Visualizing the spatial distribution of ustalic acid in the fruiting body of Tricholoma kakishimeji.

Imaging mass spectrometry (IMS) was conducted for the first time using ustalic acid (UA) and the fruiting body of Tricholoma kakishimeji to localize mushroom toxins. The mushroom materials were systematically collected in Japan, and analysis of the cross sections of the materials at a resolution of 120 µm using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-IMS) revealed the localization of UA and its biogenically related metabolites. MALDI-IMS confirmed that UA was predominantly located on the entire surface of the fruiting body and accumulated in higher amounts in younger fruiting bodies than in mature ones. UA is the first toxic secondary metabolite in the genus Tricholoma locally identified using IMS in mushrooms.

No Evidence Was Found for the Presence of Terreolides, Terreumols or Saponaceolides H-S in the Fruiting Bodies of Tricholoma terreum (Basidiomycota, Agaricales).

Two different collections of the gilled wild fungus Tricholoma terreum, collected in Italy, were subjected to phytochemical analysis. The fungal material was confidently identified by analysis of the ITS genomic sequences. Using both HR-LC-MS and NMR techniques, no evidence was found for the presence in the fruiting bodies of terreolides, terreumols or saponaceolides H-S, in striking contrast with the isolation of these terpenoids by Chinese authors from a mushroom collected in France and identified as T. terreum. The main cytotoxic terpenoid identified and isolated from the extracts of the specimens investigated in this work was the C30 derivative saponaceolide B, which had been previously isolated from T. saponaceum and other T. terreum collections. Although saponaceolide B is a rather labile molecule, easily degradable by heat or in acidic conditions, our study indicated that none of the extraction protocols used produced saponaceolide H-S or terreolide/terreumol derivatives, thus excluding the possibility that the latter compounds could be extraction artifacts. Considered together, these findings point to the need for the unambiguous identification of mushroom species belonging to the complex genus Tricholoma, characterized by high variability in the composition of metabolites. Moreover, based on our data, T. terreum must be considered an edible mushroom.

New Tricholomalides D-G from the Mushroom Tricholoma ustaloides Grown in an Italian Beech Wood.

Four novel seconeodolastane diterpenoids, named tricholomalides D-G, were isolated, together with the known tricholomalide C, from the fruiting bodies of Tricholoma ustaloides Romagn., a species belonging to the large Tricholoma genus of higher mushrooms (Basidiomycota, family Tricholomataceae). They were isolated through multiple chromatographic separations, and the structures, including the absolute configuration, were established through a detailed analysis of MS, NMR, and CD spectral data and comparison with related compounds reported in the literature, which has been thoroughly revised.

New Tricholidic Acid Triterpenoids from the Mushroom Tricholoma ustaloides Collected in an Italian Beech Wood.

The secondary metabolites produced by Tricholoma ustaloides Romagn., a mushroom species belonging to the large Tricholoma genus (Basidiomycota, Tricholomataceae), are unknown. Therefore, encouraged by the interesting results obtained in our previous chemical analyses of a few Tricholoma species collected in Italian woods, we aimed to investigate the secondary metabolites of Tricholoma ustaloides. The chemical analysis involved the isolation and characterization of secondary metabolites through an extensive chromatographic study. The structures of isolated metabolites, including the absolute configuration, were established based on a detailed analysis of MS, NMR spectroscopic, optical rotation, and circular dicroism data, and on comparison with those of related compounds reported in the literature. Two novel lanostane triterpenoids, named tricholidic acids B and C, together with triglycerides, a mixture of free fatty acids, five unidentified metabolites, and the known rare saponaceolides F and J, tricholidic acid, and tricholomenyn C, were isolated from an EtOAc extract of fruiting bodies of Tricholoma ustaloides that were collected in an Italian beech wood. This is the second example of isolation of tricholidic acid derivatives from a natural source. Saponaceolides F and J exhibited high cytotoxicity (IC50 values ≤ 10 µM) against a panel of five human cancer cell lines. The toxicity against myeloid leukemia (HL-60), lung cancer (A-549), hepatocellular cancer (HepG2), renal cancer (Caki-1), and breast cancer (MCF-7) cells was higher than that shown by the very well-known cytotoxic drug cisplatin.

