ex vivo tests using rats orally fed with the polysaccharide, an inhibitory effect on platelet aggregation was observed, as with aspirin, a well-known anti-platelet agent. The authors suggested that polysaccharides from these mushrooms may constitute a new source of compounds with action on coagulation, platelet aggregation and, perhaps, on thrombosis (Yoon et al. 2003) .

Auricularia polytricha (Jew's Ear)

An immunomodulatory protein (APP) has been purified from the fruiting body of an edible Jew's Ear mushroom, Auricularia polytricha, by extraction using 5% cold acetic acid in the presence of 0.1% 2-mercaptoethanol, followed by ammonium sulfate fractionation, DE-52 and P,MonoQ anion-exchange chromatography. The molecular mass of APP was around 13.4kDa and its pI is approximately 5.1. APP is a simple protein without carbohydrate, and can

agglutinate mouse red blood cells. APP alone activates murine splenocytes, markedly increasing their proliferation and gamma-interferon (IFN-gamma) secretion, and presented no cytotoxicity in vitro. Although murine splenocytes are stimulated by the mitogen concanavalin A (ConA), APP suppressed their proliferation in a dose-dependent manner. APP also enhanced the production of both nitric oxide (NO) and tumour necrosis factor-alpha (TNF-alpha) by LPS-induced RAW 264.7

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macrophages. The data suggest that this protein isolated from Auricularia polytricha is an immune stimulant (Sheu et al. 2004).

Cordyceps sinensis (Caterpillar mushroom)

An exo-polysaccharide fraction from cultivated Cordyceps sinensis has been shown to have immunomodulatory and anti-tumour effects on (B16 melanoma) tumour-bearing mice (Zhang et al. 2005).

Cultivated mycelium of Cordyceps sinensis, sequentially extracted by petroleum ether (PE), ethyl acetate (EtOAc), and ethanol (EtOH) showed a significant and dose-dependent inhibitory effect on the proliferation of four cancer cell lines, MCF-7 breast cancer, B16 mouse melanoma, HL-60 human pre-myelocytic leukemia and HepG2 human hepatocellular carcinoma, with IC50 values below 132 mg/ml. A hot water extract failed to

show such activity. The EtOAc extract, in particular, had the most potent effect against all four cancer cell lines, with IC50 values between 12mg/ml (on B16) and 45mg/ml (on MCF-7). In contrast, it had much lower cytotoxicity against normal mouse bone marrow cells. The EtOAc extract contained carbohydrates, adenosine, ergosterol and a trace amount of cordycepin, of which ergosterol and related compounds were identified as a major class of active constituents contributing to the in vitro cytotoxicity. In an animal test, the EtOAc extract showed a significant inhibiting effect on B16-induced melanoma in C57BL/6 mice, causing a 60% decrease of tumour size over 27 days (Wu et al. 2007).

An extract of Cordyceps sinensis (CSE) has been tested in C57BL/6 mice implanted subcutaneously with syngeneic EL-4 lymphoma cells. Oral administration of the extract led to a reduction of tumour size and prolongation of the host survival time. As for the activities of peritoneal macrophages, chemotaxis was dramatically depressed within a few days after EL-4 transplantation up to the end of life, but treatment with CSE at -14, -7, -4, +4, +7 and +10 days after the tumour transplantation augmented the activity about four times greater than that of the control. Phagocytic activity of macrophages was also decreased in tumour-bearing mice treated with cyclophosphamide (100mg/kg) 3 and 5 days after tumour transplantation, but administration of CSE restored the activity to more than the normal level. The overall efficacy of CSE was tested with protective activity against systemic infection by Salmonella enteritides. The tumour-bearing mice receiving this extract lived significantly longer than the other groups without CSE (Yamaguchi et al. 1990).

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A study has been conducted to investigate a hypocholesterolemic effect of a hot-water fraction (HW) from cultured mycelia of Cordyceps sinensis. In mice fed a cholesterol-free diet and those fed a cholesterol-enriched diet, body and liver weights were not significantly different from those of the controls. The serum total cholesterol (TC) of all mice groups administered HW (150 and 300mg/kg/d, respectively) with the cholesterol-enriched diet decreased more than in the control group. Among the mice fed the cholesterol-enriched diet, HW also increased the high-density lipoprotein (HDL) cholesterol level, but decreased the very low-density lipoprotein plus low-density lipoprotein (VLDL+LDL) cholesterol level. The changes in HDL-and VLDL+LDL-cholesterol levels consequently decreased the atherogenic value. The results indicated that HW in rats administered a cholesterol-enriched diet decreased the plasma cholesterol level. The 300mg/kg dose had a significant effect on the serum total cholesterol level (Koh et al. 2003).

Flammulina velutipes (Enoki)

It has been reported that there is currently no effective therapy for malignant estrogen-independent breast cancer. Screening of 38 species of edible mushrooms on human estrogen-receptor positive (ER+) (MCF-7) and estrogen-receptor negative (ER-) (MDA-MB-231, BT-20) breast cancer cells has been undertaken to select potential agents with broad-spectrum anti-tumour activity against breast cancer cells. Water-based

extracts of three mushroom species, Coprinellus sp., Coprinus comatus and Flammulina velutipes (CME, CCE and FVE, respectively), have been identified as anti-breast cancer agents. The anti-tumour activities included marked growth inhibition of both ER+ and ER- breast cancer cells, induction of rapid apoptosis on both ER+ and ER- cells, and significant inhibition of MCF-7 tumour colony formation in vitro. The anti-proliferative and cytotoxic activities of the three mushroom extracts were dose-dependent, regardless of the hormone receptor status of the cancer cells. The degree of produced cytotoxicity on ER- breast cancer cells was very high. Mushroom extracts CME and FVE induced a rapid (within 5 hours) apoptosis on MCF-7 and MDA-MB-231 cells. MCF-7 tumour colony formation rate was reduced by 60% in CCE- and CME-treated cells and nearly completely inhibited (99%) by FVE treatment. These results suggest that the mushroom species Coprinus comatus, Coprinellus sp.

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and Flammulina velutipes contain potent anti-tumour compounds for breast cancer. This finding is important due to the lack of chemotherapeutic and chemo-preventive agents for ER- human breast cancer (Gu and Leonard, 2006).

An anti-tumour polysaccharide, EA3 isolated from Flammulina velutipes (CURT. ex FR.) SING. has been shown to be composed of D-glucose with the chemical structure of a beta-(1 leads to 3)-glucan. Another anti-tumour polysaccharide (EA5) also isolated from F. velutipes was fractionated and among the polysaccharides isolated, the highest molecular weight polysaccharide (EA501) showed the highest anti-tumour activity. The component sugars of EA501 were found to be D-glucose 42.3%, D-galactose 17.3%, D-mannose 12.2%, D-xylose 6.7% and L-arabinose 14.7% (Ikekawa et al. 1982).

A further study by the same group has shown that proflamin, isolated from the culture mycelium of Flammulina velutipes (Curt. ex Fr.) Sing. is a weakly acidic glycoprotein containing more than 90% protein and less than 10% carbohydrate, and has a molecular weight of ~13,000Da. Proflamin has been shown to be markedly effective against the syngeneic tumours, B-16 melanoma (B-16) and adenocarcinoma 755 (Ca-755) in the mouse. The increases in median survival time of treated mice with B-16 and Ca-755 were 86% and 84%, respectively. Proflamin exhibited no cytocidal effect against the cultured cell lines in vitro. Oral administration of proflamin produced no lethal or any other apparent adverse effect in mice (Ikekawa et al. 1985).

Antioxidant activity of submerged cultured mycelium extracts of higher Basidiomycetes mushrooms has recently been reported. Antioxidant properties were studied from 28 submerged cultivated mycelium Basidiomycetes strains of 25 species. Three solvents - ethanol, water (culture liquid), and ethyl acetate were used for extraction. Water extracts from Coprinus comatus, Agaricus nevoi, and Flammulina velutipes (Enoki) showed high antioxidant activities (AA) at 2mg/ml. When the ethanol extracts were tested, the highest AA were found in Agaricus nevoi, Omphalotus olearius, and Auricularia auricula-judae extracts at a concentration of 2mg/ml. The AA of ethanol extracts from Agrocybe aegerita and C. comatus increased from 46.6% to 82.7% and from 2.4% to 62.1%, respectively, when the concentration of the extract increased from 2mg/ml to 4-8mg/ml with the authors suggesting that the extracts could be suitable as antioxidative agents and bioproducts (Asatiani et al. 2007a).

Plasma cholesterol concentration in rats has been shown to be reduced by feeding of mushroom Enokitake (Flammulina velutipes) fiber. The

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results demonstrated that mushroom fiber lowered the serum total cholesterol level by enhancement of the hepatic low density lipoprotein (LDL) receptor mRNA (Fukushima et al. 2001).

Angiotensin-converting enzyme (ACE) inhibitory activity (which has an effect on blood pressure reduction) has been demonstrated in the culture broth from Flammulina velutipes (strain 414). Nutritional requirements for the production of ACE inhibitory activity from F. velutipes were shown to include sucrose, ammonium acetate, and glutamic acid (Kim et al. 2002).

Two cuparene-type sesquiterpenes, enokipodins C (1) and D (2), have been isolated from culture medium of Flammulina velutipes (Enoki), along with enokipodins A (3) and B (4). All the metabolites showed anti-microbial activity against the fungus Cladosporium herbarum, and gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus (Ishikawa et al. 2001).

Proliferation of human leukemic U937 cells has been shown to be significantly inhibited by conditioned medium of human peripheral blood mononuclear cells stimulated with cold-water extracts (10-800mg/mL of medium) of dietary mushrooms, Hypsizigus mamoreus, Agrocybe aegerite and Flammulina velutipes (Ou et al. 2005).

Ganoderma lucidum (Reishi, Lingzhi)

A series of trials evaluating Ganoderma lucidum in several disease states have been carried out. The trials evaluated effects on cancer, Type II diabetes, coronary heart disease, chronic hepatitis B, and neurasthenia. Treatment with Ganopoly for 12 weeks showed hypoglycemic activity and produced

some anti-viral and liver protective effects in patients with chronic hepatitis B infection. However, the same treatment regimen did not result in any objective response in late-stage cancer patients (Zhou et al. 2005). Overall, the findings suggest that Ganopoly may have some pharmacological activities, although clinical proof is lacking.

A double-blind, placebo-controlled, randomized, and dose-ranging study has been carried out in men with lower urinary tract symptoms (LUTS) to

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evaluate the safety and efficacy of an extract of Ganoderma lucidum that shows the strongest 5-alpha-reductase inhibitory activity among the extracts of 19 edible and medicinal mushrooms. In this trial, 88 men over the age of 49 years who had slight-to-moderate LUTS were randomly assigned to 12 weeks of treatment with G. lucidum extract (6mg once per day) or placebo. The primary outcome measures were changes in the International Prostate Symptom Score (IPSS) and variables of uroflowmetry. Secondary outcome measures included changes in prostate size, residual urinary volume after voiding, laboratory values, and the reported adverse effects. G. lucidum was effective and significantly superior to placebo for improving total IPSS with 2.1 points decreasing at the end of treatment. No changes were observed with respect to quality of life scores, peak urinary flow, mean urinary flow, residual urine, prostate volume, serum prostate-specific antigen, or testosterone levels. Overall treatment was well tolerated with no severe adverse effects (Noguchi et al. 2008)

Ganoderma lucidum (Reishi, Lingzhi) has been reported to suppress the invasive behaviour of breast cancer cells by inhibiting the transcription factor NF-kappaB and to inhibit the growth of MDA-MB-231 breast cancer cells by modulating Akt/NF-kappaB signaling (Jiang et al. 2004).

