Preventive effects of inotodiol on polyinosinic-polycytidylic acid-induced inflammation in human dermal fibroblasts.

Double-strand RNA(dsRNA), which can induce inflammation, can be generated by necrotic keratinocytes in the skin environment. As an analog of dsRNA, polyinosinic-polycytidylic acid (poly(I:C)) is used to induce inflammation via the Toll-like Receptor 3 (TLR3) signaling pathway. Inotodiol, isolated from Inonotus obliquus, known as Chaga mushroom, is a natural lanostane-type triterpenoid with significant pharmacological activity and notable anti-inflammatory effects. However, the functions of inotodiol on dsRNA-induced inflammation in human dermal fibroblast (HDFs) remains unclear. In this study, we evaluated the anti-inflammatory effects of inotodiol inflammation induced on by poly(I:C) in HDFs. After pre-treatment with inotodiol, poly (I:C) was used to induce inflammation. Subsequently, mRNA expression and protein secretion of inflammatory cytokines, as well as TLR3 signaling protein levels were assessed. Inflammatory cytokines IL-1ß, IL-6, and TNF-α's increased mRNA expression by poly(I:C) in HDFs was significantly suppressed in the inotodiol pre-treatment group in a dose-dependent manner. A similar pattern was evaluated in the protein levels of these three cytokines. The inflammatory signals of TLR3 via p-IKK, p-p38, and NF-κB was reduced by inotodiol pre-treatment. Taken together, inotodiol possesses strong anti-inflammatory activity against poly(I:C)-induced inflammation in HDFs. Therefore, our findings support potential application of inotodiol as an effective anti-inflammatory agent in cosmetics.

Protective Effects of Lanostane Triterpenoids from Chaga Mushroom in Human Keratinocytes, HaCaT Cells, against Inflammatory and Oxidative Stresses.

Inotodiol, a lanostane-type triterpenoid, and many phytochemicals from Chaga mushrooms have been investigated for various allergic diseases. However, the anti-aging and anti-inflammatory activities of inotodiol under different types of oxidative stress and the impact of inotodiol on collagen and hyaluronan synthesis have not been sufficiently studied. Lanostane triterpenoids-rich concentrate, which contained 10% inotodiol as major (inotodiol concentrate), was prepared from Chaga and compared with pure inotodiol in terms of anti-inflammatory activities on a human keratinocyte cell line, HaCaT cells, under various stimulations such as stimulation with ultraviolet (UV) B or tumor necrosis factor (TNF)-α. In stimulation with TNF-α, interleukin (IL)-1ß, IL-6, and IL-8 genes were significantly repressed by 0.44~4.0 µg/mL of pure inotodiol. UVB irradiation induced the overexpression of pro-inflammatory cytokines, but those genes were significantly suppressed by pure inotodiol or inotodiol concentrate. Moreover, pure inotodiol/inotodiol concentrate could also modulate the synthesis of collagen and hyaluronic acid by controlling COL1A2 and HAS2/3 expression, which implies a crucial role for pure inotodiol/inotodiol concentrate in the prevention of skin aging. These results illuminate the anti-inflammatory and anti-aging effects of pure inotodiol/inotodiol concentrate, and it is highly conceivable that pure inotodiol and inotodiol concentrate could be promising natural bioactive substances to be incorporated in therapeutic and beautifying applications.

Isolation, Physicochemical Characterization, and Biological Properties of Inotodiol, the Potent Pharmaceutical Oxysterol from Chaga Mushroom.