Insights into health promoting effects and myochemical profiles of pine mushroom Tricholoma matsutake.

Tricholoma matsutake (TM) is a valuable edible mushroom that has attracted increasing attention due to its potential medicinal values and functional uses. However, the chemical composition and molecular mechanisms behinds TM are not specifically summarized yet. Hence, this review aims to systematically analyze the research progress on the characterization of chemical compositions and the reported health effects of TM in the last 20 years. The myochemical profiles of TM consist of proteins with amino acids, fatty acids, nucleic acids with their derivatives, polysaccharides, minerals, volatile components, phenolic compounds, and steroids. The bioactive substances in TM exert their health effects mainly by regulating body immunity and restoring the balance of the redox system. NF-κB signaling pathway and its downstream cytokines such as TNF-α and IL-6 are the key molecular mechanisms. In addition, MAPK, PI3K-Akt, and JAK-STAT are also involved. NF-κB, MAPK, and PI3K-Akt are also highly related to cancer regulation and thus TM has great anticancer potential. Considering that most studies have only investigated the dosage and inhibition rate of TM on cancer cell lines, more extensive studies need to focus on the specific molecular mechanisms behind these anticancer effects in the future.

Tricholosterols A-D, four new ergosterol derivatives from the mushroom Tricholoma terreum.

Four ergosterol derivatives, named tricholosterols A-D (1-4), have been isolated from the fruiting bodies of Tricholoma terreum. Their chemical structures have been determined using a combination of spectroscopic analysis as well as computational methods. Compound 1 possesses a rare D-ring opening ergosterol skeleton, while compounds 2-4 are rare degraded ergosterols. Compounds 1 and 4 exhibited moderate inhibitory activity against NO production with IC50 values of 27.6 and 31.8 µM, respectively. This is the first report of steroids from T. terreum.

Quantitative transcriptomic and metabolomic analyses reveal the changes in Tricholoma matsutake fruiting bodies during cold storage.

A combination of transcriptomic and metabolomic analyses was performed to systematically understand the metabolic changes in Tricholoma matsutake fruiting bodies during cold storage. In total, 800 metabolites were identified and 19,964 annotated unigenes were quantified. The unigenes related to the catabolism of proteins, carbohydrates, and lipids were mainly upregulated during cold storage, but the related primary metabolites were not accumulated, which indicated complete degradation and loss of nutrients. Concurrently, the synthesis and metabolism of the main components of the cell wall, chitin and ß-1,3-glucan, were regulated, indicating the dynamic remodeling of the T. matsutake cell wall structure. Additionally, indole-3-acetic acid and components of its synthesis pathway were found in T. matsutake, indicating their potential role as a communicator between T. matsutake and its symbiotic plants. The results provide new information to improve the understanding of the metabolic mechanism of T. matsutake fruit bodies during postharvest cold storage.

Fomes fomentarius and Tricholoma anatolicum (Agaricomycetes) Extracts Exhibit Significant Multiple Drug-Resistant Modulation Activity in Drug-Resistant Breast Cancer Cells.

Multiple drug resistance is one of the main problems that hinder successful cancer chemotherapy. Investigations on the development of effective chemotherapeutic agents and drug resistance inhibitors motivate studies on the effects of natural compounds on drug-resistant cancer cells. For this purpose, aqueous, methanol, and ethanol extracts of Fomes fomentarius and Tricholoma anatolicum were prepared. The extracts were evaluated to assess their anticancer and multiple drug resistance modulation activities. Cytotoxic effects of F. fomentarius and T. anatolicum extracts on paclitaxel and vincristine resistant P-glycoprotein over-expressing MCF-7 cell lines were investigated by cytotoxicity test (XTT). P-glycoprotein reversing ability and MDR modulation effects of the extracts were determined by flow cytometry through Rhodamine 123 exclusion assay. Furthermore, 11 phenolic compounds in the extracts were characterized by HPLC. As a result of the cytotoxicity assay, IC50 values of the extracts for MCF-7/Vinc were between 1.08 and 1.80 mg/mL, and IC50 values for MCF-7/Pac were found between 1.11 and 2.83 mg/mL. Strikingly, methanol extract of F. fomentarius and ethanol extract of T. anatolicum have potential value to become MDR reversing agents for drug-resistant breast cancer cells.