A subsequent study by the same group on the proliferation of human estrogen-dependent (MCF-7) and estrogen-independent (MDA-MB-231) breast cancer cells has reported that G. lucidum inhibits proliferation of human breast cancer cells and contains biologically active compounds with specificity against the estrogen receptor and NF-kappaB (transcription factor) signalling (Jiang et al. 2006).

Aqueous extracts of fruiting bodies of Ganoderma lucidum, G. sinense, and G. tsugae have been reported to have anti-tumour activities in human breast cancer cells and immunomodulatory activities in murine lymphocytes. In addition, it has also been suggested that the stipes of fruiting bodies of Ganoderma species should be included in the preparation of extracts of these fungi in order to obtain the most comprehensive active ingredients (Yue et al. 2006).

The effect of G. lucidum on oxidative stress-induced metastatic behaviour of poorly-invasive MCF-7 breast cancer cells has also been studied and it has been shown that G. lucidum inhibited oxidative stress-induced migration of MCF-7 cells by the down-regulation of mitogen activated protein kinase (MAPK) signalling, which is involved in hormonal signalling cascades. G. lucidum suppressed oxidative stress stimulated phosphorylation of extracellular signal-regulated protein kinases (Erk1/2), which resulted in the down-regulation of expression of c-fos,

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and in the inhibition of transcription factors AP-1 and NF-kappaB. The biological effect of G. lucidum on cell migration was mediated by the suppression of secretion of interleukin-8 from MCF-7 cells exposed to oxidative stress. These results suggest that G. lucidum inhibited the oxidative stress-induced invasive behaviour of breast cancer cells by modulating Erk1/2 signaling and could possibly be considered as an antioxidant in adjuvant cancer therapy (Thyagarajan et al. 2006).

A further study by the same group has also shown that an extract from green tea (GTE) increased the anti-cancer effect of G. lucidum extract (GLE) on cell proliferation (anchorage-dependent growth) as well as colony formation (anchorage-independent growth) of breast cancer cells. The effect was mediated by the down-regulation of expression of the oncogene c-myc in MDA-MB-231 cells. Although individual GTE and GLE independently inhibited adhesion, migration and invasion of MDA-MB-231 cells, their combination demonstrated a synergistic effect, which was mediated by the suppression of secretion of urokinase plasminogen activator (uPA) from breast cancer cells suggesting a potential use of combined green tea and G. lucidum extracts for the suppression of growth and invasiveness of metastatic breast cancers (Thyagarajan et al. 2007).

The effects of Ganoderma lucidum (Basidiomycetes) polysaccharide (GL-PS) extract on tumour volume and T(CD4+/CD8+) ratio of tumour infiltrating lymphocytes (TILs) in breast cancer bearing mice have been studied. The results indicated that GL-PS (100mg/kg/day) could effectively increase the delayed type hypersensitivity response against sRBC in BALB/c mice. Furthermore, intraperitoneal injection of this extract in breast cancer bearing mice could increase T-cell infiltration into the tumour. The authors concluded that GL-PS can exhibit a potent immunomodulatory effect and may be used for potentiation of the immune system against diseases such as cancer and other conditions in which the immune response has been compromised (Mojadadi et al. 2006).

An alcohol extract from the spore of Ganoderma lucidum has also been shown to inhibit the in vitro proliferation of human umbilical vein endothelial cells and MDA-MB-231 human breast cancer cells. Further fractionation of the alcohol extract revealed that the ethyl acetate fraction inhibited both cell lines in a dose-dependent manner from 2 to 40mg/ml (Lu et al. 2004).

Ganoderma lucidum has also been shown to inhibit proliferation in a dose- and time-dependent manner and induce apoptosis in human prostate cancer cells PC-3 (Jiang et al. 2004).

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The effects of Ganoderma lucidum on SW 480 human colorectal cancer cells have been evaluated. A fraction containing mainly polysaccharides (GLE-1), and a triterpenoid fraction without polysaccharides (GLE-2) were analyzed. The data showed that both GLE-1 and GLE-2 significantly inhibited the proliferation of SW 480 cells. The inhibitory effect of GLE-2 was much stronger than that of GLE-1. GLE-1 inhibited DNA synthesis in the cells and reduced the formation of DPPH radicals indicating that G. lucidum extracts inhibit proliferation of human colorectal cancer cells and possesses antioxidant activity (Xie et al. 2006).

Aqueous extracts of the sporophores of eight mushroom species have been assessed for their ability to prevent H2O2-induced oxidative damage to cellular DNA using the single-cell gel electrophoresis ("Comet") assay. The highest genoprotective effects were obtained with cold (20ºC) and hot (100ºC) water extracts of Agaricus bisporus and Ganoderma lucidum fruit bodies, respectively. These edible mushrooms therefore represent a valuable source of biologically active compounds with potential for protecting cellular DNA from oxidative damage (Rocha et al. 2002).

Protein extracts from selenium-enriched Ganoderma lucidum (Se-GLPr) have been reported to possess strong DNA protective effects from oxidative damage, which increased with the increase of Se content as suggested by chemiluminescence analysis, indicating indirectly that Se plays an important role in increasing the antioxidant activities of protein extracts. This was confirmed by spin-trapping experiments showing that Se-GLPr exhibited higher activities of scavenging superoxide and hydroxyl radicals than its analog, common Ganoderma lucidum extract. All Se-GLPr samples showed stronger activities of attenuating the production of superoxide radical than that of hydroxyl radical (Zhao et al. 2004). Polysaccharide extracts from Se-enriched G. lucidum have also been shown to protect DNA from hydroxyl radical oxidative damage in a dose dependent manner (Zhao et al. 2008).

A hot water extract from Ganoderma lucidum has been shown to have an antioxidative effect against heart toxicity in mice. Ganoderma lucidum exhibited a dose-dependent antioxidative effect on lipid peroxidation and superoxide scavenging activity in mouse heart homogenate. Furthermore, this result indicated that heart damage induced by ethanol showed a higher malonic dialdehyde level compared with heart homogenate treated with Ganoderma lucidum. The authors concluded that this effect of Ganoderma lucidum may protect the heart from superoxide induced damage (Wong et al. 2004).

A human toxicological study has evaluated the consumption of Lingzhi (Ganoderma lucidum) in a double-blinded, placebo-controlled, cross-over

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intervention study on a range of biomarkers for human health. The study investigated the effects of 4 weeks Lingzhi supplementation (1.44g Lingzhi/d; equivalent to 13.2g fresh mushroom/d) on a range of biomarkers for antioxidant status, cardiovascular disease (CHD) risk, DNA damage, immune status, and inflammation, as well as markers of liver and renal toxicity. No significant change in any of the biomarkers was found. The results showed no evidence of liver, renal or DNA toxicity with Lingzhi intake (Wachtel-Galor et al. 2004).

A recent study has investigated the anti-invasive effect of lucidenic acids isolated from a Ganoderma lucidum strain (YK-02) against human hepatoma carcinoma cells. The results indicated that the lucidenic acids isolated from G. lucidum (YK-02) were anti-invasive bioactive components on human hepatoma carcinoma cells (Weng et al. 2007).

Triterpene-enriched extracts from Ganoderma lucidum have been shown to inhibit growth of human hepatoma Huh-7 cells via suppression of protein kinase C, activating mitogen-activated protein kinases (intermediates in hormonal signalling pathways) and G2-phase cell cycle arrest. In contrast, the extracts did not inhibit growth of Chang liver cells, a normal human liver cell line (Lin et al. 2003).

Three triterpene aldehydes, lucialdehydes A - C, from the fruiting bodies of Ganoderma lucidum, have been shown to have cytotoxicity against murine and human tumour cells (Lewis lung carcinoma (LLC), T-47D, Sarcoma 180, and Meth-A tumour cell lines) (Gao et al. 2002).

Polysaccharide fractions of Ganoderma lucidum have been shown to have potent immunomodulating effects in pre-clinical trials. A clinical study of healthy volunteers demonstrated that G. lucidum did not affect their immune functions. Subsequently, an open-labeled study (i.e. not double blind or placebo controlled) aimed to evaluate the effects of water-soluble G. lucidum polysaccharides (Ganopoly) in patients with advanced colorectal cancer. Forty-seven patients were enrolled and treated with Ganopoly at 5.4 g/day for 12 weeks. In 41 assessable cancer patients, treatment with Ganopoly tended to increase mitogenic reactivity to phytohemagglutinin (Gao et al. 2005). Larger double blind trials are required to show if this is a real effect.

High immunomodulatory and protective effects against sarcoma 180 in mice fed with Ling Zhi or Reishi mushroom Ganoderma lucidum (W. Curt.: Fr.) P. Karst. (Aphyllophoromycetideae) mycelium has also been recently reported (Rubel et al. 2008). Phenolic compounds present in mushroom extracts from G. lucidum have also been shown to strongly

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generate reactive oxygen species (suggesting immunomodulatory effects) in human PBMCs and K 562 cells in vitro (Wei et al. 2008).

The polysaccharide (PS) fractions from several medicinal herbs have been reported to have anti-ulcer effects against experimental ulcers in the rat. The water-soluble PS fractions from Ganoderma lucidum (Reishi mushroom) have been shown to inhibit indomethacin-induced gastric mucosal lesions in rats. The effect of the PS fraction from G. lucidum on the healing of gastric ulcers induced by acetic acid in the rat has subsequently been studied. The results indicated that oral administration of G. lucidum PS at 0.5 and 1.0g/kg for 2 weeks caused a significant acceleration of ulcer healing by 40.1% and 55.9%, respectively. In mechanistic studies, additional rats were treated with 10M acetic acid to induce acute ulcers, and then treated with G. lucidum PS (1.0g/kg) for 3, 7, 10, or 14 days. Treatment with G. lucidum PS at 1.0 g/kg significantly suppressed or restored the decreased gastric mucus levels and increased gastric prostaglandin concentrations compared with the control group. The results indicated that G. lucidum PS is an active component with healing efficacy on acetic acid-induced ulcers in the rat, which may represent a useful preparation for the prevention and treatment of peptic ulcers (Gao et al. 2004).

Ganoderma lucidum, as well as Phellinus rimosus, Pleurotus florida and Pleurotus pulmonaris, have been reported to have significant antioxidant activities (Ajith and Janardhanan, 2007).

