Anticlastogenic effect of aqueous extracts of Agaricus blazei on CHO-k1 cells, studying different developmental phases of the mushroom.

The Agaricus blazei Murill (ABM) mushroom, known as the sun mushroom, is native to Brazil and has become known for its medicinal properties. This study examined the anticlastogenic effect of Agaricus blazei in Chinese hamster ovary cells, CHO-k1, by means of a chromosome aberration test using methyl methanesulphonate (MMS, 10(-4)M) as the DNA damage inducing agent. Two mushroom lines were used, ABM 99/26 and ABM 97/11, and the latter was used in the young (Y) and sporulating (S) developmental phases. The cells were treated for 12 h with MMS alone or combined with aqueous extracts of A. blazei at a final concentration of 0.15%, which were prepared at three different temperatures: (a) hot (60 degrees C), (b) room temperature (25 degrees C) and (c) chilled (4 degrees C). Mushroom extracts showed a marked anticlastogenic effect against DNA damage, as evidenced by a decrease in the number of cells with breaks, regardless of the line used, or the developmental stage or the temperature at which the extract was prepared. Generally, the extracts were more effective in reducing the isochromatid type breaks. The data obtained suggest that extracts of A. blazei mushroom are anticlastogenic under the conditions tested, mainly during the G1 and S stages of the cell cycle, where chromosome breaks of the isochromatid type are produced by the MMS agent.     

Anti-genotoxic effect of aqueous extracts of sun mushroom (Agaricus blazei Murill lineage 99/26) in mammalian cells in vitro

The “sun mushroom” is the popular name for the Agaricus blazei Murill fungus, a mushroom native to south-eastern Brazil, which has been frequently used in popular medicine mainly in the form of tea to treat various ailments (stress, diabetes, etc.). In the present study, the genotoxic and/or anti-genotoxic effects ofA. blazei on mammalian cells in culture was assessed by checking the increase or reduction of micronucleus (MN) frequency and comets. The sun mushroom (lineage 99/26) was used as aqueous extracts prepared (2.5%) at three different temperatures (60, 25 and 4°C). The in vitro micronucleus (MN) test in binucleated cells and comet assay were used in V79 cells cultivated in HAM-F10+DMEM medium (1:1), supplemented with 10% of fetal bovine serum. The experiments were divided into four treatment types: 1. Negative control; 2. Positive control with MMS; 3. Treatments with the three forms of extracts (60, 25 and 4°C); and 4. Treatments with the extracts in different associations (simultaneous, pre-treatment, post-treatment and simultaneous after pre-incubation for 1 h) with MMS. None of the A. blazei extracts show genotoxic activity. In the comet assay no protecting effect was found. The results obtained in the MN test showed that the three forms of extracts used had protective activity, suggesting that the compound or active ingredients of A. blazei are always present in these extracts. The greater protective efficiency of the simultaneous treatment and simultaneous treatment with pre-incubation mixture with MMS suggests that the extracts have an antimutagenic action of the desmutagenic type.     

Lack of carcinogenicity of lyophilized Agaricus blazei Murill in a F344 rat two year bioassay.

The Brazilian mushroom Agaricus blazei Murill has antimutagenic, antioxidant, immunostimulatory and antitumorigenic activities, and is increasingly consumed as a health food worldwide. We undertook the present study to evaluate the chronic toxicity and oncogenicity of A. blazei Murill in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), four treatment groups of 100 rats each (50 males and 50 females) were fed a powder diet containing lyophilized A. blazei aqueous extract at 0, 6250, 12,500, and 25,000 ppm for up to 2 years. During this period, there was 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 groups (26%, 16%, and 30%), however, was significantly lower than in controls (48%). Histopathological studies showed no increased incidence of tumors in any treatment group, and total tumor incidence across all groups was comparable to historical data. In conclusion, an A. blazei Murill lyophilized powder diet even at 25,000 ppm (1176 mg/kgb x w x /day for male rats and 1518 mg/kgb.w./day for female rats) resulted in no remarkable carcinogenic effects in F344 rats over a 2-year period. Therefore, the dietary NOAEL is 25,000 ppm.     