Arsenic speciation in mushrooms using dimensional chromatography coupled to ICP-MS detector.

This study concerns total arsenic (TAs) and arsenic species determination in three species of mushrooms collected in Yunnan, China. The purpose of this study was to check concentration level of arsenic in Boletus edulis, Tricholoma matsutake and Suillellus luridus, estimate arsenic bioaccessibility and find out which arsenic species occur in mushrooms to assess if they may pose a threat to human health. An analytical methodology based on ion chromatography (IC) hyphenated to inductively coupled plasma mass spectrometry (ICP-MS) with dynamic reaction cell (DRC) and size exclusion chromatography (SEC) with UV-Vis detection and ICP-DRC-MS detection. Ultrasound assisted extraction (UAE), microwave assisted extraction (MAE) and enzymatic assisted extraction (EAE) were applied. Quantification of As species in extracts was performed by IC/ICP-DRC-MS in the first dimension. Slightly better extraction efficiencies were obtained for MAE (from 75% to 90%) then for UAE. EAE was used for estimation of bioaccessibility by application of a modified BARGE bioaccessibility method (UBM) for in vitro studies. Bioaccessibility values were in the ranges of 73%-102%, 74%-115% and 18%-87% for step 1 (S1), for step 2 (S2) and for step 3 (S3) of EAE, respectively. Extracts obtained after EAE were subjected to SEC-UV-Vis/ICP-DRC-MS analysis as the second dimension. The main signal was obtained in the area of a molecular mass of ∼5 kDa for all mushroom extracts. Monitoring of an 50SO+ ion confirmed that this signal comes from As-protein. In sample of Boletus edulis additional signal occurred which is classified as unknown As-compound. Both signals require identification with another analytical technique.

Protein chip analysis of cytokines reveals a key mechanism of the antitumor and immunostimulatory activities of Tricholoma matsutake polysaccharide.

Polysaccharide has been widely used in medical and health field because of its function of immune regulation. The aim of present study was to use protein chip to test the 200 cytokines secreted by macrophages which were induced by the polysaccharides of Tricholoma matsutake (TMP-A) to study the role of TMP-A acting on macrophages and its mechanism, further understanding the mechanism of the TMP-A effect on immune activity. The results of the analysis indicated that among all of these cytokines, including IL-1ß, IL-10, IL-23, TNF-α, CD40L, G-CSF, etc. there are 73 up-regulated and 43 down-regulated cytokines. The KEGG analysis indicated that T. matsutake polysaccharide can influence the immune response of macrophages through a series of signaling pathways, and the three major signaling pathways are Jak-STAT signaling pathway, PI3K-Akt signaling pathway and NF-kappa B signaling pathway. Those three signaling pathway are closely related to the pathogenesis of many diseases. The results showed that TMP-A can activate immune cells to regulate the immunity.

Development of a flavor fingerprint by HS-GC-IMS with PCA for volatile compounds of Tricholoma matsutake Singer.

The flavor fingerprint of Tricholoma matsutake Singer was developed and volatile compounds were investigated by HS-GC-IMS fingerprinting combining with PCA. A total of 25 typical target compounds were identified. The differences of flavor compounds in pileus and stipe of Tricholoma matsutake Singer from different areas were observed. After drying, the concentration of C8 compounds, including 3-octanone, 3-octanol, 1-octen-3-one and 1-octanol, decreased, while the concentration of methanol and 1-pentanol increased. Subsequently, PCA was performed based on the signal intensity of the identified violate compounds, and the results clearly showed that the samples in a relatively independent space would be well distinguished. Combining the signal intensity of the identified violate compounds by HS-GC-IMS and PCA, the samples from different portions of Tricholoma matsutake Singer and different areas were distinguished. The results showed that the flavor fingerprint of Tricholoma matsutake Singer were established by HS-GC-IMS and PCA based on the identified volatile compounds.

Quality Evaluation of Tricholoma matsutake Based on the Nucleic Acid Compounds by UPLC-TOF/MS and UPLC-QqQ/MS.