Cholesterol-lowering properties of Ganoderma lucidum have been demonstrated in vitro, ex vivo, and in hamsters and mini-pigs. Organic fractions containing oxygenated lanosterol derivatives inhibited cholesterol synthesis in T9A4 hepatocytes. In hamsters, 5% Ganoderma lucidum did not affect low density lipoprotein (LDL) but decreased total cholesterol (TC) by 9.8%, and high density lipoprotein (HDL) by11.2%. Ganoderma lucidum (2.5 and 5%) had effects on several faecal neutral sterols and bile acids. In mini-pigs, 2.5% Ganoderma lucidum decreased TC, LDL- and HDL cholesterol 20, 27, and 18%, respectively, increased faecal cholestanol and coprostanol; and decreased cholate (Berger et al. 2004).

The hypolipidemic effect of the exo-biopolymer (EXBP) and endo-biopolymer (ENBP) produced from a submerged mycelial culture of Ganoderma lucidum has been investigated in dietary-induced hyperlipidemic rats. Hypolipidemic effects were achieved in both the EXBP- and ENBP-treated groups, however, the former proved to be more potent than the latter. The administration of the EXBP (100mg/kg body weight) substantially reduced the plasma total cholesterol, low-density

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lipoprotein (LDL) cholesterol, triglyceride, phospholipid levels, and atherogenic index by 31.0%, 39.0%, 35.4%, 28.1%, and 53.5%, respectively, when compared to the control group. The EXBP also lowered the liver total cholesterol, triglyceride, and phospholipid levels by 22.4%, 23.1%, and 12.9%, respectively. Furthermore, the high-density lipoprotein (HDL) cholesterol and ratio of HDL cholesterol to total cholesterol were significantly increased (Yang et al. 2002a).

Possible immuno-modulating effects of Ganoderma lucidum mycelium extract (GL-M) and spore extracts on human immune cells have been studied. Dendritic cells (DCs) are antigen-presenting cells and their role in DC-based tumour vaccines has been well defined. The differential effect of GL-M and GL spore extract (GL-S) on proliferation and Th1/Th2 cytokine mRNA expression of human peripheral blood mononuclear cells (PBMCs) and monocytes has been evaluated. The effects on the phenotypic and functional maturation of human monocyte-derived DCs were also investigated. GL-M induced the proliferation of PBMCs and monocytes, whereas GL-S showed a mild suppressive effect. Both extracts stimulated Th1 and Th2 cytokine mRNA expression, but GL-M was a relatively stronger Th1 stimulator. In contrast to GL-S, GL-M enhanced maturation of DCs in terms of up-regulation of CD40, CD80, and CD86, and also reduced fluorescein isothiocyanate-dextran endocytosis. Interestingly, GL-M-treated DCs only modestly enhanced lymphocyte proliferation in allogenic mixed lymphocyte culture with mild enhancement in Th development. The data provide evidence that GL-M has immuno-modulating effects on human immune cells and may be of use as a natural adjuvant for cancer immunotherapy with dendritic cells (Chan et al. 2005).

A polysaccharide purified from Ganoderma lucidum has also been shown to induce gene expression changes in human dendritic cells and promotes T helper 1 immune response in BALB/c mice (Lin et al. 2006).

Ganoderma lucidum mycelia (0.2-1.6mg/ml) have also been reported to stimulate tumour necrosis factor-alpha (TNF-alpha) and IL (interleukin)-6 production after 8h of treatment in human whole blood. IFN (interferon)-gamma release from human whole blood was also enhanced after 3 days of culture with Ganoderma lucidum mycelia (0.2-1.0mg/ml). However, Ganoderma lucidum mycelia did not potentiate nitric oxide production in RAW264.7 cells. An electrophoretic mobility shift assay revealed that the Ganoderma lucidum mycelia (1.6mg/ml) activated kappaB DNA binding activity in RAW264.7 cells. These results provide supporting evidence for the immunomodulatory effect of Ganoderma lucidum mycelia (Kuo et al. 2006).

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An extract from Ganoderma lucidum has been reported to have apoptotic and anti-inflammatory functions in HT-29 human colonic carcinoma cells. Ling Zhi extract (LZE) is a herbal mushroom preparation that has been shown to induce apoptosis anti-inflammatory action and differential cytokine expression during induced inflammation in the human colonic carcinoma cell line, HT-29. The extract caused no cytotoxicity in HT-29 cells at doses less than 10,000 mg/ml. Increasing concentrations reduced prostaglandin E2 production, but increased nitric oxide production. LZE treatment induced apoptosis by increasing the activity of caspase-3. RT-PCR showed that LZE at a concentration of 5,000mg/ml decreased the expression of cyclooxygenase-2 mRNA. Among 42 cytokines tested by protein array in this study, supplementation of LZE at doses of 500 and 5,000mg/ml to HT-29 cells reduced the expression of interleukin-8, macrophage inflammatory protein 1-delta, vascular epithelial growth factor, and platelet-derived growth factor. These results suggest that LZE has pro-apoptotic and anti-inflammatory functions, as well as inhibitory effects on cytokine expression during early inflammation in colonic carcinoma cells (Hong et al. 2004).

The potential of an Ganoderma lucidum extract as a radioprotector and antioxidant defense against oxygen radical-mediated damage has been studied and it was demonstrated that a hot-water extract of Ganoderma lucidum had good radioprotective ability, as well as protection against DNA damage induced by metal-catalyzed Fenton reactions and UV irradiation, although the evidence was based on in vitro tests using isolated DNA. It was also found that the water-soluble polysaccharide isolated from the fruit body of Ganoderma lucidum was as effective as a hot-water extract in protecting against hydroxyl radical-induced DNA strand breaks, indicating that the polysaccharide compound is associated with the protective properties (Kim and Kim, 1999).

The evidence for the anti-cancer effects of Ganoderma lucidum has been reviewed (Yuen and Gohel, 2005), while the active compounds in G. lucidum and their effects have also recently been reviewed (Boh et al. 2007).

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Grifola frondosa (Maitake)

A fraction extracted from Grifola frondosa (Maitake, GF-D) and its combination with human interferon alpha-2b (IFN) has been investigated for an inhibitory effect on hepatitis B virus (HBV) in HepG2 2.2.15 cells (2.2.15 cells). HBV DNA and viral antigens were analyzed by a quantitative real-time polymerase chain reaction and end-point titration in radioimmunoassays, respectively. The results showed that GF-

D or IFN alone could inhibit HBV DNA in the cells with the 50% inhibitory concentration (IC50) of 0.59mg/ml and 1399 IU/ml, respectively. The combination of GF-D and IFN for anti-HBV activity was also evaluated and it was found that they synergistically inhibited HBV replication in 2.2.15 cells. In combination with 0.45mg/ml GF-D, the apparent IC50 value for IFN was 154 IU/ml. This 9-fold increase in anti-viral activity of IFN suggested that GF-D could synergize with IFN. The results indicate that the Grifola frondosa extract, in combination with human interferon alpha-2b, might provide a potentially effective therapy against chronic hepatitis B virus infections (Gu et al. 2006).

A further study by the same group has recently reported the purification of an anti-viral protein from an extract of Grifola frondosa (Maitake) fruiting bodies. The protein inhibited herpes simplex virus type 1 (HSV-1) replication in vitro with an IC50 value of 4.1mg/ml and a therapeutic index >29.3. Higher concentrations (125 and 500 mg/ml) also significantly reduced the severity of HSV-1 induced blepharitis, neovascularization, and stromal keratitis in a murine model. Topical administration of the protein to the mouse cornea resulted in a significant decrease in virus production. It was reported that the protein directly inactivated HSV-1 while simultaneously inhibiting HSV-1 penetration into Vero cells. The N (amino)-terminal sequence of the protein consisted of an 11 amino acid peptide, NH2-REQDNAPCGLN-COOH that did not match any known amino acid sequences, indicating that the protein is likely to be a novel anti-viral protein (Gu et al. 2007).

Maitake D-fraction is a polysaccharide extracted from the Maitake mushroom (Grifola frondosa S.F. Gray). Using normal C3H/Hej mice, its effects on the natural immune system, including macrophages, dendritic cells, and natural killer (NK) cells, have been investigated. NK cells attack cells infected with pathogens such as bacteria and viruses and produce cytokines, such as interferon-gamma (IFN-g), that can modulate natural

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and specific immune responses. D-Fraction was administered to the mice intraperitoneally for 3 consecutive days. Spleen cells containing macrophages and dendritic cells were then cultured and the culture supernatants were analyzed for IL-12. The results indicated that the D-fraction stimulated the natural immunity related to the activation of NK cells indirectly through IL-12 produced by macrophages and dendritic cells, and hence administration of D-fraction to healthy individuals may serve to prevent infection (Kodama et al. 2003).

A study has also suggested that oral administration of a submerged cultivated G. frondosa mixture, by normal mice, may enhance host innate immunity against foreign pathogens without eliciting an adverse inflammatory response (Wang et al. 2008).

Anti-tumour activity induced by an extract from Grifola frondosa in a macrophage cell line, RAW264.7 has been reported to be mediated via a nitric oxide-mediated pathway (Sanzen et al. 2001). Similarly, an aqueous extract from Grifola frondosa has been reported to have immuno-modulating properties via a mechanism involving the regulation of nitric oxide (NO) production both in vivo and in vitro (Cao et al. 2006).

The changes in the content of anti-tumour polysaccharides from Grifola frondosa during storage have been investigated. When the mushrooms were stored at low temperature, the content of the anti-tumour polysaccharides showed hardly any changes, but the content decreased markedly at higher temperature (20ºC) (Mizuno, 2000).

The photo-protective potential of exopolysaccharides (EPS) from Grifola frondosa HB0071 has been tested in human dermal fibroblasts (HDF) exposed to ultraviolet-A (UVA) light. It was reported that EPS had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDF without any significant cytotoxicity. The treatment of UVA-irradiated HDF with EPS resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. The data suggested that EPS obtained from a mycelial culture of Grifola frondosa HB0071 may contribute to an inhibitory action in photo-ageing skin by reducing the MMP 1-related matrix degradation system (Bae et al. 2005).

The biological function of GFPPS1b, a polysaccharide-peptide isolated from cultured mycelia of Grifola frondosa GF9801, has been shown to suppress SGC-7901 cell growth and reduce cell survival via arresting the cell cycle in the G(2)/M phase and inducing apoptosis of tumour cells (Cui et al. 2007).

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The potential anti-tumour effect of beta-glucan, a polysaccharide of the Maitake mushroom, on human prostatic cancer PC-3 cells in vitro has been evaluated. The data showed that a bioactive beta-glucan from the Maitake mushroom has a cytotoxic effect, presumably through oxidative stress, on prostatic cancer cells in vitro, leading to apoptosis. This mushroom polysaccharide may therefore have potential as a therapeutic modality for prostate cancer (Fullerton et al. 2000). Maitake beta-glucan has also been shown to induce hematopoietic stem cell proliferation (Lin et al. 2007).

