Exploration of diverse secondary metabolites from Penicillium brasilianum by co-culturing with Armillaria mellea.

Bioinformatic analysis revealed that the genomes of ubiquitous Penicillium spp. might carry dozens of biosynthetic gene clusters (BGCs), yet many clusters have remained uncharacterized. In this study, a detailed investigation of co-culture fermentation including the basidiomycete Armillaria mellea CPCC 400891 and the P. brasilianum CGMCC 3.4402 enabled the isolation of five new compounds including two bisabolene-type sesquiterpenes (arpenibisabolanes A and B), two carotane-type sesquiterpenes (arpenicarotanes A and B), and one polyketide (arpenichorismite A) along with seven known compounds. The assignments of their structures were deduced by the extensive analyses of detailed spectroscopic data, electronic circular dichroism spectra, together with delimitation of the biogenesis. Most new compounds were not detected in monocultures under the same fermentation conditions. Arpenibisabolane A represents the first example of a 6/5-fused bicyclic bisabolene. The bioassay of these five new compounds exhibited no cytotoxic activities in vitro against three human cancer cell lines (A549, MCF-7, and HepG2). Moreover, sequence alignments and bioinformatic analysis to other metabolic pathways, two BGCs including Pb-bis and Pb-car, responsible for generating sesquiterpenoids from co-culture were identified, respectively. Furthermore, based on the chemical structures and deduced gene functions of the two clusters, a hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed. These results demonstrated that the co-culture approach would facilitate bioprospecting for new metabolites even from the well-studied microbes. Our findings would provide opportunities for further understanding of the biosynthesis of intriguing sesquiterpenoids via metabolic engineering strategies. KEY POINTS: • Penicillium and Armillaria co-culture facilitates the production of diverse secondary metabolites • Arpenibisabolane A represents the first example of 6/5-fused bicyclic bisabolenes • A hypothetic metabolic pathway for biosynthesizing induced sesquiterpenoids was proposed.

Extraction, purification, structural characteristics and biological properties of the polysaccharides from Armillaria mellea (Vahl) P. Kumm.: A review.

Armillaria mellea (Vahl) P. Kumm. is a well-known homoeopathic plant with medicinal and culinary uses. Modern phytochemical researchers have successfully extracted and purified over 40 types of A. mellea polysaccharides (AMPs) from the fruiting bodies, hyphae and fermentation broth of A. mellea, and some of them have been analyzed and identified by their chemical structures. The impressive biological activity of these polysaccharides has been recognized by scientists worldwide. Many studies show that AMPs have remarkable antioxidant, anti-diabetic, anti-tumor, anti-inflammatory, immunoregulatory, hypolipidemic, thrombectomy, anti-aging, pulmonary protective, hepatic protective, anti-Alzheimer's properties, etc. However, the current understanding of the relationships between their chemical structure and biological activity, toxicological effects and pharmacokinetics remains limited. This article provides a systematic review of the research conducted over the past decades on the extraction and purification methods, structural characteristics, biological activity and mechanism of action of AMPs. The aim is to provide a research base that will benefit the future application of AMPs as therapeutic drugs and functional foods, and also provide insights for the further development of AMPs.

Full-length genome characterization of a novel mitovirus isolated from the root rot fungus Armillaria mellea.

Members of the genus Armillaria belong to the group of pathogenic and facultative saprotrophic fungi that are generally known as one of the causative agents of white root rot in infected plants including deciduous and evergreen trees and shrubs. Although several single-stranded RNA mycoviruses were previously described in different Armillaria species, there is no report on mitoviruses (one of the simplest RNA viruses of fungal hosts) known to infect Armillaria taxa. In this study, a new mitovirus denominated "Armillaria mellea mitovirus 1" (AmMV1) was identified in the sporophore samples of Armillaria mellea, commonly known as honey mushroom. AmMV1 has a genome length of 4440 nucleotides and a G + C content of 48%. It encompasses a single open reading frame (ORF) that encodes an RNA-dependent RNA polymerase (RdRp). Comparison through BLASTp analysis revealed that the RdRp domain of AmMV1 shares a sequence identity ranging from 33.43% to 43.27% with RdRp domains of Duamitovirus genus members, having the highest similarity (43.27%) to Rhizoctonia solani mitovirus 94. According to phylogenetic analysis, AmMV1 is classified as a member of the genus Duamitovirus belonging to the Mitoviridae family. This marks the initial instance of a mitovirus identified in Armillaria spp..