So far, there has been no quality evaluation of Tricholoma matsutake. Nucleic acid compounds are a kind of functional ingredient in T. matsutake that is beneficial to human health. In this study, a UPLC-TOF/MS method was first used to scan and identify the potential nucleic acid compounds in T. matsutake. Based on the calculation of the molecular formula and subsequent confirmation by authentic standards, 15 nucleic acid compounds were unambiguously identified: adenosine, cytidine, guanosine, inosine, thymidine, uridine, xanthosine dehydrate, 2'-deoxyadenosine, 2'-deoxycytidine, 2'-deoxyguanosine, 2'-deoxyuridine, adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, and uridine 5'-monophosphate. Then, a UPLC-QqQ/MS method was developed for the subsequent quantitative analysis. After validating the limits of quantification, detection, precision, repeatability, and recovery through a calibration curve, the content of 15 nucleic acid compounds was determined by the proposed UPLC-QqQ/MS method in 80 T. matsutake samples collected from different regions in Sichuan province, Southwest China. After the statistical analysis, we suggest that the total content of nucleic acid compounds in the qualified T. matsutake should be higher than 24.49 mg/100 g. The results indicated that the combined use of UPLC-TOF/MS and UPLC-QqQ/MS is efficient for fast identification and determination of nucleic acid compounds to comprehensively evaluate the quality of T. matsutake.

Characteristic volatiles fingerprints and changes of volatile compounds in fresh and dried Tricholoma matsutake Singer by HS-GC-IMS and HS-SPME-GC-MS.

The present study was to analyze the water dynamics of Tricholoma matsutake Singer during hot air drying by low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI), and to investigate the volatile compounds in the pileus, upper stipe and lower stipe of fresh and dried Tricholoma matsutake Singer by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fresh samples were dried at 60 °C for 12 h. With the T2i relaxation times decreasing, the water migrated from the inner toward the periphery and then expelled during drying. The characteristic volatiles fingerprints of different parts of fresh and dried samples were established by HS-GC-IMS. The significant differences in volatile compounds were observed among different parts of the fresh sample and C8 compounds (70%-97%) were the principal components. After drying, the concentration of C8 compounds dramatically decreased and some volatile compounds (hexanal, heptanal, 2(5H)-furanone, acetophenone, nonanal, benzeneacetaldehyde) were formed. Thus, hot air treatment affected the volatile compounds in Tricholoma matsutake Singer.

Biomimetic synthesis of the bisindole framework present in sciodole, an alkaloid from Tricholoma sciodes.

A synthesis of the unique bisindole framework present in the mushroom-derived alkaloid sciodole has been achieved, validating a biosynthesis proposal that the C-N bisindole bond present in the natural product is forged by amination of an azafulvenium.

Lanostane triterpenoids from Tricholoma pardinum with NO production inhibitory and cytotoxic activities.

Eight undescribed lanostane triterpenoids, pardinols A‒H, along with one previously reported lanostane triterpenoid, namely saponaceol B, were isolated from the fruiting bodies of Tricholoma pardinum. Their structures and stereoconfigurations were established via combination of extensive spectroscopic analyses, alkaline methanolysis method and TDDFT/ECD calculations. Pardinols B and E-H exhibited certain inhibition activities of nitric oxide (NO) production with IC50 value ranging from 5.3 to 14.7 µM, as well as cytotoxicities against human cancer cell-lines.

Immunomodulating activity of the polysaccharide TLH-3 from Tricholomalobayense in RAW264.7 macrophages.

Polysaccharide TLH-3 extracted from Tricholoma lobayense possessed unique antioxidant and anti-aging activities, whereas its immunomodulatory properties remain unexplored. Herein in order to explore TLH-3 biological activities, the immunomodulatory effects on RAW264.7 macrophages and its molecular mechanisms were investigated. It was showed that TLH-3 could significantly enhance the phagocytic activity, releasing toxic molecules NO (nitricoxide), secretion of the cytokine TNF-?(tumor necrosis factor-?, IL-6 (interleukin-6). Further, TNF-?and IL-6 were blocked by the inhibitor of TLR4 (Toll-like receptor4), suggesting TLR4 was a receptor of TLH-3, and immunomodulatory activity of TLH-3 was mediated by TLR4. Moreover, immunofluorescence indicated that TLH-3 lead to the nuclear translocation of NF-?B (nuclear factor-?B) subunit p65. Western blotting demonstrated that NF-?B levels in nucleuses increased and cytoplasmic I?B-?(inhibitor of NF-?B) degraded after TLH-3 treatment, suggesting that TLH-3 probably stimulated macrophage by activating the IκB-α-NF-κB pathway via TLR-4. This study demonstrated that TLH-3 could be potentially used as immunomodulatory agent for healthcare and disease control.