Shiitake (Lentinula edodes (Berkeley) Pegler) extracts as a modulator of micronuclei induced in HEp-2 cells

Shiitake (Lentinula edodes (Berkeley) Pegler) is one of the most consumed mushrooms, for both therapeutic purposes and as food, therefore, the study of its biological properties is of great interest for producers and consumers. Aqueous extracts of the shiitake mushroom (L. edodes (Berkeley) Pegler) were evaluated by the micronucleus test (MN) in HEp-2 cells in vitro, to analyze their possible mutagenic and antimutagenic activities. None of the three extract concentrations tested (0.5, 1.0 and 1.5 mg/mL) presented mutagenicity at any of the preparation temperatures (4 °C, 22 ± 2 °C and 60 °C). In the antimutagenicity evaluation, all extract concentrations at all preparation temperatures presented a strong protective activity for the HEp-2 cells in response to the alkylating agent methyl methanesulfonate (MMS) in the different treatment protocols: pre-treatment, simultaneous treatment and post-treatment. The extracts prepared at 22 ± 2 °C presented the lowest frequencies of MN in the evaluations of mutagenicity and antimutagenicity, indicating these as the best option for potential therapeutic use.     

Cytotoxicity, genotoxicity and antimutagenicity of hexane extracts of Agaricus blazei determined in vitro by the comet assay and CHO/HGPRT gene mutation assay.

Agaricus blazei Murrill ss. Heinem, known as the sun mushroom or himematsutake, is a basidiomycete native to Brazil, which is popular for its medicinal properties. The aim of this study was to test hexane extracts (one fraction and its four sub-fractions) of A. blazei for bioactivity in cultured mammalian cells (CHO-K1). The comet assay, the colony forming assay (CFA) and CHO/HGPRT gene mutation assay were used respectively to determine genotoxicity, cytotoxicity and antimutagenicity of these extracts at different concentrations. The cells were incubated in culture medium and treated for 3h according to the standard protocol for each assay. The DNA damage-inducing agent ethylmethane sulfonate (EMS) was utilized as the positive control and also in combination with extracts to test for a protective effect. Statistical analysis of the data was performed using analysis of variance (ANOVA) and Tukey's test. A relationship between cytotoxicity and genotoxicity could be established and two extracts EH6B and EH6D showed a protective tendency, while the others did not, with the primary extract EH6 causing the most substantial damage to genetic material. These findings warrant more in-depth studies of the active principles of this mushroom.     

巴西蘑菇抗病毒作用研究动向

在巴西蘑菇抗癌、调节免疫功能等方面已经有不少研究,但在抗病毒方面的研究不多,为探求巴西蘑菇在抗病毒方面是否值得研究．特以国内外发表的文献为材料,介绍巴西蘑菇抗病毒作用的研究动向。可以得出巴西蘑菇具有抗病毒药理作用。并能协同机体免疫而辅助抗病毒,但作用机制还有待进一步研究。     

Antimutagenic effect of sage tea in the wing spot test of Drosophila melanogaster

The present study assayed the antimutagenic potential of Salvia officinalis (sage) in the form of tea infusion, by the somatic mutation and recombination test (SMART) on Drosophila melanogaster. The use of herbal infusions is much common in the human diet, so the aim of the present study was to estimate the antimutagenic effects of the S. officinalis tea rather than essential oils. Methyl methanesulphonate (MMS) was used as the mutagen and positive control. Several types of treatment were performed: short acute treatment with sage infusion or MMS, longer (chronic) treatment with sage solution or MMS, and two combined treatments, i.e. short treatment with sage followed by a longer treatment with MMS and vice versa. Sage infusion used in our experiments showed a clear antimutagenic effect, reducing the frequency of mutations induced by MMS. The inhibition effect of sage tea is obtained and confirmed when pre- or post-treatments with mutagen were used. The results indicate that although sage in this regime decreases the number of mutation events, it is not efficient enough in case of the 2 h sage pre-treatment. Antioxidant activity, suppression of metabolic activation, could be mechanisms through which sage or some of its components act as desmutagen.     

Lack of subchronic toxicity of an aqueous extract of Agaricus blazei Murrill in F344 rats.