UHPLC-MS-Based Metabolomics Reveal the Potential Mechanism of Armillaria mellea Acid Polysaccharide in and Its Effects on Cyclophosphamide-Induced Immunosuppressed Mice.

Armillaria mellea (Vahl) P. Kumm is commonly used for food and pharmaceutical supplements due to its immune regulatory function, and polysaccharides are one of its main components. The aim of this research is to study the immunological activity of the purified acidic polysaccharide fraction, namely, AMPA, isolated from Armillaria mellea crude polysaccharide (AMP). In this study, a combination of the immune activity of mouse macrophages in vitro and serum metabonomics in vivo was used to comprehensively explore the cell viability and metabolic changes in immune-deficient mice in the AMPA intervention, with the aim of elucidating the potential mechanisms of AMPA in the treatment of immunodeficiency. The in vitro experiments revealed that, compared with LPS-induced RAW264.7, the AMPA treatment elevated the levels of the cellular immune factors IL-2, IL-6, IgM, IgA, TNF-α, and IFN-γ; promoted the expression of immune proteins; and activated the TLR4/MyD88/NF-κB signaling pathway to produce immunological responses. The protein expression was also demonstrated in the spleen of the cyclophosphamide immunosuppressive model in vivo. The UHPLC-MS-based metabolomic analysis revealed that AMPA significantly modulated six endogenous metabolites in mice, with the associated metabolic pathways of AMPA for treating immunodeficiency selected as potential therapeutic biomarkers. The results demonstrate that phosphorylated acetyl CoA, glycolysis, and the TCA cycle were mainly activated to enhance immune factor expression and provide immune protection to the body. These experimental results are important for the development and application of AMPA as a valuable health food or drug that enhances immunity.

Honey Mushroom, Armillaria mellea (Agaricomycetes) and Its Fermentation Products Target Regulation of OAT1/OAT3 Proteins to Reduce Hyperuricemia in Mice.

BACKGROUND: Disorders of purine metabolism are the main cause of hyperuricemia. Current drugs for the treatment of hyperuricemia usually cause a degree of cardiovascular damage. METHODS: This study aimed to investigate the therapeutic effects of Armillaria mellea fruiting body (AFB), Armillaria rhizomorph (AR) and Armillaria mellea fermentation product (after rhizomorphs removal) (AFP) on hyperuricemic mice. The hyperuricemia mouse model was established by oral administration of potassium oxonate 0.9 g⋅kg-1 and hypoxanthine 0.5 g⋅kg-1 for two weeks. Starting from the third week, the intragastric administration of the intervention drug group was as follows: Allopurinol 0.013 g⋅kg-1, AFB (3.9 and 7.8 g⋅kg-1), AR (3.9 and 7.8 g⋅kg-1), AFP (1.95 and 3.9 g⋅kg-1) once daily for 14 days. RESULTS: Results showed that AFB, AR, and AFP reduced the contents of serum uric acid, serum creatinine, and blood urea nitrogen in hyperuricemic mice and the mechanism of action might be through up-regulation of the expression levels of organic anion transporter 1/organic anion transporter 3 proteins in kidney tissue. AR and AFP both exhibited better uric acid-lowering effects than AFB, which may be due to the higher purine content of AFB. CONCLUSIONS: Armillaria mellea and its fermentation products can treat hyperuricemia by up-regulating OAT1 protein and OAT3 protein, reducing uric acid content in mice.

Antidiabetic activity of Armillaria mellea polysaccharides: Joint ultrasonic and enzyme assisted extraction.

