The chaga mushroom (Inonotus obliquus) contains a wide range of excellent bioactive compounds. However, limited information exists on the antiviral activity of the compounds extracted from chaga. A number of subfractions of chaga were obtained using different solvents and different procedures. The subfractions of chaga extracted with water, alcohol, alkali were tested for their toxicity for the Vero cell culture and antiviral effect in the Vero cells infected with the Herpes simplex virus (HSV), Type 1. It was shown that most of the subfractions were not toxic for the Vero cells and had protective effect on the Vero cells infected with HSV. The subfraction IV in the concentration 5 microg/ml protected the Vero cells from cytodestructive action of HSV and no viral DNA was detected in infected cells treated with chaga extracts. Best protective effect was observed when compound was added before or within one hour after the Vero cells were infected with HSV.

Mushroom Inonotus obliquus (I. obliquus) has been used as functional food and traditional Chinese herbs for long time. An efficient method for bioassay-guided preparative isolation was used for identifying the anti-inflammatory and anticancer constituents in I. obliquus. The petroleum ether and ethyl acetate fractions were found to have significant inhibition effects on NO production and NF-κB luciferase activity in macrophage RAW 264.7 cells and cytotoxicity against human prostatic carcinoma cell PC3 and breast carcinoma cell MDA-MB-231. Six main constituents were isolated from these two fractions and they were identified as lanosterol (1), 3β-hydroxy-8,24-dien-21-al (2), ergosterol(3), inotodiol (4), ergosterol peroxide (5) and trametenolic acid (6). Compound ergosterol, ergosterol peroxide and trametenolic acid showed anti-inflammatory activities and ergosterol peroxide and trametenolic acid showed obviously cytotoxicity on human prostatic carcinoma cell PC3 and breast carcinoma MDA-MB-231 cell. The results obtained in this work might contribute to understanding the biological activity of mushroom I. obliquus for food and drug application.

Inonotus obliquus (IO) is parasitic mushroom that grows on birch and other trees in Russia, Korea, Europe and United States. However, IO is not readily available for consumption due to its high cost and difficult growth. In this regard, IO was inoculated on germinated brown rice (GBR) in the present study and the antioxidant and anti-inflammatory activities of the IO grown on germinated brown rice (IOGBR) extracts were evaluated extensively and compared with those for IO and GBR. IOGBR showed highest antioxidant activities with scavenging total intracellular ROS and MDA levels as well as increasing the antioxidant enzymes activity in the H₂O₂-stimulated mice liver. It also exhibited best inflammatory activities by suppressing the proinflammatory mediators such as NO, PGE₂, iNOS, COX-2, TNF-α, IL-1β, and IL-6 in an LPS-stimulated RAW 264.7 cell line. This study provides a comparative approach to find out an excellent natural source of antioxidants and anti-inflammatory agent as a dietary supplement.

The medicinal mushroom Chaga, Inonotus obliquus (Pers.:Fr.) Pilát (Hymenochaetaceae), has been used in folk medicine in Russia, Poland, and most of the Baltic countries, as a cleansing and disinfecting measure, and as decoctions for stomach diseases, intestinal worms, liver and heart ailments, and cancer treatment. Many reports have been published concerning the health promoting functions of this mushroom, including antibacterial, hepatoprotective, anti-inflammatory, antitumor, and antioxidant activities. The purpose of the present study was evaluation of in vitro anticancer activity of fraction IO4 isolated from I. obliquus. The effect on cell proliferation, motility and viability was assessed in a range of cancer and normal cells. Chaga fraction prepared from dried fruiting bodies was subjected to anticancer evaluation in human lung carcinoma (A549), colon adenocarcinoma (HT-29), and rat glioma (C6) cell cultures. Human skin fibroblasts (HSF), bovine aorta endothelial cells (BAEC), models of rat oligodendrocytes (OLN-93), hepatocytes (Fao), rat astroglia, and mouse neurons (P19) were applied to test toxicity in normal cells. The following methods were applied: tumor cell proliferation (MTT assay and BrdU assay), cytotoxicity (LDH assay), tumor cell motility (wound assay), tumor cell morphology (May-Grünwald-Giemsa staining), and death detection (ELISA). Chaga fraction elicited anticancer effects which were attributed to decreased tumor cell proliferation, motility and morphological changes induction. Of note is the fact that it produced no or low toxicity in tested normal cells. The data presented could open interesting paths for further investigations of fraction IO4 as a potential anticancer agent.