Agaricus blazei Murrill, an edible mushroom, is widely used as a functional food due to its possible medicinal effects. Aqueous extracts are also used as food additive to provide an agreeable bitter taste. As a part of its safety assessment, the present 90-day subchronic toxicity study was performed in F344 rats. To establish a no-observed-adverse-effect level (NOAEL), rats were fed powder diet containing A. blazei Murrill aqueous extract at dose levels of 0 (basal diet), 0.63, 1.25, 2.5 and 5% (maximum) for 90 days. During the experiment, there were no remarkable changes in general appearance and no deaths occurred in any experimental group. Although serum blood urea nitrogen was slightly but significantly increased in males of the 2.5 and 5% groups, no related histopathological changes were observed in the kidney, and serum creatinine levels were rather reduced, suggesting the increase of blood urea nitrogen to be of little toxicological significance. Hematology, organ weight measurement and histopathological observation revealed no test compound-related toxicological changes. In conclusion, A. blazei Murrill extract even at 5% in the diet (2654 mg/kgb.w./day for male rats and 2965 mg/kgb.w./day for female rats) did not cause remarkable adverse effects in F344 rats. Thus, the NOAEL was concluded to be 5% in the diet.     

Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom.

Aqueous extracts of baked and raw Agaricus bisporus (AB) mushroom were tested for mutagenic activity in Salmonella typhimurium strains TA1535 and TA1537. The extracts were studied with and without metabolic activation by Aroclor-induced rat liver S9 mix. The extracts of the baked and raw AB exhibited a dose related mutagenic activity with and without activation in strain TA1535. Similar findings were obtained in strain TA1537, although the net revertant values were of lower magnitude. Because humans mainly consume the cultivated mushroom AB in baked form, the implications of the findings are self-evident.     

Evaluation of the cytotoxicity, mutagenicity and antimutagenicity of emerging edible plants.

This study evaluates the toxic, mutagenic and antimutagenic effects of emerging edible plants that are consumed as new leafy vegetables in Taiwan. Among eight plant extracts, only the extracts of Sol (Solanum nigrum L.) showed cytotoxicity to Salmonella typhimurium TA100 in the absence of S9 mix. The toxicity of extracts from different parts of the Sol plant, such as leaf and stem, immature fruit and mature fruit, towards S. typhimurium TA100 and human lymphocytes was also assayed. The immature fruit extracts of Sol exhibited strong cytotoxicity with dose dependence and induced significant DNA damage in human lymphocytes based on the comet assay. However, no mutagenicity was found in eight plant extracts to TA98 or TA100 either with or without the S9 mixture. Sol and Sec [Sechium edule (Jacq.) Swartz] extracts showed the strongest inhibitory effect towards the mutagenicity of 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) in S. typhimurium TA98 and TA100; the ID(50) was less then 1 mg/plate. Cra [Crassocephalum creidioides (Benth.) S. Moore] extracts also expressed moderate antimutagenic activities towards IQ and benzo[a]pyrene (B[a]P) either in TA98 or in TA100; the ID(50) was 1.63-2.41 mg/plate. The extracts from Bas (Basella alba L.), Bou (Boussingaultia gracilis Miers var. pseudobaselloides Bailey), Cen (Centella asiatica L. Urban), Cor (Corchorus olitorius L.) and Por (Portulaca oleracea L.) showed weak to moderate inhibition of mutagenicity of IQ. However, the potential antimutagenicity of these plant extracts towards B[a]P was weaker than that towards IQ. For a direct mutagen, 4-nitroquinoline-N-oxide (NQNO), only the Sol extracts showed strong inhibitory effects in the TA100 system. The antimutagenic activity of water extracts of Sec was partly reduced by heating at 100 degrees C for 20 min. The heat-stable antimutagens in Sec extracts could be produced in the plant extract preparation process. Fractions with molecular weights above 30,000 showed the strongest antimutagenicity and peroxidase activity in all the fractions of the Sec extracts.     

Nerve growth factor-inducing activity of Hericium erinaceus in 1321N1 human astrocytoma cells

Neurotrophic factors are essential to maintain and organize neurons functionally; thereby neurotrophic factor-like substances or their inducers are expected to be applied to the treatment of neurodegenerative diseases such as Alzheimer's disease. In the present study, we firstly examined the effects of ethanol extracts of four edible mushrooms, Hericium erinaceus (Yamabushitake), Pleurotus eryngii (Eringi), Grifola frondosa (Maitake), and Agaricus blazei (Himematsutake), on nerve growth factor (NGF) gene expression in 1321N1 human astrocytoma cells. Among the four mushroom extracts, only H. erinaceus extract promoted NGF mRNA expression in a concentration-dependent manner. In addition, secretion of NGF protein from 1321N1 cells was enhanced by H. erinaceus extracts, and the conditioned medium of 1321N1 cells incubated with H. erinaceus extract enhanced the neurite outgrowth of PC12 cells. However, hericenones C, D and E, constituents of H. erinaceus, failed to promote NGF gene expression in 1321N1 cells. The enhancement of NGF gene expression by H. erinaceus extracts was inhibited by the c-jun N-terminal kinase (JNK) inhibitor SP600125. In addition, H. erinaceus extracts induced phosphorylation of JNK and its downstream substrate c-Jun, and increased c-fos expression, suggesting that H. erinaceus promotes NGF gene expression via JNK signaling. Furthermore we examined the efficacy of H. erinaceus in vivo. ddY mice given feed containing 5% H. erinaceus dry powder for 7 d showed an increase in the level of NGF mRNA expression in the hippocampus. In conclusion, H. erinaceus contains active compounds that stimulate NGF synthesis via activation of the JNK pathway; these compounds are not hericenones.     