Armillaria mellea polysaccharides (AMPs) were obtained by ultrasonic assisted extraction (U), enzyme assisted extraction (E) and ultrasonic-enzyme assisted extraction (UE), respectively. The yield of UE-AMPs (6.32 ± 0.14%) was 1.64 times higher than that of U-AMPs (3.86 ± 0.11%) and 1.21 times higher than that of E-AMPs (5.21 ± 0.09%); meanwhile, the highest total sugar content and the lowest protein content were found in UE-AMPs. AMPs obtained from the three extraction methods had the same monosaccharide composition but in different proportions, allowing UE-AMPs to have the most potent antioxidant activity. The antidiabetic activity of UE-AMPs was investigated in streptozotocin (STZ)-induced diabetic mice. UE-AMPs, when given by gavage, greatly prevented weight loss, increased water intake, and considerably decreased blood glucose levels in diabetic mice, which were dose-dependent (P < 0.05). In addition, UE-AMPs also had a positive effect on the reduction of lipid levels in the blood, oxidative damage and liver function impairment. The pathological observation by hematoxylin-eosin staining (HE) revealed that UE-AMPs protected the organs of mice from diabetic complications (liver disease and nephropathy). Hence, our findings demonstrate that UE-AMPs are a suitable choice for improving diabetes and its complications and have great application prospects in the fields of natural medicine and functional food.

Effects of an Armillaria mellea Polysaccharide on Learning and Memory of D-Galactose-Induced Aging Mice.

Arm illaria mellea has been known and used in traditional medicine in East Asia for hundreds of years. It has already been reported that A. mellea extracts have various pharmacological effects, and the polysaccharides of A. mellea exhibit antioxidant and anti-apoptotic activities. In this study, a water-soluble polysaccharide (AMP-N-a-1), with an average molecular weight of 17 kD, was isolated and purified from the water extract of A. mellea using DEAE-52, Sepharose CL-4B, and Sephadex G-100 column chromatography. AMP-N-a-1 was mainly composed of Man (1.65%), Glca (1.64%), Rha (1.82%), Gala (2.49%), Glc (90.48%), Gal (0.89%), Xyl (0.42%), and Ara (0.61%). AMP-N-a-1 was used to study the effect on the learning and memory of mice and its underlying mechanisms. The results showed that AMP-N-a-1 could significantly increase the activities of catalase (CAT) and superoxide dismutase (SOD) and reduce the content of nitric oxide (NO) in mouse brain tissue. Meanwhile, AMP-N-a-1 could reduce the contents of norepinephrine (NE) and dopamine (DA) but could increase the content of 5-hydroxytryptamine (5-HT) in mouse brain tissue. In addition, the immunofluorescence experiment showed that AMP-N-a-1 could promote the proliferation of hippocampal dentate gyrus neurons. The above results indicate that AMP-N-a-1 can significantly improve the learning and memory of mice, and the mechanism may be that AMP-N-a-1 can participate in the regulation of learning and memory through a variety of ways.

[Utilization of used fungus-growing materials of Gastrodia elata].

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Oral Administration of Armillaria mellea Mycelia Promotes Non-Rapid Eye Movement and Rapid Eye Movement Sleep in Rats.

The present study aimed to explore whether water and ethanol extracts of Armillaria mellea mycelia produce sedative and hypnotic effects in rats. Male Sprague-Dawley rats were surgically implanted with two electroencephalogram electrodes on the skull and an electromyogram electrode on neck muscle to evaluate the alterations in rapid eye movement (REM) and non-REM (NREM) sleep after oral administration of the water and ethanol extracts. Following post-surgical recovery, thirty-six rats were randomly divided into four treatment groups and two control groups. They were treated orally with vehicle, 75 and 150 mg/kg doses of water and ethanolic extracts 15 min prior to the onset of dark (active) period. Electroencephalography results showed that the low dose of A. mellea mycelia water extract increased REM sleep time while the high dose enhanced both REM and NREM sleep times during the subsequent light (rest) period. On the other hand, although the low dose of A. mellea mycelia ethanolic extract did not alter both NREM sleep and REM sleep during the dark and light periods, the high dose increased both REM and NREM sleep during the light periods in naive rats. The HPLC-DAD analyses of both extracts allowed the identification of GABA and seven sesquiterpenoids. Based on these findings, the present study showed for the first time that water and ethanolic extracts of A. mellea mycelia, containing a source of biologically active compounds, could increase both NREM sleep and REM sleep during the rest period and may be useful for the treatment of insomnia.

Chemical Composition, Bioactive Compounds, and Antioxidant Activity of Two Wild Edible Mushrooms Armillaria mellea and Macrolepiota procera from Two Countries (Morocco and Portugal).