Inonotus obliquus is a traditional mushroom well known for its therapeutic value. In this study, various solvent fractions of I. obliquus were preliminarily screened for their antioxidant,α-amylase and α-glucosidase inhibition properties. To improve the drug delivery, the active fraction (ethyl acetate fraction) of I. obliquus was synthesized into fungisome (ethyl acetate phophotidyl choline complex, EAPC) and its physical parameters were assessed using Fourier transform infrared spectroscopy (FTIR), High performance liquid chromatography (HPLC), Scanning electron microscope (SEM), and ς potential analysis. Then normal human hepatic L02 cells was used to evaluate the cytotoxicity of EAPC. The results showed that EA fraction possesses significant free radical scavenging, α-amylase and α-glucosidase inhibition properties. FTIR, SEM, and HPLC analysis confirmed the fungisome formation. The particle size of EAPC was 102.80 ± 0.42 nm and the ς potential was -54.30 ± 0.61 mV. The percentage of drug entrapment efficiency was 97.13% and the drug release rates of EAPC in simulated gastric fluid and simulated intestinal fluid were 75.04 ± 0.29% and 93.03 ± 0.36%, respectively. EAPC was nontoxic to L02 cells, however it could selectively fight against the HOinduced oxidative damage in L02 cells. This is the first study to provide scientific information to utilize the active fraction of I. obliquus as fungisome. PRACTICAL APPLICATIONS: Inonotus obliquus (IO) is a traditional medicinal fungus. The extracts of IO have obvious antioxidant and hypoglycemic activities. Ethyl acetate (EA) fraction of IO was encapsulated in liposomes to form EAPC. EAPC has a sustained-release effect. It has nontoxic to L02 cells and could protect L02 cells from oxidative damage caused by hydrogen peroxide. This study could provide new ideas for the treatment of diabetes.

The mushroom Inonotus obliquus has been widely used as a folk medicine in Russia, Poland and most of the Baltic countries. In this study, water-soluble and alkali-soluble crude polysaccharides (IOW and IOA) were isolated from I. obliquus, and the carbohydrate-rich fractions IOW-1 and IOA-1 were obtained respectively after deproteination and depigmentation. Their contents, such as neutral carbohydrate, uronic acid and protein, were measured. Their antioxidant properties against chemicals-induced reactive species (ROS) including 1,1'-Diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide anion radical, as well as their protective effects on H(2)O(2)-induced PC12 cell death were investigated. Results showed that I. obliquus polysaccharides can scavenge all ROS tested above in a dose-dependent manner. IOA and its product IOA-1 could rescue PC12 cell viability from 38.6% to 79.8% and 83.0% at a concentration of 20μg/mL. Similarly, IOW and its product IOW-1 at the same dose, can also increase cell viability to 84.9% and 88.6% respectively. The antioxidative activities of water-soluble and alkali-soluble polysaccharide constituents from I. obliquus might contribute to diverse medicinal and nutritional valuesof this mushroom.

We analyzed the antiproliferative activity of 6 medicinal wood-destroying mushrooms (Fomes fomentarius, Fomitopsis pinicola, Trametes versicolor, Trichaptum biforme, Inonotus obliquus, and Coniophora puteana) that are common in deciduous and mixed coniferous forests in Central Russia. Morphological identification of strains collected from the wild was confirmed based on ribosomal DNA internal transcribed spacer phylogenetic analysis. We observed cytotoxic and cell growth-inhibitory effects of hot water extracts from mycelial biomass of 5 species-T. versicolor, C. puteana, F. fomentarius, F. pinicola, and I. obliquus-on leukemia cell lines (Jukart, K562, and THP-1); the effective extract concentrations were mostly less than 50μg · mL-1. However, we observed no antiproliferative activity of dry biomass from methanol-chloroform (1:1) extracts of C. puteana and F. fomentarius. A chemosensitivity assay showed that the most effective polypore mushroom extract was the methanol extract of T. versicolor (strain It-1), which inhibited the growth of 6 various solid tumors (A-549 and SWi573 [lung], HBL-100 and T-47D [breast], HeLa [cervix], and WiDr [colon]) at concentrations below 45 μg · mL-1, with a concentration as low as 0.7-3.6 μg · mL-1 causing 50% reduction in the proliferation of cancer cells in lung and cervixtumors. Methanol extracts of F. pinicola and I. obliquus were less effective, with proliferation-inhibiting capacities at concentrations below 70 and 200 μg · mL-1, respectively. Thus, T. versicolor is a prospective candidate in the search for and production of new antiproliferative chemical compounds.