巴西蘑菇Agaricus blazei Murill水溶性多糖的分离纯化

从巴西蘑菇子实体中提取的水溶性粗多糖经过脱蛋白、脱色等步骤获得半纯品多糖。依次采用DE—AB纤维素和Sepharose CL-4B柱层析进一步纯化后，获得5种水溶性多糖组分。其中主要组分AB-I、AB-Ⅱ-b和AB-Ⅲ-b得率分别为3．3％、1．52％、0．85％。Sephadex G-l00凝胶层析、旋光度和HPLC鉴定各组分已达到层析纯。     

Effect of hot water extract from Agaricus blazei Murill on antibody-producing cells in mice

We investigated the immunopotentiating activities of boiled water-soluble extracts from desiccated Agaricus blazei Murill (ABM). Effect of ABM extract on antibody production was investigated by method of hemolytic plaque-forming cells (PFC) against sheep red blood cells (SRBC) antigen. ABM extracts significantly (p<0.01) increased the number of PFC in spleen with intraperitoneal administration at doses of 25 mg/kg as compared with control group. The populations of Mac-1- or CD25-positive cells significantly (p<0.01, p<0.001) increased, but in CD19-positive cells, there were no differences in ABM-treated mice as compared with control mice. The expressions of IL-6 and IL-1beta mRNA were augmented by ABM extract in both peritoneal macrophages and spleen cells. These results suggested that ABM extract might be an effective stimulator for T cell and macrophage to IL-1beta and IL-6 release, resulting in augmentation of antibody production against SRBC antigen.     

Investigation of extracts from (Tunisian) Cyperus rotundus as antimutagens and radical scavengers

This study evaluates mutagenic and antimutagenic effects of aqueous, total oligomers flavonoïds (TOF), ethyl acetate and methanol extracts from aerial parts of Cyperus rotundus with the Salmonella typhimurium assay system.The different extracts showed no mutagenicity when tested with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1538 either with or without the S9 mix. On the other hand, our results showed that all extracts have antimutagenic activity against Aflatoxin B1 (AFB1) in TA100 and TA98 assay system, and against sodium azide in TA100 and TA1535 assay system. TOF, ethyl acetate and methanol extracts exhibited the highest inhibition level of the Ames response induced by the indirect mutagen AFB1. Whereas, ethyl acetate and methanol extracts exhibited the highest level of protection towards the direct mutagen, sodium azide, induced response. In addition to antimutagenic activity, these extracts showed an important free radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical. TOF, ethyl acetate and methanol extracts showed IC₅₀ value of 15, 14 and 20 μg/ml, respectively.Taken together, our finding showed that C. rotundus exhibits significant antioxidant and antimutagenic activities.     

Evaluation of cytotoxic potential,oral toxicity,genotoxicity, and mutagenicity of organic extracts of Pityrocarpa moniliformis

The objective of this study was to determine the cytotoxicity of organic extracts of P. moniliformis in vitro and identify the acute toxicity and genotoxicity in vivo. The leaves were extracted using three organic solvents (cyclohexane [EP1], ethyl acetate [EP2], and methanol [EP3]). Phytochemical qualitative analysis was performed by thin layer chromatography (TLC). Cytotoxicity tests were performed on human embryonic kidney (HEK) cells and J774 murine macrophages. Acute toxicity in mice was measured after intraperitoneal (ip) administration of 2000 mg/kg, while evaluation of genotoxicity and mutagenicity were assessed using the comet assay and the micronucleus (MN) test, respectively. The TLC analysis of the extracts revealed the presence of flavonoids, triterpenes, steroids, and saponins. In the cytotoxicity assay, extracts EP1 and EP3 altered proliferation of HEK cells, and all organic extracts increased the viability of J774 cells. In the toxicity tests, no deaths or behavioral alterations were observed in mice exposed to the acute dose of the extracts. Although some extracts led to changes in hematological and histological parameters, these results did not indicate physiological changes. In relation to the MN test and comet assay, no significant changes were detected in the DNA of the animals tested with the extracts EP1, EP2, and EP3. Thus, extracts of P. moniliformis were not considered to be toxic and did not induce formation of MN or damage to cellular DNA in the genotoxicity tests.     