The present study aimed to investigate the chemical composition, bioactive compounds, and antioxidant activity of two wild edible mushrooms, the honey fungus (Armillaria mellea) and the parasol mushroom (Macrolepiota procera), collected from Northern Morocco (MA) and Portugal (PT). Those species were chosen due to their edibility, nutraceutical, and medicinal properties. Bioactive compounds (ascorbic acid, tannin, total phenolic, total flavonoid, ß-carotene, and lycopene) and their antioxidant activity were determined by spectrophotometric methods. Herein, the fruiting body of the samples revealed a significantly higher amount of bioactive compounds, and values varied between the Moroccan and the Portuguese ones. Methanolic extracts shown a strong antioxidant capacity: Using DPPH free radical-scavenging activity radicals (IC50 1.06-1.32 mg/mL); inhibition of ß-carotene bleaching radicals (IC50 0.09-0.53 mg/mL); and, reducing power radicals (IC50 0.52-1.11 mg/mL). The mushroom species with the highest antioxidant capacity was A. mellea from MA. Chemical composition was analyzed by GC-MS and LC-MS methodologies. GC-MS analysis showed that the most abundant biomolecules group was sugar compositions in the four samples (62.90%, 48.93%, 59.00%, and 53.71%) and the main components were galactitol 16.74%, petroselinic acid 19.83%, d-galactose 38.43%, and glycerol 24.43% in A. mellea (MA), A. mellea (PT), M. procera (MA), and M. procera (PT), respectively. LC-MS analysis of individual phenolic compounds revealed that vanillic acid (198.40 ± 2.82 µg/g dry weight (dw) and cinnamic acid (155.20 ± 0.97 µg/g dw) were the main compounds detected in A. mellea, while protocatechuic acid (92.52 ± 0.45 and 125.50 ± 0.89 µg/g dw) was predominated in M. procera for MA and PT samples, respectively. In general, the results of this comparative study demonstrate that the geographic and climatic conditions of the collection site can influence biomolecule compounds and antioxidant properties of wild mushrooms. This study contributes to the elaboration of nutritional, nutraceutical, and pharmaceutical databases of the worldwide consumed mushrooms.

Armillaria mellea Symbiosis Drives Metabolomic and Transcriptomic Changes in Polyporus umbellatus Sclerotia.

Sclerotia, the medicinal part of Polyporus umbellatus, play important roles in diuresis and renal protection, with steroids and polysaccharides as the main active ingredients. The sclerotia grow and develop only after symbiosis with Armillaria sp. In this study, a systematic metabolomics based on non-targeted UPLC-MS method was carried out between the infected part of the separated cavity wall of the sclerotia (QR) and the uninfected part (the control group, CK) to find and identify differential metabolites. The biosynthetic pathway of characteristic steroids in sclerotia of P. umbellatus was deduced and the content of ergosterol, polyporusterone A and B in the QR and CK groups were detected with the High Performance Liquid Chromatography (HPLC). Furthermore, the expression patterns of putative genes associated with steroid biosynthesis pathway were also performed with quantitative real-time PCR. The results showed that a total of 258 metabolites originated from fungi with the fragmentation score more than 45 and high resolution mass were identified, based on UPLC-MS metabolomic analysis, and there were 118 differentially expressed metabolites (DEMs) between both groups. The metabolic pathways indicated that steroids, fatty acid and carbohydrate were active and enriched during P. umbellatus sclerotia infected by A. mellea. The content of ergosterol, polyporusterone A and B in the QR group increased by 32.2, 75.0, and 20.0%, in comparison to that of the control group. The qRT-PCR analysis showed that series of enzymes including C-8 sterol isomerase (ERG2), sterol C-24 methyltransferase (ERG6) and sterol 22-desaturase (ERG5), which played important roles in the final steps of ergosterol biosynthesis, all presented up-regulated patterns in the QR group in P. umbellatus. The comprehensive metabolomic and transcriptomic information will contribute to further study concerning the mechanisms of P. umbellatus sclerotial formation infected by A. mellea in the future.

Water extract of Armillaria mellea (Vahl) P. Kumm. Alleviates the depression-like behaviors in acute- and chronic mild stress-induced rodent models via anti-inflammatory action.