In the current study, it was demonstrated that the hot water extract of I. obliquus (IOWE) exerts inhibitory activity against the proliferation of human colon cancer cells (HT-29). The inhibitory effect of IOWE on the growth of HT-29 cancer cells was evaluated by treating cells with IOWE at concentrations of 0.25, 0.5 and 1.0 mg/mL for 24 or 48 h. The IOWE inhibited cell growth in a dose-dependent manner, and this inhibition was accompanied by apoptotic cell death. The maximum inhibitory effect (56%) was observed when IOWE was treated at a concentration of 1.0 mg/mL for 48 h. The apoptotic effect of IOWE on HT-29 cells was also confirmed by flow cytometric analysis. In addition, the apoptotic cell percentage was closely associated with down-regulation of Bcl-2 and up-regulation of Bax and caspase-3. The results suggest that IOWE would be useful as an antitumor agent via the induction of apoptosis and inhibition of the growth of cancer cells through up-regulation of the expression of proapoptotic proteins and down-regulation of antiapoptotic proteins. Copyright (c) 2009 John Wiley & Sons, Ltd.

Fractions of aqueous and water-alcohol extracts of the birch fungus Inonotus obliquus have antiviral effect against the human immunodeficiency virus type 1 (HIV-1). Antiviral properties of low toxic extracts were manifested in the concentration of 5.0μg/ml upon simultaneous application with the virus in the lymphoblastoid cells culture MT-4. The extract of the birch fungus can be used for development of new antiviral drugs, inhibitors of HIV-replication when used both in the form of individual drugs and as a part of complex therapy.

Fractions of Inonotus obliquus fungus water extract exhibited a virucidal effect towards hepatitis C virus: it 100-fold reduced its infective properties within 10 min. The antiviral effects of fungus extracts manifested after preventive (24 h before infection) and therapeutic use (during infection of porcine embryo kidney cells). Moreover, the data indicate that the birch fungus extracts inhibit production of infective virus by porcine embryo kidney cells.

ETHNOPHARMACOLOGICAL RELEVANCE:Inonotus obliquus, also known as Chaga mushroom, is one of the most widely appreciated wild edible mushrooms in Russia and northern European countries and is renowned for its use in cancer treatment. Indeed, recently published in vitro and in vivo studies have demonstrated its anticancer activity in various types of cancer and support its potential application for therapeutic intervention in cancer. However, its activity against lung cancer, the most commonly diagnosed cancer and the leading cause of cancer death worldwide, and the underlying molecular basis of its action remain to be fully elucidated.

OBJECTIVE:This study aimed to evaluate the cytotoxic activity of I. obliquus in four human lung adenocarcinoma cell lines with different p53 status (A549, H1264, H1299, and Calu-6) and identify its active constituents by bioactivity-based analysis and the underlying molecular basis of their cytotoxicity on lung cancer cells.

MATERIALS AND METHODS:Bioactivity-guided fractionation and preparative/semi-preparative HPLC purification were used with LC/MS analysis to separate the bioactive constituents. Cell viability and apoptosis in human lung cancer cell lines (A549, H1264, H1299, and Calu-6) were assessed using the WST-1 assay and TUNEL staining, respectively. Caspase activation was assessed by detecting its surrogate markers, cleaved poly (ADP-ribose) polymerase (PARP) and caspase-3, using an immunoblot assay.

RESULTS:The MeOH extract of I. obliquus reduced cell viability in all lung cancer cell lines tested through induction of apoptosis accompanied by caspase-3 cleavage. Bioactivity-guided fractionation of the MeOH extract and chemical investigation of its cytotoxic hexane-soluble and CHCl-soluble fractions led to the isolation of eight triterpenoids (1-8), including a new lanostane-type triterpenoid named chagabusone A (7). The structures of the isolates were elucidated based on spectroscopic analysis, including 1D and 2D NMR and high-resolution ESIMS. Among isolated compounds, compounds 1, 6, and 7 showed the most potent cytotoxic activity in all human lung cancer cell lines examined, with ICvalues ranging from 75.1 to 227.4 μM. Cytotoxicity of these compounds was mediated by apoptosis with caspase-3 activation.

CONCLUSION:These findings provide experimental evidence supporting the potential application of I. obliquus in lung cancer treatment and reveal the molecular basis underlying its cytotoxic activity against human lung cancer cells.