Inotodiol, an oxysterol found only in Chaga mushroom, has received attention from the pharmaceutical industry due to its strong antioxidant and anti-allergic activities. However, the production of inotodiol is still challenging, and its fundamental properties have yet to be investigated. This study aims to develop an efficient method to produce high-purity inotodiol from Chaga mushroom. Then, pure inotodiol was used to assess its physicochemical properties and biological activities. By optimizing the solvent used for extraction and purification, a new method to produce inotodiol was developed with high purity (>97%) and purification yield (33.6%). Inotodiol exhibited a melting point (192.06 °C) much higher than lanosterol and cholesterol. However, the solubility of inotodiol in organic solvents was notably lower than those of the other two sterols. The difference in the hydroxyl group at C-22 of inotodiol has shown the distinctive physicochemical properties of inotodiol compared with cholesterol and lanosterol. Based on those findings, a nonionic surfactant-based delivery system for inotodiol was developed to improve its bioavailability. The inotodiol microemulsion prepared with 1-2% Tween-80 exhibited homogenous droplets with an acceptable diameter (354 to 217 nm) and encapsulation efficiency (85.6-86.9%). The pharmacokinetic analysis of inotodiol microemulsion in oral administration of 4.5 mg/kg exhibited AUC0-24h = 341.81 (ng·h/mL), and Cmax = 88.05 (ng/mL). Notably, when the dose increased from 4.5 to 8.0 mg/kg, the bioavailability of inotodiol decreased from 41.32% to 33.28%. In a mouse model of sepsis, the serum level of interleukin-6 significantly decreased, and the rectal temperature of mice was recovered in the inotodiol emulsion group, indicating that inotodiol microemulsion is an effective oral delivery method. These results could provide valuable information for applying inotodiol in functional food, cosmetic, and pharmaceutical industries.

Inotodiol, an antiasthmatic agent with efficacy and safety, preferentially impairs membrane-proximal signaling for mast cell activation.

While inhaled corticosteroids (ICSs) are the mainstay of asthma treatment, due to compliance, drug safety, and resistance issues, new medications to replace ICSs are in high demand. Inotodiol, a fungal triterpenoid, showed a unique immunosuppressive property with a preference for mast cells. It exerted a mast cell-stabilizing activity equally potent to dexamethasone in mouse anaphylaxis models when orally administered in a lipid-based formulation, upgrading bioavailability. However, it was four to over ten times less effective in suppressing other immune cell subsets, depending on the subsets, than dexamethasone showing invariably potent inhibition. Accordingly, inotodiol affected the membrane-proximal signaling for activating mast cell functions more profoundly than other subsets. Inotodiol also effectively prevented asthma exacerbation. Importantly, considering the no-observed-adverse-effect level of inotodiol was over 15 times higher than dexamethasone, its therapeutic index would be at least eight times better,implying that inotodiol is a viable option for replacing CSs in treating asthma.

Inotodiol suppresses allergic inflammation in allergic rhinitis mice.

BACKGROUND: Inotodiol has been proven to have antitumor, antiviral, anti-inflammatory, and antiallergic properties. This study investigated the immunomodulatory capability of inotodiol in allergic rhinitis (AR) mice. METHODS: Forty BALB/c mice were divided into four groups, 10 mice each: control (CON), AR with phosphate-buffered saline (PBS) treatment (AR), inotodiol treatment (AR+Ino), and dexamethasone treatment (AR+Dex). Episodes of sneezing and nose rubbing were counted. Cytokines in nasal lavage fluid (NLF) and immunoglobulin in blood serum were measured. Nasal mucosae from each group were used for protein, reverse transcriptase-polymerase chain reaction (RT-PCR), and histological analyses. Splenocytes were cultured for evaluation of cytokine production in each group. RESULTS: Symptoms of rubbing and sneezing improved in the group of AR+Ino and AR+Dex than in the AR. NLF in the AR+Ino and AR+Dex also showed a significant decrease in interleukin (IL)-5, IL-10, and IL-13 compared to the AR. In addition, the number of eosinophils, goblet cells, and mast cells were notably lower in the nasal mucosae of the AR+Ino and AR+Dex. IL-4 and IL-17A in the AR+Ino and AR+Dex groups were decreased compared to the AR. Chemokines related to mast cell degradation were also decreased in the AR+Ino and AR+Dex groups. Total immunoglobulin (Ig)E, specific IgE and ovalbumin (OVA)-specific IgG1, and histamine levels were also significantly lower in the AR+Ino and AR+Dex groups. IL-10 and IL-13 were notably increased in the splenocytes of the AR after OVA stimulation, whereas the other groups showed no change. CONCLUSION: These results indicate inotodiol can help suppress allergic responses by immunomodulation activities.