ETHNOPHARMACOLOGY RELEVANCE: Armillaria mellea (Vahl) P. Kumm. (AM) is an edible mushroom that has been reported as treatment for several neurological disorders, such as dizziness and epilepsy in Asia. Importantly, AM shares a symbiotic relationship with Gastrodia elata Blume (GE), a medicinal herb with antidepressant-like properties. Researchers believe that AM may possess pharmacological properties similar to GE due to their symbiosis, however, few studies have investigated the pharmacological effect of AM. AIM OF THE STUDY: The aim of this study was to explore the potential of AM as an antidepressant in forced-swimming test (FST) and unpredictable chronic mild stress (UCMS) rodent models and investigate its possible underlying mechanism. MATERIALS AND METHODS: Rats were orally administrated with 250, 500, and 1000 mg/kg body weight (bw) water extract of AM (WAM) for 28 and 35 consecutive days prior to the FST and UCMS protocols, respectively. The cerebral serotonin (5-HT) and the metabolites in the frontal cortex of rats were measured. The brain was dissected and the blood was collected to investigate the levels of inflammatory-related signaling pathway. RESULTS: All doses of WAM reduced the immobility time in the FST without disturbing autonomic locomotion. All doses of WAM prevented stress-induced abnormal behaviors in the UCMS model, including decreased sucrose preference and hypoactivity. 500 and 1000 mg/kg bw WAM attenuated the stress-induced increases in IL-1ß and TNF-α in the serum and cerebrum. 1000 mg/kg bw WAM alleviated brain inflammation by reducing the protein expression of ionized calcium binding adaptor molecule 1. CONCLUSION: WAM exhibited acute and chronic antidepressant-like effects, and may result from the anti-inflammatory actions. Therefore, the development of AM as a dietary therapy or adjuvant for depression treatment should be considered.

Study on antidepressant-like effect of protoilludane sesquiterpenoid aromatic esters from Armillaria Mellea.

Armillaria mellea, also known as Hazel mushroom, is a delicious food material and traditional herbal medicine in East Asia. Protoilludane sesquiterpenoid aromatic esters from A. mellea (PSAM) are the main active components with antibacterial and anticancer activities. This study explored the antidepressant-like activities of PSAM and its possible mechanisms of action using the open field test (OFT), tail suspension test (TST) and forced swimming test (FST) in mice for the first time. The results revealed that PSAM (1 mg/kg, i.p.) exhibited markedly antidepressant-like activity, which could be reversed by pretreatment with haloperidol (a non-selective D2 receptor antagonist), bicuculline (a competitive GABA antagonist), NMDA (an agonist at the glutamate site). Meanwhile, PSAM also effectively increased the hippocampus dopamine (DA) and γ-aminobutyric acid (GABA) and decreased the hippocampus glutamate (Glu) levels of mice, indicating that the antidepressant-like effect of PSAM might be mediated by the DAergic, GABAergic and Gluergic systems.

Identification of native endophytic Trichoderma spp. for investigation of in vitro antagonism towards Armillaria mellea using synthetic- and plant-based substrates.

AIMS: To isolate endophytic Trichoderma species and investigate the potential for biological control of the root rot pathogen Armillaria mellea. METHODS AND RESULTS: In all, 40 Trichoderma isolates were obtained from a range of host plants and identities were confirmed by ITS, rpb2 and tef1 sequence. When tested in dual culture assays for antagonism against A. mellea, Trichoderma isolates overgrew the A. mellea colonies within four days and by eight days 38 Trichoderma isolates significantly reduced A. mellea colony size. Armillaria mellea was unable to be recovered from five of eight co-cultivations tested, suggesting Trichoderma had killed the A. mellea in these cases. Pre-colonized hazel disks were used to determine what happens in a more heterogeneous situation with A. mellea and a refined set of eight Trichoderma isolates. Similar to plate-based assays, Trichoderma quickly covered A. mellea stopping any further growth and two Trichoderma isolates were able to eradicate A. mellea. CONCLUSIONS: Of the Trichoderma spp. tested, endophytic isolates of Trichoderma virens and T. hamatum offered the greatest antagonism towards A. mellea. Using pre-colonized hazel disks was of great importance for this work to demonstrate the fungal interactions in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: Controlling Armillaria root rot is difficult with chemical treatments, thus an environmentally benign and cost-effective alternative is required. This study highlights the prospect of biological control as an effective, environmentally friendly alternative to chemicals.