Oxidation of the Meroterpenoid (-)-Terreumol C from the Mushroom Tricholoma terreum: Discovery of Cytotoxic Analogues.

Aiming at the discovery of new cytotoxic meroterpenoids, the chemical reactivity of the natural product (-)-terreumol C from the edible mushroom Tricholoma terreum was investigated. A series of high-yielding oxygenations and brominations at the 10-membered ring were discovered. The regioselectivity of hydroxylation could be guided by installing protecting groups at the hydroquinone section. Dihydroxylation led to the stereoselective formation of a novel tricycle containing an 11-oxabicyclo[5.3.1]undecane system. Several of the compounds proved to be as cytotoxic against cancer cell lines as the natural products terreumols A and C in the single-digit micromolar range. Interestingly, functionalization of the southern rim formed by carbons C5-C6-C7-C8 is tolerated without much loss of cytotoxicity.

Investigation of the immunomodulatory activity of Tricholoma matsutake mycelium in cyclophosphamide-induced immunosuppressed mice.

Tricholoma matsutake, a popular food and biopharmaceutical resource in Asia, possesses various pharmacological activities. Although T. matsutake mycelium (TM) may enhance immunity, previous studies, to the best of our knowledge, have been performed on normal animals or cells alone. The present study aimed to evaluate the immunomodulatory activity of TM at doses of 0.3, 1.0 and 2.0 g/kg in cyclophosphamide (CTX)­induced immunosuppressed mouse models. TM treatment for 2 weeks markedly improved the gain in bodyweight, increased organ indices, reduced hind paw swelling and positively regulated the cytotoxicity of natural killer cells and the proliferation of lymphocytes. These effects are similar to that of thymosin α1 (0.16 mg/kg) which served as the positive control. In CTX­induced immunosuppressed mice, TM demonstrated marked effects on the modulation of the production of immunoglobulin (Ig)G and IgA, and the levels of interleukin­2, 6, 10 and 12, interferon­α and γ and tumor necrosis factor­α in serum. Compared with CTX mice, the reduced activity of nuclear factor (NF)­κB in serum and spleen, and phosphorylation of inhibitor of NF­κB kinase α/ß in spleen were observed in TM­treated mice. Taken together, TM effectively improved immune function in immunosuppressed mice via modulation of ILs and inflammatory factors associated with the NF­κB signaling pathway.

Structure elucidation and antitumor activity of a new polysaccharide from Maerkang Tricholoma matsutake.

A new heteropolysaccharide was isolated from the fruiting bodies of Tricholoma matsutake which had a molecular weight of 12078 Da. The results of structural features analysis showed that T. matsutake polysaccharide, here named TMP-B, was mainly composed of α - D - glucose and α - D - galactose which ratios were 7:2 and had a backbone of 1, 4 - linked α - D - glucose which branches were mainly composed of two 6 - linked α - D - galactose residue, and the α - D - galactose was 1, 6 - linked. Antitumor activity results showed that heteropolysaccharide TMP-B could inhibit the growth of S180 tumor in vivo and promote the apoptosis of L929 cells in vitro. Immunoregulatory activity results showed that TMP-B could promote the proliferation of macrophages by affecting G0/G1 phase, S phases and G2/M phases and promote cytokines release and gene expression. The result of this study introduced Maerkang T. matsutake as a possible valuable source which helped to exhibit unique antitumor and immunoregulatory properties.

Matsutakone and Matsutoic Acid, Two (Nor)steroids with Unusual Skeletons from the Edible Mushroom Tricholoma matsutake.

Matsutakone (1), a novel sterol with an unprecedented polycyclic ring system, together with a new norsteroid matsutoic acid (2) were isolated from the fruiting bodies of Tricholoma matsutake. Their structures and absolute configurations were assigned by extensive spectroscopic analyses and computational methods. Bioassay results showed that compounds 1 and 2 exhibited inhibitory activities against acetylcholinesterase (IC50 20.9 µM for 1).