A beta-glucan extracted from the fruiting body of Grifola frondosa has been reported to activate cellular immunity and expresses anti-tumour effects, with the anti-tumour effects relating to its control of the balance between T lymphocyte subsets Th-1 and Th-2. The fraction decreased the activation of B cells and potentiated the activation of helper T cells, resulting in enhanced cellular immunity. It also induced the production of interferon (IFN)-gamma, interleukin (IL)-12 p70, and IL-18 by whole spleen cells and lymph node cells, but suppressed that of IL-4. These results suggest that this fraction establishes Th-1 dominance which induces cellular immunity in the population that was Th-2 dominant due to carcinoma (Inoue et al. 2002).

The anti-diabetic effect of an alpha-glucan (MT-alpha-glucan) from the fruit body of Maitake mushrooms (Grifola frondosa) on KK-Ay mice (a type 2 diabetes animal model) has been evaluated. Treatment with MT-alpha-glucan significantly decreased body weight, level of fasting plasma glucose, glycosylated serum protein, serum insulin, triglycerides, cholesterol, free fatty acid and malondialdehyde content in liver. Treatment with MT-alpha-glucan significantly increased the content of hepatic glycogen, reduced glutathione and the activity of liver superoxide dismutase and glutathione peroxidase. Furthermore, the insulin binding capacity to liver crude plasma membranes increased and histopathological changes in the pancreas were ameliorated in the treatment group. The data suggested that MT-alpha-glucan has an anti-diabetic effect on KK-Ay mice, which may be related to its effect on insulin receptors by increasing insulin sensitivity and ameliorating insulin resistance of peripheral target tissues (Lei et al. 2007).

Enhanced insulin-hypoglycaemic activity (improvement in insulin sensitivity) has also been reported in spontaneously hypertensive rats consuming a glycoprotein extracted from Maitake mushrooms (Preuss et al. 2007).

The effect of Grifola frondosa total water extract on two osteoblastic cell cultures (HOS58 and SaOS-2) has been investigated to determine if this

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mushroom has osteo-inductive properties. The activity of alkaline phosphate and mineralization were used as indicators for the vitality and maturation of bone cells. The cultivation of human osteosarcoma cells HOS58 for 5 days in the presence of an aqueous extract of G. frondosa resulted in a significant elevation of alkaline phosphatase activity of the cells in comparison to untreated cells. SaOS-2 cells, incubated with GFfor 21 days, showed a nearly two-fold higher mineralization than cells cultured with a positive control, demonstrating the activity of Grifola frondosa extract as a bone-inducing agent (Saif et al. 2007).

Plasma cholesterol concentration in rats has been shown to be reduced by feeding of mushroom Maitake (Grifola frondosa) fiber. The results demonstrated that mushroom fiber lowered the serum total cholesterol level by enhancement of the hepatic low density lipoprotein (LDL) receptor mRNA (Fukushima et al. 2001).

Maitake mushroom consumption has also been shown, in Sprague-Dawley rats, to have the ability to alter lipid metabolism by inhibiting both the accumulation of liver lipids and the elevation of serum lipids. Further studies are needed to determine the mechanism of activity of Maitake mushrooms and to establish whether their action in humans is similar to that observed in the rat model (Kubo and Nanba, 1996). A further study by the same group, using the same rat model system, has also shown that consumption of dried Maitake powder (mixed with a basic high-cholesterol rat chow) cholesterol, triglyceride and phospholipids in the serum of rats in the Maitake-feed group were suppressed by 0.3-0.8 times those in animals fed the basic feed.

Weights of liver and epididymal fat-pads were significantly lower (0.6-0.7 times) than those in the basic feed group, indicating that Maitake inhibited lipid accumulation in the body. Liver lipids were also measured and the values were found to be decreased by Maitake administration. Measurement of the amount of total cholesterol and bile acid in faeces showed the ratio of cholesterol-excretion had increased 1.8 fold and bile acid-excretion 3 fold by Maitake treatment suggesting that Maitake consumption may help to improve the lipid metabolism as it inhibits both liver lipid and serum lipids which were increased by the ingestion of high-fat feed (Kubo and Nanba, 1997).

Blood pressure of spontaneously hypertensive rats (SHR) has been shown to be significantly reduced by Maitake feeding for 8 weeks, beginning at a time when the animals were 10 weeks of age with well-established high blood pressure. There was no difference in the plasma total and free cholesterol, triglyceride and phospholipid levels between the Maitake fed animals and the control (Kabir and Kimura, 1989).

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A single oral dose of an extract of powdered Grifola frondosa, at dosage levels of 500 and 2,000mg/kg has been given to 5 Crj:CD(SD) IGS strain of rats of each sex for 1 day, and its toxicity was examined. The control group was treated with water by injection. No abnormal signs were noted in either sex of any group. No effects of powdered Grifola Frondosa were reported in either sex by body weight measurement or necropsy finding (Koike et al. 2003).

Hexane extracts of the cultured mycelia of Grifola frondosa have been shown to contain ergosterol (1), ergostra-4,6,8(14),22-tetraen-3-one (2), and 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (3) and a fatty acid fraction containing palmitic, oleic, and linoleic acids. The fatty acid fraction and compounds 1-3 showed cyclooxygenase (COX) enzyme inhibitory and antioxidant activities. The inhibition of COX-1 enzyme by the fatty acid fraction and compounds 1-3 at 250mg/mL were 98, 37, 55, and 67%, respectively. Similarly, COX-2 enzyme activity was reduced by the fatty acid fraction and compounds 1-3 at 250mg/mL by 99, 37, 70, and 4%, respectively. The inhibition of liposome peroxidation by the fatty acid fraction and compounds 1 and 2 at 100 mg/mL was 79, 48, and 42%, respectively (Zhang et al. 2002).

The active compounds in Grifola frondosa (Maitake) and their effects have recently been reviewed (Boh and Berovic, 2007).

Hericium erinaceum (Pom Pom, Lion’s Mane)

Anti-dementia compounds have recently been reported from the mushroom Hericium erinaceum (Kawagishi, 2007; Kawagishi and Zhuang, 2008). Hericium erinaceum extracts have also been reported to induce neurite outgrowth from neural (NG108-15) cells. Maximum stimulation of neurite outgrowth was recorded with mycelial extracts, and the least stimulation was observed with an oven-dried fruit body extract. Aqueous

extracts of H. erinaceus therefore contain neuroactive compounds that stimulate neurite outgrowth in vitro suggesting some value in the treatment of neurodegenerative disease (Wong et al. 2007)

Immuno-regulatory functions of Hericium erinaceum have been demonstrated in an aqueous extract by a stimulation of inducible nitric oxide gene expression followed by nitric oxide production in macrophages

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via enhancement of activation of the transcription factor, NF-kappaB (Son et al. 2006).

Polysaccharides from Hericium erinaceum and Hericium laciniatum have been extracted from culture broth and the polysaccharide components were mainly glucose in H. erinaceus and galactose in H. laciniatum. Both polysaccharides had significant anti-artificial pulmonary metastatic tumour effects in mice with the polysaccharide from H. erinaceus being more effective than that from H. laciniatum. However, both of the polysaccharides enhanced the increase of T cells and macrophages (immuno-enhancing activity) (Wang et al. 2001).

The hypolipidemic effect of exo-polymers produced in submerged mycelial cultures of Hericium erinaceus (HE), Auricularia auricula judae (AA), Flammulina velutipes (FV), Phellinus pini (PP), and Grifola frondosa (GF) has been investigated in dietary-induced hyperlipidemic rats. The animals were administered with exo-polymers at the level of 100mg/kg body weight daily for four weeks. A hypolipidemic effect was achieved in all the experimental groups, however, HE exo-polymer proved to be the most potent, significantly reducing plasma triglyceride (28.9%), total cholesterol (29.7%), low-density lipoprotein (LDL) cholesterol (39.6%), phospholipid (16.0%), and liver total cholesterol (28.9%) levels, compared to the saline administered (control) group. The results demonstrated the potential of Hericium erinaceus exo-polymer in treating hyperlipidemia in dietary-induced hyperlipidemic rat (Yang et al. 2002c; Yang et al. 2003).

A methanol extract of the fruiting bodies of Hericium erinaceus has been fed to rats and shown to result in a significantly lower elevation rate of blood glucose level than control rats. The effects on blood glucose, serum triglyceride and total cholesterol levels were more significant in the rats fed daily with the Hericium erinaceus extract at doses of 100mg/kg body weight rather than 20mg/kg body weight (Wang et al. 2005).

Hypsizigus marmoreus (Buna-shimeji)

Anti-tubercular activity and an inhibitory effect on Epstein-Barr virus activation of sterols and polyisoprenepolyols from an edible mushroom, Hypsizigus marmoreus (Buna-shimeji) have been reported. Seven sterols and eight polyisoprenepolyols, isolated from the non-saponifiable lipid fraction of the

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dichloromethane extract of Hypsizigus marmoreus, have been tested for their anti-tubercular activity against Mycobacterium tuberculosis strain H37Rv using the Microplate Alamar Blue Assay (MABA). Six of the sterols and two of the polyisoprenepolyols showed a minimum inhibitory concentration (MIC) in the range of 1-51 mg/ml, while the others were inactive. The seven sterols and three polyisoprenepolyols were further evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. Two of the sterols showed a potent inhibitory effect while preserving the high viability of the Raji cells (Akihisa et al. 2005).

Anti-tumour-promoting activity has been found in methanol and ethyl acetate extracts of the mushroom 'Buna-shimeji', Hypsizigus marmoreus (Tricholomataceae). From the active fractions of the extracts, two sterols, ergosterol and ergosterol peroxide, were isolated. The isolates showed inhibitory activity against 12-O-tetradecanoylphorbol-13-acetate induced ear inflammation in mice, and ergosterol markedly inhibited tumour promotion in a two-stage carcinogenesis experiment (Yasukawa et al. 1994).

Aqueous and methanol extracts of Hypsizigus marmoreus have been tested against allogeneic tumour, solid sarcoma 180 and syngeneic tumour and Meth A fibrosarcoma. The aqueous extract was highly active in inhibiting growth of solid sarcoma 180, but not as effective for Meth A fibrosarcoma. Fractionation of anti-tumour substances of the aqueous extract isolated four polysaccharides. Chemical analysis revealed one of them to be beta-(1-3)-glucan with a significant inhibitory effect against tumour-growth of sarcoma 180 (Ikekawa et al. 1992).

Chloroform extracts of the fruit bodies of Hypsizigus marmoreus have been shown to exhibit moderate cytotoxicity towards cultured human colon carcinoma (HT-29), human breast carcinoma (MCF-7) and human hepatoblastoma (HepG-2) cell lines (Xu et al. 2007).

Anti-proliferative activities of fractions of Hypsizigus marmoreus have been examined using HepG2 cells in vitro. The methanol extract of H.marmoreus markedly induced anti-proliferative activity and an active compound from this mushroom has been identified as hypsiziprenol A9. Hypsiziprenol A9 inhibited cell proliferation in a time- and concentration-dependent manner by up to 80% on HepG2 cells by inducing arrest of the G1 phase. Hypsiziprenol A9 also decreased expression of phosphorylated retinoblastoma protein (ppRb), cyclin D1, and cyclin E in a dose-dependent manner. The results suggested that hypsiziprenol A9 can

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inhibit the growth of HepG2 cells through inducing G1 phase cell cycle arrest due to the inhibition of pRb phosphorylation (Chang et al. 2004).