Susceptibility of Garden Trees and Shrubs to Armillaria Root Rot.

Armillaria root rot (ARR) is a serious disease of woody plants caused by several species of Armillaria. Armillaria isolates from diagnostic samples received in 2017 were identified by genus- and species-specific PCR and compared with isolates from an earlier survey (2004 to 2007). The results were comparable and, therefore, were combined for further analysis. Three species were identified: Armillaria mellea (83%), A. gallica (15%), and A. ostoyae (2%). Their wide host range makes choice of resistant plants in management of the disease difficult. We used the Royal Horticultural Society diagnostic dataset of ARR records from U.K. gardens to compare the susceptibility of different host genera to the disease. The dataset was compared with an earlier experiment at the University of California. An index-based approach was used to separate genera into three categories: 77 low-index (<0.99), 37 medium-index (0.99 to 1.76), and 56 high-index (>1.76) genera were recorded. All three species were associated with both angiosperms and gymnosperms; moreover, A. ostoyae did not show the host preference for gymnosperms that has been reported elsewhere. A. gallica was particularly common on herbaceous perennials and showed a trend to occur on resistant hosts that may be under other stress, supporting its description as an opportunistic pathogen. Four monocotyledons grown as trees or shrubs in U.K. gardens had a very low ARR index according to indices associated with A. mellea and A. ostoyae. Genera in the order Myrtales were almost always low index, while those in the Saxifragales and Fagales were mostly high index. These results provide confidence in the use of host resistance as part of the integrated management of ARR.

Chloroanisoles and Other Chlorinated Compounds in Cork from Different Geographical Areas.

Cork quality is crucial for the fabrication of corks intended to be used to seal wine bottles. This work has focused on the determination of chloroanisoles (CAs)-exogenous compounds with a low perception threshold-in cork. The identification and quantification of these compounds was carried out with Bond Elut-ENV solid phase extraction and gas chromatography with mass spectrometry detection. Cork samples were obtained from oaks from Catalonia, Extremadura and Italy, and the presence of CAs was evaluated. Moreover, cork affected by the presence of yellow stains (a defect present in cork, mainly originated from the growth of the fungus Armillaria mellea) was analysed separately. The results obtained from cork macerates revealed the presence of trichloroanisole (TCA) in Catalan and Italian cork. Furthermore, TCA concentration was not statistically different when comparing cork affected and non-affected by the growth of A. mellea. Other chlorinated compounds were identified by comparison of their mass spectra with the data from the NIST library.

Induction of Autophagic Death of Human Hepatocellular Carcinoma Cells by Armillaridin from Armillaria mellea.

The honey mushroom, Armillaria mellea, is known to have medicinal qualities and has been used in recent years as a health food and dietary supplement worldwide. In Asia, it is commonly consumed as an herbal medicine, being a key component of the Chinese preparation "Tien-ma". Here, we examined the antitumor effects of armillaridin, a bioactive compound isolated from A. mellea, on human hepatocellular carcinoma (HCC) cells. Armillaridin inhibited the growth of human Huh7, HepG2, and HA22T HCC cells, and its cytotoxicity was confirmed by observations of its induction of mitochondrial transmembrane potential collapse. However, armillaridin treatment did not result in large numbers of cells with fragmented chromosomal DNA, suggesting that apoptosis was not responsible for these effects. We therefore tested for signs of autophagic cell death following armillaridin administration. Armillaridin induced LC3 aggregation in green fluorescent protein-LC3-overexpressing cells. Moreover, flow cytometry and immunoblotting revealed that it increased the number of acridine orange-positive cells and upregulated autophagy-related proteins, respectively. Furthermore, armillaridin cytotoxicity was suppressed by the autophagy inhibitor 3-methyladenine. In summary, our results indicated that armillaridin induces HCC cell death by autophagy, and demonstrated the potential of armillaridin as an antihepatoma agent.

Microbial Asymmetric Functionalization of ß-Cyclocitral-Derived Tetramethyl-Substituted γ-Lactone.