Comparative Analyses of Bioactive Compounds in Inonotus obliquus Conks Growing on Alnus and Betula.

Inonotus obliquus grows in the Northern Hemisphere on some living broadleaved tree species as a pathogen, causing stem rot. In Estonia, the fungus is well known in the Betula species but can also be found on Alnus. Sterile conks of I. obliquus contain different bioactive compounds, but the quantitative and comparative research of these compounds in conks on different host species is limited. In the current work, I. obliquus was isolated and, evidently, determined from Alnus incana (L.) Moench., Alnus glutinosa (L.) Gaertn., and Betula pendula Roth, and the content of bioactive compounds in conks on these hosts were analysed. All the analysed conks sampled from A. incana and B. pendula contained betulin that varied from 111 to 159 µg/g. A significantly (p < 0.05) higher betulinic acid content was found in conks sampled from A. incana when compared with B. pendula: 474−635 and 20−132 µg/g, respectively. However, the conks from Betula were richer in total polyphenols, flavonols, and glucans. The content of inotodiol was quite similar in the conks from A. incana (7455−8961 µg/g) and B. pendula (7881−9057 µg/g). Also, no significant differences in the lanosterol content were found between the samples from these two tree species. To the best of our knowledge, this study is the first investigation of the chemical composition of I. obliquus parasitizing on Alnus. The results demonstrate that the bioactive compounds are promising in conks of I. obliquus growing not only on Betula but also on the Alnus species. It supports the opportunity to cultivate I. obliquus, also on the Alnus species, thus increasing the economic value of growing this tree species in forestry.

Evaluation of Toxicity and Efficacy of Inotodiol as an Anti-Inflammatory Agent Using Animal Model.

Chaga mushroom (Inonotus obliquus) comprises polyphenolic compounds, triterpenoids, polysaccharides, and sterols. Among the triterpenoid components, inotodiol has been broadly examined because of its various biological activities. The purpose of this study is to examine inotodiol from a safety point of view and to present the potential possibilities of inotodiol for medical usage. From chaga mushroom extract, crude inotodiol (INO20) and pure inotodiol (INO95) were produced. Mice were treated with either INO20 or INO95 once daily using oral administration for repeated dose toxicity evaluation. Serum biochemistry parameters were analyzed, and the level of pro-inflammatory cytokines in the serum was quantified. In parallel, the effect of inotodiol on food allergic symptoms was investigated. Repeated administration of inotodiol did not show any mortality or abnormalities in organs. In food allergy studies, the symptoms of diarrhea were ameliorated by administration with INO95 and INO20. Furthermore, the level of MCPT-1 decreased by treatment with inotodiol. In this study, we demonstrated for the first time that inotodiol does not cause any detrimental effect by showing anti-allergic activities in vivo by inhibiting mast cell function. Our data highlight the potential to use inotodiol as an immune modulator for diseases related to inflammation.

Antiaging effect of inotodiol on oxidative stress in human dermal fibroblasts.

Oxidative damage is one of the major causes of human skin aging. Inotodiol is a lanostane triterpenoid that demonstrates antiviral, anticancer, and anti-inflammatory activities. Previous studies have reported that inotodiol also has antiallergic effects. However, whether inotodiol inhibits oxidative stress-induced human skin aging is not known. Stimulation of human dermal fibroblast cells with hydrogen peroxide is related to skin aging. Inotodiol inhibited the expression of mitogen-activated protein kinase (MAPK) and NADPH Oxidase 5 (NOX5). Moreover, inotodiol effectively decreased nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), as well as nitric oxide (NO), reactive oxygen species (ROS), cyclooxygenase-2 (COX-2), and cytokines such as IL-1ß, IL-6, and TNF-α. Based on our results, inotodiol protects human dermal fibroblast by preventing MAPK-NOX5 and NF-κB activation and attenuates the expression of aging genes. Inotodiol may therefore be considered a potential candidate for developing natural antiaging products, because it protects the human skin from oxidative stress-induced skin aging by inhibiting the MAPK-NOX5 and NF-κB signaling pathways.