A novel ribonuclease, from fresh fruiting bodies of Hypsizigus marmoreus,with anti-proliferative activity against the L1210 leukemia cell line has also been purified (Guan et al. 2007). A thermostable ribosome-inactivating protein with a molecular weight of 20kDa, isolated from fruiting bodies of Hypsizigus marmoreus has also been shown to have anti-proliferative activity against mouse leukemia cells and human leukemia and hepatoma cells (Lam and Ng, 2001).

The antioxidant effects of Hypsizygus marmoreus have been studied for peroxyl and alkoxyl radicals by ordinary, non-tumour-bearing and tumour-bearing mice. Oral administration of the fruit body of H. marmoreus exhibited potent anti-tumour or cancer-preventive effects and caused a significant decrease in lipid peroxide levels, which were determined as thiobarbituric acid reactive substances. These results showed that the intake of H. marmoreus fruit body could induce an antioxidant effect, and the increase of antioxidant activity in the plasma of tumour-bearing mice was an important mechanism in cancer prevention. It was also suggested that the mushroom might play a role in the decrease of lipid peroxides through antioxidant activity induction (Matsuzawa, 2006).

Proliferation of human leukemic U937 cells has been shown to be significantly inhibited by conditioned medium of human peripheral blood mononuclear cells stimulated with cold-water extracts (10-800 mg/mL of medium) of Hypsizigus mamoreus, Agrocybe aegerite and Flammulina velutipes (Ou et al. 2005).

The isolation of a collagen-binding protein from Hypsizigus marmoreus, which inhibits the Lewis lung carcinoma cell adhesion to type IV collagen has been reported. A type IV collagen-binding protein of 23kDa was isolated from the mushroom, Hypsizigus marmoreus. This protein, HM 23, bound to type IV and type I collagens and gelatin, and to much lesser extent to fibronectin, but not to laminin or bovine serum albumin. The adhesion of Lewis lung carcinoma cells was inhibited when the type IV collagen substratum was pretreated with HM 23 (Tsuchida et al. 1995).

Bunashimeji-related hypersensitivity pneumonitis has been reported in workers who cultivate this mushroom in indoor facilities in Japan. An evaluation of protective measures concluded that complete cessation was the best treatment for hypersensitivity pneumonitis. The use of a mask was ineffective for patients with a high serum Krebs von der Lungen-6 (KL-6), surfactant protein-D (SP-D) concentration and severe ground-

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glass opacity on chest high-resolution computed tomography. Initial treatment with oral prednisolone was recommended for patients with high levels of total cell counts in bronchoalveolar lavage fluid (Tsushima et al. 2006).

Lentinula edodes (Shiitake)

Extracts from Lentinula edodes (Shiitake) have been widely reported to have anti-tumour activity. However, this activity has been shown to be host-mediated and not by direct cytotoxic activity to cancer cells. A very recent study (Israilides et al. 2008) has now demonstrated cytotoxic and cell growth inhibitory (cytostatic) effects of aqueous extracts of the mushroom on the MCF-7 human breast adenocarcinoma cell line. The effect was demonstrated with fruit body and mycelial extracts, the difference being that there was no significant suppression on normal

cells with the latter. Furthermore, mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively low IC50 values and the simultaneous higher respective values on normal fibroblast cells. In addition to the direct inhibition of the proliferation of human breast cancer cells in vitro, the Lentinula edodes extract had immunostimulatory properties in terms of mitogenic and co-mitogenic activity in vitro.

The effects of diets containing dry powdered Shiitake mushroom on frequency of azoxymethane-induced colon aberrant crypt foci (ACF) and intestinal tumours in male Sprague-Dawley rats have been studied. Pregnant rat dams and their progeny were fed AIN-93G diets containing casein (20%; control diet) or casein supplemented with Shiitake (1% or 4% wt/wt). Casein- and 1% Shiitake-fed rats exhibited identical growth curves, whereas those fed the 4% Shiitake diet were of slightly reduced body weight. The 4% Shiitake diet elicited increased active energy expenditure and reduced adiposity of rats. Small bowel and colon tumours and colon ACF were evaluated in the male progeny at 18 weeks after azoxymethane treatment. Aberrant crypt foci and tumours were most prevalent in the mid and distal regions of the colon. Shiitake intake had no effect on the relative incidence of tumours in the colon or small intestine (duodenum). Consumption of 1% Shiitake stimulated growth of invasive adenocarcinomas in the mid colon and favoured a non-significant

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increase in median frequency of ACF in this same region. In contrast, Shiitake at 4% intake elicited a reduction in colon tumour multiplicity. The authors suggested a stimulatory action of 1% Shiitake on rat colon tumourigenesis which is puzzling as the data were not statistically significant. However, the inhibitory actions of 4% Shiitake mushroom on the indices of rat colon tumourigenesis where statistically validated (Frank et al. 2006).

Shiitake extracts have been dispersed with lecithin micelles to prepare superfine particles (0.05 to 0.2 microns in diameter) of beta-1,3-glucan (micellary mushroom extracts). When mice were fed with these micelles of beta-glucan (0.75mg/day/mouse, smaller amounts of beta-glucan), the number of lymphocytes yielded by the small intestine increased by up to 40% and tumour cytotoxicity against P815 cells and cytokine production was also augmented, suggesting that smaller amounts of micellary beta-glucan might be useful for the potentiation of intestinal immunity (Shen et al. 2007).

The effects of protein-bound polysaccharides (A-PBP and L-PBP), extracted from the mycelia of Agaricus blazei and Lentinus edodes, on serum cholesterol and body weight have been investigated in 90 female volunteers. The data demonstrated a weight-controlling and hypolipidemic effect of both A-PBP and L-PBP via a mechanism involving absorption of cholesterol (Kweon et al. 2002).

The effect of Shiitake (Lentinus edodes, LE) and autolyzed- (fermented-) Shiitake (autolyzed-LE) on blood pressure and serum fat levels of spontaneously hypertensive rats (SHR) have been studied. The animals of the autolyzed-LE group showed significantly lower blood pressure compared to the control or LE group. The serum levels of total cholesterol (TC), triglyceride and phospholipid of the groups fed with LE and autolyzed-LE were lower than those of the control group, and atherogenic index [(TC-HDL-C)/HDL-C] improved significantly in 21 days. It was suggested that the serum TC decline is the action of eritadenine that is contained in the Shiitake mushroom. An inhibitory activity of the angiotensin I-converting enzyme (ACE) was compared between of LE and autolyzed-LE. Autolyzed-LE showed higher inhibitory activity than LE against the ACE. The results suggested that the hypotensive action of autolyzed-LE was due to concomitant ACE inhibitory activities of peptides and gamma-aminobutyric acid contained in higher amounts during the autolysis of LE (Watanabe et al. 2002).

The changes in the content of an anti-tumour polysaccharide from Lentinus edodes (lentinan) during storage have been investigated. When the mushrooms were stored at low temperature, the content of their anti-

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tumour polysaccharides showed hardly any changes, but the content decreased markedly at higher temperature (20ºC) (Mizuno, 2000).

The Shiitake mushroom (Lentinus edodes) contains the hypocholesterolemic agent eritadenine, 2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid. A study has recently been conducted to quantify the amount of the cholesterol reducing agent eritadenine in Shiitake mushrooms. The amounts of eritadenine in the fruit bodies of four different shiitake mushrooms, Le-1, Le-2, Le-A, and Le-B, were investigated. Methanol extraction was used to recover as much eritadenine as possible from the fungal cells, and enzymes that degrade the fungal cell walls were also used to elucidate if the extraction could be further enhanced. The Shiitake strains under investigation exhibited up to 10-fold higher levels of eritadenine than previously reported for other Shiitake strains. Pre-treatment of mushrooms with hydrolytic enzymes before methanol extraction resulted in an insignificant increase in the amount of eritadenine released. The results suggested the potential for delivery of therapeutic amounts of eritadenine from the ingestion of extracts or dried concentrates of Shiitake mushroom strains (Enman et al. 2007).

Plasma cholesterol concentration in rats has been shown to be reduced by feeding of mushroom Shiitake (Lentinus edodes) fiber. The results demonstrated that mushroom fiber lowered the serum total cholesterol level by enhancement of the hepatic low density lipoprotein (LDL) receptor mRNA (Fukushima et al. 2001).

Methanol and water extracts from Lentinus edodes have been shown to have antioxidant activity against lipid peroxidation of rat brain homogenate. The antioxidant activity against lipid peroxidation was found to correlate with the phenolic content in different sub-fractions of the mushroom extracts (Cheung and Cheung, 2005).

The effect of heat treatment on the changes in the overall antioxidant activity and polyphenolic compounds of Shiitake extract has been investigated. Raw Shiitake was heated at 100 and 121ºC for 15 or 30 min using an autoclave. After heat treatment, the free and bound polyphenolics and flavonoids in the mushroom extracts were analyzed. 2,2-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical and 1,1-diphenyl-2-picrythydrazyl (DPPH) radical scavenging activities were measured to evaluate antioxidant activity of the extracts. The polyphenolic contents and antioxidant activities in the extracts increased as heating temperature and time increased. The free polyphenolic content in the extract heated at 121ºC for 30 min was increased by 1.9-fold compared to that in the extract from the raw sample. The ABTS and

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DPPH radical scavenging activities were increased by 2.0-fold and 2.2-fold compared to the raw sample, respectively. The data showed that heat treatment significantly enhanced the overall antioxidant activities of Shiitake mushrooms (Choi et al. 2006a).

High-molecular-weight polysaccharides (HMWP), including lentinan, in Shiitake mushrooms may promote human health. A study has been conducted to determine if management protocols influence the HMWP of Shiitake (Lentinula edodes (Berk.) Pegler) mushrooms. The results indicated that measuring the total carbohydrate content of water-extractable, ethanol-insoluble polysaccharides was a simple way to estimate HMWP. The results also indicated that log-grown Shiitake contained more HMWP than did substrate-grown Shiitake. Among log-grown Shiitake, both mushroom strain and tree species influenced HMWP content. The results suggested that there is considerable variation among Shiitake mushrooms in HMWP content and that production protocols influenced the HMWP content of mushrooms (Brauer et al. 2002).

Supplemental amounts of a polysaccharide/oligosaccharide complex obtained from a Shiitake mushroom extract have been evaluated for the ability to lower the prostate-specific antigen level in patients (n=62) with prostate cancer. The data showed that the Shiitake mushroom extract alone was ineffective in the treatment of clinical prostate cancer (White et al. 2002). A high genistein, Shiitake mushroom extract has also been reported to have anti-tumour effects on prostate cancer (Hackman et al. 2001).