Previously, we studied the antioxidant potential of Chaga mushroom [Inonotus obliquus (persoon) Pilat] extracts and isolated several small (poly)phenolic compounds as the major antioxidant components in the 80% methanol (MeOH) extract. In the present study, these isolated phenolic ingredients together with several other types of Chaga extracts were examined for cytotoxic effects against normal (IMR90) and cancer (A549, PA-1, U937, and HL-60) cell lines. Results revealed decoctions from both the fruiting body (FB) and sclerotium (ST) parts of Chaga, especially the ST part, showed considerable cytotoxicity toward tumor cells, but the cytotoxicity appeared to be stronger against normal cells than cancer cells. The 80% MeOH ST extract also showed the same trend. On the other hand, the 80% MeOH extract of FB showed significant cytotoxicity towards tumor cell lines without affecting normal cells, for example, the 50% lethal dose was 49.4 +/- 2.9 microg/mL for PA-1 cells versus 123.6 +/- 13.8 microg/mL for normal cells. The phenolic components isolated from the 80% MeOH extracts had markedly greater cancer cell toxicity than the extracts themselves. In particular, two out of seven compounds showed strong cytotoxicity towards several tumor cell lines without giving rise to significant cell toxicity toward normal cells. For example, the 50% lethal dose for 3,4-dihydroxybenzalacetone was 12.2 micromol/L in PA-1 cells but was 272.8 micromol/L in IMR90 cells. Fluorescence-activated cell sorting analysis further revealed these phenolic ingredients have high potentiality for apoptosis induction in PA-1 cells.

BACKGROUND:Inonotus obliquus, also known as Chaga, is a parasitic fungus growing on birches and used in traditional medicine (especially by Khanty people) to treat various health problems. In this study, we aimed to quantify the 3 metabolites frequently cited in literature, that is, betulin, betulinic acid, and inotodiol in the Chaga recently discovered in forests located in Normandy (France), and to compare their concentrations with Ukrainian and Canadian Chaga. This study also explores the cytotoxicity of the French Chaga against cancer-derived cells and transformed cells.

METHODS:A quantification method by HPLC-MS-MS (high-performance liquid chromatography-tandem mass spectrometry) of betulin, betulinic acid, and inotodiol was developed to study the French Chaga and compare the concentration of these metabolites with extracts provided from Chaga growing in Canada and Ukraine. This method was also used to identify and quantify those 3 compounds in other traditional preparations of Chaga (aqueous extract, infusion, and decoction). Among these preparations, the aqueous extract that contains betulin, betulinic acid, and inotodiol was chosen to evaluate and compare its cytotoxic activity toward human lung adenocarcinoma cells (A549 line) and human bronchial epithelial cells (BEAS-2B line).

RESULTS:French Chaga contains betulin and betulinic acid at higher levels than in other Chaga, whereas the concentration of inotodiol is greater in the Canadian Chaga. Moreover, the results highlighted a cytotoxic activity of the Chaga's aqueous extract after 48 and 72 hours of exposure with a higher effect on cancer-derived cells A549 than on normal transformed cells BEAS-2B ( P = 0.025 after 48 hours of exposure and P = 0.004 after 72 hours of exposure).

AIM: To investigate the anti-proliferative and apoptotic effects of Chaga mushroom (Inonotus obliquus) water extract on human hepatoma cell lines, HepG2 and Hep3B cells. METHODS: The cytotoxicity of Chaga extract was screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, flow cytometry analysis, Western blot were employed to elucidate the cytotoxic mechanism of Chaga extract. RESULTS: HepG2 cells were more sensitive to Chaga extract than Hep3B cells, as demonstrated by markedly reduced cell viability. Chaga extract inhibited the cell growth in a dose-dependent manner, which was accompanied with G0/G1-phase arrest and apoptotic cell death. In addition, G0/G1 arrest in the cell cycle was closely associated with down-regulation of p53, pRb, p27, cyclins D1, D2, E, cyclin-dependent kinase (Cdk) 2, Cdk4, and Cdk6 expression. CONCLUSION: Chaga mushroom may provide a new therapeutic option, as a potential anticancer agent, in the treatment of hepatoma.

The Chaga mushroom (Inonotus obliquus) is claimed to have beneficial properties for human health, such as anti-bacterial, anti-allergic, anti-inflammatory and antioxidant activities. The antioxidant effects of the mushroom may be partly explained by protection of cell components against free radicals. We evaluated the effect of aqueous Chaga mushroom extracts for their potential for protecting against oxidative damage to DNA in human lymphocytes. Cells were pretreated with various concentrations (10, 50, 100 and 500 microg/mL) of the extract for 1 h at 37 degrees C. Cells were then treated with 100 microM of H2O2 for 5 min as an oxidative stress. Evaluation of oxidative damage was performed using single-cell gel electrophoresis for DNA fragmentation (Comet assay). Using image analysis, the degree of DNA damage was evaluated as the DNA tail moment. Cells pretreated with Chaga extract showed over 40% reduction in DNA fragmentation compared with the positive control (100 micromol H2O2 treatment). Thus, Chaga mushroom treatment affords cellular protection against endogenous DNA damage produced by H2O2.