Inonotus obliquus and its bioactive compounds alleviate non-alcoholic fatty liver disease via regulating FXR/SHP/SREBP-1c axis.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease worldwide. However, there is still lack of specific drugs for treating NAFLD in clinic. Inonotus obliquus (IO), a folk medicinal fungus, has long been used to prevent against metabolic syndrome related diseases, such as hypertension and diabetes, etc. However, the study of IO anti-NAFLD effect has been reported rarely. This study aimed to investigate whether IO has an inhibitory effect on NAFLD, identify the active compounds in IO and clarify the underlying mechanisms of its anti-NAFLD effects. The results of Oil Red O(ORO) and Hematoxylin-Eosin (HE) staining, lipid extraction and determination showed that IO and its extracts, including inotodiol (Ino), lanosterol (Lan) and trametenolic acid (TA), could remarkably ameliorate lipid accumulation in MCD diet-induced mouse livers or OA-induced LO2 hepatocytes. Moreover, qPCR analysis revealed that IO and its compounds significantly downregulated the mRNA levels of lipogenic genes, such as SREBP-1c, ACC1 and FASN, and upregulated the mRNA levels of FXR and SHP. We found that the administration of guggulsterone (GS), a FXR inhibitor, abolished the inhibitory effect of Ino on lipid deposition in OA-induced LO2 cells. In conclusion, IO and its compounds attenuate hepatic lipid accumulation in NAFLD by inhibiting liver lipogenesis. The anti-NAFLD effects of Ino, a bioactive compound in IO, are through regulating FXR/SHP/SREBP-1c pathway. Our results suggested that IO and its bioactive compound Ino may become promising drugs to treat NAFLD.

Supercritical CO2 Extraction of Triterpenoids from Chaga Sterile Conk of Inonotus obliquus.

Triterpenoids are among the bioactive components of Chaga, the sterile conk of the medicinal fungus Inonotus obliquus. Supercritical fluid extraction of Chaga triterpenoids was carried out with supercritical CO2, while a modified Folch method was used as a comparison. Three temperature-pressure combinations were tested varying between 314-324 K (40-50 °C) and 281-350 bars, using time- and volume-limited extractions. Six triterpenoids were identified with GC-MS and quantified with GC-FID: ergosterol, lanosterol, ß-sitosterol, stigmastanol, betulin, and inotodiol. The Folch extraction resulted in recovery of trametenolic acid, which was not extracted by supercritical CO2. Inotodiol was the major triterpenoid of all the extracts, with a yield of 87-101 mg/100 g and 139 mg/100 g, for SFEs and the Folch method, respectively. The contents of other major triterpenoids, lanosterol and ergosterol, varied in the ranges 59-63 mg/100 g and 17-18 mg/100 g by SFE, respectively. With the Folch method, the yields were 81 mg/100 g and 40 mg/100 g, respectively. The highest recovery of triterpenoids with SFE in relation to Folch was 56% and it was obtained at 324 K (50 °C) and 350 bar, regardless of extraction time or volume of CO2. The recoveries of lanosterol and stigmastanol were unaffected by SFE conditions. Despite the lower yield, SFE showed several advantages including shorter extraction time and less impact on the environment. This work could be a starting point for further studies on green extraction methods of bioactive triterpenoids from Chaga.

A Novel Bioanalytical Method for Determination of Inotodiol Isolated from Inonotus Obliquus and Its Application to Pharmacokinetic Study.

In this study, we developed a bioanalytical method using liquid chromatography coupled to triple quadrupole tandem mass spectrometry (LC-MS/MS) to apply to a pharmacokinetic study of inotodiol, which is known for its anti-cancer activity. Plasma samples were prepared with alkaline hydrolysis, liquid-liquid extraction, and solid-phase extraction. Inotodiol was detected in positive mode with atmospheric pressure chemical ionization by multiple-reaction monitoring mode using LC-MS/MS. The developed method was validated with linearity, accuracy, and precision. Accuracy ranged from 97.8% to 111.9%, and the coefficient of variation for precision was 1.8% to 4.4%. The developed method was applied for pharmacokinetic study, and the mean pharmacokinetic parameters administration were calculated as follows: λz 0.016 min-1; T1/2 49.35 min; Cmax 2582 ng/mL; Cl 0.004 ng/min; AUC0-t 109,500 ng×min/mL; MRT0-t 32.30 min; Vd 0.281 mL after intravenous administration at dose of 2 mg/kg and λz 0.005 min-1; T1/2 138.6 min; Tmax 40 min; Cmax 49.56 ng/mL; AUC0-t 6176 ng×in/mL; MRT0-t 103.7 min after oral administration. The absolute oral bioavailability of inotodiol was 0.45%, similar to nonpolar phytosterols. Collectively, this is the first bioanalytical method and pharmacokinetic study for inotodiol.