An ethyl acetate fraction from Shiitake (Lentinus edodes) mushrooms has been investigated using two human breast carcinoma cell lines (MDA-MB-453 and MCF-7), one human non-malignant breast epithelial cell line (MCF-10F), and two myeloma cell lines (RPMI-8226 and IM-9). Concentration-dependent anti-proliferative effects of the fraction were observed in all cell lines. Approximately 50mg/L of the fraction induced apoptosis in 50% of the population of four human tumour cell lines and the fraction-induced apoptosis may have been mediated through the pro-apoptotic bax protein which was up-regulated. Cell cycle analysis revealed that the fraction induced cell cycle arrest by a significant decrease of the S phase, which was associated with the induction of cdk inhibitors (p21) and the suppression of cdk4 and cyclin D1 activity. Compared to malignant tumour cells, non-malignant cells were less sensitive to the fraction for the suppression of cell growth and regulation of bax, p21, cyclin D1, and cdk4 expression. A 51% anti-proliferative effect occurred at the highest concentration of the fraction (800mg/L). The data suggest that inhibition of growth in tumour cells by the Shiitake

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mushroom extract may result from an induction of apoptosis (Fang et al. 2006).

The isolation and characterization of an anti-tumour polysaccharide, KS-2, extracted from culture mycelia of Lentinus edodes has also been reported. KS-2 suppressed the growth of EHRLICH as well as Sarcoma-180 tumours in mice when given either orally or intraperitoneally (Fujii et al. 1978).

Extracts from fermentation broth and mycelium of 15 strains of Lentinus edodes have been shown to be active against gram positive and gram negative bacteria, yeasts and mycelial fungi, including dermatophytes and phytopathogens. The strains differed by the set of the organisms susceptible to the action of the extracts. Strains of L. edodes combining marked anti-bacterial properties and high yields of water soluble polysaccharides were screened. The active compounds were detected by preparative thin layer chromatography. Two were identified with UV- and mass spectrometry as lentinamycin B and erytadenine (lentinacin). Lentinamycin B was found to be the main component responsible for the anti-bacterial activity of the L. edodes strains (Soboleva et al. 2006).

The anti-microbial activity of the culture fluid of Lentinus edodes mycelium grown in submerged liquid culture has been demonstrated against Streptococcus pyogenes, Staphylococcus aureus and Bacillus megaterium. The substance responsible for the activity was heat-stable and was suggested to be lenthionine, an anti-bacterial and anti-fungal sulphur-containing compound (Hatvani, 2001).

A study on the action of lentinan (extracted from Shiitake mushrooms (Lentinus edodes) has been conducted using murine lymphoma (K36) cells in a AKR mouse model. Further investigation on the effectiveness of the extracted lentinan was then performed using human colon-carcinoma cell lines in mice. Six established human colon-carcinoma cell lines segregated into three groups of different degrees of differentiation were used in this study. One group was not fed (control) and the second group was prefed with lentinan for 7 days prior to inoculations with the cancer cells. The size of the tumours that developed was rated after 1 month. Significant regression in tumour formation was observed in prefed mice compared to control (unfed) mice when K36 or human colon-carcinoma cells were used. Significant reductions in the size of the tumours were observed in mice prefed with lentinan. Follow-up investigation proceeded with the use of nude mice (athymic). Lymphocytes extracted from AKR mice prefed with lentinan for 7 days were inoculated into the nude mice. This was then followed by inoculation of the human colon-carcinoma cell lines into these mice. Much smaller tumours were formed in nude mice

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inoculated with lymphocytes, in contrast to the larger tumour formed in nude mice without lymphocyte inoculation. The study concluded that the anti-tumour property of lentinan was maintained with oral administration. In addition, "primed" lymphocytes, when given passively to immuno-deficient mice, were able to retard the development of tumours in these mice (Ng and Yap, 2002).

The hypoglycemic effect of exo-polymers (EPs) produced from submerged mycelial cultures of five varieties of mushrooms on streptozotocin (STZ)-induced diabetic rats have been investigated. The five experimental groups were fed with EPs (50mg/kg body weight) for 7 days. Significant reductions in plasma glucose, total cholesterol (TC), and triglyceride (TG) levels were observed in rats fed with Lentinus edodes and Cordyceps militaris EPs. Plasma glucose and TC were also reduced by administration of Phellinus linteus EPs, but the TG level was not changed significantly. The EPs of the three mushroom species also demonstrated a marked reduction in the level of plasma glutamate-pyruvate transaminase (GPT). The result demonstrated the hypoglycemic activity of EPs of three mushroom varieties in STZ-induced diabetic rats and suggests some potential in the control of diabetes mellitus (Kim et al. 2001).

A subsequent study by the same group, using higher concentrations (200mg/kg body weight in streptozotocin-induced diabetic rats) of exo-polymers from a submerged mycelial culture of Lentinus edodes has shown that the administration of the exo-polymer reduced the plasma glucose level by as much as 21.5%, and increased plasma insulin by 22.1% compared to the control group. It was also shown to lower the plasma total cholesterol and triglyceride levels by 25.1 and 44.5%, respectively (Yang et al. 2002b).

Three anti-bacterial compounds extracted by chloroform, ethylacetate or water from dried Shiitake mushrooms (Lentinus edodes) have been reported which possessed efficient anti-bacterial activities against Streptococcus spp., Actinomyces spp., Lactobacillus spp., Prevotella spp., and Porphyromonas spp. of oral origin. In contrast, other general bacteria, such as Enterococcus spp., Staphylococcus spp., Escherichia spp., Bacillus spp., and Candida spp. were relatively resistant to these compounds. The anti-bacterial activity of chloroform extracts and ethylacetate extracts were relatively heat-stable, while the water extract was heat-labile (Hirasawa et al. 1999).

The action of the juice of Shiitake mushrooms (L. edodes) at a concentration of 5% from the volume of the nutrient medium was found to produce a pronounced anti-microbial effect with respect to Escherichia

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coli O-114, Staphylococcus aureus, Enterococcus faecalis, Candida albicans and to stimulate the growth of E. coli M-17. Bifidobacteria and Lactobacteria exhibited resistance to the action of L. edodes juice (Kuznetsov et al. 2005).

Shiitake dermatitis after the ingestion of raw Shiitake mushrooms has been reported, primarily in Japan, and it has been suggested that this dermatitis may be photosensitive as nearly half of the patients studied developed the dermatitis on skin exposed to sunlight (Hanada and Hashimoto, 1998). A study in Korea has also reported dermatitis effects, but in contrast to the previous reports in Japan, cases with Shiitake dermatitis occurred after eating boiled or cooked Shiitake mushrooms suggesting that a non-thermolabile factor/component may be involved (Ha et al. 2003).

A study has been conducted with 10 people where each participant ingested 4g of Shiitake powder daily for 10 weeks (trial 1), and the protocol was repeated in the same subjects after 3 to 6 months (trial 2). Gastrointestinal symptoms coincided with eosinophilia in two subjects. Symptoms and eosinophilia resolved after discontinuing Shiitake ingestion. The authors reported that daily ingestion of Shiitake mushroom powder in five of 10 healthy persons provoked blood eosinophilia, increased eosinophil granule proteins in serum and stool, and increased gastrointestinal symptoms (Levy et al. 1998).

A single oral dose of an extract of cultured Lentinus edodes mycelia, at dosage levels of 500 and 2,000mg/kg has been given to 5 Crj:CD(SD) IGS strain of rats of each sex for 1 day, and its toxicity was examined. The control group was treated with water by injection. No abnormal signs were noted in either sex of any group. No effects of Lentinus edodes mycelia were reported in either sex by body weight measurement or necropsy finding (Koike et al. 2002a). A follow on study by the same authors that extended the treatments for 28 days reported no effects in either sex by body weight measurement, food consumption measurement, urinalysis, ophthalmological examination, hematological examination, blood chemical analysis, necropsy finding, organ weight measurement, or histopathological examination (Koike et al. 2002b).

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Lyophyllum connatum

A new ergothioneine derivative, beta-hydroxyergothioneine has been isolated from the mushroom Lyophyllum connatum. Ergothioneine, N-hydroxy-N',N'-dimethylurea, and connatin (N-hydroxy-N',N'-dimethylcitrulline) were also isolated. All the compounds displayed the ability to scavenge free radicals, based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Structural determination, including the absolute stereochemistry of beta-hydroxyergothioneine, was achieved by spectroscopic analysis and X-ray crystallography. The radical scavenging activity of beta-

hydroxyergothioneine was almost the same as that of ergothioneine. Beta-hydroxyergothioneine showed the greatest protective activity against carbon tetrachloride-induced injury in primary culture hepatocytes (Kimura et al. 2005).

Phellinus linteus

Ethanol extracts of Phellinus linteus have been shown to have antioxidant activities comparable to Vitamin C in scavenging the stable free radical 1,1-diphenyl-2-picrylhyrazyl (DPPH). The extracts also inhibited lipid peroxidation (LPO) in a concentration-dependent manner. The study also reported anti-angiogenic activities of Phellinus linteus (Song et al. 2003).

Phellinus linteus has been reported to sensitise apoptosis induced by doxorubicin (an anti-cancer drug) in prostate cancer LNCaP cells suggesting that Phellinus linteus may have therapeutic potential to augment the magnitude of apoptosis induced by anti-prostate cancer drugs (Collins et al. 2006). Phellinus linteus has also been shown to mediate cell-cycle arrest at a low concentration and apoptosis in response to a high dose in mouse and human lung cancer cells (Guo et al. 2007).

Phellinus linteus has been reported to contain constituents that exhibit potent anti-tumour effects through activation of immune cells. A recent study in mice has reported that boiling water soluble fractions from

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mycelium of P.linteus contain anti-allergic and immuno-potentiating properties (Inagaki et al. 2005).

An acidic polysaccharide from Phellinus linteus has been shown to markedly inhibit melanoma cell metastasis in mice, and directly inhibit cancer cell adhesion to, and invasion through, the extracellular matrix, but that it had no direct effect on cancer cell growth. In addition, the authors reported that PL increased macrophage NO production. These results suggest that Phellinus linteus has two anti-metastatic functions - it acts as an immuno-potentiator and as a direct inhibitor of cancer cell adhesion (Han et al. 2006).

An extract from Phellinus linteus has been shown to have anti-inflammatory activity (Kim et al. 2004) via mediation of heme oxygenase-1 in an in vitro inflammation (macrophage) model (Kim et al. 2006).

The effect of a mushroom extract of Phellinus linteus on non-cancerous prostate cells using an experimentally developed rat benign prostatic hyperplasia model has been studied. The results showed that prostate weight increased significantly by 37% owing to treatment with the mushroom extract, and in particular, the stromal component of the prostate increased significantly by 80%. A suppression of transforming growth factor-beta1 expression by 56% was observed with the mushroom extract treatment. It was found that the mushroom extract enlarged the prostate and therefore administration of Phellinus linteus extract should be considered carefully by those with an enlarged prostate (Shibata et al. 2005).

Phellinus linteus has also been shown to suppress growth, angiogenesis and invasive behaviour of breast cancer cells (Sliva et al. 2008).