Four new lanostane-type triterpenes (inonotusanes D-G, 1-4), including a 24,25,26,27-tetranorlanostane, together with 11 known compounds (5-15), including 7 lanostane derivatives, 2 steroids and 2 aromatic compounds, were isolated from the sclerotia of Inonotus obliquus. Their structures were elucidated by 1D and 2D NMR spectroscopy and HRMS. To our knowledge, 1 is the first 24,25,26,27-tetranorlanostane-type triterpenoid from fungus, and this is the first time that 31-member lanostane-type triterpenes, 5 and 6, have been isolated from the sclerotia of I. obliquus instead of from its submerged culture. 7 and 8 are also new isolates of this genus. Compounds 1, 8, 12 and 13 exhibited strong cytotoxicity against the 4T1 (mouse breast cancer) cell line, with IC50 9.40, 7.79, 9.06 and 9.31 μM, respectively. 8, 12 and 13 also exhibited strong cytotoxicity against the the MCF-7 (human breast cancer) cell line, with IC50 8.35-9.01 μM.

Seven new triterpenes, inonotusol A-G (1-7), one new diterpene, inonotusic acid (8), and 22 known compounds were isolated from Inonotus obliquus. Their structures were elucidated on the basis of spectroscopic analysis, including homonuclear and heteronuclear correlation NMR ((1)H-(1)H COSY, ROESY, HSQC, and HMBC) experiments. In in vitro assays, compounds 6 and 8-16 showed hepatoprotective effects against d-galactosamine-induced WB-F344 cell damage, with inhibitory effects from 34.4% to 81.2%. Compounds 7, 17, and 18 exhibited selective cytotoxicities against KB, Bel-7402, or A-549 cell lines. Compounds 16 and 17 showed inhibitory effects against protein tyrosine kinases, with IC50 values of 24.6 and 7.7μM, respectively.

The medicinal mushroom Inonotus obliquus is a traditional and widely used multi-functional fungus. Hot water (50 degrees C, 70 degrees C, and 80 degrees C) and ethanol crude extracts of I. obliquus were investigated for their antioxidant activity with superoxide dismutase (SOD) and (1,1-diphenyl-2-picryhydrazyl) (DPPH) radical-scavenging activity assays. We also investigated the antiproliferative effects and ability of the extracts to induce apoptosis in human colon cancer DLD-1 cells. Among the four extracts, the ethanol extract (EE) exhibited the strongest SOD-like activity and antiproliferative effect on DLD-1 cells, and exposure to the EE resulted in the induction of apoptosis, whereas no apoptosis was observed in DLD-1 cells exposed to the hot water extracts (HWEs). HWE at 70 degrees C (HWE70) exhibited the strongest DPPH radical-scavenging activity (EC50, 126 microg/ml), whereas the EE showed the weakest activity (EC50, 224 microg/ml). The different biological activities among the four extracts may be attributed to differences in their chemical composition, partially supported by polysaccharide, protein and phenolic content, and the 1H-NMR spectra.

Data on the chemical composition of triterpenic and steroid compounds, isolated from the chaga mushroom grown in natural environment or in a synthetic culture have been summarized. Special attention has been paid to the biological activity of chaga mushroom extracts and these particular compounds against various cancer cell lines in vitro and in vivo. This analysis has demonstrated some common features in inhibition of growth of various cell lines by chaga mushroom components. In this context, the most active are triterpene compounds containing OH group at C-22 and a side chain unsaturated bond.

To investigate effects of light wavelengths and coherence on growth of liquid-cultured Inonotus obliquus mycelia, melanin accumulation and enzymes activity, culture condition as light of different wavelengths and coherence were studied. Short-term exposure of the vegetative mycelium by low-intensity coherent blue light was optimal for stimulation of growth, melanin synthesis, and increase in extracellular and intracellular activities of tyrosinase and polyphenoloxidase and extracellular catalase. Red coherent light, in the same mode, can effectively be used to stimulate the growth of mycelium and to increase intracellular and extracellular activity of polyphenoloxidase, extracellular catalase and tyrosinase, and intracellular peroxidase. Low-coherent light had less stimulating effect on the biosynthetic activity of I.оbliquus. It should be used in the cultivation directed at the obtaining endomelanin, polyphenoloxidase, and extracellular tyrosinase.