Searching for the new anticancer compounds we prepared three new ß-cyclocitral-derived hydroxyl-γ-lactones by microbial hydroxylation of tetramethyl-substituted bicyclic γ-lactone. The substrate was transformed by the enzymatic system of filamentous fungi. Three out of fifteen strains were selected as effective biocatalysts (Fusarium culmorum AM10, Armillaria mellea AM296, Trametes versicolor AM536). The hydroxylation processes were not only regioselective but also stereoselective. The hydroxylation products of each secondary carbon atom in the cyclohexane ring were obtained by the application of the selected fungal strains. The Fusarium culmorum AM10 introduced the hydroxy function at C-3 and C-4, Armillaria mellea AM296 incorporated the hydroxy function at C-3 and C-5 and Trametes versicolor AM536 transformed the substrate to the mixture of C-3, C-4 and C-5 hydroxylactones. The hydroxylactones obtained were enantiomericaly enriched (ee values in the range 17⁻99%). The in vitro antiproliferative activities of the functionalization products were also evaluated. Regardless of the hydroxy substituent location all tested lactones exhibited similar, significant activity towards selected cancer cell lines (IC50 in the range 22.8⁻33.9 µg/mL).

Bioluminescence expression during the transition from mycelium to mushroom in three North American Armillaria and Desarmillaria species.

Unlike most bioluminescent fungi, mycelia of Armillaria and Desarmillaria are constitutively bioluminescent while mature mushrooms are not. The absence of the luciferin, 3-hydroxyhispidin, and its precursor hispidin in mature mushrooms have been proposed to explain the lack of bioluminescence from Armillaria mushrooms. Using three North American species, A. gallica, A. mellea and D. tabescens (syn., Armillaria tabescens), we documented a decline in luminescence of ten fold during the transition from mycelia to, immature mushrooms (i.e., pins) for the two Armillaria species. As pins matured, luminescence declined by an additional two or three orders of magnitude. Lower initial luminescence of D. tabescens mycelia declined to negligible levels during mushroom development. Further, light production was localized in the gills and lower stipe of A. mellea mushrooms. The decline in luminescence during mushroom formation was reversed by addition of hispidin to stipe or gills which significantly enhanced luminescence by one and three orders of magnitude, respectively. We conclude that the modulation of Armillaria and Desarmillaria luminescence is achieved by luciferin availability early in mushroom development. However, since the temporal regulation of bioluminescence differs between Armillaria species and other genera, we conclude that bioluminescence in Armillaria is under unique selective pressures.

Highly regioselective biotransformation of ginsenoside Rb2 into compound Y and compound K by ß-glycosidase purified from Armillaria mellea mycelia.

BACKGROUND: The biological activities of ginseng saponins (ginsenosides) are associated with type, number, and position of sugar moieties linked to aglycone skeletons. Deglycosylated minor ginsenosides are known to be more biologically active than major ginsenosides. Accordingly, the deglycosylation of major ginsenosides can provide the multibioactive effects of ginsenosides. The purpose of this study was to transform ginsenoside Rb2, one of the protopanaxadiol-type major ginsenosides, into minor ginsenosides using ß-glycosidase (BG-1) purified from Armillaria mellea mycelium. METHODS: Ginsenoside Rb2 was hydrolyzed by using BG-1; the hydrolytic properties of Rb2 by BG-1 were also characterized. In addition, the influence of reaction conditions such as reaction time, pH, and temperature, and transformation pathways of Rb2, Rd, F2, compound O (C-O), and C-Y by treatment with BG-1 were investigated. RESULTS: BG-1 first hydrolyzes 3-O-outer ß-d-glucoside of Rb2, then 3-O-ß-d-glucoside of C-O into C-Y. C-Y was gradually converted into C-K with a prolonged reaction time, but the pathway of Rb2 → Rd → F2 → C-K was not observed. The optimum reaction conditions for C-Y and C-K formation from Rb2 by BG-1 were pH 4.0-4.5, temperature 45-60°C, and reaction time 72-96 h. CONCLUSION: ß-Glycosidase purified from A. mellea mycelium can be efficiently used to transform Rb2 into C-Y and C-K. To our best knowledge, this is the first result of transformation from Rb2 into C-Y and C-K by basidiomycete mushroom enzyme.