Phellinus robustus

It has been reported that melanins from the medicinal mushroom Phellinus robustus have high antioxidant and geno-protective properties (Babitskaya et al. 2007).

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Pholiota nameko (Nameko)

Hypersensitivity pneumonitis to spores of Pholiota nameko has been reported in a mushroom farmer, although separation from the antigen along with corticosteroid therapy, resulted in the symptoms and inflammatory effects quickly subsiding (Inage et al. 1996).

Pleurotus eryngii

The effects of Pleurotus eryngii extracts (PEX) on bone metabolism have been studied. PEX treatment showed an increase in the alkaline phosphatase activity of osteoblasts and in the osteocalcin mRNA expression from primary osteoblasts. PEX also increased the expression of the Runx2 gene, and the secretion of osteoprotegerin from the osteoblasts showed marked increases after treatment with PEX. In vivo studies, using rats with ovariectomy-induced

osteoporosis revealed that PEX alleviated the decrease in the trabecular bone mineral density (Kim et al. 2006).

The ergothioneine content of mushrooms has been reported to be in the range of 0.4-2.0mg/g (dry weight). The white Agaricus bisporus contained the least ergothioneine and portabellas (brown) contained the highest within the varieties of A. bisporus studied. The specialty mushrooms tested (Lentinus edodes, Pleurotus ostreatus, P. eryngii, Grifola frondosa) all contained a statistically significant greater amount of ergothioneine compared to A. bisporus, however, no significant difference was found between the specialty mushrooms (Dubost et al. 2006).

The antioxidative potency of commercially available mushrooms in Taiwan has been studied. The order of inhibitory activity of mushroom extracts on oxidation in emulsion system was Agaricus bisporus > Hypsizigus marmoreus > Volvariella volvacea > Flammulina velutipes > Pleurotus eryngii > Pleurotus ostreatus > Hericium erinaceus > Lentinula

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edodes. In a thermal oxidative stability test, using lard, the order of antioxidative activity of the mushroom extracts showed similar tendencies, except for the extract of Lentinula edodes (Fui et al. 2002).

Mycelial extracts (ethyl acetate and 70% ethanol) of 20 higher Basidiomycetes edible or medicinal mushrooms and culture media extracts (ethyl acetate and Butan-1-ol) have been evaluated for in vitro anti-inflammatory activity using the cyclooxygenase-1 and -2 enzymes (COX-1 and -2). In general, 70% ethanolic extracts showed lower inhibitory activity against both enzymes compared to ethyl acetate extracts. Of the mushrooms tested, the ethyl acetate extracts of Ganoderma applanata, Naematoloma sublateritium, Pleurotus eryngii, and P. salmoneostramineus showed the highest COX-2 inhibitory effects compared to COX-1 inhibition. Of the culture media tested in this study, only the ethyl acetate extracts of the culture medium of Agrocybe cylindracea exhibited high inhibition of the COX-2 enzyme (Elgorashi et al. 2008). Ceramide from Agrocybe aegerita has also been reported to inhibit the cyclooxygenase enzymes, COX-1 and -2 (Diyabalanage et al. 2008).

Hypersensitivity pneumonitis induced by Pleurotus eryngii spores has been reported in a worker in an Eringi (Pleurotus eryngii) mushroom factory who had worked there for 6 years. Chest radiography showed diffuse fine nodular shadows. Chest computed tomography demonstrated centrilobular nodules and increased attenuation in both lungs. The patient suffered from hypoxemia while breathing room air. The lymphocyte count in the bronchoalveolar lavage fluid was increased, and transbronchial lung biopsy specimens showed lymphocyte alveolitis with epithelioid cell granulomas in the alveolar spaces. After admission, the patient's symptoms improved rapidly without medication. However, on his return to work, fever and hypoxemia appeared again. The lymphocyte stimulating test was positive against extracts of Eringi spores. Precipitins against the extracts of Eringi spores were detected by the double immunodiffusion test. The diagnosis was hypersensitivity pneumonitis (HP) caused by Eringi spores. In Japan, more than 30 cases of HP induced by mushroom spores have been reported and therefore this is an occupational health and safety issue, related to air quality in mushroom factories that needs to be addressed. The symptoms appear to improve without medication (Miyazaki et al. 2003).

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Pleurotus ferulae

Ethanol and hot water extracts of Pleurotus ferulae have been shown to have anti-tumourigenic properties in human cervical cancer and human lung cancer cell lines. When A549, SiHa and HeLa cells were incubated with different concentrations of ethanol and hot water extracts, the ethanol extracts showed strong cytotoxicity against A549 cells at

concentrations over 10 mg/mL and against SiHa and HeLa cells at concentrations over 40 mg/mL. The ethanol extracts were the most prominent anti-tumour agents (of those studied) toward A549 human lung cancer cells (Choi et al. 2004a).

Pleurotus Ostreatus (Oyster mushrooms)

The effect of Oyster mushrooms on reduction of blood glucose, cholesterol and triglycerides in diabetic patients has been evaluated in a clinical investigation of 89 subjects. Mushroom consumption significantly reduced systolic and diastolic blood pressure, lowered plasma glucose, total cholesterol and triglycerides significantly, whereas there was no significant change in body weight. There were no deleterious effects on liver or kidney function (Khatun et al. 2007).

A hypo-cholesterolemic effect has been shown with Oyster mushrooms (Pleurotus Ostreatus) in rats with Streptozotocin-induced diabetes. Oyster mushroom (4% dry oyster mushroom fruit body) lowered cholesterol content by more than 60% in the liver although it did not significantly affect either the serum triacylglycerol level or the content in liver (Bobek et al. 1991). Similar results have been observed in rats with a hereditary hypersensitivity to dietary cholesterol (Bobek et al. 1990). The hypo-cholesterolemic effects of Oyster mushrooms has been demonstrated to be dose-dependent (Bobek et al. 1997). A similar hypocholesterolemic effect of the oyster mushroom (Pleurotus ostreatus) was also observed in hamsters (Bobek et al. 1993) and in rabbits (Bobek and Galbavy, 1999).

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The addition of 4% dried whole oyster mushroom (Pleurotus Ostreatus) to the diet of Wistar rats has been reported to have led to a reduced level of serum and liver cholesterol at the end of the 10th week of the experiment. The level of serum triacylglycerols was not influenced by the mushroom, but was significantly reduced by 13% in liver. The decrease in serum cholesterol level was a consequence of the decreased cholesterol concentration in very-low-density lipoproteins (VLDL) and in low-density lipoproteins (LDL). The content of cholesterol in high-density lipoproteins (HDL) was not influenced by the mushroom. Dietary Pleurotus ostreatus increased the fractional turnover rate of LDL (by 28%) and HDL (by 31%) as determined by the analysis of decay curves of 125I-labelled lipoproteins. The increase in the rate of LDL and HDL catabolism is one of the mechanisms which mediates the hypocholesterolemic effect of mushrooms in the rat model (Bobek et al. 1993).

Feeding of 5% powder of the fruiting bodies of the Oyster mushroom (Pleurotus ostreatus) to hyper-cholesterolaemic rats reduced their plasma total cholesterol by ~28%, low-density lipoprotein-cholesterol by ~55%, triglyceride by ~34%, non-esterified fatty acid by ~30% and total liver cholesterol levels by > 34%, with a concurrent increase in plasma high-density lipoprotein-cholesterol concentration of > 21%. However, these effects were not observed in mushroom-fed normocholesterolaemic rats. Mushroom feeding significantly increased plasma fatty acid unsaturation in both normo- and hypercholesterolaemic rats, while plasma total antioxidant status was significantly decreased in mushroom-fed hypercholesterolaemic rats, concomitant with a decrease in plasma total cholesterol. The study concluded that 5% P. ostreatus supplementation provides health benefits, at least partially, by acting on the atherogenic lipid profile in the hyper-cholesterolaemic condition (Hossain et al. 2003).

The effects of pleuran, a beta-glucan isolated from Pleurotus ostreatus, have been studied in a model of acute colitis in rats. Pleuran was given either as a 2% food component or as a 0.44% pleuran hydrogel drink over 4 weeks. Colitis was induced by intraluminal instillation of 4% acetic acid and after 48h the extent of colonic damage and several biochemical parameters were examined. Pleuran supplementation both in food and in drinking fluid significantly decreased the disposition to colitis. The enhanced activity of myeloperoxidase in the inflamed colonic segment was reduced by pleuran diets, reflecting decreased neutrophil infiltration. The mechanism of the described protective effect of pleuran is not yet clear, but the authors suggest that the pleuran-enhanced antioxidant

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defence of the colonic wall against the inflammatory attack maybe a factor (Bobek et al. 2001).

In vivo injection of three water-soluble proteoglycan fractions from Pleurotus ostreatus mycelia, which had polysaccharide to protein ratios 14.2, 26.4 and 18.3 respectively, into Sarcoma-180-bearing mice decreased the number of tumour cells and cell cycle analysis showed that most of the cells were found to be arrested in pre-G(0)/G(1) phase of the cell cycle. All of the three proteoglycans elevated mouse natural killer (NK) cell cytotoxicity and stimulated macrophages to produce nitric oxide. Fourier transform infra red (FTIR) spectra suggested the presence of a beta-glycosidic bond in all the fractions (Sarangi et al. 2006).

Anti-proliferative and pro-apoptotic activities of fractions of Pleurotus ostreatus have been evaluated in HT-29 colon cancer cells in vitro. A hot-water-soluble fraction of the mycelium of the liquid cultured mushroom was partially isolated and chemically characterized as a low-molecular-weight alpha-glucan. This low-molecular-weight alpha-glucan possessed anti-tumourigenic properties, and demonstrated its direct effect on colon cancer cell proliferation via induction of apoptosis - programmed cell death (Lavi et al. 2006).

A dimeric lectin isolated from fresh fruiting bodies of Pleurotus ostreatus has been shown to possess potent anti-tumour activity in mice bearing sarcoma S-180 and hepatoma H-22. Survival in these mice was prolonged and body weight increase reduced after lectin treatment (Wang et al. 2000).

Pleurotus pulmonarius

Hypoglycaemic activity of an aqueous extract of Pleurotus pulmonarius in alloxan-induced diabetic mice has been reported. Pleurotus pulmonarius extract was administrated orally at doses of 250, 500, and 1,000mg/kg to separate groups of mice (normal and alloxan-treated mice), and serum glucose and body weight were measured. In the separate group of mice, an oral glucose tolerance test

was carried out. Acute oral toxicity data showed no mortality in the normal mice up to 5,000mg/kg, while oral administration of extracts reduced the serum glucose level in alloxan-treated diabetic mice at all the

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doses tested after acute and chronic (28 days) administration. The extract also showed increased glucose tolerance in both normal and diabetic mice. The data suggest that the extract possesses hypoglycaemic activity (Badole et al. 2006).