Chaga mushroom extract induces autophagy via the AMPK-mTOR signaling pathway in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaga mushrooms (Inonotus obliquus) are commonly used in traditional treatments in Eastern Europe and Asia due to their diverse pharmacological effects, including anti-tumor and immunologic effects. Thus, many cancer patients take Chaga mushrooms as a complementary medicine, even during chemotherapy or radiotherapy. However, few studies have investigated the effects or molecular targets of Chaga mushrooms in breast cancer. AIM OF THE STUDY: Herein, we examined the anticancer effects of Chaga mushrooms in different types of breast cancer cell lines, and explored the underlying molecular mechanism to better understand their effects and benefits. MATERIALS AND METHODS: Chaga mushroom extract (CME) was prepared by extracting Chaga mushrooms with 70% ethanol. The cytotoxic effects of CME were assessed by MTT assay and protein expressions were evaluated by western blotting. To evaluate in vivo anti-tumor effects of CME, CME (2 g/kg) was orally administered to 4T1 tumor-bearing BALB/c mice every other day over 30 days (15 administrations), and tumor sizes were measured. Silica gel column chromatography was used to fractionate CME, and major constituents responsible for cytotoxic effects of CME were identified by 1H/13C-NMR and LC-MS. RESULTS: CME inhibited the proliferation of 4T1 mouse breast cancer cells in a dose and time-dependent manner. The expression of LC3 and phosphorylation of AMPK were increased by CME, while the phosphorylation of mTOR, S6, and S6K1 were suppressed, suggesting that CME induced autophagy by activating AMPK and inhibiting mTOR signaling pathways. Consistent with its observed cytotoxic effect in vitro, CME effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition, inotodiol and trametenolic acid were identified as the major constituents responsible for the cytotoxic effects of CME on breast cancer cells. Moreover, inotodiol and trametenolic acid-enriched fractions both exhibited cytotoxic effects regardless of breast cancer cell subtypes and did not interfere with the cytotoxic effects of conventional drugs. CONCLUSIONS: Taken together, Chaga mushroom extract induced autophagy by activating AMPK and inhibiting the mTOR signaling pathway. Our data suggest Chaga mushrooms may be a beneficial complementary medicine for breast cancer patients.

Inotodiol From Inonotus obliquus Chaga Mushroom Induces Atypical Maturation in Dendritic Cells.

Dendritic cells (DCs) have the ability to stimulate naïve T cells that coordinate subsequent adaptive response toward an inflammatory response or tolerance depending on the DC differentiation level. Inotodiol, a lanostane triterpenoid found in Inonotus obliquus (wild Chaga mushroom), is a natural compound with a wide range of biological activities. In this study, we investigated whether inotodiol promotes the maturation of bone marrow-derived DCs (BMDCs) and inotodiol-treated BMDCs induce T cell activation. Inotodiol increased the expression of surface maturation markers, including MHC-I, MHC-II, CD86, and CD40, on BMDCs without affecting the production of various cytokines, including TNF-α and IL-12p40 in these cells. T cells primed with inotodiol-treated BMDCs proliferated and produced IL-2, without producing other cytokines, including IL-12p40 and IFN-γ. Injection of inotodiol into mice induced maturation of splenic DCs and IL-2 production, and the administration of inotodiol and inotodiol-treated BMDCs induced the proliferation of adoptively transferred CD8+ T cells in vivo. The phosphatidylinositol-3-kinase inhibitor wortmannin abrogated the upregulation of Akt phosphorylation and CD86 and MHC-II expression induced by inotodiol. However, inotodiol failed to induce phosphorylation of the IκB kinase and degradation of IκB-α, and increased expression of CD86 induced by inotodiol was not blocked by an IκB kinase inhibitor. These results suggest that inotodiol induces a characteristic type of maturation in DCs through phosphatidylinositol-3-kinase activation independent of NF-κB, and inotodiol-treated DCs enhance T cell proliferation and IL-2 secretion.