Genotoxic and antigenotoxic evaluation of extracts from Arenosclera brasiliensis, a Brazilian marine sponge

The marine environment is a rich source of biological active compounds and the sponges can be considered the most productive one. This diversity gives rise to unique chemical compounds with potential pharmacological properties. Our study is focused on the genotoxic and antigenotoxic evaluation of two crude extracts obtained from the Brazilian endemic marine sponge Arenosclera brasiliensis. Salmonella typhimurium reverse mutation test with TA97, TA98, TA100 and TA102 strains were performed. For antimutagenic analysis, a pre-, co-, and post-treatment to evaluate, respectively, intracellular and extracellular reactions and possible modulation on DNA repair. Additionally, in order to verify the influence of the crude extracts on DNA damage induction, a plasmid-DNA treatment was assayed. No mutagenicity was observed in Salmonella reverse mutation test, neither DNA strand induced damage. Antimutagenic activity was observed in pre-, co-, and post-treatment. A significant antigenotoxic effect was observed in the crude extract, which suggests that A. brasiliensis extract has the potential to protect DNA from the action of 4NQO, 2-aminofluorene, sodium azide and mitomycin C.     

Antigenotoxicity of Agaricus blazei mushroom organic and aqueous extracts in chromosomal aberration and cytokinesis block micronucleus assays in CHO-k1 and HTC cells.

Agaricus blazei (Ab) has become popularly known for its medicinal properties. Scientifically, it has been tested with regard to its capacity to protect genetic material against damage. We examined different organic extracts (methanolic extract -- ME, hexanic extract -- HE and n-butanolic extract -- BE) and an aqueous extract (AE) of Ab, for their capacity to induce DNA damage as well as for their protective effect. Genetic damage was determined by the chromosomal aberration assay (CA) in CHO-k1 cells for all extracts and the cytokinesis block micronucleus assay (CBMN) in non drug-metabolizing (CHO-k1) and drug-metabolizing (HTC) cell lines for extract BE only. The extracts did not show clastogenicity but showed anticlastogenicity. The greatest percent reduction obtained were with BE (105%) and AE (126%) treatments in CA. BE treatment did not display genotoxicity in CHO-k1, but was genotoxic in HTC. However, BE was shown to be antigenotoxic causing decreased micronucleus frequency in HTC and CHO-k1 cells. These results suggest that all the extracts contained protective substances, but in some cases they could show a genotoxic effect with regard to metabolism. Therefore, these findings warrant caution in the use of this mushroom by the population.     

Podophyllum hexandrum ameliorates endosulfan-induced genotoxicity and mutagenicity in freshwater cyprinid fish crucian carp

Context: Medicinal plants continue to act as a repository for novel drug leads with novel mechanisms of action. Podophyllum hexandrum Royale (Berberideceae) treats diverse conditions in folk medicine.

Objective: The antimutagenic potential of P. hexandrum was evaluated against endosulfan-induced clastogenicity in a piscine model by cytogenetic endpoints.

Materials and methods: Podophyllum hexandrum rhizomes were subjected to successive solvent extraction. Fish were exposed to hexane, chloroform, ethyl acetate, methanol and aqueous extracts (15?mg/L each) of plant and endosulfan (0.05?mg/L) alone followed by their combination for antimutagenicity estimates. Chromosomal aberrations (CA) were made from kidney cells and micronuclei (MN) slides from peripheral blood erythrocytes at 48, 72 and 96?h. Antioxidant activity was analyzed by the DPPH assay. Phytochemical analyses were carried out using chromatographic and spectroscopic techniques.

Results: Endosulfan induced significant (p?R^2?=^?.900) with antimutagenic activity, whereas EE-F4 seemed to act through a different mechanism.

Discussion and conclusion: This study confirms the antimutagenic potential of the subject plant with the identification of some novel compounds, justifying their use in folk medicine, and their corresponding benefit to mankind.