In a subsequent study by the same group, the interaction of an aqueous extract of Pleurotus pulmonarius with acarbose on serum glucose levels, and on an oral glucose-tolerance test in alloxan induced diabetic mice was studied. The anti-hyperglycaemic effects of aqueous extract and acarbose alone were similar but combination treatment of the Pleurotus pulmonarius extract with acarbose produced a more synergistic anti-hyperglycaemic effect than either agent alone (Badole and Bodhankar, 2007).

Podaxis pistillaris

Anti-bacterial components of the mushroom Podaxis pistillaris have recently been reported. Podaxis pistillaris (Podaxales, Podaxaceae, Basidiomycetes) was found to exhibit anti-bacterial activity against Staphylococcus aureus, Micrococcus flavus, Bacillus subtilis, Proteus mirabilis, Serratia marcescens and Escherichia coli. In a culture medium of P. pistillaris, three epidithiodiketopiperazines were identified by activity-guided isolation. Based on spectral data their identity was established as epicorazine A(1), epicorazine B(2) and epicorazine C (3, antibiotic F 3822), which have not

previously been reported as constituents of P. pistillaris (Al-Fatimi et al. 2006).

Schizophyllum commune (Split Gills Mushroom)

A case of mucoid impaction of the bronchi due to a hypersensitivity reaction to the monokaryotic mycelium of Schizophyllum commune has been reported. The patient was hospitalized because of mild asthma attacks, persistent cough, peripheral eosinophilia, and "gloved finger" shadows on a chest roentgenogram. Cultures of the mucous plugs and sputum samples yielded white, felt-like mycelial colonies that were later identified as the monokaryotic mycelium of S. commune by

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use of mating tests with established monokaryotic and dikaryotic strains of S. commune. The results of tests for serum antibody to S. commune cytosol antigen were positive. Bronchoscopies were effective in removing the mucous plugs and relieving the patient's symptoms. The authors suggested that the monokaryotic mycelium of S. commune should be considered as one of the fungi that can cause hypersensitivity-related lung diseases (Amitani et al. 1996). The incidence of Schizophyllum commune related effects in patients suffering from diseases of the nasal sinuses also appears to be on the increase (Buzina et al. 2003).

A lectin from the split gill mushroom Schizophyllum commune has been shown to exhibit potent mitogenic activity toward mouse splenocytes, anti-proliferative activity toward tumour cell lines, and inhibitory activity toward HIV-1 reverse transcriptase, but did not possess any anti-fungal activity (Han et al. 2005).

Tremella fuciformis (White Wood Ear, White Jelly Leaf)

Tremella fuciformis has been shown to have hypo-cholesterolemic properties in rats (Cheung, 1996b).

Tremetes (Coriolus) versicolor (Turkey tail, Yunzhi)

Yunzhi (Coriolus versicolor) has been reported to modulate various immunological functions in vitro, in vivo, and in human clinical trials, while Danshen (Salvia miltiorrhiza) has been shown to benefit the circulatory system by its vasodilating activity. A clinical trial has been carried out to evaluate the

immunomodulatory effects of Yunzhi-Danshen capsules in post-treatment breast cancer patients. Eighty-two patients with breast cancer were recruited to take Yunzhi and Danshen capsules with the data showing that the percentage and the absolute counts of B-lymphocytes were significantly elevated in patients with breast cancer after taking Yunzhi-Danshen capsules, while plasma sIL-2R concentration was significantly

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decreased (Wong et al. 2005). The significance of these findings is not yet clear.

A polysaccharopeptide from the Turkey Tail fungus Trametes (=Coriolus) versicolor has been reported to be capable of inhibiting human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and protease, the two enzymes of paramount importance to the life cycle of the HIV. The polysaccharopeptide inhibited other proteases including trypsin, chymotrypsin, proteinase K, subtilisin, and elastase to a lesser extent. The anti-HIV enzyme and immuno-stimulatory activities of the mushroom polysaccharopeptide make it an interesting potential candidate for the therapeutic treatment of AIDS (Tzi et al. 2006), although clearly further studies are required to confirm such effects. A recent report has also suggested that the culture (harvest) duration affects the immunomodulatory and anti-cancer effects of polysaccharopeptides derived from Coriolus versicolor (Lee et al. 2006)

Coriolus versicolor (CV), also known as Trametes versicolor, has been evaluated for its cytotoxic activities on a B-cell lymphoma (Raji) and two human promyelocytic leukemia (HL-60, NB-4) cell lines. The results demonstrated that CV extract at 50 to 800mg/ml dose-dependently suppressed the proliferation of Raji, NB-4, and HL-60 cells by more than 90%. The extract however did not exert any significant cytotoxic effect on the normal liver cell line WRL when compared with a chemotherapeutic anti-cancer drug, mitomycin C, confirming the tumour-selective cytotoxicity. Nucleosome production in HL-60, NB-4 and Raji cells was significantly increased by 3.6-, 3.6- and 5.6-fold respectively upon the treatment of CV extract, while no significant nucleosome production was detected in extract-treated WRL cells. The CV extract was found to selectively and dose-dependently inhibit the proliferation of lymphoma and leukemic cells possibly via an apoptosis-dependent pathway (Lau et al. 2004).

Coriolus versicolor polysaccharide (CVP) extracted from C. versicolor dry fruit bodies by hot-water extraction and ethanol precipitation has been evaluated against four human liver cancer (7703, HepG2, 7721, PLC) and four human breast cancer (Bcap37, ZR75-30, MCF-7, T-47D) cell lines using a cytotoxicity assay. The results showed that the CVP inhibited the proliferation of 7703, Bcap37, T-47D in low concentration and also inhibited the proliferation of MCF-7 and ZR75-30, but at high concentrations. The CVP did not inhibit the proliferation of HepG2, 7721, PLC and human normal liver cell line (WRL). The CVP was found to selectively inhibit the proliferation of human liver cancer and human breast cancer (Zhou et al. 2007).

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The effect of dietary supplementation with a protein-bound polysaccharide (PSP)-containing extract derived from mycelia of Coriolus versicolor on in vitro and in vivo indices of immune function of young and old C57BL/6NIA mice has been studied. Young (5 month) and old (23 month) mice were fed purified diets containing 0%, 0.1%, 0.5% or 1.0% PSP for 1 month after which time indices of immune function were measured. PSP supplementation had no significant effect on mitogenic response to concanavalin A (Con A), phytohemagglutinin (PHA) or lipopolysaccharide (LPS), or on the production of interleukin (IL)-1, IL-2, IL-4 and prostaglandin E-2 (PGE(2)). Of the in vivo indices of immune function tested, old mice fed 1.0% PSP had significantly higher delayed-type hypersensitivity (DTH) response than those fed 0% PSP. No significant effect of PSP was observed on the DTH response of young mice. The antibody response to sheep red blood cells was not significantly influenced by PSP in young or old mice, suggesting that the PSP-containing extract from mycelia of Coriolus versicolor might have a modest immuno-enhancing effect in aged mice, but not in young mice (Wu et al. 1998).

Toth et al. have recently reported the inhibition of intestinal cancer by a hot water extract of the Coriolus versicolor (Turkey Tail) mushroom in C57bl/6j-Apc(Min) mice (Toth et al. 2007).

A highly N-methylated cyclic heptapeptide, isolated from the mushroom Coriolus versicolor, has been shown to have an inhibitory effect on fat accumulation by 3T3-L1 murine adipocytes (EC50 = 0.02mg/mL) (Shimokawa et al. 2008).

Volvariella volvacea (Straw Mushroom)

Methanol and water extracts from Volvariella volvacea have been shown to have antioxidant activity against lipid peroxidation of rat brain homogenate. The ethyl acetate sub-fraction of the methanol extract of V. volvacea was found to have comparable antioxidant activity to caffeic acid against the oxidation of human low-density lipoprotein (LDL). The antioxidant activities against lipid peroxidation were found to correlate with the phenolic content in different sub-fractions

of the mushroom extracts (Cheung and Cheung, 2005).

A report on the fractionation of extracts of the edible mushroom, Volvariella volvacea, has shown the isolation of two heterocyclic

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carboxylic acids, namely pyridine-3-carboxylic acid [nicotinic acid] and pyrazole-3(5)-carboxylic acid and four steroidal metabolites ergosterol, 5-dihydroergosterol, ergosterol peroxide, and cerevisterol. In light of the structural similarity of pyrazole-3(5)-carboxylic acid to pyrazole-3-carboxylic acids, which act as agonists for nicotinic acid receptors, the levels of pyridine-3-carboxylic acid and pyrazole-3(5)-carboxylic acid were estimated in V. volvacea and two other edible mushrooms, namely Agaricus bisporus and Calocybe indica. Significant levels of pyridine-3-carboxylic acid (nicotinic acid) were found in C. indica, and pyrazole-3(5)-carboxylic acid was found in abundance in A. bisporus. Correlations have been suggested between the occurrence of these compounds in mushrooms and consumption as well as beneficial health effects (Mallavadhani et al. 2006).

Volvariella volvacea has been reported to produce a hypotensive response in animals. An aqueous extract of the mushroom (SME) was prepared and given through intravenous injections to normotensive rats. An i.v. injection of SME produced a hypotensive effect in rats with an ED50 of 25mg dry weight/kg body weight. SME did not increase urinary excretion or sodium diuresis. It produced positive chronotropic and inotropic effects on isolated right atria and induced contraction of isolated tail artery strips. This latter contractile response was inhibited by antagonists of serotonin and alpha-adrenoceptor, ketanserin and phentolamine respectively. Partial purification using dialysis and liquid chromatography revealed that the hypotensive active substances had molecular weights between 8,000 and 12,000 daltons. These substances were heat stable and resistant to trypsin digestion (Chiu et al. 1995).

Volvariella volvacea has also been shown to have hypo-cholesterolemic properties (Cheung, 1996a; Cheung, 1998).

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Wild Edible Fungi

While fungi are a good source of digestible proteins and fibre, are low in fat and energy and make a useful contribution to vitamin and mineral intake, nevertheless, there are some safety concerns with wild fungi. Edible species might be mistaken for poisonous ones, high heavy-metal concentrations (in some regions and countries) in wild edible fungi (WEF) are a known source of chronic poisoning and the consumption of WEF can contribute markedly to the radiocaesium intake of human subjects (reported for the UK). Some regions of Europe have a strong WEF tradition, particularly eastern Europe. Only one-third of adults consume fungi (cultivated species and WEF) throughout the UK;

the average intake of fungi in the UK being estimated to be 0.12 kg fresh weight per capita per year. At least eighty-two species of wild fungi are recorded as being consumed in the UK, although certain species (e.g. chanterelle (Cantharellus cibarius), cep (Boletus edulis), Oyster mushroom (Pleurotus ostreatus)) are favoured over others. Although wild edible fungi are not essential components in the daily diet, they have been reported to be a nutritionally-valuable addition to the range of vegetables consumed (de Roman et al. 2006).

In contrast to the above study, a recent analysis of a large variety (7 different families) of wild edible mushrooms in Greece has shown high mineral contents and low contents of heavy metals (Pb, Cd and As) suggesting that the mushroom species studied can be consumed without a risk to health (Ouzouni et al. 2007)