Anti-allergic effect of inotodiol, a lanostane triterpenoid from Chaga mushroom, via selective inhibition of mast cell function.

Inotodiol is a lanostane triterpenoid found only in Chaga mushroom. In the previous study investigating anti-allergic effects of fractionated Chaga mushroom extracts, we have found evidence that purified inotodiol holds an activity to suppress the mast cell function in vivo. To address the therapeutic relevance of the finding, in this study, we investigated whether inotodiol could also alleviate allergy symptoms observed in a chicken ovalbumin (cOVA)-induced mouse model of food allergy. Like the crude 70% ethanol extract of Chaga mushroom (320 mg/kg), oral administration of inotodiol (20 mg/kg), regardless of whether that was for preventive or treatment purpose, resulted in a significant improvement in allergic symptoms and inflammatory lesions in the small intestine appearing after repeated oral challenge with cOVA. Despite the results that inotodiol (20 mg/kg) and the Chaga mushroom extract (320 mg/kg) took effect to a similar extent, immunological mechanisms underlying those effects were found to be distinct from each other. That is, the results obtained from several in vivo assays, including mast cell-mediated passive systemic anaphylaxis, activation/proliferation of adoptively transferred antigen-specific T cells and immunoglobulin (IgG1, IgE, IgA) production by antigen-specific B cells, illustrated that inotodiol selectively inhibited the mast cell function without having any noticeable effect on other immune responses while the crude Chaga mushroom extract indiscriminately suppressed diverse immune responses. The strong anti-allergic activity of inotodiol, along with its remarkable selectivity to mast cell, makes it an excellent therapeutic candidate for food allergy with both high efficacy and outstanding safety.

Inotodiol inhibits cells migration and invasion and induces apoptosis via p53-dependent pathway in HeLa cells.

BACKGROUND: Inonotus obliquus, namely as Chaga mushroom, is a medicinal and edible fungus, which is widely used in food and medical fields. Inotodiol, a natural lanostane-type triterpenoid with remarkable pharmacological activities, was isolated from Inonotus obliquus, which its potential anti-tumor molecular mechanism was elaborated poorly. PURPOSE: The aim of the present study was to investigate the effect of Inotodiol on HeLa cell migration, invasion and apoptosis through p53-dependent pathway. STUDY DESIGN AND METHODS: The potential mechanisms of Inotodiol on HeLa cell anti-metastatic and pro-apoptosis via wound healing assay, trans-well invasion assay, flow cytometry, caspase-3 activity assay and western blot analysis were studied, as well as the involvement of p53 signaling pathway in anti-metastatic and pro-apoptosis of Inotodiol. Besides, the function of tumor suppressor p53 was further verified by small interfering RNA. RESULTS: Firstly, the cell viability assay showed that low-concentration of Inotodiol had no cytotoxicity to HeLa cells and whereas the concentration above 25 µM significantly inhibited HeLa cell growth and even induced apoptosis. This result was further demonstrated by cell proliferation and morphology assay. Secondly, in vitro wound healing and trans-well invasion assays reported that low-concentration treatment of Inotodiol significantly inhibited cells migration and invasion in a dose-dependent manner, the western blot analysis of matrix mettalloprotinase-2 (MMP2) and matrix mettalloprotinase-9 (MMP9) levels were also decreased. Moreover, Inotodiol notably induced tumor cell apoptosis by Annexin-V-FITC apoptosis assay, which is associated with activation pro-apoptotic proteins of PARP, cleaved caspase-3 and Bax expression, inhibition anti-apoptotic protein Bcl-2 expression. Finally, the anti-tumor activity of Inotodiol was attenuated by silencing p53 tumor suppressor, the result revealed that pre-treatment with p53-specific small interfering RNA (si-p53) markedly inhibited Intodiol-indeuced HeLa cell apoptosis and decreased the caspase-3 activity. What is more, the inhibitory effect of Inotodiol on tumor migration and invasion was blocked under p53 knockdown. CONCLUSION: To sum up, the present study indicated that Inotodiol possessed the potential to prevent malignant tumor migration and invasion, and it might be a natural active compound candidate for clinical treatment of human cervical cancer.

