A very recent study has demonstrated that agaritine purified from Agaricus blazei Murrill exerts anti-tumour activity against leukemic cells (Endo et al., 2010). In this study, a hot water extract of Agaricus blazei Murrill (ABM) powder was fractionated by HPLC based on the anti-tumour activity against leukemic cells in vitro. The purified substance was identified as agaritine, beta-N-(gamma- l(+)-glutamyl)-4-(hydroxymethyl) phenylhydrazine, having a molecular mass of 267Da. This compound inhibited the proliferation of leukemic cell lines such as U937, MOLT4, HL60 and K562, but showed no significant effect on normal lymphatic cells. The authors concluded that agaritine has direct anti-tumour activity against leukemic tumor cells in vitro which is in contrast to the carcinogenic activity previously ascribed to this compound. The data also showed that this activity was distinct from that of beta-glucan, which indirectly suppresses proliferation of tumour cells.
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-α), 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).
The effect of Agaricus blazei Murill (AbM) on the release of several cytokines in human whole blood both after stimulation ex vivo and in vivo after oral intake over several days has been studied in healthy volunteers (Johnson et al., 2009). After stimulation of whole blood ex vivo with 0.5-5.0% of a mushroom extract, mainly containing AbM, there was a dose-dependent increase in all the cytokines studied, ranging from two to 399-fold (TNF-alpha). However, in vivo in the eight volunteers who completed the daily intake (60 ml) of this AbM extract for 12 days, a significant reduction was observed in levels of IL-1-beta (97%), TNF-alpha (84%), IL-17 (50%) and IL-2 (46%). Another nine cytokines were measured but they were unaltered. The discrepant results on cytokine release ex vivo and in vivo may partly be explained by the antioxidant activity of AbM in vivo and limited absorption of its large beta-glucans across the intestinal mucosa to the reticuloendothelial system and blood.
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-2deficient mice, but not from interferon-gamma (IFN-gamma) deficient 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-12mediated 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 antitumour activity against the solid form of Sarcoma 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-betaglucanase, 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-betaglucan 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 their enzymatically hydrolyzed oligosaccharides from Agaricus blazei have antihyperglycemic, 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., 2005b).
Beta-glucans from Agaricus blazei have also been reported to not exert a genotoxic or mutagenic effect, but that it does protect against DNA damage caused by benzo[a]pyrene in the human hepatoma cell line HepG2. The data suggest that beta-glucan acts through binding to benzo[a]pyrene or the capture of free radicals produced during its activation (Angeli et al., 2009a).
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 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).
The effects of Agaricus blazei Murill on the growth of human prostate cancer have been examined in vitro and in vivo. A. blazei, particularly in a broth fraction, inhibited cell proliferation in both androgen-dependent and androgen-independent prostate cancer cell lines. The broth of A. blazei induced lactate dehydrogenase leakage in three cancer cell lines, whereas the activities of caspase 3 and the DNA fragmentation were enhanced the most in androgen-independent PC3 cells. Oral supplementation with the broth of A. blazei (with the higher ratio of beta-glucan) significantly suppressed tumour growth without inducing adverse effects in severe combined immunodeficient mice with PC3 tumor xenograft. The data suggested that the broth of A. blazei may directly inhibit the growth of prostate cancer cell via an apoptotic pathway and suppress prostate tumour growth via antiproliferative and antiangiogenic mechanisms (Yu et al., 2009a).
The efficacy and safety of Senseiro (containing extracts from Agaricus blazei Murill) and Rokkaku Reishi, (containing the Ganoderma lucidum mushroom) have been evaluated over 6 months in patients with prostate cancer in Japan (Yoshimura et al., 2010). Patients with biochemical failure after radical treatment for non-metastasized prostate cancer were enrolled in this open-label study. No partial response in terms of serum prostate-specific antigen was observed. Alteration of serum prostate-specific antigen doubling time did not correlate with that of serum testosterone levels. Serious adverse effects were not observed and no significant anticancer effects were observed with the intake of these two mushrooms in the study population.
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 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). Inhibitory effects of Agaricus blazei extracts on human myeloid leukemia cells NB-4 and K-562 cells have also been reported (Kim et al., 2009a).
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).
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. Chemopreventative 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., 2005a).
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 recently 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).
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).