Bioactivity-based analysis and chemical characterization of cytotoxic constituents from Chaga mushroom (Inonotus obliquus) that induce apoptosis in human lung adenocarcinoma 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 CH2Cl2-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 IC50 values 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.

Chaga ( Inonotus obliquus), a Future Potential Medicinal Fungus in Oncology? A Chemical Study and a Comparison of the Cytotoxicity Against Human Lung Adenocarcinoma Cells (A549) and Human Bronchial Epithelial Cells (BEAS-2B).

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).

The mast cell stabilizing activity of Chaga mushroom critical for its therapeutic effect on food allergy is derived from inotodiol.

While an anti-allergic effect of Chaga mushroom (Inonotus obliquus) has been indicated, its therapeutic effect on allergy and immunoregulatory mechanisms and chemical constituents directly responsible for that are hardly known. We examined the effect of 70% ethanol extract of Chaga mushroom (EE) and its dichloromethane (DF) and aqueous (AF) fractions using a mouse model of chicken ovalbumin (cOVA)-induced food allergy, and found that only EE and DF ameliorated allergy symptoms to a significant extent. The in vivo mast cell-stabilizing activity was also found only in EE and DF whereas the activities to suppress Th2 and Th17 immune responses and cOVA-specific IgE production in the small intestine were observed in all three treatment regimens, implying that inhibition of the mast cell function by lipophilic compounds was vital for the therapeutic effect. Results also indicated that inotodiol, a triterpenoid predominantly present in DF, played an active role as a mast cell stabilizer.

Induction of human MCF-7 breast cancer cell apoptosis by Inonotus obliquus and study on its mechanism / 中草药

Objective: To study the induction of human MCF-7 breast cancer cell apoptosis by inotodiol isolated from Inonotus obliquus, a traditional Chinese medicinal fungi. Methods: Inotodiol was isolated from the crude ethanol extract of I. obliquus and purified. Inotodiol at different concentration and human breast cancer MCF-7 cell line were co-incubated. Relative cell viabilities were determined by MTT method; apoptosis was analyzed by Annexin V/PI dual staining with flow cytometry; The protein expression levels of Caspase-3 and Poly ADP ribose polymerase (PARP) were detected by Western blotting. Results: Inotodiol reduced the viability of MCF-7 cells dose-and time-dependently. Moreover, inotodiol induced the apoptosis in MCF-7 cells. The data of Western blotting showed that inotodiol up-regulated the expression levels of cleaved Caspase-3 and PARP, in a time-dependent manner. Conclusion: Inotodiol could induce apoptosis in human breast cancer cells, which provides novel information for clinical application of inotodiol and its preparations for the breast cancer patients.

Triterpenoids from Inonotus obliquus and their antitumor activities.

Three new lanostane-type triterpenes, inonotusanes A-C (1-3), and a new naturally occurring one, 3ß-hydroxy-25,26,27-trinorlanosta-8,22E-dien-24-oic acid (4), together with sixteen known triterpenoids (5-20), including 13 lanostane derivatives, 2 lupanes and 1 oleanane-type triterpene were isolated from the sclerotia of Inonotus obliquus. Their structures were elucidated by 1D and 2D NMR spectroscopy and HRMS. Compounds 6, 8, 18 and 20 exhibited strong cytotoxicity against A549 tumor cell lines, with IC50 values of 2.34, 1.63, 8.39 and 5.39µM, respectively. Seven compounds (3, 9, 10, 12, 18-20) exhibited moderate cytotoxicity against A549, HT29, Hela or L1210 tumor cell lines.